Rapid tests for the diagnosis of visceral leishmaniasis in patients with suspected disease

  • Review
  • Diagnostic

Authors


Abstract

Background

The diagnosis of visceral leishmaniasis (VL) in patients with fever and a large spleen relies on showing Leishmania parasites in tissue samples and on serological tests. Parasitological techniques are invasive, require sophisticated laboratories, consume time, or lack accuracy. Recently, rapid diagnostic tests that are easy to perform have become available.

Objectives

To determine the diagnostic accuracy of rapid tests for diagnosing VL in patients with suspected disease presenting at health services in endemic areas.

Search methods

We searched MEDLINE, EMBASE, LILACS, CIDG SR, CENTRAL, SCI-expanded, Medion, Arif, CCT, and the WHO trials register on 3 December 2013, without applying language or date limits.

Selection criteria

This review includes original, phase III, diagnostic accuracy studies of rapid tests in patients clinically suspected to have VL. As reference standards, we accepted: (1) direct smear or culture of spleen aspirate; (2) composite reference standard based on one or more of the following: parasitology, serology, or response to treatment; and (3) latent class analysis.

Data collection and analysis

Two review authors independently extracted data and assessed quality of included studies using the QUADAS-2 tool. Discrepancies were resolved by a third author. We carried out a meta-analysis to estimate sensitivity and specificity of rapid tests, using a bivariate normal model with a complementary log-log link function. We analysed each index test separately. As possible sources of heterogeneity, we explored: geographical area, commercial brand of index test, type of reference standard, disease prevalence, study size, and risk of bias (QUADAS-2). We also undertook a sensitivity analysis to assess the influence of imperfect reference standards.

Main results

Twenty-four studies containing information about five index tests (rK39 immunochromatographic test (ICT), KAtex latex agglutination test in urine, FAST agglutination test, rK26 ICT, and rKE16 ICT) recruiting 4271 participants (2605 with VL) were included. We carried out a meta-analysis for the rK39 ICT (including 18 studies; 3622 participants) and the latex agglutination test (six studies; 1374 participants). The results showed considerable heterogeneity. For the rK39 ICT, the overall sensitivity was 91.9% (95% confidence interval (95% CI) 84.8 to 96.5) and the specificity 92.4% (95% CI 85.6 to 96.8). The sensitivity was lower in East Africa (85.3%; 95% CI 74.5 to 93.2) than in the Indian subcontinent (97.0%; 95% CI 90.0 to 99.5). For the latex agglutination test, overall sensitivity was 63.6% (95% CI 40.9 to 85.6) and specificity 92.9% (95% CI 76.7 to 99.2).

Authors' conclusions

The rK39 ICT shows high sensitivity and specificity for the diagnosis of visceral leishmaniasis in patients with febrile splenomegaly and no previous history of the disease, but the sensitivity is notably lower in east Africa than in the Indian subcontinent. Other rapid tests lack accuracy, validation, or both.

Résumé scientifique

Les tests rapides pour le diagnostic de la leishmaniose viscérale chez les patients suspectés d'être atteints de la maladie

Contexte

Le diagnostic de la leishmaniose viscérale (LV) chez des patients avec de la fièvre et une grosse rate repose sur la mise en évidence des parasites Leishmania dans des prélèvements tissulaires et sur des examens sérologiques. Les techniques parasitologiques sont invasives, nécessitent des laboratoires sophistiqués, prennent du temps ou manquent de précision. Récemment, des tests diagnostiques rapides, faciles à réaliser, sont devenus disponibles.

Objectifs

Déterminer la précision des tests rapides dans le diagnostic de la LV chez des patients suspectés d'être atteints de la maladie, se présentant dans des services de santé de zones d'endémie.

Stratégie de recherche documentaire

Nous avons effectué des recherches dans MEDLINE, EMBASE, LILACS, CIDG SR, CENTRAL, SCI-expanded, Medion, Arif, CCT et le registre des essais cliniques de l'OMS le 3 décembre 2013, sans appliquer de restriction de langue ni de limite de date.

Critères de sélection

Cette revue inclut des études originales, phase III, portant sur la précision diagnostique de tests rapides chez des patients suspectés cliniquement d'être atteints de LV. Comme étalon de référence, nous avons accepté : (1) l'analyse microscopique directe ou la culture de l'aspiration splénique; (2) un composé d'éléments de référence basé sur un ou plusieurs éléments suivants: la parasitologie, la sérologie ou la réponse au traitement; et (3) l'analyse de classes latentes.

Recueil et analyse des données

Deux auteurs de la revue ont extrait les données et évalué la qualité des études incluses à l'aide de l'outil QUADAS-2. Les divergences ont été résolues par un troisième auteur. Nous avons réalisé une méta-analyse pour estimer la sensibilité et la spécificité des tests rapides, à l'aide d'un modèle normal bivarié avec une fonction de lien log-log complémentaire. Nous avons analysé séparément chacun des tests évalués. Comme possibles sources d'hétérogénéité, nous avons exploré : la zone géographique, la marque commerciale du test, le type de l'étalon de référence, la prévalence de la maladie, la taille de l'étude et le risque de biais (QUADAS -2). Nous avons également entrepris une analyse de sensibilité pour évaluer l'influence des étalons de référence imparfaits.

Résultats principaux

Vingt-quatre études ont été incluses, elles contenaient des informations sur cinq tests évalués (test immunochromatographique (TIC) rK39, test KAtex d'agglutination au latex dans l'urine, test d'agglutination FAST, TIC rK26 et TIC rKE16) et avaient recruté 4 271 participant (2 605 avec une LV). Nous avons réalisé une méta-analyse pour le TIC rK39 (incluant 18 études ; 3 622 participants) et le test d'agglutination au latex (six études ; 1 374 participants). Les résultats ont montré une hétérogénéité considérable. Pour le TIC rK39, la sensibilité globale était de 91,9 % (intervalle de confiance à 95 % (IC à 95 %) de 84,8 à 96,5) et la spécificité de 92,4 % (IC à 95 % de 85,6 à 96,8). La sensibilité était plus faible dans les pays d'Afrique de l'Est (85,3 %; IC à 95 % de 74,5 à 93.2) que dans le sous-continent indien (97,0 % ; IC à 95 % de 90,0 à 99,5). Pour le test d'agglutination au latex, la sensibilité globale était de 63,6 % (IC à 95 % de 40,9 à 85,6) et la spécificité de 92,9 % (IC à 95 % de 76,7 à 99,2).

Conclusions des auteurs

Le TIC rK39 montre une sensibilité et une spécificité élevées pour le diagnostic de la leishmaniose viscérale chez les patients présentant une splénomégalie fébrile et sans antécédents de la maladie, mais la sensibilité est notablement plus faible en Afrique de l'Est que dans le sous-continent indien. D'autres tests rapides manquent de précision, de validation ou des deux.

Resumo

Testes rápidos para o diagnóstico da leishmaniose visceral em pacientes com suspeita de doença

Introdução

O diagnóstico da leishmaniose visceral (LV) em pacientes com febre e baço aumentado depende da identificação dos parasitas da Leishmania em amostras de tecidos e em testes sorológicos. As técnicas parasitológicas são invasivas, exigem laboratórios sofisticados, consomem tempo, e carecem de precisão. Recentemente, os testes de diagnóstico rápido, que são fáceis de executar, tornaram-se disponíveis.

Objetivos

Avaliar a acurácia de testes rápidos para diagnosticar LV em pacientes com suspeita da doença atendidos nos serviços de saúde em áreas endêmicas.

Métodos de busca

Realizamos buscas em 3 de dezembro de 2013 nas seguintes bases de dados: MEDLINE, EMBASE, LILACS, CIDG SR, CENTRAL, SCI-expanded, Medion, Arif, CCT, e no WHO Trials Register. Não houve restrição quanto ao idioma ou à data de publicação dos estudos.

Critério de seleção

Esta revisão inclui estudos originais, de fase III, de acurácia diagnóstica dos testes rápidos em pacientes com suspeita clínica de LV. Os padrões de referência foram: (1) exame microscópico direto (esfregaço) ou cultura do aspirado esplênico; (2) padrão de referência combinado baseado em um ou mais dos seguintes: parasitologia, sorologia ou resposta ao tratamento; e (3) a análise de classe latente.

Coleta dos dados e análises

Dois autores, de modo independente, extraíram os dados e avaliaram a qualidade dos estudos incluídos utilizando a ferramenta QUADAS-2. As discrepâncias foram resolvidas por um terceiro autor. Realizou-se uma metanálise para estimar a sensibilidade e especificidade dos testes rápidos utilizando um modelo de distribuição normal bivariada com uma função de ligação complementar log-log. Analisamos cada teste separadamente. Avaliamos as seguintes variáveis como possíveis fontes de heterogeneidade: a área geográfica, a marca comercial do teste, o tipo de padrão de referência, a prevalência da doença, o tamanho do estudo e o risco de viés (QUADAS-2). Nós também realizamos uma análise de sensibilidade para avaliar a influência de padrões de referência imperfeitos.

Principais resultados

Um total de 24 estudos foram incluídos. Esses estudos apresentavam informações sobre cinco testes: imunocromatográfia rK39 (TIC), aglutinação em látex na urina (Katex), aglutinação rápida (FAST), o TIC rK26 e o TIC rKE16. Esses 24 estudos recrutaram 4.271 participantes (2.605 com LV). Realizamos meta-análises para o TIC rK39 (incluindo 18 estudos; 3.622 participantes) e para o teste de aglutinação em látex (6 estudos; 1.374 participantes). Houve heterogeneidade considerável. Para o TIC rK39, a sensibilidade global foi de 91,9% (intervalo de confiança de 95% [CI 95%] 84,8 - 96,5) e a especificidade global foi de 92,4% (CI 95% 85,6 - 96,8). A sensibilidade foi menor no leste da África (85,3%; CI 95% 74,5 - 93,2) do que no subcontinente indiano (97,0%; CI 95% 90,0 - 99,5). Para o teste de aglutinação em látex, a sensibilidade global foi 63,6% (CI 95% 40,9 - 85,6) e a especificidade global foi de 92,9% (CI 95% 76,7 - 99,2).

Conclusão dos autores

O TIC rK39 apresenta alta sensibilidade e especificidade para o diagnóstico da leishmaniose visceral em pacientes com esplenomegalia febril e sem história prévia de doença, mas a sensibilidade é consideravelmente menor no leste da África do que no subcontinente indiano. Os demais testes rápidos carecem de acurácia, validação ou ambos.

Notas de tradução

Tradução do Centro Cochrane do Brasil (Aline de Gregori Adami)

Plain language summary

Rapid diagnostic tests for visceral leishmaniasis

Visceral leishmaniasis (or kala-azar) is caused by a parasite, results in fever, a large spleen and other health problems, occuring in India, Bangladesh and Nepal, east Africa, the Mediterranean region and Brazil. Without treatment people die, and proper treatment can result in cure, so diagnosis is important. Many of the tests that are used to determine if a person has visceral leishmaniasis are complicated, costly, painful and sometimes dangerous for the patients. Now rapid diagnostic tests that are safe and easy to perform are available.

This Cochrane review describes how accurate these rapid diagnostic tests are for diagnosing visceral leishmaniasis. We summarize those studies that evaluated the rapid tests in people who, according to their physicians, could have the disease. We only included studies in which the researchers had used established methods to distinguish the people with visceral leishmaniasis from those who did not have the disease.

We found 24 studies, which contained information about five different rapid tests. A total of 4271 people participated in these studies. One of the rapid tests (called the rK39 immunochromatographic test) gave correct, positive results in 92% of the people with visceral leishmaniasis and it gave correct, negative results in 92% of the people who did not have the disease. This test worked better in India and Nepal than in east Africa. In India and Nepal, it gave correct, positive results in 97% of the people with the disease. In east Africa, it gave correct, positive results in only 85% of the people with the disease.

A second rapid test (called latex agglutination test) gave correct, positive results in 64% of the people with the disease and it gave correct, negative results in 93% of the people without the disease. For the other rapid tests evaluated, there are too few studies to know how accurate they are.

Résumé simplifié

Les tests de diagnostic rapide pour la leishmaniose viscérale

La leishmaniose viscérale (ou kala-azar) est causée par un parasite, et il en résulte de la fièvre, une grosse rate et d'autres problèmes de santé. Elle est présente en Inde, au Bangladesh et au Népal, en Afrique de l'Est, dans le bassin méditerranéen et au Brésil. Sans traitement, les personnes infectées en meurent, tandis qu'un traitement adéquat peut entraîner la guérison, de sorte que le diagnostic est important. De nombreux examens utilisés pour déterminer si une personne est atteinte de leishmaniose viscérale sont compliqués, coûteux, douloureux et parfois dangereux pour les patients. Aujourd'hui, des tests diagnostiques rapides sûrs et faciles à réaliser sont disponibles.

Cette revue Cochrane décrit quelle est l'efficacité de ces tests diagnostiques rapides pour diagnostiquer la leishmaniose viscérale. Nous résumons les études qui ont évalué les tests rapide chez des personnes qui, selon leurs médecins, pouvaient avoir la maladie. Nous avons uniquement inclus les études dans lesquelles les chercheurs avaient utilisé des méthodes établies pour distinguer les personnes atteintes de leishmaniose viscérale de celles qui n'avaient pas la maladie.

Nous avons trouvé 24 études, qui contenaient des informations sur cinq différents tests rapides. Un total de 4 271 patients ont été recrutés dans ces études. L'un des tests rapides (dit test immunochromatographique rK39) donnait des résultats positifs corrects chez 92 % des personnes atteintes de leishmaniose viscérale et des résultats négatifs corrects chez 92 % des personnes n'ayant pas eu la maladie. Ce test a mieux fonctionné en Inde et au Népal qu'en Afrique de l'Est. En Inde et au Népal, il donnait des résultats positifs corrects pour 97 % des personnes atteintes de la maladie. En Afrique de l'Est, il donnait des résultats positifs corrects pour seulement 85 % des personnes atteintes de la maladie.

Un deuxième test rapide (dit test d'agglutination au latex) a donné des résultats positifs corrects chez 64% des personnes atteintes de la maladie et des résultats négatifs corrects chez 93 % des personnes sans la maladie. Pour les autres tests rapides évalués, il existe trop peu d'études pour savoir quelle est leur efficacité diagnostique.

Notes de traduction

Traduit par: French Cochrane Centre 9th November, 2014
Traduction financée par: Financeurs pour le Canada : Instituts de Recherche en Santé du Canada, Ministère de la Santé et des Services Sociaux du Québec, Fonds de recherche du Québec-Santé et Institut National d'Excellence en Santé et en Services Sociaux; pour la France : Ministère en charge de la Santé

Resumo para leigos

Testes rápidos para o diagnóstico da leishmaniose visceral

A leishmaniose visceral (ou calazar) é uma doença causada por um parasita e os sintomas incluem febre, baço aumentado e outros problemas de saúde. A leishmaniose visceral ocorre na Índia, Bangladesh e Nepal, leste da África, na região do Mediterrâneo e no Brasil. O diagnóstico é importante, pois sem tratamento, as pessoas morrem e o tratamento adequado pode levar a cura. Muitos dos testes usados para saber se uma pessoa tem leishmaniose visceral são complicados, caros, dolorosos e às vezes perigosos para o paciente. Recentemente, foram lançados no mercado os testes rápidos de diagnóstico, que são seguros e fáceis de fazer.

Esta revisão Cochrane avaliou a acurácia desses testes rápidos (ou seja, o quanto eles acertam o diagnóstico) para detectar a leishmaniose visceral. Nós resumimos os estudos que avaliaram os testes rápidos em pessoas que, de acordo com seus médicos, poderiam ter a doença. Incluímos apenas estudos em que os pesquisadores também usaram outros métodos diagnósticos tradicionais para concluir quem tinha e quem não tinha a doença.

Encontramos 24 estudos que traziam informações sobre cinco testes rápidos diferentes. Um total de 4.271 pessoas participou desses estudos. Um dos testes rápidos (denominado teste imunocromatográfico rK39) teve resultados positivos corretos em 92% das pessoas com leishmaniose visceral e teve resultados negativos corretos em 92% das pessoas que não tinham a doença. Esse teste apresentou melhores resultados na Índia e Nepal do que no leste da África. Na Índia e no Nepal, ele teve resultados positivos corretos em 97% das pessoas com a doença. No leste da África, teve resultados positivos corretos em apenas 85% das pessoas com a doença.

Um segundo teste rápido (denominado teste de aglutinação em látex) teve resultados positivos corretos em 64% das pessoas com a doença e resultados negativos corretos em 93% das pessoas que não tinham a doença. Não há estudos suficientes disponíveis para saber qual é a acurácia dos outros testes rápidos existentes.

Notas de tradução

Tradução do Centro Cochrane do Brasil (Aline de Gregori Adami)

Background

Target condition being diagnosed

Visceral leishmaniasis (VL), also known as kala-azar, is a life-threatening systemic disease caused by the obligate intracellular protozoan, Leishmania, and transmitted through the bites of phlebotomine sand flies (Herwaldt 1999). Leishmanial infection can cause a diverse spectrum of diseases, among which VL is the most severe form and which is almost always fatal without adequate, timely treatment. The species that causes VL isLeishmania donovani in Asia and eastern Africa, and L. infantum in Europe, North Africa and Latin America (Boelaert 2000). The geographical distribution of VL is often limited to well-identified endemic foci, but it has also emerged as epidemics (Seaman 1996) and as one of the opportunistic infections in human immunodeficiency virus (HIV)-positive patients (Alvar 2008; Pasquau 2005). VL is a neglected disease that affects the poorest and most vulnerable people in rural, remote settings where there is limited access to health care. The estimated incidence is 200,000 to 400,000 cases per year, of which more than 90% are reported from India, Bangladesh, Ethiopia, Sudan, South Sudan, and Brazil (Alvar 2012). The two main transmission modes of VL are anthroponotic and zoonotic. The human-to-human transmission is predominant in the Indian subcontinent and in eastern Africa, while the zoonotic transmission mode is found in the Mediterranean region and the Americas. The zoonotic transmission mode involves dogs as the main parasite reservoir (Chappuis 2007).

The pathogenesis of VL entails a complex interaction between the characteristics of the parasite and the host (Rittig 2000). The parasite life cycle involves the replication of the promastigote form in the gut of the female sand fly and is completed when she takes a blood meal on a non-immune host. In the human body, the Leishmania promastigote evolves into an amastigote form (without flagellum), which colonizes the macrophages in the liver, spleen and bone marrow. The control of the infection depends on the characteristics of the host, especially an intact cell-mediated immunity in the form of a T-helper type 1 response, involving the secretion of interleukin 12 and interferon-gamma. The failure of this cell-mediated immunity in some, but not all, of the infected individuals ultimately leads to the clinical manifestations of VL disease (Murray 2005).

The main clinical manifestations of VL are fever of insidious onset, weakness, loss of appetite, weight loss, abdominal distension due to enlargement of the spleen or the liver or both, lymph node enlargement, and a low blood cell count (pancytopenia). In children, other symptoms include diarrhoea, coughing, abdominal pain, and growth retardation. In any age group, if left untreated, the disease will progress with time, causing debilitation, bleeding, susceptibility to secondary infection and, eventually, death. Anti-leishmanial treatment is life-saving but non-response or relapse can occur. The pentavalent antimonials, sodium stibogluconate and meglumine antimoniate, despite their toxicity, have been the mainstay of VL treatment in many areas for decades. Alternatives include (liposomal) amphotericin B, paromomycin, and miltefosine. Combination therapy is the suggested way forward to increase treatment efficacy, prevent resistance, and reduce treatment duration and cost (van Griensven 2010). It is important that diagnostic tests for VL do not give false-negative results because VL may be fatal. Neither should they give false-positive results in order to prevent people without VL from receiving the toxic VL treatment.

As stated above, asymptomatic or subclinical infections also occur, and in cross-sectional surveys in endemic areas, a significant proportion of the healthy individuals present with anti-leishmanial antibodies. In longitudinal studies, the ratio between incident clinical cases and incident asymptomatic infections ranged from 1:5 in Kenya (Ho 1982) to 1:8 in Brazil (Evans 1992), 1:9 in Ethiopia (Hailu 2009), 1:8.9 in India and Nepal (Ostyn 2011), and 1:11 and 2.6:1 in Sudan (Zijlstra 1994). Furthermore, antibodies (seropositivity) in VL patients tend to persist for many years after cure (De Almeida 2006; Hailu 1990).

Index test(s)

Rapid diagnostic tests (RDTs) are defined as equipment-free diagnostic devices that do not require highly skilled laboratory staff. The results of an RDT can be read easily within minutes, or at most an hour or two (PATH 2008). Most RDTs work by capturing either an antigen or an antibody on a solid surface and then attaching molecules to them that allow detection by the naked eye. The technology used is mainly immunochromatography with a dipstick or lateral flow format. These immunochromatographic tests (ICTs) are used for VL diagnosis in dipstick or cassette format, using protein isolated from Leishmania sp as the antigen. The recombinant form of the 39 amino-acid-sequence from L. chagasi is the most widely used and is known as rK39. Other recombinant antigens such as rK9, rK16, rK26 and rK28 have also been evaluated. Currently, there are several on-going projects to develop a VL test based on antigen detection, for example, the latex agglutination test detecting a urinary leishmanial antigen.

The rK39 antigen was first used in an enzyme-linked immunosorbent assay (ELISA) format, but the newer dipstick or strip formats are increasingly being used in the field or away from the main centres. These rK39 ICTs give an immediate result (typically between 10 and 20 minutes) and give a binary reading (positive or negative). The test procedure involves adding the patient’s blood or serum with diluent buffer on the strip. When present in the blood or serum, specific antibodies against rK39 antigens, which are bound to the strip, can be read with the naked eye in the test window. There is a limited number of commercially available RDTs for VL, such as the IT-LEISH® (DiaMed AG, Switzerland – now Biorad, France), Kalazar Detect® (InBios International, USA), Onsite Leishmania Ab Rapid Test (CTK Biotech, USA) as well as a number of prototypes under various formats (Cunningham 2012).

rK39 ICTs cannot be used to diagnose VL in people with a past history of VL due to the persistence of antibodies after cure. In addition, to avoid diagnosis of asymptomatic infections, these tests must be applied in patients suspected to have VL, ie patients with prolonged fever and splenomegaly (WHO 2010).

Clinical pathway

Prior test(s)

The RDTs are meant to be used in patients with a clinical suspicion of VL without previous history of VL. According to the case definition proposed by the World Health Organization (WHO), VL is an illness with prolonged irregular fever, splenomegaly and weight loss as its main symptoms (WHO 2010).

Role of index test(s)

The RDTs were specifically developed for field use in VL-endemic areas. If they are sufficiently accurate, they could be used for the early diagnosis of VL at both peripheral and central levels. A positive RDT result would then confirm the diagnosis of VL in clinically suspected patients and allow the start of treatment (WHO 2010).

Alternative test(s)

A definite VL diagnosis is provided by the demonstration of the parasite through a microscopic visualization from spleen, bone marrow or lymph node aspirates. These invasive procedures are not always feasible in field settings, and using them is by no means free of risk: spleen aspirates are the most sensitive (93% to 99%) but the aspiration carries a rare but fatal risk of bleeding (Zijlstra 1992). Splenic aspiration should only be performed if there is rapid access to blood transfusion in case of bleeding and if there are no contraindications. Clinical contraindications include signs of active bleeding, jaundice, pregnancy, a barely palpable spleen and a bad general condition. Biological contraindications include severe anaemia, a prolonged prothrombin time and a low platelet count (WHO 2010). Bone marrow and lymph node aspiration are safer, but both have lower sensitivity (Sarker 2004; Babiker 2007). Parasitological confirmation through culture or molecular techniques such as a polymerase chain reaction (PCR) is possible, but their complexity restricts their use as a routine diagnosis method. In addition, in endemic areas, a substantial proportion of healthy individuals have parasite DNA in the blood, which is detected by PCR (Bhattarai 2009).

Serological tests based on indirect fluorescence antibody, ELISA and Western blot were developed but their use is limited in the first-line health services of endemic areas as they require a too sophisticated laboratory infrastructure. A more useful antibody detection test is the direct agglutination test (DAT), a semi-quantitative method based on visual agglutinations obtained by the increased dilution of blood or serum mixed with stained, killed parasites in V-shaped wells (Harith 1986). It has been extensively validated and used in the field. Nonetheless, it cannot be categorized as an RDT because it requires a degree of laboratory skills, facilities and equipment, and the result is ready only after overnight incubation (Boelaert 1999).

Rationale

Correctly diagnosing VL disease is crucial as the signs and symptoms of VL are not specific enough to differentiate the condition from chronic malaria, schistosomiasis or other systemic infections. VL should be suspected in patients presenting with fever and splenomegaly, but confirmation is needed. The classical method relying on microscopic smears from tissue aspirates (spleen, bone marrow, lymph node) are unsuitable for settings with limited resources. Other methods such as antibody detection are more feasible, most notably the DAT and the rK39 antigen-based ICT. Both have been specifically developed in the past two decades for use in such contexts and have shown high diagnostic accuracy in most endemic areas (Boelaert 2004; Chappuis 2006).

The major drawbacks of these serological methods are linked to the antibodies that remain detectable after a cure and those that are due to past or present asymptomatic infection present in a sizeable proportion of residents of endemic areas. The use of a clinical case definition and adequate medical history taking should help to avoid false positives, yet it underscores the need for better VL diagnosis tests that are specific to acute stage disease. Ideally, the test should be highly sensitive, as VL is potentially fatal, and it should also be specific, as presumptive treatment cannot be fully justified with the current treatment regimens (den Boer 2006).

The RDTs for VL, mainly but not exclusively the rK39-based ICTs, seem to be the current solution for field diagnosis in remote settings: their ease of use, convenience and cost make them potentially advantageous to increase patients' access to VL diagnosis and treatment. The current WHO recommendation for VL case management includes the use of the rK39-based ICT as the basis for initiating treatment and it has also been adopted as the diagnostic tool in first-line services in the VL elimination initiative in the Indian subcontinent (WHO 2005). In other areas, the RDTs have been used as the first test in diagnosis-treatment algorithms that often incorporate other test(s), such as the DAT or tissue aspiration (Raguenaud 2007; Veeken 2003;).

Despite its operational advantages, some regional variability in rK39-based ICT performance has been observed (Chappuis 2006). Other prototype tests have been proposed as RDTs, but their real value in clinical practice is unclear. Furthermore, in the published literature, many phase II studies (with a sub-optimal, case-control design) might overshadow the more limited information of better quality based on phase III studies (including consecutive patients suspected to have VL). Thus, it is of utmost importance to synthesize the available evidence in this rapidly evolving field. Knowing the diagnostic accuracy of these RDTs would help inform policy makers and stakeholders on how best to use them in VL control in diverse settings. The role of RDTs in the different epidemiological contexts could be better defined, giving clearer evidence on their accuracy, and this review aims to contribute to exactly this goal.

Objectives

To determine the diagnostic accuracy of rapid tests for the diagnosis of visceral leishmaniasis in patients with suspected disease presenting at health services in endemic areas.

Secondary objectives

Investigation of sources of heterogeneity

We investigated differences in diagnostic accuracy in relation to test conditions (index and reference: commercial brand of index test, type of reference standard), geographical region (the Indian subcontinent, eastern Africa, Latin America and the Mediterranean region), disease prevalence in the sample, and study quality.

Methods

Criteria for considering studies for this review

Types of studies

Only original diagnostic accuracy studies of the phase III type (Zhou 2002) were included in the review, that is, prospective or retrospective cohort studies (meaning studies with patients who were consecutively enrolled, be it prospectively or retrospectively, and in which all patients were given the index test and reference standard), or studies that enrolled a random selection of patients from a series of patients. Randomized controlled trials in which patients were randomized to one of several index tests and all received the reference test could also be included.

Participants

Patients with a clinical suspicion of VL, that is, those who are febrile for more than two weeks, and present with splenomegaly; according to the WHO case definition (WHO 2010) presenting at health services in endemic areas.

We excluded studies with participants:

  • who were previously treated for VL (non-responders or relapsed cases); and

  • who had signs and symptoms of other forms of leishmaniasis, such as post kala-azar dermal leishmaniasis (PKDL).

Studies in which only a subgroup of participants was eligible for the review were included if it was possible to extract relevant data specific to that subgroup.

Studies of patients with HIV or other co-infections were eligible for this review.

Index tests

All types of RDTs for VL, with results that are read out within one hour, regardless of the manufacturer. The RDTs could be assessed alone or in comparison with other tests.

Target conditions

The target condition was restricted to current clinical VL, and did not include asymptomatic leishmanial infection or VL in the past. 

Reference standards

We accepted the following reference standards for the diagnosis of active VL (adapted from Boelaert 2007):

  • reference standard including direct smears or culture of splenic aspirate;

  • composite reference standard (Sullivan 2003) based on a combination of several tests. Test algorithms could include smears or culture of tissue aspirate, serology (other than the index test), or clinical arguments; and

  • latent class analysis (LCA) (Zhou 2002) based on one or more of the following: smears or culture of tissue aspirates, serology (other than the index test), clinical response to antimonial treatment; or specific clinical signs (pancytopenia, darkened skin). To be selected, the studies had to assess the conditional independence assumption between the tests, and, if conditional dependence was expected, they had to use appropriate statistical methods (Dendukuri 2001). More information about LCA is given in Appendix 1.

Search methods for identification of studies

We attempted to identify all relevant studies regardless of their publication status (published, unpublished, in press, ongoing). No language or date limits were applied. We limited our searches to studies conducted in humans.

Electronic searches

To identify all relevant studies, we searched the following databases using the search terms and strategy described in Appendix 2: MEDLINE, EMBASE, LILACS, CIDG SR, the Cochrane Central Register of Controlled Trials (CENTRAL) published in The Cochrane Library, ISI Web of Knowledge (Science Citation Index Expanded), Medion, Arif, and CCT.

We also searched the metaRegister of Controlled Trials (mRCT) and the WHO Clinical Trials Registry Platform using "leishman*" and "diagnos* OR RDT*" as search terms.

MeSH and other search terms included: visceral leishmaniasis, kala-azar, Leishmania donovani, Leishmania infantum,Leishmania chagasi, K39, immunoassay, chromatography, dipstick, immunochromatography, rapid diagnostic tests; RDT; antibody detection, serology.

Searching other resources

We checked the reference lists of the studies identified by the above methods. We also checked conference proceedings and reports from the WHO.

Data collection and analysis

Selection of studies

Two review authors assessed the titles and abstracts identified through the search strategy. All potentially relevant articles were retrieved in full and two authors (MB and TS or MB and KV) independently examined them for inclusion in the present review using an assessment form. Any discrepancies were resolved by discussion with a third author (JvG).

Data extraction and management

We extracted a standard set of data from each study, using a specifically modified data extraction form. If only a subgroup of participants in a study was eligible, we extracted the data for that particular subgroup.

Two authors independently extracted the data (MB and JvG). The discrepancies were resolved by discussion with a third author (KV).

For each study, data were extracted on:

Study IDFirst author, year of publication.
Clinical features and settingsPresenting signs and symptoms, clinical setting.
ParticipantsSample size, summary statistics of age (mean or median), sex (% male or female), HIV status (% positive, if available), country.
Study design

Whether the sample was consecutive or random.

Were consecutive patients enrolled retrospectively or prospectively?

If the study evaluated more than one RDT, how were tests allocated to individuals, or did each individual receive all tests?

Target condition Leishmania species, clinical features.
Reference standard

The reference standard test(s) used.

Who performed the reference standard test(s) and where?

How many observers or repeats were used?

How were discrepancies between observers resolved?

If not all patients received the reference tests, how many did not (and what proportion were they of the total)?

If any participant received a different reference test, what reasons were stated for this, and how many participants were involved?

Index test

The commercial name, with batch number, if provided.

Transport and storage conditions.

Details of the test operators.

Index test results and reference standard results

Number of missing, not interpretable or doubtful results (for both index and reference tests).

For each index test (and for each acceptable reference standard, if more than one reference standard was reported), the number of true and false positives, false and true negatives (2 x 2 contingency table); or for studies that used LCA as the reference standard: estimates and 95% confidence intervals (CI) for the prevalence and for the sensitivity and specificity of each index test.

NotesAnything else of relevance

Assessment of methodological quality

We first assessed the standard signalling questions of the QUADAS-2 tool (Whiting 2011) and decided to omit one question (“If a threshold was used, was it pre-specified?”) and not to add any specific signalling questions for this review.

The QUADAS-2 tool comprises four domains: patient selection, index test, reference standard, and flow and timing. We considered that the risk of bias for a certain domain was low if all the signalling questions for that domain indicated a low risk of bias. If the answer to at least one signalling question for a certain domain was 'no', the risk of bias for that domain was considered to be high. If none of the answers was 'no', but the answer to at least one question was 'unclear', the risk of bias for that domain was considered to be unclear.

Two review authors (JVG and MB) then independently applied the tool to the included records. For each record, they assessed risk of bias and concerns about applicability. Several of the original studies included in this review were conducted by the review team. In order to ensure an objective assessment of all the included records, the judgements about eligibility, bias and applicability were entirely based on the published documents and not on unpublished background information. In addition, two of the three review authors who made the judgements about bias and applicability (JVG and KV) had not been involved in any of the original studies. In case of discrepant judgements, KV took the final decision.

Statistical analysis and data synthesis

The aim of the analysis was to estimate the diagnostic accuracy, that is, the sensitivity (Se) and specificity (Sp), of the index tests (RDTs for VL). We analysed each RDT separately and did not plan to make comparisons between different index tests.

The analysis was performed using R (R Development Team 2010) and WinBugs (Spiegelhalter 2003) because of these programs’ flexibility in model fitting, in line with recommendations in the Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy - Chapter 10 Analysing and Presenting Results (Macaskill 2010). More technical detail on the statistical methods can be found in Appendix 1 and Menten 2013.

1) Outcome data

The statistical analysis was based on summary data as provided in the source publications: either the number of true and false positive or negative test results compared to the reference test, or the Sensitivity (Se) and Specificity (Sp) of the index tests and 95% CI or credible intervals obtained from latent class analysis.

For those studies that reported data using several reference standards (Table 1), we extracted and summarized all 2 x 2 tables or Se and Sp estimates. The main analysis used a single 2 x 2 table or set of Se and Sp estimates for each study. The primary data for each study was selected based on a predefined ranking of reference standards: (1) latent class analysis, (2) parasitology and serology, (3) parasitology including spleen aspirate without serology, and (4) parasitology not including spleen aspirate without serology. The influence of the possible selection of alternative data sets was explored in sensitivity analyses.

Table 1. Studies on rK39 ICT presenting two sets of Se and Sp estimates based on different reference standards: estimates included in primary analysis and sensitivity analysis
  1. * Cota 2013 describes HIV-infected patients only and is not included in the formal meta-analysis

Study Selected for primary analysisSelected for sensitivity analysis
Boelaert 2004 - LCA & Boelaert 2004 - classicLCAParasitology including spleen aspirate – no serology
Boelaert 2008 - Ethiopia & Diro 2007LCAParasitology including spleen aspirate – no serology
Boelaert 2008 - India & Sundar 2007LCAParasitology and serology
Cota 2013 - composite 1 & Cota 2013 - composite 2 *Parasitology and serologyParasitology not including spleen aspirate - no serology
Machado de Assis 2012 & Machado de Assis 2011LCAParasitology not including spleen aspirate - no serology
Veeken 2003 - composite & Veeken 2003 - spleenParasitology and serologyParasitology including spleen aspirate – no serology
2) Basic data presentation

The basic study results were summarized using coupled forest plots, presenting the crude Se and Sp estimates and 95% CIs as presented in the original publications. A summary receiver operating characteristic (ROC) plot was presented to assess the need for a ROC-based analysis rather than the bivariate logistic normal model, planned and described below.

3) Basic statistical model

The primary analysis was performed using the bivariate model with a hierarchical approach (Reitsma 2005). The bivariate, rather than a ROC-based, approach was chosen based on the prior experience of the author team in a VL diagnostic meta-analysis (Chappuis 2006), where no threshold effects were observed. In addition, the rapid tests report the results as positive or negative, which implies a common cut-off point for test positivity and which indicates, on prior grounds, that the bivariate model is the most appropriate. We determined the most appropriate link function for the bivariate model using the deviance information criterion (DIC, Verde 2010) and graphical assessment of model fit. Based on this analysis, we selected the complementary log-log (cloglog) function (see Appendix 1) as providing the best and most parsimonious fit to the data. All results are presented on the probability scale allowing direct comparison with results obtained using the logit link function.

For the studies estimating Se and Sp compared to a reference standard, at the lower level the cell counts in the 2 x 2 tables extracted from each study were modelled using binomial distributions (Macaskill 2010). 

For the studies estimating Se and Sp through LCA, the cloglog transforms of the Se and Sp and their standard errors were derived from the estimates and CIs in the source publications. They were then entered in the lower level of the hierarchical model using the normal distribution.

The Se and Sp, irrespective of their source, were, in the higher level of the hierarchical model, presumed to come from the same bivariate normal, that is, the model proposed corresponds to a hybrid approach of the bivariate model of Reitsma (Reitsma 2005) and standard random effects meta-analyses (Whitehead 2002). This model was estimated using Markov Chain Monte Carlo (MCMC) methods as implemented by WinBugs with uniform ]0,1[ priors for the index test Se and Sp. The WinBugs code for the model can be found in Appendix 1.

4) Model summary

Results from the models were provided as point estimates of test Se and Sp and joint 95% CI and prediction regions. The confidence regions and prediction regions were plotted on the Se versus 1-Sp diagram (that is, the ROC space), together with the crude or model-based point estimates of Se and Sp, or both, for each study. In tables, we presented marginal CIs obtained from these confidence regions.

Investigations of heterogeneity

The amount of between-study heterogeneity was graphically assessed in coupled forest plots.

To explain the between-study variability, we assessed the following possible sources of heterogeneity:

  • geographical area: Indian subcontinent, eastern Africa, Latin America, Mediterranean region (note: this is related to the Leishmania species diversity and distribution: L. donovani complex in Asia and eastern Africa, and L. infantum in the Mediterranean region and Latin America);

  • commercial brand or type of test: categories determined based on extracted data;

  • type of reference standard: (a) parasitology including spleen aspirate – no serology; (b) parasitology not including spleen aspirate – no serology; (c) parasitology and serology; (d) latent class analysis;

  • disease prevalence (in the sample): below versus equal to or above the median (65%) across all studies; and

  • quality assessment: a first indicator of quality was based on the QUADAS-2 assessment. Here, we used an overall, study-level assessment of the risk of bias. If the risk of bias in a study was low in all four QUADAS-2 domains, we defined the overall risk of bias in that study as 'low'. If the risk of bias was high in at least one of the QUADAS-2 domains, we defined the overall risk of bias as 'high'. In all other cases, the overall risk of bias was labelled 'uncertain'. A second indicator of quality was the size of the study (below versus equal to or above median [250]), because in order to ensure reasonably precise estimates of sensitivity and specificity, investigators are expected to consider sample size issues during the planning of the study (Bachmann 2006; Peeling 2007). Larger studies may have been subjected to closer scrutiny and may have recruited a more representative sample of the clinical suspect population. Yet, the precision of Se and Sp estimates in original studies does not only depend on sample size, but also on disease prevalence (Deeks 2005).

We informally assessed the influence of these factors by presenting summary Sp and Se for subgroups of studies (categories of covariate). In a formal analysis, these sources were then individually added as fixed-effect predictors of Se and Sp in the statistical model described above. We assessed the estimates and 95% CI of the effects of each of the predictors on Se and Sp. If warranted by the results of these analyses and the amount of studies available, we planned to consider developing this model further using multiple predictors or assuming separate bivariate normal distributions in different subgroups of studies.

Sensitivity analyses

Analysis using secondary reference standards

As a sensitivity analysis to the choice of the set of estimates of a given study, we made an alternative selection of the reference standard for those studies presenting results for two reference standards (Table 1).

Analysis allowing for imperfect reference standards

The model described above estimated the Se and Sp by comparison to a reference standard presuming that this reference standard was perfect, that is, that its Se and Sp were both equal to 1. However, in VL, it can be expected that reference standards have less than perfect Se or Sp, or both.

To allow for possible imperfect reference standards, we used the following approach:

  • during data extraction, each study was classified according to the type of reference standard: (a) parasitology including spleen aspirate – no serology; (b) parasitology not including spleen aspirate – no serology; (c) parasitology and serology; or (d) latent class analysis;

  • for these reference standards, expert opinion for the Se and Sp was elicited from seven experts (three of the authors FC, MB and SR, and four from the WHO technical expert panel on leishmaniasis);

  • the expert opinion on the reference test Se and Sp was then entered as priors, using the beta distribution, in an extension of the bivariate model described above as a Bayesian sensitivity analysis (Greenland 2006).

The bivariate model was extended using a multinomial distribution for the cell counts in the 2 x 2 tables, as in the latent class analysis of diagnostic tests (Black 2002). The Se and Sp of the index test were modelled through a bivariate normal distribution as in the basic model described above. The Se and Sp of the reference tests were assumed to be equal across studies using the same reference standard, with informative priors for the Se and Sp derived from expert opinion as described above. Prevalences were estimated for each study separately by providing a uniform ]0,1[ for the study-specific prevalences. Data from studies that performed LCA in the source publication were included as in the basic model, as they already allowed for uncertainty in the true disease status in the source publication. The model was fitted using MCMC methods. Care was taken in assessing the fit and identifiability of the model using posterior predictive checks (Gelman 1995). It could be expected that the model would remain identifiable through the use of informative priors on the Se and Sp of the reference test and the assumption that the Se and Sp of the index test across studies arose from the same normal bivariate distribution (Enoe 2000), but, if needed, further constraints on the model parameters could be added (for example, by constraining the Sp of some of the reference standards to 1).

The results from this model were compared with those obtained in the primary analysis, described above. We also contrasted the assumptions of our model against the assumption of perfect ascertainment underlying the primary analysis approach. The performance of the model was evaluated through simulation studies. Details are given in Appendix 1 and Menten 2013.

Assessment of structure of hierarchical model

We compared the primary results with those obtained using the standard logit link function for hierarchical model. In addition, we relaxed the assumption of normally distributed random-effects, by using the t-distribution instead of the normal distribution.

Assessment of reporting bias

It is well known that reporting or publication bias in studies of diagnostic test accuracy may be difficult to detect and that formal tests of funnel plot asymmetry are biased (Macaskill 2010). We did not formally test for publication bias but explored the relationship between the logit Se, logit Sp, and log diagnostic odds ratio and the effective sample size (Deeks 2005) using funnel plots. However, the presence or absence of funnel plot asymmetry was not interpreted as definite proof of the presence or absence of publication bias.

Results

Results of the search

The date of the search was 3 December 2013. The search identified 1758 records, each record corresponding to a published article (Figure 1). After screening titles and abstracts, 1648 irrelevant records were removed. We were unable to obtain the full-text article of one record and excluded one other article because it was written in Chinese language and no translators were available. We retrieved the full text of the remaining 108 records and excluded another 87 for the following reasons: publication of the same study in more than one record (we kept the record that was published most recently and excluded the others; n = 4); no original research (n = 6); index test not a rapid test (n = 1); target condition not clinical visceral leishmaniasis (n = 5); not a phase III diagnostic accuracy study (n = 66); and reference standard not according to criteria (n = 5; Figure 1 and Characteristics of excluded studies). The remaining 21 records were included in this review.

Figure 1.

Study flow diagram showing the process of selection of records and studies for the review and for the meta-analyses

Several records reported multi-country, multicentre or stratified data. If a ‘study’ is defined as a phase III design leading to a sensitivity and specificity estimate of one or more RDTs in a specific patient population, then three of the 21 included records contained results of more than one unique study. One article described RDT performance in patient populations in five countries (Boelaert 2008, henceforth distinguished as Boelaert 2008 - Ethiopia; Boelaert 2008 - India; Boelaert 2008 - Kenya; Boelaert 2008 - Nepal; Boelaert 2008 - Sudan); Ter Horst et al. reported data separately for HIV-positive and HIV-negative patient populations (ter Horst 2009 - HIV neg; ter Horst 2009 - HIV pos); and Diro 2007 included data from clinically suspected patients presenting at the study site and from people identified through active case finding in the community. Furthermore, in one record, two different brands of the rK39 immunochromatographic test (ICT) were evaluated in the same population (Chappuis 2005 - DiaMed; Chappuis 2005 - InBios), and we treated this record as two different studies. On the other hand, three patient populations were presented in more than one record. Boelaert 2008 - Ethiopia re-analysed the same patient population data as Diro 2007, but using a different reference standard. Similarly, Boelaert 2008 - India described the same patient population as Sundar 2007 but again, analysed it with a different reference standard. This also occurred in Machado de Assis 2011 and Machado de Assis 2012. We have treated these records as one single study in each country with a primary and a secondary analysis (see below).  Altogether, the 21 included records contained information about 25 unique studies.  We excluded the study based on active case finding (part of Diro 2007) from the review as it did not comply with the eligibility criteria of our protocol and, therefore, we finally included 24 studies.

These 24 studies included 4271 participants of whom 2606 were classified as having VL. Four studies used a reference standard including direct smears or culture of splenic aspirate, or both, 11 used a composite reference standard, three used latent class analysis, and six presented two sets of accuracy estimates using two different reference standard categories (Table 1).

The 24 studies contained information about five index tests: the rK39 ICT, the latex agglutination test in urine, the FAST agglutination test, an rK26 ICT and an rKE16 ICT. Six studies assessed the accuracy of more than one type of index test in the same patient population: four studies evaluated the rK39 ICT and the latex agglutination test; one study evaluated the rK39 and the rKE16 ICT; and one study evaluated the rK39 ICT, the rK26 ICT, and the latex agglutination test (Sundar 2007). Overall, the rK39 ICT test was evaluated in 20 studies including a total of 3806 participants of whom 2370 had VL. The latex agglutination test was evaluated in seven studies, which corresponds to 1459 participants including 873 people with VL. The FAST agglutination screening test was evaluated in two studies with a total of 148 participants including 69 with VL. The rK26 ICT test was assessed in one study with 352 participants of whom 282 had VL, and the rKE16 test was evaluated in one study with 219 participants of whom 131 had VL.

For the meta-analysis of the accuracy of the rK39 ICT test, 18 out of 20 studies were included (Table 2). At this stage, we excluded two studies because they only described HIV-positive patients (ter Horst 2009 - HIV pos and Cota 2013). In all the other studies using the rK39 ICT test, the included patients were HIV negative or the HIV status was unknown but considered of no importance in the study population. Six out of the 18 included studies generated more than one set of sensitivity and specificity estimates by using different reference standards in a primary and a secondary analysis. We selected one of the two estimates for the primary meta-analysis and explored the effects of this choice in the sensitivity analysis (Table 1).

Table 2. Overall Analysis Summary
 Number of studiesSe (95% CI)Sp (95% CI)
rK39 immunochromatographic test   
Overall1891.9 (84.8 to 96.5)92.4 (85.6 to 96.8)
Indian subcontinent697.0 (90.0 to 99.5)90.2 (76.1 to 97.7)
East Africa985.3 (74.5 to 93.2)91.1 (80.3 to 97.3)
Latex agglutination test   
Overall663.6 (40.9 to 85.6)92.9 (76.7 to 99.2)

For the meta-analysis of the accuracy of the latex agglutination test, we included six studies (Table 2). We excluded one study because it described mostly HIV-positive patients (Vilaplana 2004).

Methodological quality of included studies

The QUADAS-2 tool was used to assess the quality of the 21 included records in terms of the risk of bias and of concerns about applicability. Figure 2 summarizes the overall methodological quality and Figure 3 gives the ratings for each of the included records. In the domain of patient selection, we had high concerns about the risk of bias and the applicability of one record (Veeken 2003 - composite) because the inclusion of participants was conditional on the availability of stored serum samples and of diagnostic information from another serological test that could have correlated with the index test. In another record (Cota 2013), we had high concerns about the applicability to the review question because a considerable proportion of the study population (21%) had a previous history of VL. In the domains of the index test and the reference standard, the risk of bias was unclear for 14 of the 21 included records. The most frequent underlying reason was that the publication did not report whether the index test results were interpreted without knowledge of the reference standard and vice versa. We considered that there was a high risk of bias in those studies that used bone marrow aspirate smear tests and culture with or without clinical information as a reference standard due to the low sensitivity of these techniques (Kilic 2008 and Cota 2013 - composite 2). In the domain of flow and timing, we considered that there was a high risk of bias in five records, because not all patients were included in the analysis (Rijal 2004; ter Horst 2009; Veeken 2003; and Peruhype-Magalhaes 2012) or because not all patients received the same reference standard (Sundar 1998).

Figure 2.

Risk of bias and applicability concerns graph: review authors' judgements about each domain presented as percentages across included studies

Footnote

Figure 2 represents studies but our quality assessment was done per record. Some of the records include more than one study. Figure 2 shows all the estimates used in this review, including the estimates for primary and sensitivity analyses of the same study.

Figure 3.

Risk of bias and applicability concerns summary: review authors' judgements about each domain for each included study

Footnote

Figure 3 represents studies but our quality assessment was done per record. Some of the records include more than one study. Figure 3 shows all the estimates used in this review, including the estimates for primary and sensitivity analyses of the same study.

Findings

rK39-based rapid diagnostic tests

Data summary

Twenty studies reported Se and Sp estimates for the rK39 ICT. Six of these studies gave two sets of Se and Sp estimates, based on alternative reference standards (Table 1). A forest plot of the 26 available Se/Sp estimates is given in Figure 4. Figure 5 presents the available Se/Sp pairs according to the reference standard used; the results referring to the same data analysed with different reference standards are connected with a line.

Figure 4.

rK39 ICT: forest plot of all the available estimates of sensitivity and specificity (n = 20 studies; 26 sets of estimates)

Footnote

For studies that use latent class analysis, the counts of true positive, false positive, false negative and true negative results are imputed from the Se and Sp estimates and the overall sample size. The estimates and confidence intervals are subsequently calculated from these imputed values.

Figure 5.

rK39 ICT: summary of sensitivity-specificity pairs according to the reference standard (n = 20 studies; 26 sets of estimates). The type of reference standard is classified as: (a) parasitology including spleen aspirate – no serology; (b) parasitology not including spleen aspirate – no serology; (c) parasitology and serology; or (d) latent class analysis. The data points that are connected with a line refer to the same data analysed with different reference standards.

Meta-analysis

A formal meta-analysis was performed on 18 of the 26 available data points: studies including mainly HIV-infected patients were reported separately (ter Horst 2009 - HIV pos, Cota 2013 - composite 1 and Cota 2013 - composite 2) and for the five remaining studies with multiple Se and Sp estimates, we used one set of estimates for the primary analysis (Table 1). Combining data from all studies without accounting for possible covariates explaining heterogeneity and using the bivariate model (Reitsma 2005), the average (95% CI) Se was 91.9% (84.8 to 96.5) and the average Sp was 92.4% (85.6 to 96.8)

The 95% prediction interval (PI) for the diagnostic accuracy that would be observed in a new study was (52.3 to 100)% for the Se and (54.9 to 100)% for the Sp (Figure 6).

Figure 6.

rK39 ICT: basic summary using the bivariate normal model with a complementary log-log link function. This analysis combines data from all studies without accounting for covariates. The average sensitivity is 91.9% (95% confidence interval: 84.8, to 96.5) and the average specificity is 92.4% (95% confidence interval: 85.6, to 96.8). The confidence region is indicated with a full line, the prediction region with a dotted line.

Of note is the fact that, in contrast to what is presumed in the HROC model, the estimated correlation between the transformed Se and Sp was positive: 0.16 (95% CI: -0.40 to 0.65). Given these observations, we only report results from the bivariate model using the complementary log-log, and not from the ROC-based analysis.

Assessment of heterogeneity

A summary of the heterogeneity assessment through meta-analysis can be found in Figure 7 and Table 3.

Figure 7.

rK39 ICT; summary of the heterogeneity assessment through meta-analysis: sensitivity and specificity estimates by covariates. Rectangles indicate significant differences.

Table 3. Heterogeneity assessment of the rK39 ICT meta-analysis
  1. * The categories "parasitology including spleen aspirate - no serology" and "parasitology not including spleen aspirate - no serology" were combined because the latter category contained only one study.

 SensitivitySpecificity
 Estimate(95% CI)Estimate(95% CI)
Geographic region
Indian subcontinent97.0(90.0 to 99.5)90.2(76.1 to 97.7)
East Africa85.3(74.5 to 93.2)91.1(80.3 to 97.3)
Commercial brand
DiaMed86.4(70.7 to 95.9)94.4(80.5 to 99.2)
InBios91.3(83.8 to 96.3)91.2(83.1 to 96.3)
Other97.8(88.7 to 99.992.5(75.5 to 99.0)
Disease prevalence in sample
Low (< 65%)91.0(82.2 to 96.9)94.4(87.2 to 98.3)
High (≥ 65%)92.4(84.5 to 97.4)89.8(79.7 to 95.8)
Study size
Small (< 250)91.5(82.7 to 96.7)89.8(80.6 to 95.6)
Large (≥ 250)92.3(83.4 to 97.4)94.5(87.8 to 98.3)
QUADAS-2: risk of bias
Low89.5(69.5 to 98.3)96.0(82.8 to 99.7)
Unclear91.9(83.7 to 97.1)92.0(84.0 to 96.7)
High93.0(77.9 to 99.1)87.8(70.7 to 97.2)
Reference standard
Parasitology - no serology *95.6(84.3 to 99.6)89.1(72.4 to 97.4)
Parasitology and serology89.6(78.6 to 96.7)94.6(85.2 to 98.7)
Latent class analysis91.0(79.8 to 97.2)91.5(80.7 to 97.3)
Geographic region

We assessed the difference in diagnostic accuracy effect between East Africa and the Indian subcontinent. There were nine data points from East Africa (two from Ethiopia, two from Kenya, three from Sudan, and two from Uganda), and six from the Indian subcontinent (two from India and four from Nepal). Three studies from other regions (two from Brazil and one from Turkey) were not considered in this analysis. The Se was significantly lower in East Africa (85.3%; 95% CI: 74.5 to 93.2) than in the Indian subcontinent (97.0%; 95% CI: 90.0 to 99.5). There was no significant difference in Sp: the Sp (95% CI) in east Africa was 91.1% (80.3 to 97.3) and in the Indian subcontinent 90.2% (76.1 to 97.7) (Summary of findings 1 and Summary of findings 2). Confidence and prediction regions by geographic region are given in Figure 8.

Figure 8.

rK39 ICT: summary of the meta-regression model with effects for geographic region using the bivariate normal model with a complementary log-log link function. The sensitivity was significantly lower in East Africa (85.3%; 95% confidence interval: 74.5 to 93.2) than in the Indian subcontinent (97.0%; 95% confidence interval: 90.0 to 99.5). There was no significant difference in specificity: the specificity (95% confidence interval) in East Africa was 91.1% (80.3 to 97.3) and in the Indian subcontinent 90.2% (76.1 to 97.7). The confidence region is indicated with a full line, the prediction region with a dotted line.

Commercial brand or type of test

The majority of studies used the InBios rK39 ICT test (11 studies); four assessed the DiaMed rK39 ICT test; and three evaluated other tests (DiaMed DUAL-IT L/M ICT test, Amrad ICT rK39 ICT test, and Arista Biologicals rK39 ICT test). There were no significant differences in diagnostic accuracy between commercial brands.

Disease prevalence

We separated studies by estimated prevalence of VL in the study sample: nine studies reported prevalence rates < 65% and nine reported prevalence rates ≥ 65%. There were no significant differences in diagnostic accuracy between studies with low and high prior probability of disease in the patient sample.

Quality assessment

We used study size and risk of bias according to QUADAS-2 as indicators of the quality of the primary studies.

Study size

We categorized studies according to the total number of study participants (true cases + non-cases) in small (< 250) and large (≥ 250) studies. There were 10 small and eight large studies. There were no significant differences in diagnostic accuracy between small and large studies, but there was a trend for larger studies to show a higher Sp estimate.

QUADAS-2: risk of bias

Studies were categorized according to the risk of bias assessed with QUADAS-2: low risk (three studies), high risk (four studies), and unclear risk of bias (11 studies). There were no significant differences among these categories, but there was a trend for studies with high risk of bias to show a low Sp and studies with low risk of bias to show a higher Sp.

Type of reference standard

Studies were categorized according to the reference standard used in the primary publication. There were no significant differences in accuracy among these categories. Further exploration of the influence of the reference standard is assessed in the sensitivity analysis below.

Diagnostic accuracy in HIV-positive participants

Two studies evaluated the rK39 ICT in HIV-positive participants and were not included in the meta-analysis.

The first study reported on the accuracy of the DiaMed rK39 ICT in 71 HIV-positive participants in Ethiopia (ter Horst 2009 - HIV pos). Based on a composite reference standard that combined direct parasitological examination of spleen aspirate with DAT serology, 65 participants had a diagnosis of VL. In this setting, the sensitivity of the rK39 ICT was 64.6% and the specificity 66.7%.

The second study evaluated the InBios rK39 ICT in 113 HIV-infected people in Brazil. Data were extracted according to two different reference standards: (1) the decision of an adjudication committee after clinical follow-up and taking into account all available information (including parasitology and serology) (Cota 2013 - composite 1; VL prevalence: 40.7%); and (2) direct smear and culture of bone marrow aspirate (Cota 2013 - composite 2; VL prevalence: 36.9%). The choice of the reference standard was of little influence. The Se of the rK39 ICT was low: 45.7% with the first and 46.3% with the second reference standard; and the Sp was high: 97.0% with the first and 97.1% with the second reference standard.

Sensitivity analyses

We performed sensitivity analyses of the influence of the choice of reference standard and possible biases resulting from imperfect reference standards. Given the impact of geographic region on the diagnostic accuracy of rK39 ICT, we corrected the sensitivity analyses for region effects and limited the analyses to 15 studies performed in the Indian subcontinent and East Africa. An overview of the results of the sensitivity analyses is given in Table 4.

Table 4. Sensitivity analysis results of the rK39 ICT meta-analysis
 SensitivitySpecificity
 Estimate(95% CI)Estimate(95% CI)
Main Analysis
Indian subcontinent97.0(90.0 to 99.5)90.2(76.1 to 97.7)
East Africa85.3(74.5 to 93.2)91.1(80.3 to 97.3)
Alternative Analysis Set
Indian subcontinent96.1(88.9 to 99.2)86.7(69.2 to 99.2)
East Africa85.2(75.0 to 92.7)90.1(77.0 to 97.4)
Bayesian analysis allowing for imperfect reference standards: expert priors - using main analysis set
Indian subcontinent97.3(91.9 to 99.5)93.7(74.9 to 99.7)
East Africa86.7(77.5 to 94.0)95.7(84.3 to 99.8)
Bayesian analysis allowing for imperfect reference standards: vague priors - using main analysis set
Indian subcontinent97.3(91.9 to 99.5)93.0(77.8 to 99.3)
East Africa86.1(77.2 to 93.3)94.3(83.8 to 99.6)
Analysis using secondary reference standards

As a sensitivity analysis to the choice of the set of estimates of a given study, we made an alternative selection of the reference standard for those studies presenting results for two reference standards (Table 1). The estimated Sp was slightly lower compared to the main analysis set: 86.7% versus 90.2% for the Indian subcontinent and 90.1% versus 91.1% for east Africa. This may be related to a lower Se of the reference standards included in this analysis, which would result in an underestimation of the Sp of the index test. Estimated Se was similar in the sensitivity and main analysis.

Analysis allowing for imperfect reference standards

In the studies available for this sensitivity analysis, two types of reference standards were used: parasitology including spleen aspirate – no serology; (three studies) and a combined reference standard of parasitology and serology (six studies), in addition to the six studies analysed using latent class analysis. We elicited the opinion from seven VL experts on the diagnostic accuracy and possible variation across the two reference standards (Figure 9). We used these expert opinions as prior information in a Bayesian statistical model to estimate the diagnostic accuracy of the rK39 ICT. This analysis indicated that due to lack of sensitivity of the reference standard, the Sp of rK39 ICT may be somewhat underestimated. The estimated Sp correcting for this bias was 93.7% in the Indian subcontinent, and 95.7% in east Africa, compared to 90.2% and 91.1% assuming perfect reference standards. The estimates of Se did not change significantly when we corrected for imperfect reference standards. In this model, the Se of spleen aspirate was estimated to be 96.0% (95% CI: 92.5 to 98.8) and the Sp was estimated to be 99.4% (95% CI: 98.1 to 99.9). The estimated Se of the combined reference standard of spleen parasitology and a serological test was estimated to be 98.6% (95% CI: 97.1 to 99.4) and the estimated Sp was 96.5% (95% CI: 91.5 to 99.2).

Figure 9.

Opinion from seven experts on visceral leishmaniasis regarding the diagnostic accuracy and possible variation of two reference standards: parasitology including spleen aspirate without serology and any combination of parasitology with serology. These expert opinions were used as prior information in a Bayesian statistical model to estimate the diagnostic accuracy of the rK39 ICT.

Similar results were obtained when we used non-informative priors instead of expert opinion to estimate the Bayesian model.

Assessment of structure of hierarchical model

Using the more standard logit link, in comparison to the better fitting complementary log-log link increased the DIC from 171.3 to 199.0. The logit link resulted in similar estimates of Se and Sp: the average (95% CI) Se was 91.9% (83.6 to 96.2) and the average Sp was 92.4% (84.9 to 96.3). However, the remaining heterogeneity was considerably larger resulting in wider prediction regions: 28.3 to 99.7% for Se and 31.1 to 99.7% for Sp (Figure 10) compared to the primary analysis.

Figure 10.

rK39 ICT: basic model summary using the bivariate logistic normal model. This analysis combines data from all studies without accounting for covariates. The average sensitivity is 91.9% (95% confidence interval: 83.6, to 96.2) and the average specificity is 92.4% (95% confidence interval: 84.9, to 96.3). The confidence region is indicated with a full line, the prediction region with a dotted line. In this analysis, the prediction intervals are wide: 28.3 to 99.7% for the sensitivity and 31.1 to 99.7% for the specificity.

Using a bivariate t-distribution for the random-effects did not improve model fit compared to the normal model.

Assessment of reporting bias

Reporting bias was assessed through the robust funnel plot proposed by Deeks 2005. This robust funnel plot presents the log of the diagnostic odds ratio by a robust measure of the study size. No indication of reporting bias was observed in this plot (Figure 11).

Figure 11.

rK39 ICT; assessment of reporting bias: funnel plot for log diagnostic odds ratio

Latex agglutination test

Data summary

Seven studies were identified that reported Se or Sp results of the latex agglutination test in urine (KAtex). For two of these studies, two sets of Se and Sp estimates were available. A forest plot of the nine available Se/Sp estimates is given in Figure 12.

Figure 12.

Latex agglutination test: forest plot of all the available estimates of sensitivity and specificity (n = 7 studies; 9 sets of estimates).

Meta-analysis

One study was not pooled with the others in the formal meta-analysis because, contrary to the other studies, this study included a majority of HIV-positive participants (Vilaplana 2004). For the two studies that presented Se/Sp estimates using LCA and a reference standard on the same data (Boelaert 2008 - Ethiopia and Diro 2007, Boelaert 2008 - India and Sundar 2007), the results from the two analysis approaches were very similar. We used the results from LCA for the primary meta-analysis.

Combining data from six studies without accounting for possible covariates explaining heterogeneity, and using the bivariate normal model with complementary log-log link, the average (95% CI) Se was 63.6% (40.9 to 85.6) and the average Sp was 92.9% (76.7 to 99.2) (Figure 13 and Summary of findings 3). The 95% prediction intervals were very wide: (16.5 to 99.6)% for the Se and (41.3 to 100)% for the Sp (Figure 13). The estimated correlation between the transformed Se and Sp was -0.39 (95% CI: -0.90 to 0.70).

Figure 13.

Latex agglutination test: basic summary using the bivariate normal model with a complementary log-log link function. This analysis combines data from all studies without accounting for covariates. The average sensitivity (95% confidence interval) is 63.6% (40.9 to 85.6) and the average specificity is 92.9% (76.7 to 99.2). The confidence region is indicated with a full line, the prediction region with a dotted line. The 95% prediction intervals are very wide: 16.5% to 99.6% for the sensitivity and 41.3% to 100% for the specificity.

Assessment of heterogeneity

Of the predefined sources of heterogeneity, geographic region, disease prevalence, and study size showed sufficient variability across studies to warrant a meta-regression. All studies used the KAtex latex agglutination test manufactured by Kalon biological. Five of the six studies had an unclear risk of bias. All studies in east Africa reported prevalences of VL < 65% and all studies in the Indian subcontinent reported prevalences ≥ 65%. Consequently, the effects of region and disease prevalence in the sample could not be separated. There were no significant differences in diagnostic accuracy of the latex agglutination test among regions or study sizes (Figure 14 and Table 5).

Figure 14.

Latex agglutination test; summary of the heterogeneity assessment through meta-analysis: sensitivity and specificity estimates by covariates.

Table 5. Heterogeneity assessment of the Latex agglutination test meta-analysis
 SensitivitySpecificity
 Estimate(95% CI)Estimate(95% CI)
Geographic region
Indian subcontinent50.8(34.1 to 69.3)95.3(73.6 to 99.8)
East Africa77.9(58.2 to 92.3)88.6(59.5 to 92.3)
Study size
Small (< 200)52.9(27.6 to 84.1)86.2(52.5 to 99.2)
Large (≥ 200)68.5(45.2 to 88.2)94.5(49.0 to 100.0)
Sensitivity analysis

As a sensitivity analysis to the choice of the set of estimates of a given study, we made an alternative selection of the reference standard for those studies presenting results for two reference standards. The results were very similar to the primary analysis: the average (95% CI) Se was 63.4% (40.8 to 85.4) and the average Sp was 92.8% (76.3 to 99.2) (Table 6).

Table 6. Sensitivity analysis results of the Latex agglutination test meta-analysis
 SensitivitySpecificity
 Estimate(95% CI)Estimate(95% CI)
Main Analysis63.6(40.9 to 85.6)92.9(76.7 to 99.2)
Alternative Analysis Set63.4(40.8 to 85.4)92.8(76.3 to 99.2)
Diagnostic accuracy in HIV-positive participants

One study assessed the accuracy of the latex agglutination test in a population of 85 mostly HIV-positive participants (93%) in Spain (Vilaplana 2004). According to a reference standard based on culture or direct parasitological examination of bone marrow aspirate, 12 participants were classified as having VL. The estimated Se and Sp of the KAtex test were very high: the Se was 100% (12/12; 95% CI: 74 to 100) and the Sp was 96% (70/73; 95% CI: 88 to 99).

FAST

Two studies assessed the accuracy of the Fast agglutination screening test (FAST), one in Ethiopia (Hailu 2006) and one in Turkey (Kilic 2008). Both studies were small (89 participants in Ethiopia and 59 in Turkey). The prevalence of VL in the study population was 51% in Ethiopia and 41% in Turkey. The reference standard was the culture or direct parasitological examination of spleen or lymph node samples (Ethiopia) or of bone marrow samples (Turkey). In Ethiopia, the estimated Se of FAST was 91.1% and the Sp 70.5%. In Turkey, the estimated Se was 95.8% and the Sp 85.7%.

rK26-based rapid diagnostic test

One study assessed the accuracy of an rK26-based rapid diagnostic test manufactured by InBios International (Seattle, Washington, USA) in a population of 352 patients with suspected VL in India (Sundar 2007). According to a reference standard combining direct parasitological examination of spleen aspirate with DAT serology and response to VL treatment, 282 participants had a diagnosis of VL. The sensitivity of the rK26 ICT was 21.3% and the specificity 100%.

rKE16 immunochromatographic test

One study evaluated an rKE16-based ICT (Signal KA, Span Diagnostics, India) in Kenya (Mbui 2013). This study included 219 patients suspected to have VL. Based on a reference standard consisting of direct smear examination of a splenic aspirate sample, 131 participants were classified as having VL. The Se of the rKE16 ICT was 77.1% and the Sp 95.5%.

Summary of findings

Summary of findings 1. rK39 immunochromatographic test for visceral leishmaniasis in the Indian subcontinent
  1. 1. The rK39 immunochromatographic test must be used in combination with a clinical case definition (fever and splenomegaly for more than two weeks and no previous history of visceral leishmaniasis). Studies with mainly HIV-positive patients were not included in the pooled analyses.

    2. The results of the meta-analysis showed considerable heterogeneity, which was partly explained by the geographic region.

    3. This rapid diagnostic test has been developed specifically for field use. It is less invasive, less time-consuming, and easier to perform than the alternative parasitological or serological tests.

    4. Latent class analysis is a modelling technique that allows us to estimate the sensitivity and specificity of a set of diagnostic tests in situations in which there is no good reference standard.

    5. Two hypothetical situations: a peripheral health centre and a referral centre with a different prior probability of disease

    6. A narrative explanation of the predictive values is given in Appendix 3.

    7. QUADAS-2 is a tool for the assessment of the quality of diagnostic accuracy studies. The tool comprises four domains: patient selection, index test, reference standard, and flow and timing. Each domain is assessed in terms of risk of bias, and the first three domains are also assessed in terms of concerns regarding applicability.

Population: Patients suspected to have visceral leishmaniasis disease 1

Setting: Health services in endemic areas of the Indian subcontinent 2

New test: rK39 immunochromatographic test 3

Reference standard: (1) direct smear test or culture of splenic aspirate; (2) composite reference standard based on one or more of the following: parasitology, serology, or response to treatment; or (3) latent class analysis 4

Pooled sensitivity: 0.97 (95% CI 0.90 to 1.00) | Pooled specificity : 0.90 (95% CI 0.76 to 0.98)
Setting 5 Positive predictive value6 Negative predictive value6 Number of participants (studies) Quality of the evidence (QUADAS-2) 7
Peripheral health centre with a prior probability of disease of 40% 87% 98%

1468

(6 studies)

Risk of bias: none of the studies had a low risk of bias in all domains. One study had a high risk of bias (domain of flow and timing). Five studies had an unclear risk of bias (domains of index test or reference standard).

Applicability: low concerns in all studies and in all domains.

Referral centre with a prior probability of disease of 60% 94% 95%

Interpretation: When the rK39 ICT is used 1 in the Indian subcontinent, in a setting where the prior probability of VL among clinical suspects is 40%, which is typically seen in a peripheral health centre in an endemic area, the positive predictive value of the test is 87%. This means that out of 100 patients with a positive rK39 result, 87 would have VL (true positive result) and 13 would have another disease (false positive). The negative predictive value is 98%, meaning that out of 100 patients with a negative rK39 ICT result, 98 would have another disease (true negative) and 2 would have VL (false negative).

When the same test is used in a setting with a prior probability of VL of 60%, which is more typical for a referral centre in an endemic area, the positive predictive value is 94% and the negative predictive value is 95%.

A likelihood ratio is another way of expressing how informative a diagnostic test is: it indicates to what extent the rK39 ICT result changes the odds that a patient has VL. The likelihood ratio of a positive rK39 ICT result is 9.90, and the likelihood ratio of a negative test result is 0.03. This means that in the Indian subcontinent, a positive rK39 ICT result is a strong argument in favour of VL (ruling in) and that a negative rK39 ICT result is a strong argument against VL (ruling out).

CI: confidence interval
Boelaert M, Verdonck K, Menten J, Sunyoto T, van Griensven J, Chappuis F, Rijal S. Rapid diagnostic tests for visceral leishmaniasis. Cochrane Database of Systematic Reviews 2011, Issue 6. Art. No.: CD009135. DOI: 10.1002/14651858.CD009135.
Summary of findings 2. rK39 immunochromatographic test for visceral leishmaniasis in east Africa
  1. 1. The rK39 immunochromatographic test must be used in combination with a clinical case definition (fever and splenomegaly for more than two weeks and no previous history of visceral leishmaniasis). Studies with mainly HIV-positive patients were not included in the pooled analyses.

    2. The results of the meta-analysis showed considerable heterogeneity, which was partly explained by the geographic region.

    3. This rapid diagnostic test has been developed specifically for field use. It is less invasive, less time-consuming, and easier to perform than the alternative parasitological or serological tests.

    4. Latent class analysis is a modelling technique that allows to estimate the sensitivity and specificity of a set of diagnostic tests in situations in which there is no good reference standard.

    5. Two hypothetical situations: a peripheral health centre and a referral centre with a different prior probability of disease

    6. A narrative explanation of the predictive values is given in Appendix 3.

    7. QUADAS-2 is a tool for the assessment of the quality of diagnostic accuracy studies. The tool comprises four domains: patient selection, index test, reference standard, and flow and timing. Each domain is assessed in terms of risk of bias, and the first three domains are also assessed in terms of concerns regarding applicability.

Population: Patients suspected to have visceral leishmaniasis disease 1

Setting: Health services in endemic areas of east Africa 2

New test: rK39 immunochromatographic test 3

Reference standard: (1) direct smear test or culture of splenic aspirate; (2) composite reference standard based on one or more of the following: parasitology, serology, or response to treatment; or (3) latent class analysis 4

Pooled sensitivity: 0.85 (95% CI 0.75 to 0.93) ; Pooled specificity : 0.91 (95% CI 0.80 to 0.97)
Setting 5 Positive predictive value 6 Negative predictive value6 Number of participants (studies)

Quality of the evidence

(QUADAS-2) 7

Peripheral health centre with a prior probability of disease of 40% 86% 90%

1692

(9 studies)

Risk of bias: three studies had a low risk of bias across all domains; four studies had an unclear risk of bias (domains of index test or reference standard); two studies had a high risk of bias (domains of patient selection or flow and timing, or both).

Applicability: one study with high concerns about applicability (domain of patient selection); low concerns for all other studies and all other domains.

Referral centre with a prior probability of disease of 60% 93% 81%

Interpretation: When the rK39 ICT is used 1in east Africa, in a setting where the prior probability of VL is 40%, which is typically seen in a peripheral health centre in an endemic area, the positive predictive value of the test is 86%. This means that out of 100 patients with a positive rK39 ICT result, 86 would have VL (true positive result) and 14 would have another disease (false positive). The negative predictive value is 90%, meaning that out of 100 patients with a negative rK39 ICT result, 90 would have another disease (true negative) and 10 would have VL (false negative).

When the same test is used in a setting with a prior probability of VL of 60%, which is more typical for a referral centre in an endemic area, the positive predictive value is 93% and the negative predictive value is 81%.

In east Africa, the likelihood ratio of a positive rK39 ICT result is 9.58, and the likelihood of a negative rk39 ICT result is 0.16. This means that a positive rK39 ICT result is strong argument in favour of VL (ruling in), and that a negative rK39 ICT result is not an absolute argument against VL (does not allow to rule out VL completely).

CI: confidence interval
Boelaert M, Verdonck K, Menten J, Sunyoto T, van Griensven J, Chappuis F, Rijal S. Rapid diagnostic tests for visceral leishmaniasis. Cochrane Database of Systematic Reviews 2011, Issue 6. Art. No.: CD009135. DOI: 10.1002/14651858.CD009135.
Summary of findings 3. Latex agglutination test in urine for the diagnosis of visceral leishmaniasis
  1. 1. Studies with mainly HIV-positive patients were not included in the pooled analyses.

    2. The studies included in this review were conducted in Ethiopia, Kenya, Sudan, India and Nepal.

    3. This rapid diagnostic test has been developed specifically for field use. It is less invasive, less time-consuming, and easier to perform than the alternative parasitological or serological tests.

    4. Latent class analysis is a modelling technique that allows to estimate the sensitivity and specificity of a set of diagnostic tests in situations in which there is no good reference standard.

    5. The results of the meta-analysis showed considerable heterogeneity.

    6. Two hypothetical situations: a peripheral health centre and a referral centre with a different prior probability of disease.

    7. QUADAS-2 is a tool for the assessment of the quality of diagnostic accuracy studies. The tool comprises four domains: patient selection, index test, reference standard, and flow and timing. Each domain is assessed in terms of risk of bias, and the first three domains are also assessed in terms of concerns regarding applicability.

Population: Patients suspected to have visceral leishmaniasis disease 1

Setting: Health services in endemic areas 2

New test: Latex agglutination test in urine 3

Reference standard: (1) direct smear test or culture of splenic aspirate; (2) composite reference standard based on one or more of the following: parasitology, serology, or response to treatment; or (3) latent class analysis 4

Pooled sensitivity : 0.64 (95% CI 0.41 to 0.86) ; Pooled specificity : 0.93 (95% CI 0.77 to 0.99) 5
Setting 6 Positive predictive value Negative predictive value Number of participants (studies)

Quality of the evidence

(QUADAS-2) 7

Peripheral health centre with a prior probability of disease of 40% 86% 79%

1374

(6 studies)

Risk of bias: none of the studies had a low risk of bias in all domains. One study had a high risk of bias (domain of flow and timing). Five studies had an unclear risk of bias (domain of reference standard).

Applicability: low concerns in all studies and in all domains.

Referral centre with a prior probability of disease of 60% 93% 63%
CI: confidence interval
Boelaert M, Verdonck K, Menten J, Sunyoto T, van Griensven J, Chappuis F, Rijal S. Rapid diagnostic tests for visceral leishmaniasis. Cochrane Database of Systematic Reviews 2011, Issue 6. Art. No.: CD009135. DOI: 10.1002/14651858.CD009135.

Discussion

Summary of main results

The rK39 ICT, developed in 1998, is the rapid diagnostic test for VL that has been most thoroughly evaluated so far. We retrieved 21 publications that corresponded to our inclusion criteria for the review, and from these, ultimately 18 independent sets of sensitivity (Se) and specificity (Sp) estimates could be included in a formal meta-analysis of diagnostic accuracy of the rK39 ICT. In patients with febrile splenomegaly and no previous history of VL, the overall sensitivity of the rK39 ICT was 91.9% (95% CI 84.8 to 96.5) and the specificity 92.4% (95% CI 85.6 to 96.8). Sensitivity was significantly lower in East Africa (84.3%; 95% CI 74.5 to 93.2) than in the Indian subcontinent (97.0%; 95% CI 90.0 to 99.5), but there was no significant difference in specificity between geographic regions (Summary of findings 1, Summary of findings 2, and Appendix 3). This assessment of heterogeneity did not include the Latin American and Mediterranean region as the number of studies from those regions was too low. There was no significant difference according to prevalence of disease in the sample or manufacturer of the rK39 ICT, but the number of estimates in some categories of the latter was low (11/18 InBios, 4/18 DiaMed, and 3/18 other brands). Generally, care should be taken when interpreting this heterogeneity assessment due to the limited number of studies included in the meta-analysis. Apart from the lack of power, several risk factors may be correlated inducing confounding between different covariates.

For the KAtex test, a latex agglutination test in urine, when used in a clinical setting in patients with febrile splenomegaly, overall sensitivity was 63.6% (95% CI 40.9 to 85.6) and specificity 92.9 % (95% CI 76.7 to 99.2) (Summary of findings 3). Seven studies were included in the review and six in the meta-analysis. Because of the limited number of estimates, no complete heterogeneity assessment could be made. There were no significant differences in diagnostic accuracy of the KAtex test among regions or study sizes, but the number of included studies was too small to allow for definite conclusions.

The number of studies addressing three other rapid diagnostic tests (FAST, rK26 ICT, and rKE16 ICT) was too low to allow for a meta-analysis. Our conclusions do not apply to HIV-positive patients. In summary, completeness of evidence was most satisfactory for the rK39 ICT, only partially complete for the latex agglutination test in urine, and too incomplete for the other three tests.

The methodological quality of the evidence on the rK39 ICT and the latex agglutination test was acceptable, although we had concerns about the risk of bias in some studies (see below).

Strengths and weaknesses of the review

Our review protocol explicitly set out to select clinical evaluations in a phase III design, based on consecutively recruited patients all suspect for VL, as this type of evidence level is required for making policy recommendations for clinical practice. Therefore, we had to exclude many records using a phase II (case control) design. However, in some records, the study design was not clearly reported, which made it difficult to distinguish between a phase III and a case control design. Because of this poor reporting, we may have missed some true phase III studies.

We believe that the procedures used for study selection were quite exhaustive, as we did not impose any a priori language barriers (excluding only one study in Chinese ex-post), and we searched several databases including regional ones such as LILACS. Therefore, we believe that the information presented here reflects the body of evidence accumulating over the past 15 years rather well. In addition, assessment of possible publication bias did not indicate that smaller studies without favourable results for the RDTs were less likely to be reported. 

Most important was the striking heterogeneity in methods across the studies. Although the included studies all complied with the criteria for an acceptable reference standard set out in our protocol, there was a remarkable variety in the reference standards that were used. These reference standards were based on different tests, done in different orders, with different cut-off values, and making different use of clinical information. We formally assessed the risk of bias in each record using the revised QUADAS-2 instrument. In five out of the 21 included records, we had clear concerns related to the patient and sample flow and the timing. For 14 records, the risk of bias was difficult to assess in the domains of the index test and the reference standard mainly because authors failed to report explicitly if and how blinding was applied.  When we categorized records into low (n = 3), unclear (n = 11), and high (n = 7) risk of bias, there was no significant difference in Se and Sp of the rK39 ICT, but high-risk studies tended to have a higher sensitivity and a lower specificity. One possible explanation is that the reference standards in studies classified as at high risk of bias were of sub-optimal sensitivity. This could result in a selection of cases being primarily severe cases which may be easy to diagnose with rapid diagnostic tests, while the control group may contain some undiagnosed true VL cases (falsely negative in the reference standard).

We tried to assess the influence of this quality in study design on the estimates of accuracy in a sensitivity analysis in the case of the rK39 ICT. Neither the use of a secondary analysis set that was based on a different reference standard, nor an analysis allowing for an imperfect reference standard affected the main conclusions of the meta-analysis on the rK39 ICT, although the secondary analysis using an alternative reference standard in five studies tended to give a lower specificity estimate. This may be related to a lower Se of the reference standards included in this analysis, which would result in an underestimation of the Sp of the index test.

Finally, the findings seemed sufficiently homogeneous to allow for a meaningful summary estimate for the specificity of rK39 ICT as well as the Se and Sp of the latex agglutination test in urine. However, the sensitivity of rK39 ICT was different according to geographic region, and seems substantially lower in East Africa than in the Indian subcontinent. This finding is in line with an earlier meta-analysis conducted by our team (Chappuis 2006) and with a multi-country study conducted by WHO/TDR (data not included) (Cunningham 2012). Several hypotheses have been raised to explain this lower sensitivity in east Africa: a lower level of circulating antibodies, different age group, or parasitological differences (Bhattacharyya 2013; Harhay 2011).

Applicability of findings to the review question

Our review focused on clinical studies (phase III) of diagnostic accuracy, recruiting participants representative for the spectrum of patients encountered in clinical practice in whom a rapid diagnostic test for VL would be warranted. In summary, those patients would be in line with the WHO case definition and defined as “patients with a clinical suspicion of VL, that is, those who are febrile for more than two weeks and have splenomegaly, presenting at health services in endemic areas.” (Source: protocol). Patients who were previously treated for VL (non-responders or relapsed cases), and those who had signs and symptoms of other forms of leishmaniasis, such as post kala-azar dermal leishmaniasis (PKDL) were excluded. All clinical studies included in the review clearly corresponded with these inclusion criteria. Nevertheless, the settings of these clinical studies varied: some were conducted in tertiary care centres, and some in smaller hospitals of an intermediary level. Few studies recruited patients at the first-contact point, the most peripheral level of the health service, ie health centres or health posts. Although the level of health service leads to variable prior probability of disease in the study sample, and as such is of influence in diagnostic accuracy studies, we believe that our findings are applicable across the several levels of the health system as an analysis of heterogeneity according to disease prevalence did not reveal any difference in the Se or Sp estimates.

Our review does not apply to HIV-positive patients, and caution should be taken in areas with high HIV-prevalence. Because HIV-status is known to influence diagnostic accuracy (ter Horst 2009 - HIV neg; ter Horst 2009 - HIV pos; Alvar 2008), we originally intended to analyse the accuracy of the RDTs in HIV-positive and HIV-negative patients separately. Unfortunately, the number of included studies of HIV-positive people was too low (n = 3) for a separate analysis or a comparison. Furthermore, the information from the Mediterranean region and from Latin America was too limited to draw robust conclusions. Finally, it was not possible to precisely evaluate the importance of the type of sample (serum, plasma or blood; fresh or frozen urine) or the manufacturer or version of a certain test, because these parameters did not vary enough among the included studies. In particular, Cunningham et al. pointed to important variations of sensitivity in a head-to-head comparison of five brands of RDT where two tests based on rK16 antigen performed substantially less well in east Africa and Brazil (Cunningham 2012).

In summary, we conclude that for current practice of VL control, there is one rapid diagnostic test, the rK39 ICT, which has been sufficiently evaluated, showing high sensitivity and specificity on average, but with a notably lower sensitivity in east Africa compared to the Indian subcontinent. In the Indian subcontinent, the rK39 ICT clearly complies with the normative requirements put forward before, of a sensitivity above 95% and a specificity above 90% (Boelaert 2007). In east Africa, the sensitivity of the rK39 ICT does not comply with these criteria, and can therefore not be used as a stand-alone test to reliably rule out VL in a patient who is suspected to have VL. This does not mean that the use of an rK39 ICT is precluded, but it should be embedded in a test algorithm with a clear instruction on what to do in case of a negative rK39 ICT (second test, referral, come back after two weeks for repeat testing or other).

For the latex agglutination test, although there were only six studies included in the meta-analysis, we can conclude that the low sensitivity makes it unsuitable for use in current clinical practice, though it does not preclude that its performance could be further improved in the future.

No recommendations can be made concerning the FAST test, the rK26 ICT or the rKE16 ICT because of paucity of evidence. 

Authors' conclusions

Implications for practice

The rK39 ICT can be clearly recommended as a rapid diagnostic test for use in clinical care in the Indian subcontinent in patients with febrile splenomegaly and no previous history of VL.

In east Africa, the rK39 ICT can replace the DAT and other diagnostic tests as the basis for the therapeutic decision in patients suspected to have VL. However, because of the low sensitivity of the rK39 ICT, a negative test result does not rule out VL. Therefore, additional actions are needed in case of a negative result (eg second or different test, referral, coming back after two weeks for repeat testing or other). Too little evidence has accrued so far from other regions to make specific recommendations.

It is important to remember that this antibody-based test has to be used in combination with a clinical case definition (fever of more than two weeks, splenomegaly and no previous history of VL) that needs to be strictly adhered to.

The sensitivity of the KAtex latex agglutination test in urine is too low to recommend it for standard practice guidelines for detection of VL in similar settings.

Implications for research

Although this review yielded solid evidence for recommending the rK39 ICT for clinical practice in the Indian subcontinent and East Africa, it would be helpful to conduct in the future more head-to-head comparisons of available tests by region and by test format, as was done by Cunningham et al. (Cunningham 2012), on a regular basis, as this would inform policy makers for their purchasing policies and quality assurance.

Several rapid diagnostic tests such as the latex agglutination test and the FAST should be further developed and evaluated before any recommendation on wide-scale use can be made. Furthermore, better tests are needed, ie tests that are more specific for the acute stage of disease and more sensitive in all geographic regions, especially east Africa.

Last but not least, phase III clinical prospective studies are an essential element in the research and development process of a diagnostic device because they provide the basis for clinical guidelines. More studies of this type are needed, although in the case of VL, they are more complex in terms of reference standards. In addition, further standardization of evaluation methodology and a broader awareness of the QUADAS-2 and STARD criteria by investigators would be beneficial for the quality of future evidence in this field.

Acknowledgements

We thank Anne-Marie Trooskens from the Institute of Tropical Medicine in Antwerp for expert administrative support and Vittoria Lutje, Information Specialist of the Cochrane Infectious Diseases Group for the literature searches.

The editorial base of the Cochrane Infectious Diseases Group (CIDG) is funded by UKaid from the UK Government for the benefit of low- and middle-income countries.

Data

Presented below are all the data for all of the tests entered into the review.

Table Tests. Data tables by test
TestNo. of studiesNo. of participants
1 rK39 immunochromatographic test265544
2 KAtex91848
3 FAST2148
4 rK26 immunochromatographic test1352
5 rK39 Primary Analysis163574
6 rKE16 immunochromatographic test1219
Test 1.

rK39 immunochromatographic test.

Test 2.

KAtex.

Test 3.

FAST.

Test 4.

rK26 immunochromatographic test.

Test 5.

rK39 Primary Analysis.

Test 6.

rKE16 immunochromatographic test.

Appendices

Appendix 1. Statistical methods

A short summary of some less common statistical methods used in this review is given below. A more extensive description of the model used to correct for imperfect reference standards can be found in Menten 2013.

1) Latent class analysis (LCA)

LCA is a modelling technique that can be used in situations in which there is no good reference standard. It assumes that the true disease status in a study population is unknown (or latent). The LCA model estimates the sensitivity and specificity of a set of diagnostic tests (A, B, C, …) on the basis of observed frequencies in test patterns (ABC+++, ABC++-, ABC+-+,…). As such, the LCA model provides a model-based estimate of the gold standard classification; ie the best way to group study participants in diseased or non-diseased.

The basic latent class model assumes that the observed variables are conditionally independent. This means that there should be no associations between the results of the diagnostic tests within each category of the latent variable (disease status). If this assumption does not hold, more advanced techniques (eg based on Bayesian statistical methodology) can be used. To be selected for this review, studies using LCA had to assess the conditional independence assumption between the diagnostic tests, and if conditional dependence was expected, they had to use appropriate statistical methods to take this into account. If a study was selected, the sensitivity and specificity estimates derived from the final LCA model were included in this review.

More information can be found in Hui 1980, Black 2002, Branscum 2005, and Baughman 2008 among other references.

2) The complementary Log-Log function

In the bivariate model a "link" function g(y) is used to allow the use of the Normal (Gaussian) distribution to model the underlying study-specific sensitivity (Se) and specificity (Sp) of each study included in the meta-analysis. The standard link function used in the bivariate model is the logit link:

g(y) = log (y/(1-y))

An alternative link function is the complementary log log (cloglog) link:

g(y) = log(-log(1-y))

Both link functions transform Se and Sp, which are in the interval [0,1], to any real number between minus infinity and plus infinity.

The advantage of the cloglog link with our data is that it approaches infinity less quickly when y approaches 1 and consequently it mitigates the influence of studies that report 100% Se or Sp. This also reduces the inflation of the random-effects standard deviations as is apparent from comparison of Figure 6 and Figure 10. With the logit link, the prediction region extends to below the line of no diagnostic value (Se + Sp < 1), while the study which reports the lowest diagnostic value has Se = 0.75 and Sp = 0.70 (Figure 10). On the other hand, some observed data with high Se and Sp are not contained within the prediction region. The prediction region of the model with the cloglog link contains all observed data points, while not extending far beyond the studies with lowest observed Se and Sp (Figure 6). This is reflected in a lower deviance information criterion (DIC), a measure of model fit, for the cloglog model formulation compared to the logit formulation. The model with the lowest DIC shows the best fit to the data.

3) WinBUGS code for the primary model

WinBUGS is a statistical software for Bayesian analysis using Markov chain Monte Carlo methods. WinBUGS (or its recent open-source version OpenBUGS) provides a flexible Bayesian framework for model fitting. It can be used to fit both the bivariate and HSROC models.

Below is the code to fit the basic bivariate model, allowing for data from studies that use a reference standard and for studies that use latent class analysis.

Assuming there are N=N1+N2 studies:

  • for the N1 studies that use a reference standard, the data extracted is:

    • NDiseased[i] and NNotDiseased[i]: the (true) number of diseased and non-diseased subjects in study i

    • TP[i], TN[i]: the number of true positives and true negatives in study i

  • for the N2 studies that use latent class analysis, the data extracted is:

    • Y1[i] and Y2[i]: the estimates of g(Se) and g(Sp) obtained from the results of the LCA reported in the publication for study i

    • W1[i] and W2[i]: the estimates of the standard error of Y1[i] and Y2[i] for study i

    • with g() the link function (logit or cloglog)

The code is as follows:

model{

# Binomials for Studies that Use a Reference Standard

# (where the reference standard is presumed to be perfect)

for(i in 1:N1){

TP[i] ˜ dbin(SE[i],NDiseased[i])

TN[i] ˜ dbin(SP[i],NNotDiseased[i])

}

# Normals for Studies that Use LCA

for(i in 1:N2){

Y1[i] ˜ dnorm(alpha[i+N1,1],W1[i])

Y2[i] ˜ dnorm(alpha[i+N1,2],W2[i])

}

# Bivariate normals for g(Se) and g(Sp)

for(i in 1:N){

# Implement the link function g()

# If logit is used

logit(SE[i]) <- alpha[i,1]

logit(SP[i]) <- alpha[i,2]

OR

# If cloglog is used

SE[i] <- 1-exp(-exp(alpha[i,1]))

SP[i] <- 1-exp(-exp(alpha[i,2]))

# Specify the bivariate normal

alpha[i,1:2] ˜ dmnorm(mu[],R[,])

}

# Specify Non-Informative Priors

# for means:

mu[1] ˜ dnorm(0.0,.37)

mu[2] ˜ dnorm(0.0,.37)

# for variance-covariance matrix

R[1:2,1:2] ˜ dwish(Omega[,], 2) }

Note: Prior for Omega are provided as data:

Omega = matrix(c(.001,0,0,.001),nrow=2,byrow=T)

Appendix 2. Search strategy

Detailed search strategy

Search set MEDLINE EMBASE
1Exp Leishmaniasis, visceral   [MeSH]Exp Visceral leishmaniasis   [Emtree]
2Exp Leishmania donovani  [MeSH]Exp Leishmania donovani [Emtree]
3Exp Leishmania infantum   [MeSH]Exp Leishmania infantum   [Emtree]
4Kala azar OR kala-azar  ti, abExp Leishmania chagasi   [Emtree]
5Leishmania chagasi ti, abKala azar OR kala-azar  ti, ab
6Visceral leishmania* ti, abVisceral leishmania* ti, ab
71-6/OR1-6/OR
8Rapid diagnostic test* ti, abRapid diagnostic test* ti, ab
9RDT ti, abRDT ti, ab
10Antigen* detect*  ti, abAntigen detection [Emtree]
11Antibod* detect* ti, abAntibody detection [Emtree]
12Latex Fixation Tests [MeSH]Latex agglutination test [Emtree]
13Lateral flow test ti, abLateral flow test ti, ab
14Enzyme-Linked Immunosorbent Assay [MeSH]Latex fixation test* ti, ab
15Serodiagnostic test* ti, abEnzyme-linked immunosorbent assay [Emtree]
16ELISA  ti, abELISA  ti, ab
17Direct agglutination test* ti, abAgglutination test [Emtree]
18Dipstick* ti, abDipstick* ti, ab
19K39 antigen, Leishmania [Substance Name]Test strip [Emtree]
20K26 antigen, Leishmania [Substance Name]K39 Or rK39 ti, ab
21K39 Or rK39 ti, abK26 or rK26 ti, ab
22Strip test* ti, abSerodiagnosis [Emtree]
23Reagent kits, diagnostic [MeSH]Immunoblotting [Emtree]
24Immunoblotting [MeSH]8-12/OR
25Serological tests [MeSH]7 AND 24
268-25/OR Limit 25 to humans
277 AND 26 
28Limit 27 to humans 
27  
28  

Appendix 3. Interpretation of the clinical relevance of the findings of this review - predictive values and likelihood ratios

What follows is an interpretation of the clinical relevance of the findings of this review regarding the rK39 immunochromatographic test (ICT) when it is used to diagnose visceral leishmaniasis (VL) among patients with febrile splenomegaly and no previous history of VL.

Indian subcontinent

When the rK39 ICT is used in the Indian subcontinent, in a setting where the prior probability of VL among clinical suspects is 40%, which is typically seen in a peripheral health centre in an endemic area, the positive predictive value of the test is 87%. This means that out of 100 patients with a positive rK39 result, 87 would have VL (true positive result) and 13 would have another disease (false positive). The negative predictive value is 98%, meaning that out of 100 patients with a negative rK39 ICT result, 98 would have another disease (true negative) and 2 would have VL (false negative).

When the same test is used in a setting with a prior probability of VL of 60%, which is more typical for a referral centre in an endemic area, the positive predictive value is 94% and the negative predictive value is 95%.

A likelihood ratio is another way of expressing how informative a diagnostic test is: it indicates to what extent the rK39 ICT result changes the odds that a patient has VL. The likelihood ratio of a positive rK39 ICT result is 9.90, and the likelihood ratio of a negative test result is 0.03. This means that in the Indian subcontinent, a positive rK39 ICT result is a strong argument in favour of VL (ruling in) and that a negative rK39 ICT result is a strong argument against VL (ruling out).

East Africa

When the rK39 ICT is used in east Africa, in a setting where the prior probability of VL is 40%, which is typically seen in a peripheral health centre in an endemic area, the positive predictive value of the test is 86%. This means that out of 100 patients with a positive rK39 ICT result, 86 would have VL (true positive result) and 14 would have another disease (false positive). The negative predictive value is 90%, meaning that out of 100 patients with a negative rK39 ICT result, 90 would have another disease (true negative) and 10 would have VL (false negative).

When the same test is used in a setting with a prior probability of VL of 60%, which is more typical for a referral centre in an endemic area, the positive predictive value is 93% and the negative predictive value is 81%.

In east Africa, the likelihood ratio of a positive rK39 ICT result is 9.58, and the likelihood of a negative rk39 ICT result is 0.16. This means that a positive rK39 ICT result is strong argument in favour of VL (ruling in), and that a negative rK39 ICT result is not an absolute argument against VL (does not allow to rule out VL completely).

Contributions of authors

Marleen Boelaert, Joris Menten, François Chappuis and Suman Rijal performed previous work that was the foundation of the current study. Marleen Boelaert, Joris Menten and Temmy Sunyoto conceived and designed the review. Marleen Boelaert, Temmy Sunyoto, Kristien Verdonck and Johan van Griensven screened the retrieved papers against the inclusion criteria. Marleen Boelaert, Johan van Griensven and Kristien Verdonck extracted data and appraised the quality of the papers. Data management for the review was carried out by Joris Menten, Kristien Verdonck and Temmy Sunyoto. Joris Menten did the statistical analysis. Marleen Boelaert, Joris Menten, Temmy Sunyoto and Kristien Verdonck wrote the first draft of the review. All authors contributed to the final manuscript. Marleen Boelaert is the guarantor of the review.

Declarations of interest

Marleen Boelaert, Joris Menten, François Chappuis and Suman Rijal are authors of studies included in this review. There are no other known conflicts of interest.

Sources of support

Internal sources

  • Institute of Tropical Medicine, Antwerp, Belgium.

External sources

  • Belgian Development Cooperation, Belgium.

    Grant to Temmy Sunyoto

  • Department of Economy, Science and Innovation of the Flemish Government, Belgium.

    Funding for the Clinical Trials Unit at the Institute of Tropical Medicine, Antwerp

Differences between protocol and review

For the assessment of the quality of the included records, we planned and started to work with QUADAS. When QUADAS-2 became available, we switched to this new tool (Whiting 2011). In the review, we only report the information obtained using QUADAS-2.

In order to avoid loss of information, we used a somewhat wider interpretation of the composite reference standard. For the sake of clarity, we slightly reformulated the accepted reference standards in the section Criteria for considering studies for this review.

In the statistical modelling, we used a complementary log-log, rather than a logit link function in the bivariate model. The choice was based on an improved fit of the model to the data as described in the findings section.

Characteristics of studies

Characteristics of included studies [ordered by study ID]

Boelaert 2004 - classic

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 310

Age (reported for 181 new VL cases): median 25, interquartile range 13-36

Sex (reported for 181 new VL cases): male:female ratio 1.7:1

Presenting signs and symptoms: fever of 14 days or more and splenomegaly

Frequency of VL: 59%

HIV: not reported

Clinical setting: tertiary care centre (B.P. Koirala Institute of Health Sciences in Dharan)

Country: Nepal

Endemic Leishmania species: L. donovani

Index tests

Type: rK39 immunochromatographic test

Brand: InSure Rapid test for Visceral Leishmaniasis, InBios International, Washington, USA

Sample: serum

Target condition and reference standard(s)

Target condition: clinical VL (3/310 participants were relapse cases)

Sample: bone marrow; if negative, possible and no malaria: spleen

Technique: direct smear (Giemsa stain)

Definition of VL: bone marrow or spleen parasitology positive

Definition of non-VL: bone marrow negative, and spleen negative or spleen aspirate not done

Reference standard category: parasitology including spleen aspirate - no serology

Flow and timingone participant was excluded because of a missing serum sample
Comparative 
NotesSame study as Boelaert 2004 - LCA but different analysis
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Boelaert 2004 - LCA

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 310

Age (reported for 181 new VL cases): median 25, interquartile range 13-36

Sex (reported for 181 new VL cases): male:female ratio 1.7:1

Presenting signs and symptoms: fever of 14 days or more and splenomegaly

Frequency of VL: 59%

HIV: not reported

Clinical setting: tertiary care centre (B.P. Koirala Institute of Health Sciences in Dharan)

Country: Nepal

EndemicLeishmania species: L. donovani

Index tests

Type: rK39 immunochromatographic test

Brand: InSure Rapid test for Visceral Leishmaniasis, InBios International, Washington, USA

Sample: serum

Target condition and reference standard(s)

Target condition: clinical VL (3/310 participants were relapse cases)

Approach: latent class analysis

Tests included in latent class analysis: rK39 immunochromatographic test, pancytopenia, IFAT, DAT, formol-gel test, and parasitology (direct smear; Giemsa stain) of bone marrow or spleen samples

Flow and timingOne participant was excluded because of a missing serum sample
Comparative 
NotesSame study as Boelaert 2004 - classic but different analysis
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Boelaert 2008 - Ethiopia

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 38

Age: median 25 years; range 16-49. No children ≤ 12 years

Sex: 95% men

Presenting signs and symptoms: fever of 2 weeks or more, and splenomegaly or lymphadenopathy or both

Pregnant women were excluded. Patients with a thick-film-positive malaria episode were excluded.

Estimated frequency of VL: 57%

HIV: not tested

Clinical setting: university hospital (Kahsay Abera Hospital in Humera and Gondar College of Medical Sciences)

Country: Ethiopia

Endemic Leishmania species: L. donovani

Index tests

Type: rK39 immunochromatographic test; Brand: Kalazar Detect, InBios International, Washington, USA; Sample: serum

Type: latex agglutination test in urine; Brand: KAtex, Kalon Biological Ltd, Guildford, UK; Sample: fresh urine

Target condition and reference standard(s)

Target condition: clinical VL

Approach: latent class analysis

Tests included in latent class analysis: rK39 immunochromatographic test, latex agglutination test, DAT, and parasitology (culture and direct smear (Giemsa stain)) of lymph node, bone marrow or spleen sample

Flow and timingThree participants were excluded because of missing data.
Comparative 
NotesSame study as Diro 2007 but different analysis.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Boelaert 2008 - India

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 352

Age (computed on 260/352 participants): median 15 years; range 2-65; 44% of the study population was ≤ 12 years old

Sex (computed on 260/352 participants): 55% men

Presenting signs and symptoms: fever (more or less than 2 weeks), and splenomegaly or lymphadenopathy or both

No children < 2 years old; no pregnant women

Estimated frequency of VL: 80%

HIV: not reported

Clinical setting: research centre (Kala-Azar Medical Research and Training Centre in Muzaffarpur)

Country: India

Endemic Leishmania species: L. donovani

Index tests

Type: rK39 immunochromatographic test; Brand: Kalazar Detect, InBios International, Washington, USA; Sample: serum

Type: latex agglutination test in urine; Brand: KAtex, Kalon Biological Ltd, Guildford, UK; Sample: fresh urine

Target condition and reference standard(s)

Target condition: clinical VL

Approach: latent class analysis

Tests included in latent class analysis: rK39 immunochromatographic test, latex agglutination test, DAT, and parasitology (direct smear (Giemsa stain)) of lymph node, bone marrow or spleen sample

Flow and timingNo withdrawals
Comparative 
NotesSame study as Sundar 2007 but different analysis
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Boelaert 2008 - Kenya

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 308

Age: median 13 years; range 1-75; 45% of the study population was ≤12 years old

Sex: 65% men

Presenting signs and symptoms: fever for 2 weeks or more, and splenomegaly or lymphadenopathy or both;

no children < 2 years old; no pregnant women; no patients with a thick-film-positive malaria episode

Estimated frequency of VL: 61%

HIV: not reported

Clinical setting: hospital (Kabarnet hospital in Baringo district)

Country: Kenya

Endemic Leishmania species: L. donovani

Index tests

Type: rK39 immunochromatographic test; Brand: Kalazar Detect, InBios International, Washington, USA; Sample: serum

Type: latex agglutination test in urine; Brand: KAtex, Kalon Biological Ltd, Guildford, UK; Sample: fresh urine

Target condition and reference standard(s)

Target condition: clinical VL

Approach: latent class analysis

Tests included in latent class analysis: rK39 immunochromatographic test, latex agglutination test, DAT, and parasitology (direct smear (Giemsa stain)) of lymph node, bone marrow or spleen sample

Flow and timingOne participant was excluded because of missing data.
Comparative 
Notes 
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Boelaert 2008 - Nepal

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 158

Age: median 23 years; range 2-68; 25% of the study population was ≤ 12 years old

Sex: 59% men

Presenting signs and symptoms: fever for 2 weeks or more, and splenomegaly or lymphadenopathy or both;

No children < 2 years old; no pregnant women

Estimated frequency of VL: 71%

HIV: not reported

Clinical setting: tertiary care centre (B.P. Koirala Institute of Health Sciences in Dharan)

Country: Nepal

Endemic Leishmania species: L. donovani

Index tests

Type: rK39 immunochromatographic test; Brand: Kalazar Detect, InBios International, Washington, USA; Sample: serum

Type: latex agglutination test in urine; Brand: KAtex, Kalon Biological Ltd, Guildford, UK; Sample: fresh urine

Target condition and reference standard(s)

Target condition: clinical VL

Approach: latent class analysis

Tests included in latent class analysis: rK39 immunochromatographic test, latex agglutination test, DAT, and parasitology (direct smear (Giemsa stain)) of lymph node, bone marrow or spleen sample

Flow and timingNo withdrawals
Comparative 
Notes 
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Boelaert 2008 - Sudan

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 294

Age: median 20 years; range 2-72; 36% of the study population was ≤12 years old

Sex: 64% men

Presenting signs and symptoms: fever for 2 weeks or more, and splenomegaly or lymphadenopathy or both

No children < 2 years old; no pregnant women; no patients with a thick-film-positive malaria episode

Estimated frequency of VL: 37%

HIV: not reported

Clinical setting: two health centres (Umalkhair and Tabarakelleh)

Country: Sudan

Endemic Leishmania species: L. donovani

Index tests

Type: rK39 immunochromatographic test; Brand: Kalazar Detect, InBios International, Washington, USA; Sample: serum

Type: latex agglutination test in urine; Brand: KAtex, Kalon Biological Ltd, Guildford, UK; Sample: fresh urine

Target condition and reference standard(s)

Target condition: clinical VL

Approach: latent class analysis

Tests included in latent class analysis: rK39 immunochromatographic test, latex agglutination, DAT, and parasitology (direct smear (Giemsa stain)) of lymph node, bone marrow or spleen sample

Flow and timingThree participants were excluded because of missing data.
Comparative 
Notes 
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Chappuis 2003

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 195

Age (data available for 184 included participants): mean 23 years

Sex (data available for 184 included participants): 59% men

Presenting signs and symptoms: fever for 2 weeks or more and clinical splenomegaly

Frequency of VL: 76%

HIV: 0%

Clinical setting: tertiary care centre (B.P. Koirala Institute of Health Sciences in Dharan)

Country: Nepal

Endemic Leishmania species: L. donovani

Index tests Type: rK39 immunochromatographic test; Brand: InSure Rapid Test for Visceral Leishmaniasis, InBios Interational, Washington, USA; Sample: serum
Target condition and reference standard(s)

Target condition: clinical VL

Sample: bone marrow; if bone marrow negative and spleen aspiration possible: spleen

Technique: direct smear examination

Reference standard category: parasitology including spleen aspirate - no serology

Flow and timingEleven participants with an uncertain diagnosis were excluded.
Comparative 
Notes 
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Unclear  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Chappuis 2005 - DiaMed

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 255

Age (among 131 VL patients): mean age 14 years (standard deviation 11)

Age (among 112 non-VL patients): mean age 16 years (standard deviation 13)

Sex (among 131 VL patients): 73% men;

Sex (among 112 non-VL patients): 53% men

Presenting signs and symptoms: fever for 2 weeks or more and either clinical splenomegaly or wasting syndrome

Frequency of VL: 54%

6/112 non-VL patients had previous history of treatment for VL

HIV: not reported

Clinical setting: a 120-bed district hospital (Amudat Hospital)

Country: Uganda

Endemic Leishmania species: L. donovani

Index tests Type: rK39 immunochromatographic test (dual test for visceral leishmaniasis and malaria); Brand: DUAL-IT L/M dipstick, Diamed AG, Switzerland; Sample: blood
Target condition and reference standard(s)

Target condition: clinical VL

Combination of parasitology of spleen sample (direct smear, Giemsa stain), PCR of spleen sample, serology (DAT), and response to VL therapy

Definition of VL: (1) positive spleen aspirate in two laboratories (by microscopic reading in local or regional laboratory and by PCR in reference laboratory) or (2) DAT >1:12800 and response to VL therapy

Definition of non-VL: (1) DAT titres of <1:1600; or (2) borderline DAT titres with negative spleen aspirate in two laboratories with no diagnosis of VL made during the following 6 months

Reference standard category: combination of parasitology and serology

Flow and timingTwelve participants were excluded: 2 persons died and 10 had discrepant results for the reference standard
Comparative 
NotesSame patient population as Chappuis 2005 - InBios but different brand of rK39 immunochromatographic test. Considered as two studies in meta-analysis.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Yes  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Chappuis 2005 - InBios

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 255

Age (among 131 VL patients): mean age 14 years (standard deviation 11)

Age (among 112 non-VL patients): mean age 16 years (standard deviation 13)

Sex (among 131 VL patients): 73% men;

Sex (among 112 non-VL patients): 53% men

Presenting signs and symptoms: fever for 2 weeks or more and either clinical splenomegaly or wasting syndrome

Frequency of VL: 54%

6/112 non-VL patients had previous history of treatment for VL

HIV: not reported

Clinical setting: a 120-bed district hospital (Amudat Hospital)

Country: Uganda

Endemic Leishmania species: L. donovani

Index tests Type: rK39 immunochromatographic test; Brand: InBios International, Washington, USA; Sample: plasma
Target condition and reference standard(s)

Target condition: clinical VL

Combination of parasitology of spleen sample (direct smear, Giemsa stain), PCR of spleen sample, serology (DAT), and response to VL therapy

Definition of VL: (1) positive spleen aspirate in two laboratories (by microscopic reading in local or regional laboratory and by PCR in reference laboratory) or (2) DAT >1:12800 and response to VL therapy

Definition of non-VL: (1) DAT titres of <1:1600; or (2) borderline DAT titres with negative spleen aspirate in two laboratories with no diagnosis of VL made during the following 6 months

Reference standard category: combination of parasitology and serology

Flow and timingTwelve participants were excluded: 2 persons died and 10 had discrepant results for the reference standard
Comparative 
NotesSame patient population as Chappuis 2005 - DiaMed but different brand of rK39 immunochromatographic test. Considered as two studies in meta-analysis.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Yes  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Chappuis 2006b

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 154

Age (among 85 VL patients): mean age 22 years (standard deviation 13)

Age (among 57 non-VL patients): mean age 26 (standard deviation 15)

Sex (among 85 VL patients): 57% men

Age (among 57 non-VL patients): 65% men

Presenting signs and symptoms: fever for 2 weeks or more and clinical splenomegaly

All patients received malaria treatment

12/142 participants had previous history of VL

Frequency of VL: 60%

HIV: not tested

Clinical setting: 15-bed first-referral government hospital (Rangeli District Hospital)

Country: Nepal

Endemic Leishmania species: L. donovani

Index tests Type: rK39 immunochromatographic test; Brand: InSure One-Step Rapid Test for Visceral Leishmaniasis, InBios International, Washington, USA; Sample: serum
Target condition and reference standard(s)

Target condition: clinical VL

Combination of parasitology of bone marrow sample (direct smear) and serology (DAT) and response to therapy

Definition of VL: (1) positive bone marrow aspirate during initial evaluation or follow-up; or (2) DAT titre ≥1:3200 and absence of response to antimalarial treatment but successful response to anti-leishmanial therapy

Definition of non-VL: negative bone marrow aspirate with (1) DAT titre < 1:3200 or with (2) positive DAT but a definite cure with non-VL specific therapy

Reference standard category: combination of parasitology and serology

Flow and timingTwelve patients were excluded: 4 with incomplete diagnostic work-up and 8 with an uncertain diagnosis
Comparative 
NotesExclusion of patients with previous history of VL had no significant effect on Se and Sp estimates (data not reported). Data on accuracy of latex agglutination test were incomplete and not included.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Cota 2013 - composite 1

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 113

Age (VL cases): mean 41.0 years (standard deviation 10.8)

Age (non-VL cases): mean 40.0 years (standard deviation 9.8)

Sex (VL cases): 24% men (11 men out of 46)

Sex (non-VL cases): 42% men (28 men out of 67)

Presenting signs and symptoms: clinical suspicion of VL, ie more than 14 days of fever or splenomegaly or cytopenia

Previous VL diagnosis: 24 out of 113 participants had a previous history of VL

Frequency of VL: 41%

HIV: 100%

Clinical setting: HIV cohort in reference hospital in Belo Horizonte (Eduardo de Menezes Hospital, Fundação Hospitalar do Estado de Minos Gerais)

Country: Brazil

Endemic Leishmania species: L. infantum

Index tests Type: rK39 immunochromatographic test; Brand: Kalazar Detect, InBios International, Washington, USA; sample: serum
Target condition and reference standard(s)

Target condition: clinical VL

Samples: bone marrow aspirate and venous blood

Techniques: direct smear and culture of bone marrow aspirate; PCR on peripheral blood; and three serological tests: index test, indirect fluorescent antibody test (IFI, Bio-Manguinhos) and direct agglutination test (prototype kit)

Definition of VL and non-VL: decided after clinical follow-up by an adjudication committee with four members. They evaluated results of all tests available, including tests performed for other diagnostic possibilities

Reference standard category: parasitology and serology

Flow and timing 
Comparative 
NotesAnother estimate of Se and Sp based on latent class analysis in the same study population was not included because there was no information about the assessment of the conditional independence assumption (explained in the Criteria for considering studies for this review and in Appendix 1).
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   High
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?No  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Cota 2013 - composite 2

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 113

Age (VL cases): mean 41.0 years (standard deviation 10.8)

Age (non-VL cases): mean 40.0 years (standard deviation 9.8)

Sex (VL cases): 24% men (11 men out of 46)

Sex (non-VL cases): 42% men (28 men out of 67)

Presenting signs and symptoms: clinical suspicion of VL, ie more than 14 days of fever or splenomegaly or cytopenia

Previous VL diagnosis: 24 out of 113 participants had a previous history of VL

Frequency of VL: 37%

HIV: 100%

Clinical setting: HIV cohort in reference hospital in Belo Horizonte (Eduardo de Menezes Hospital, Fundação Hospitalar do Estado de Minos Gerais)

Country: Brazil

Endemic Leishmania species: L. infantum

Index tests Type: rK39 immunochromatographic test; Brand: Kalazar Detect, InBios International, Washington, USA; sample: serum
Target condition and reference standard(s)

Target condition: clinical VL

Samples: bone marrow aspirate

Techniques: direct smear and culture

Reference standard category: parasitology not including spleen aspirate - no serology

Flow and timingTwo patients were not included in the analysis because bone marrow aspiration was not done
Comparative 
NotesAnother estimate of Se and Sp based on latent class analysis in the same study population was not included because there was no information about the assessment of the conditional independence assumption (explained in the Criteria for considering studies for this review and in Appendix 1).
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   High
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?No  
Were the reference standard results interpreted without knowledge of the results of the index tests?Yes  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Diro 2007

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 38

Age: median 25 years; range 16-49. No children ≤ 12 years

Sex: 95% men

Presenting signs and symptoms: fever of 2 weeks or more, and splenomegaly or lymphadenopathy or both

Pregnant women and people with a previous history of VL treatment were not included. Patients with a thick-film-positive malaria episode were excluded.

Frequency of VL: 61%

HIV: not tested

Clinical setting: university hospital (Kahsay Abera Hospital in Humera and Gondar College of Medical Sciences)

Country: Ethiopia

Endemic Leishmania species: L. donovani

Index tests

Type: rK39 immunochromatographic test; Brand: Kalazar Detect, InBios International, Washington, USA; Sample: serum

Type: Latex agglutination test in urine; Brand: KAtex, Kalon Biologicals Ltd, Guildford, UK; Sample: fresh urine

Target condition and reference standard(s)

Target condition: clinical VL

Sample: spleen or lymph node aspirate

Technique: parasitology: culture or direct smear (Giemsa stain)

Reference standard category: parasitology including spleen aspirate - no serology

Flow and timing 
Comparative 
NotesFindings in context of active case detection were not included in this review. Same study as Boelaert 2008 - Ethiopia but different analysis.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Hailu 2006

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 103

Age: not reported

Sex: not reported

Presenting signs and symptoms: fever for 2 weeks or more and splenomegaly

Not clear whether VL relapse suspects were excluded

Frequency of VL: 51% (45/89)

HIV: not tested

Clinical setting: health centres in rural northern Ethiopia (Humera and Gondar)

Country: Ethiopia

Endemic Leishmania species: L. donovani

Index tests Type: FAST; Brand: not applicable; Sample: not reported
Target condition and reference standard(s)

Target condition: clinical VL

Sample: lymph node or spleen

Technique: direct smear (Giemsa stain) or culture

Reference standard category: parasitology including spleen aspirate - no serology

Flow and timingFourteen patients were excluded, probably because the FAST results could not be read under field conditions.
Comparative 
Notes 
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Kilic 2008

Study characteristics
Patient samplingConsecutive enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 59

Age (data available for 24 VL patients): median age 7 years; 22/24 cases <14 years

Sex: 71% men

Presenting signs and symptoms: prolonged fever + splenomegaly or hepatomegaly + anaemia/pancytopenia

Frequency of VL: 41%

HIV: not reported

Clinical setting: tertiary care centre

Country: Turkey

Endemic Leishmania species: L. infantum

Index tests Type: FAST; Brand: not applicable; Sample: serum
Target condition and reference standard(s)

Target condition: clinical VL

Sample: bone marrow

Technique: direct smear (Giemsa stain) and culture

Reference standard category: parasitology not including spleen aspirate - no serology

Flow and timingNo information about exclusion
Comparative 
Notes 
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Unclear  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Unclear  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?No  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Unclear  
Did all patients receive a reference standard?Yes  
    

Machado de Assis 2011

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 332

Age: median 13 years; range 1 month - 76 years

Sex: 58% men

Presenting signs and symptoms: fever + coming from endemic area + one of the following: splenomegaly, hepatomegaly, anaemia, leucopenia, and thrombocytopenia

Frequency of VL: 64%

HIV: people with known immunodeficiency were excluded

Clinical setting: two university hospitals and two research centres in four different states

Country: Brazil

Endemic Leishmania species: L. infantum

Index tests Type: rK39 immunochromatographic test; Brand: IT-LEISH, DiaMed Latino-America S.A, Cressier sur Morat, Switzerland; Sample: blood
Target condition and reference standard(s)

Target condition: clinical VL

Sample: bone marrow

Technique: culture or direct smear (Giemsa stain); at least 2 smears per patient

Definition of VL: positive parasitological test

Definition of non-VL: negative parasitological test and firm diagnosis of another disease

Reference standard category: parasitology not including spleen aspirate - no serology

Flow and timingPeople with missing data or uncertain diagnosis were not mentioned
Comparative 
NotesSame study as Machado de Assis 2012 but different analysis. Same study as Peruhype-Magalhaes 2012 but different brand of index test.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Unclear  
Did the study avoid inappropriate exclusions?Unclear  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Unclear  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Unclear  
Did all patients receive a reference standard?Yes  
    

Machado de Assis 2012

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 404

Age: median 13 years; range 1 month - 77 years; standard deviation 17 years

Sex: 58% men

Presenting signs and symptoms: fever + coming from endemic area + one of the following: splenomegaly, hepatomegaly, anaemia, leucopenia, and thrombocytopaenia

Frequency of VL: 67%

HIV: people with known immunodeficiency were excluded

Clinical setting: two university hospitals and two research centres in four different states

Country: Brazil

Endemic Leishmania species: L. infantum

Index tests Type: rK39 immunochromatographic test; Brand: IT-LEISH, DiaMed Latino-America S.A, Cressier sur Morat, Switzerland; Sample: finger prick sample of blood
Target condition and reference standard(s)

Target condition: clinical VL

Sample: bone marrow

Approach: latent class analysis

Tests included in latent class analysis: rK39 immunochromatographic test, rK39-ELISA, IFAT, DAT, microscopy of bone marrow sample

Flow and timingPeople with missing data or inconclusive test results were not mentioned
Comparative 
NotesSame study as Machado de Assis 2011 but different analysis. Same study as Peruhype-Magalhaes 2012 but different brand of index test.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Unclear  
Did the study avoid inappropriate exclusions?Unclear  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Unclear  
Did all patients receive a reference standard?Yes  
    

Mbui 2013

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL ≥ 5 years with suspected VL and presenting at outpatient department
Patient characteristics and setting

Sample size: 249 (total number of participants)

Age (VL cases): median 16 years (interquartile range 10-25 years)

Age (non-VL cases): median 16.5 years (interquartile range 11-25 years)

Sex (VL cases): 66.4% men

Sex (non-VL cases): 79.6% men

Presenting signs and symptoms: fever for 2 weeks or more + clinical splenomegaly + malaria ruled out by a negative rapid test (Paracheck)

Frequency of VL: 59.8%

HIV: 0.5% (195 participants were tested; 1 non-VL case was HIV positive)

Clinical setting: Kimalel Health Centre, Baringo district and Kacheliba Kala-azar Treatment Centre, Pokot North district

Country: Kenya

Endemic Leishmania species: L. donovani

Index tests

Type: rK39 immunochromatographic test; Brand: DiaMed-IT LEISH, DiaMed AG, Switzerland; Sample: serum

Type: rKE16 immunochromatographic test; Brand: Signal KA, Span Diagnostics Ltd, India; Sample: serum

Target condition and reference standard(s)

Target condition: clinical VL

Sample: spleen aspirate

Technique: direct smear examination (Giemsa stain)

Definition of VL: positive splenic aspirate

Definition of non-VL: negative splenic aspirate

Reference standard category: parasitology including spleen aspirate - no serology

Flow and timingThirty participants were excluded: 27 had contra-indications for spleen aspiration and 3 had an inconclusive smear reading
Comparative 
NotesUnclear if study population includes people with previous history of VL
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Yes  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Peruhype-Magalhaes 2012

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 278

Age: mean 12 years; range 1 month - 77 years

Sex: 58% (161/278) men

Presenting signs and symptoms: fever + coming from endemic area + one of the following: splenomegaly, hepatomegaly, anaemia, leucopenia, and thrombocytopaenia

Frequency of VL: 69% (193/278)

HIV: people with known immunodeficiency were excluded

Clinical setting: two university hospitals and two research centres in four different states

Country: Brazil

Endemic Leishmania species: L. infantum

Index tests Type: rK39 immunochromatographic test; Brand: Kalazar Detect, InBios International, Washington, USA; Sample: stored serum
Target condition and reference standard(s)

Target condition: clinical VL

Sample: bone marrow

Technique: culture or direct smear (Giemsa stain); at least 2 smears per patient

Definition of VL: positive parasitological test

Definition of non-VL: negative parasitological test and firm diagnosis of another disease

Reference standard category: parasitology not including spleen aspirate - no serology

Flow and timingUnclear why there are 54 participants less than in Machado de Assis 2011
Comparative 
NotesSame study population as Machado de Assis 2011 and Machado de Assis 2012. Evaluation of a new index test on stored serum samples.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Unclear  
Did the study avoid inappropriate exclusions?Unclear  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Unclear  
Were the reference standard results interpreted without knowledge of the results of the index tests?Yes  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Unclear  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?No  
Did all patients receive a reference standard?Yes  
    

Rijal 2004

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 269

Age (155 VL patients): median age 23 (interquartile range 13-26)

Age (77 non-VL patients): median age 20 (interquartile range 10-30)

Sex: not reported

Presenting signs and symptoms: fever for 2 weeks or more and clinical splenomegaly

Frequency of VL: 67%

HIV: done but not reported; at least 3 HIV-positive participants among non-VL patients

Clinical setting: tertiary care centre (B.P. Koirala Institute of Health Sciences in Dharan)

Country: Nepal

Endemic Leishmania species: L. donovani

Index tests Type: latex agglutination test in urine; Brand: KAtex, Kalon Biological Ltd, Aldershot, UK; Sample: frozen urine
Target condition and reference standard(s)

Target condition: clinical VL

Combination of parasitology (direct smear, Giemsa stain) of bone marrow or spleen aspirate and serology (DAT)

Definition of VL: parasitology positive

Definition of non-VL: parasitology negative and DAT ≤1:3200

Reference standard category: combination of parasitology and serology

Flow and timingThirty-seven patients were not included: the urine samples of 8 patients were lost; 1 person left against medical advice; and 28 had an uncertain diagnosis (parasitology negative and DAT positive)
Comparative 
NotesNot clear whether VL relapse suspects were excluded
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Yes  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?No  
Did all patients receive a reference standard?Yes  
    

Ritmeijer 2006

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 356

Age: not reported

Sex: not reported

Presenting signs and symptoms: fever for more than 2 weeks + malaria ruled out + wasting + splenomegaly or lymphadenopathy

Frequency of VL: 67% (228/341)

HIV: not reported

Clinical setting: three Kala-azar treatment centres in eastern and southern Sudan, supported by Médecins sans frontières

Country: Sudan

Endemic Leishmania species: L. donovani

Index tests Type: rK39 immunochromatographic test; Brand: DiaMed-IT Leish, DiaMed AG, Cressier sur Morat, Switzerland; Sample: blood
Target condition and reference standard(s)

Target condition: clinical VL

Combination of parasitology (direct smear, Giemsa stain) of lymph node or spleen aspirate sample and serology (DAT)

Definition of VL: parasitology positive or DAT positive ≥1:6400

Definition of non-VL: DAT≤1/400 or borderline DAT with negative aspirate
Reference standard category: combination of parasitology and serology

Flow and timingFifteen patients were excluded because of an uncertain diagnosis
Comparative 
Notes 
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

Sundar 1998

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 323

Age: mean age of VL patients: 25 years (standard error 1);

among non-VL patients, age is reported in subgroups: 73 patients with documented other infections: mean age 26 (standard error 2); 83 patients with presumed other infections: mean age 23 (standard error 2); and 40 patients without final diagnosis: mean age 25 years (standard error 2)

Sex: 69% men

Frequency of VL: 39%

Presenting signs and symptoms: persistent fever with or without splenomegaly and suspected VL

Some patients had been empirically treated with antimalarial or antibacterial agents before referral, without response

HIV: not reported

Clinical setting: kala-azar units in Varanasi and Muzaffarpur, linked to research centre

Country: India

Endemic Leishmania species: L. donovani

Index tests Type: rK39 immunochromatographic test; Brand: not applicable;Sample: blood
Target condition and reference standard(s)

Target condition: clinical VL

Combination of parasitology (direct smear Giemsa stain) of spleen aspirate sample and diagnosis of other diseases

Reference standard category: parasitology including spleen aspirate - no serology

Flow and timingReference standard was conditional on result of index test.
Comparative 
Notes 
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Yes  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?No  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?No  
    

Sundar 2007

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 352

Age: median 15 years; interquartile range 2-65

Sex: 55% men

Presenting signs and symptoms: fever for 2 weeks or more and splenomegaly

No children < 1 year old; no pregnant women; no people with past kala-azar history

Frequency of VL: 80%

HIV: known HIV infection exclusion criterion

Clinical setting: two research centres, in Muzaffarpur and Patna

Country: India

Endemic Leishmania species: L. donovani

Index tests

Type: rK39 immunochromatographic test; Brand: InBios International, Washington, USA; Sample: serum or blood

Type: rK26 immunochromatographic test; Brand: InBios International, Washington, USA; Sample: serum or blood

Type: latex agglutination test in urine; Brand: KAtex, Kalon Biologicals, Aldershot, UK; Sample: fresh urine

Target condition and reference standard(s)

Target condition: clinical VL

Combination of parasitology (direct smear, Giemsa stain) of spleen aspirate sample and serology (DAT) and clinical diagnosis / follow-up / response to therapy

Definition of VL: (1) spleen parasitology positive; or (2) clinical diagnosis and DAT positive ≥ 1:3200 and response to anti-leishmanial therapy

Definition of non-VL: spleen parasitology negative and alternative diagnosis and no VL during follow-up of six months

Reference standard category: combination of parasitology and serology

Flow and timingNo withdrawals explained.
Comparative 
NotesSame study as Boelaert 2008 - India but different analysis and additional test (rK26 immunochromatographic test). Group of 100 healthy endemic controls not included in this review.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Yes  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Yes  
Did all patients receive a reference standard?Yes  
    

ter Horst 2009 - HIV neg

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 179

Age: reported for larger population (n = 699): mean age is 25,4 (standard deviation 10,1)

Sex: reported for larger population (n = 699): ratio men:women is 15:1

Presenting signs and symptoms: clinical VL case definition (not specified)

No patients with previous history of VL treatment.

Frequency of VL: 94%

HIV: all HIV negative

Clinical setting: kala-azar treatment centre supported by Médecins sans frontières (Kahsay Abera Hospital in Humera)

Country: Ethiopia

Endemic Leishmania species: L. donovani

Index tests Type: rK39 immunochromatographic test; Brand: DiaMed AG, Switzerland; Sample: blood
Target condition and reference standard(s)

Target condition: clinical VL

Combination of parasitology (direct smear, Giemsa stain) of spleen aspirate sample and serology (DAT)

Definition of VL: (1) spleen parasitology positive; or (2) DAT positive ≥ 1:3200

Definition of non-VL: (1) DAT ≤ 1:400; or (2) DAT≤ 1:3200 and spleen parasitology negative

Reference standard category: combination of parasitology and serology

Flow and timingMany patients with unknown HIV status were not included.
Comparative 
Notes 
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Unclear  
Were the reference standard results interpreted without knowledge of the results of the index tests?Yes  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Unclear  
Were all patients included in the analysis?No  
Did all patients receive a reference standard?Yes  
    

ter Horst 2009 - HIV pos

Study characteristics
Patient samplingConsecutive and prospective enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 71

Age: reported for larger population (n = 699): mean age is 25.4 (standard deviation 10.1)

Sex: reported for larger population (n = 699): ratio men:women is 15:1

Presenting signs and symptoms: clinical VL case definition (not specified)

No patients with previous history of VL treatment.

Frequency of VL: 92%

HIV: all HIV positive

Clinical setting: kala-azar treatment centre supported by Médecins sans frontières (Kahsay Abera Hospital in Humera)

Country: Ethiopia

Endemic Leishmania species: L. donovani

Index tests Type: rK39 immunochromatographic test; Brand: DiaMed AG, Switzerland; Sample: blood
Target condition and reference standard(s)

Target condition: clinical VL

Combination of parasitology (direct smear, Giemsa stain) of spleen aspirate sample and serology (DAT)

Definition of VL: (1) spleen parasitology positive; or (2) DAT positive ≥ 1:3200

Definition of non-VL: (1) DAT ≤ 1:400; or (2) DAT≤ 1:3200 and spleen parasitology negative

Reference standard category: combination of parasitology and serology

Flow and timingMany patients with unknown HIV status were not included.
Comparative 
NotesNot included in meta-analysis because this is the only evaluation of the rK39 immunochromatographic test in a population of HIV-positive patients.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Yes  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Yes  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Unclear  
Were the reference standard results interpreted without knowledge of the results of the index tests?Yes  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Unclear  
Were all patients included in the analysis?No  
Did all patients receive a reference standard?Yes  
    

Toz 2004

Study characteristics
Patient samplingConsecutive enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 58

Age: only reported for 13 cases with all tests positive: 12 children between 1 and 15 years and 1 adult of 48 years

Sex: not reported

Frequency of VL: 28%

Presenting signs and symptoms: ≥1 of the following signs: fever, anaemia, thrombocytopenia, leucopenia, splenomegaly, hepatomegaly, and weight loss

HIV: not reported

Clinical setting: various regional hospitals

Country: Turkey

Endemic Leishmania species: L. infantum

Index tests Type: rK39 immunochromatographic test; Brand: InBios International, Washington, USA; Sample: serum or plasma
Target condition and reference standard(s)

Target condition: clinical VL

Combination of parasitology (direct smear, Giemsa stain) of lymph node or bone marrow aspirate sample and serology (IFAT)

Definition of VL: (1) lymph node or bone marrow parasitology positive; or (2) IFAT positive ≥ 1:128

Definition of non-VL: parasitology and IFAT negative

Reference standard category: combination of parasitology and serology

Flow and timingNo information about excluded patients.
Comparative 
Notes 
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Unclear  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Unclear  
   Low
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Unclear  
Did all patients receive a reference standard?Yes  
    

Veeken 2003 - composite

Study characteristics
Patient samplingRetrospective selection of patients with suspected VL
Patient characteristics and setting

Sample size: 77

Age: median 11; range 4-66 years

Sex: 53% men

Presenting signs and symptoms: fever, and splenomegaly or wasting

Frequency of VL: 70%

HIV: not reported

Clinical setting: kala-azar treatment centre at Um el Kher supported by Médecins sans frontières

Country: Sudan

Endemic Leishmania species: L. donovani

Index tests Type: rK39 immunochromatographic test; Brand: Amrad ICT, Brookdale, Australia; Sample: serum
Target condition and reference standard(s)

Target condition: clinical VL

Combination of parasitology (direct smear, Giemsa stain) of spleen aspirate sample and serology (DAT)

Definition of VL: spleen parasitology positive or DAT positive ≥ 1:6400

Definition of non-VL: all other patients

Reference standard category: combination of parasitology and serology

Flow and timingOnly people with stored serum and available results of DAT and parasitological test of spleen aspirate were included. According to the protocol by Médecins sans frontières, splenic aspiration was done in patients with suspected VL who were critically ill and in those with borderline DAT titres.
Comparative 
NotesSame study as Veeken 2003 - spleen but different analysis.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?No  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Unclear  
   High
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Unclear  
Were all patients included in the analysis?No  
Did all patients receive a reference standard?Unclear  
    

Veeken 2003 - spleen

Study characteristics
Patient samplingRetrospective selection of patients with suspected VL
Patient characteristics and setting

Sample size: 77

Age: median 11; range 4-66 years

Sex: 53% men

Presenting signs and symptoms: fever, and splenomegaly or wasting

Frequency of VL: 65%

HIV: not reported

Clinical setting: kala-azar treatment centre at Um el Kher supported by Médecins sans frontières

Country: Sudan

Endemic Leishmania species: L. donovani

Index tests Type: rK39 immunochromatographic test; Brand: Amrad ICT, Brookdale, Australia; Sample: serum
Target condition and reference standard(s)

Target condition: clinical VL

Sample: spleen

Technique: parasitology (direct smear, Giemsa stain)

Reference standard category: parasitology including spleen aspirate - no serology

Flow and timingOnly people with stored serum and available results of DAT and parasitological test of spleen aspirate were included. According to the protocol by Médecins sans frontières, splenic aspiration was done in patients with suspected VL who were critically ill and in those with borderline DAT titres.
Comparative 
NotesSame study as Veeken 2003 - composite but different analysis.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?No  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Unclear  
   High
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Yes  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Unclear  
Were all patients included in the analysis?No  
Did all patients receive a reference standard?Unclear  
    

Vilaplana 2004

  1. a

    DAT: direct agglutination test
    ELISA: enzyme-linked immuno-sorbent assay
    FAST: fast agglutination screening test
    IFAT: indirect fluorescence antibody test
    LCA: latent class analysis
    PCR: polymerase chain reaction
    Se: sensitivity
    Sp: specificity
    VL: visceral leishmaniasis

Study characteristics
Patient samplingConsecutive enrolment of patients with suspected VL
Patient characteristics and setting

Sample size: 89

Age: not reported

Sex: not reported

Presenting signs and symptoms: not specified (suggestive clinical signs and symptoms of VL)

Frequency of VL: 14% (12/85)

HIV: 100% of cases and 91% of non-cases

Clinical setting: not reported

Country: Spain

Endemic Leishmania species: L. infantum

Index tests Type: latex agglutination test in urine; Brand: KAtex, Kalon Biological Ltd, Aldershot, UK
Target condition and reference standard(s)

Target condition: clinical VL

Sample: bone marrow

Technique: parasitology: culture or direct smear (Giemsa stain)

Reference standard category: parasitology not including spleen aspirate - no serology

Flow and timingThree patients are not included in the analysis
Comparative 
NotesNot included in meta-analysis because only evaluation of latex agglutination test in population with mainly HIV-positive patients.
Methodological quality
ItemAuthors' judgementRisk of biasApplicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?Yes  
Was a case-control design avoided?Yes  
Did the study avoid inappropriate exclusions?Unclear  
   Unclear
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?Unclear  
   Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?Unclear  
Were the reference standard results interpreted without knowledge of the results of the index tests?Unclear  
   Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?Yes  
Did all patients receive the same reference standard?Yes  
Were all patients included in the analysis?Unclear  
Did all patients receive a reference standard?Yes  
    

Characteristics of excluded studies [ordered by study ID]

StudyReason for exclusion
Abeijon 2013index test is not a rapid test
Ahsan 2010not phase III diagnostic accuracy study
Akhoundi 2010not phase III diagnostic accuracy study
Al-Nahhas 2003target condition is not clinical visceral leishmaniasis
Al-Nahhas 2008not phase III diagnostic accuracy study
Alam 2008not phase III diagnostic accuracy study
Alborzi 2006not phase III diagnostic accuracy study
Amato 2009not phase III diagnostic accuracy study
Arya 1997not original research article
Arya 2001not original research article
Attar 2001not phase III diagnostic accuracy study
Azazy 2004not phase III diagnostic accuracy study
Bagchi 1998not phase III diagnostic accuracy study
Bern 2000not phase III diagnostic accuracy study
Brandonisio 2002not phase III diagnostic accuracy study
Canavate 2011not phase III diagnostic accuracy study
Carvalho 2003not phase III diagnostic accuracy study
Cruz 2006not phase III diagnostic accuracy study
de Assis 2008publication of the same study in more than one record
Delgado 2001not phase III diagnostic accuracy study
Edrissian 2003not phase III diagnostic accuracy study
El-Safi 2003not phase III diagnostic accuracy study
Ferreira Dourado 2007not original research article
Gavgani 2008not phase III diagnostic accuracy study
Goswami 2003not phase III diagnostic accuracy study
Goswami 2007not phase III diagnostic accuracy study
Goswami 2012not phase III diagnostic accuracy study
Gupta 1994not phase III diagnostic accuracy study
Hatam 2009not phase III diagnostic accuracy study
Hommel 2001not original research article
Iqbal 2002not phase III diagnostic accuracy study
Jelinek 1999target condition is not clinical visceral leishmaniasis
Khan 2009not phase III diagnostic accuracy study
Khan 2010not phase III diagnostic accuracy study
Kumar 2006not phase III diagnostic accuracy study
Lemos 2003not phase III diagnostic accuracy study
López Corbalán 2012not phase III diagnostic accuracy study
Mandal 2008reference standard is not according to criteria
Mansour 2009not phase III diagnostic accuracy study
Marty 2007not phase III diagnostic accuracy study
Mathur 2005not phase III diagnostic accuracy study
Matlashewski 2013reference standard is not according to criteria
Mbui 2011publication of the same study in more than one record
Mohapatra 2010not phase III diagnostic accuracy study
Monno 2009not phase III diagnostic accuracy study
Moura 2013reference standard is not according to criteria
Mueller 2013not phase III diagnostic accuracy study
Ozerdem 2009reference standard is not according to criteria
Ozkan 2008not phase III diagnostic accuracy study
Pappas 1983not phase III diagnostic accuracy study
Pappas 1984not phase III diagnostic accuracy study
Pappas 1984anot phase III diagnostic accuracy study
Pattabhi 2010not phase III diagnostic accuracy study
Qu 1987target condition is not clinical visceral leishmaniasis
Qu 2000not phase III diagnostic accuracy study
Riera 2004not phase III diagnostic accuracy study
Rouf 2009not phase III diagnostic accuracy study
Saghrouni 2009not phase III diagnostic accuracy study
Saha 2011not phase III diagnostic accuracy study
Salam 2008not phase III diagnostic accuracy study
Salam 2011target condition is not clinical visceral leishmaniasis
Salotra 2005not original research article
Sarkari 2005not phase III diagnostic accuracy study
Sarker 2003not phase III diagnostic accuracy study
Schallig 2002not phase III diagnostic accuracy study
Schoone 2001not phase III diagnostic accuracy study
Scott 1991not phase III diagnostic accuracy study
Senaldi 1996not phase III diagnostic accuracy study
Shamsuzzaman 2003not phase III diagnostic accuracy study
Sharma 2009target condition is not clinical visceral leishmaniasis
Silva 2005not phase III diagnostic accuracy study
Singh 2009not phase III diagnostic accuracy study
Singh 2010not phase III diagnostic accuracy study
Sinha 2008not phase III diagnostic accuracy study
Srivastava 1988not phase III diagnostic accuracy study
Sundar 2002reference standard is not according to criteria
Sundar 2002anot phase III diagnostic accuracy study
Sundar 2002bnot original research article
Sundar 2005not phase III diagnostic accuracy study
Sundar 2005apublication of the same study in more than one record
Sundar 2006not phase III diagnostic accuracy study
Sundar 2006anot phase III diagnostic accuracy study
Teran-Angel 2010not phase III diagnostic accuracy study
Walton 1986not phase III diagnostic accuracy study
Walton 1987publication of the same study in more than one record
Welch 2008not phase III diagnostic accuracy study
Zijlstra 2001not phase III diagnostic accuracy study

Ancillary