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Keywords:

  • visceral leishmaniasis;
  • diagnosis;
  • polymerase chain reaction;
  • nucleic acid sequence-based amplification;
  • sensitivity;
  • specificity
  • leishmaniose viscérale;
  • diagnostic;
  • PCR;
  • NASBA;
  • sensibilité;
  • spécificité
  • leishmaniasis visceral;
  • diagnóstico;
  • PCR;
  • NASBA;
  • sensibilidad;
  • especificidad

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective  To estimate the sensitivity and specificity of the OligoC-TesT and nucleic acid sequence-based amplification coupled to oligochromatography (NASBA-OC) for molecular detection of Leishmania in blood from patients with confirmed visceral leishmaniasis (VL) and healthy endemic controls from Kenya.

Methods  Blood specimens of 84 patients with confirmed VL and 98 endemic healthy controls from Baringo district in Kenya were submitted to both assays.

Results  The Leishmania OligoC-TesT showed a sensitivity of 96.4% (95% confidence interval [CI]: 90–98.8%) and a specificity of 88.8% (95% CI: 81–93.6%), while the sensitivity and specificity of the NASBA-OC were 79.8% (95% CI: 67–87%) and 100% (95% CI: 96.3–100%), respectively.

Conclusion  Our findings indicate high sensitivity of the Leishmania OligoC-TesT on blood while the NASBA-OC is a better marker for active disease.

Sensibilité et spécificité des tests Leishmania OligoC et NASBA-Oligochromatographie pour le diagnostic de la leishmaniose viscérale au Kenya

Objectif:  Estimer la sensibilité et la spécificité des tests OligoC et NASBA-OC pour la détection moléculaire de Leishmania dans le sang de patients LV confirmés et contrôles sains de zones endémiques du Kenya.

Méthodes:  Des échantillons de sang de 84 patients LV confirmés et 98 contrôles sains de zones endémiques dans le district de Baringo au Kenya ont été soumis aux deux tests.

Résultats:  Le test Leishmania OligoC a révélé une sensibilité de 96,4% (intervalle de confiance à 95% [IC]: 90% -98,8%) et une spécificité de 88,8% (IC95%: 81% -93,6%) tandis que la sensibilité et la spécificité du test NASBA-OC étaient de 79,8% (IC95%: 67% -87%) et 100% (IC95%: 96,3% -100%), respectivement.

Conclusion:  Nos résultats indiquent une sensibilitéélevée du test Leishmania OligoC sur le sang tandis que le test NASBA-OC est un meilleur marqueur de la maladie active.

Sensibilidad y especificidad de la prueba Leishmania OligoC-TesT y NASBA-Oligochromatografía para el diagnóstico de la leishmaniasis visceral en Kenia

Objetivo:  Calcular la sensibilidad y especificidad de la prueba OligoC-TesT y NASBA-OC para la detección molecular de Leishmania en sangre de pacientes con confirmación de LV y controles sanos provenientes de áreas endémicas de Kenia.

Métodos:  Ambas pruebas se realizaron con muestras de sangre de 84 pacientes con LV confirmada y 98 controles sanos provenientes de áreas endémicas del distrito de Baringo, en Kenia.

Resultados:  La prueba Leishmania OligoC-TesT mostró una sensibilidad del 96.4% (95% intervalo de confianza [IC]: 90%-98.8%) y una especificidad del 88.8% (95% IC: 81%-93.6%) mientras que la sensibilidad y la especificidad del NASBA-OC fue del 79.8% (95% CI: 67%-87%) y 100% (95% CI: 96.3%-100%), respectivamente.

Conclusión:  Nuestros hallazgos indican una alta sensibilidad del Leishmania OligoC-TesT en sangre, mientras que el NASBA-OC es un mejor marcador para la enfermedad activa.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Visceral leishmaniasis (VL) is a major health problem on the African continent, where it mainly affects rural populations (Hunt et al. 2007). In East Africa, VL is exclusively linked with the Leishmania donovani species, and parasites mainly infect the reticuloendothelial organs like the liver, spleen and bone marrow. Antibody detection tests such as the direct agglutination test (DAT) (Harith et al. 1988; Boelaert et al. 1999) and the rK39 immunochromatographic strip test (Chappuis et al. 2005; Ritmeijer et al. 2006; ter Horst et al. 2009) play a key role in diagnosis of the disease, but parasite demonstration by microscopic analysis of lymph, bone marrow or splenic aspirates is still frequently used in East Africa as a confirmation test.

Molecular techniques such as polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) are powerful tools for parasite detection as they combine sensitivity and specificity (Antinori et al. 2007; Reithinger & Dujardin 2007) and can be applied to blood and other types of specimens. Hence, molecular detection of Leishmania parasites’ DNA or RNA in blood might lead to less invasive diagnosis of VL than conventional parasite detection in bone marrow or spleen aspirates. Several test formats are available, which can be classified into ‘high-tech’, ‘mid-tech’ and ‘low-tech’ approaches. High-tech approaches are methods that require elaborate and rather expensive equipment such as real-time PCR. Mid-tech approaches are probably the most widely used and comprise conventional PCR assays, in which PCR amplicons are resolved by electrophoresis in agarose gels. Low-tech approaches refer to simplified amplification and/or detection methods for use in laboratory settings with minimal molecular biology equipment.

Recently, the Leishmania OligoC-TesT and nucleic acid sequence-based amplification coupled to oligochromatography (NASBA-OC) were introduced as standardised low-tech molecular diagnostics for leishmaniasis (Deborggraeve et al. 2008a; Mugasa et al. 2010b). These assays amplify a part of the 18S ribosomal DNA or RNA by PCR (OligoC-TesT) or NASBA (NASBA-OC) where after the amplification products are detected by a simple and rapid dipstick method based on oligochromatography. Both assays showed high sensitivity and specificity for Leishmania detection during the phase I evaluations (Deborggraeve et al. 2008a; Mugasa et al. 2010b) and satisfactory repeatability and reproducibility in a multicentre evaluation study (Mugasa et al. 2010a). In this study, we subjected both tests to a phase II evaluation (Boelaert et al. 2007) on blood specimens collected from patients with confirmed VL and healthy endemic controls in Baringo district in Kenya.

Methodology

  1. Top of page
  2. Summary
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Ethical considerations

Ethical clearance for the study was obtained from the institutional ethical committee of Kenya Medical Research Institute (KEMRI). Informed consent was obtained from the patients or their guardians and from the healthy endemic controls. Patients with confirmed VL were referred to the Centre for Clinical Research (CCR) at KEMRI or to Kabarnet District Hospital, where medication was provided to them at no cost.

Study participants

This phase II diagnostic study was carried out between 2007 and 2008, during which patients with VL were recruited in villages of the VL endemic areas in Baringo district (Kenya). Healthy endemic controls were volunteers recruited from the same villages or other villages in the endemic areas.

Participant classification and reference tests

A participant was classified as a confirmed VL case if there was clinical suspicion for VL, if DAT titre on serum was 1:12 000 or more and if parasites were observed during microscopic analysis of splenic aspirate. A participant was classified as a healthy endemic control if there was no previous history of VL, if there was no clinical suspicion for VL and if DAT on serum showed a titre of 1:6400 or below. Clinical suspicion for VL was defined as a history of fever for 2 weeks or more and splenomegaly or lymphadenopathy. The reference tests were performed at the collection site as described in the WHO manual on VL (WHO 1996) except for splenic aspirate taking. Individuals suspected of VL and with a positive DAT titre were transported to the Centre for Clinical Research (CCR) at KEMRI in Nairobi for microscopic analysis of splenic aspirates.

Index tests

Two hundred microlitres of blood was collected from the patients and healthy endemic controls and mixed with the same volume of L3 buffer (University of Amsterdam, confidential). This guanidium-based buffer allows storage of the blood without loss of DNA and RNA quality. The stabilised blood specimens were shipped at 4 °C from the collection site to KEMRI and stored at 4 °C for a maximum of 2 weeks. The nucleic acids of the blood specimens were extracted by the method described by Boom et al. (1990). Elution was performed in 50 μl RNAse-free water by incubating at 56 °C for 10 min, and the purified nucleic acids were stored at −80 °C until further analysis. The extracts were tested with the Leishmania OligoC-TesT and NASBA-OC as described by Deborggraeve et al. (2008a) and Mugasa et al. (2010b). Test kits were provided by Coris BioConcept (Gembloux, Belgium). In brief, the Leishmania OligoC-TesT amplifies a short sequence of the Leishmania 18S ribosomal DNA by PCR, and the NASBA-OC amplifies a part of the 18S ribosomal RNA by NASBA. For both assays, the amplification products are mixed with migration buffer pre-heated to 55 °C after which the dipstick is dipped into the solution. Migration is performed at 55 °C, and test results are read after 10 min. The two tests contain internal controls to validate the migration as well as the amplification reaction. Nucleic acid extraction was performed at KEMRI within 2 weeks of arrival of the specimen from the collection site. The extracts were analysed with the index tests between October 2008 and February 2009 at Khartoum University (Sudan) in the frame of a collaborative consortium. No external quality control confirming the reference test or index test results could be performed during the study. The executors of the index tests were not blinded to the participant classification and thus the results of the reference tests.

Statistical analysis

The sensitivity and specificity of the Leishmania OligoC-TesT and NASBA-OC were calculated from data entered into contingency tables. Differences in sensitivity and specificity between the two tests were estimated by the McNemar test. Concordances between the two tests were determined using the kappa index. All calculations were estimated at a 95% confidence interval (95% CI).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Eighty-four patients with VL and 98 endemic healthy controls were recruited in Baringo district in Kenya. An overview of the sensitivity and specificity of the OligoC-TesT and NASBA-OC on the study population is presented in Table 1.

Table 1.   Diagnostic accuracy of the Leishmania OligoC-TesT and NASBA-OC on blood from confirmed VL cases and healthy endemic controls from Baringo district, Kenya
ParticipantsNumber of specimensOligoC-TesTNASBA-OC
Number of positivesSensitivity % (95% CI)Specificity % (95% CI)Number of positivesSensitivity % (95% CI)Specificity % (95% CI)
  1. CI, confidence interval; VL, visceral leishmaniasis; NASBA-OC, nucleic acid sequence-based amplification coupled to oligochromatography.

Confirmed VL cases848196.4 (90–98.8) 6779.8 (67–87) 
Healthy endemic controls9811 88.8 (81–93.6)0 100 (96.3–100)

Sensitivity of the Leishmania OligoC-TesT and NASBA-OC

The Leishmania OligoC-TesT and NASBA-OC on blood showed a positive test result in 81 and 67 of the patients with VL, respectively. Hence, the sensitivity of the Leishmania OligoC-TesT is 96.4% (95% CI: 90–98.8%) and of the NASBA-OC 79.8% (95% CI: 67–87%), which is significantly lower (< 0.05).

Specificity of the Leishmania OligoC-TesT and NASBA-OC

Eleven out of 98 healthy endemic controls were positive with the Leishmania OligoC-TesT, while no positive test results were observed with the NASBA-OC, indicating a specificity of 88.8% (95% CI: 81–93.6%) and 100% (95% CI: 96.3–100%), respectively. The McNemar test indicated a significant difference in specificity of both tests (< 0.05).

Test agreement

For the 182 blood specimens analysed, the kappa index was 0.73 (95% CI: 0.59–0.87), indicating substantial agreement in results of the two index tests.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

When analysing blood from the confirmed VL cases, we observed a significantly higher sensitivity of the Leishmania OligoC-TesT (96.4%) than the NASBA-OC (79.8%). As VL diagnosis was confirmed by microscopic analysis of spleen aspirates, the high sensitivity of the OligoC-TesT on blood is promising. Indeed, this means that the OligoC-TesT on blood could indicate the infection status of VL-suspected cases. Our findings are in agreement with the sensitivities shown by conventional PCR techniques for Leishmania, which generally range from 92% to 100% (Anderson et al. 1994; Salotra et al. 2001; Maurya et al. 2005). The lower sensitivity of NASBA-OC was unexpected because this test showed similar sensitivity as the OligoC-TesT in a multicentre evaluation study on Leishmania parasite-spiked blood (Mugasa et al. 2010a). The lower sensitivity observed in the current phase II study might be because of the fact that NASBA-OC targets the parasite’s RNA, which is known to be more liable to degradation compared to DNA. Nucleic acid quality might have been decreased during specimen storage and transportation from the collection sites in the field to the reference centre in Nairobi.

In contrast, the NASBA-OC showed a significant higher specificity than the OligoC-TesT when analysing blood from the healthy endemic controls (100%vs. 88.8%). Because the controls taken along during specimen analysis did not indicate contamination of the PCR-based OligoC-TesT, the detection of Leishmania DNA in this control group is probably associated with asymptomatic Leishmania infections, which are known to be common in VL endemic areas (Deborggraeve et al. 2008b; Bhattarai et al. 2009). The inclusion of healthy parasite carriers in the control group can be explained by the low concordance between negative DAT status and PCR outcome on blood from endemic control persons, as described by Deborggraeve et al. (2008b) and Bhattarai et al. (2009). The observation that these healthy parasite carriers are not picked up by the NASBA-OC is probably because of its lower sensitivity, as reflected in the results on the confirmed VL cases. Patients with confirmed VL are individuals presenting symptoms and thus probably have higher parasite loads than asymptomatic parasite carriers. The sensitivity and specificity of both tests should also be evaluated in settings that do not need specimen storage and transport. It might be that in the present study, the sensitivity of the NASBA-OC is underestimated, and the specificity overestimated because of RNA degradation during specimen storage or transport.

The low specificity of the Leishmania OligoC-TesT in this study confirms that PCR-based diagnostic tests are a marker of infection rather than disease, as described by Deborggraeve et al. (2008b). As only diseased persons are treated, PCR alone is of less value for VL diagnosis in endemic regions. However, one could state the same for diagnosis based on antibody detection tests alone or on clinical data alone because none of them represent a gold standard for acute disease. Hence, laboratory tests should always be interpreted in combination with a standardised clinical case definition. Although molecular diagnostics are not yet applicable in most operational settings because of their complexity, they can be useful in hospitals with basic molecular biology facilities. Molecular diagnostics should not replace the existing immunodiagnostics but give an added value as a marker of infection.

It is important to emphasise that the Leishmania OligoC-Test and NASBA-OC are important steps forward to improved and standardised molecular detection of the parasite, but they are still restricted to use in reference centres with basic molecular biology facilities. Efforts towards further simplification of molecular diagnostics should therefore be strongly encouraged. In addition, although still to be confirmed by large-scale studies, molecular detection of the parasite might be useful for cure assessment. While antibodies remain detectable for years after successful treatment (Hailu 1990), the parasite’s RNA and DNA is rapidly degraded following parasite death (Prina et al. 2007).

In conclusion, we successfully evaluated two innovative molecular diagnostics for VL in a phase II study on Kenyan patients with VL and healthy endemic controls. The Leishmania OligoC-TesT showed high sensitivity on blood but a rather low specificity for active disease. In contrast, the sensitivity of RNA detection by NASBA-OC in blood was lower but the specificity was 100%, indicating that it might be a better marker for active VL.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The study received financial support from the commission of the European communities’ Sixth Framework Programme, priority INCO-DEV, project TRYLEIDIAG, contract 015379. S. Deborggraeve is a postdoctoral fellow of the Research Foundation Flanders (FWO). OligoC-TesT and NASBA-OC kits used in this evaluation study were provided by Coris Bioconcept, Gembloux, Belgium. We further acknowledge Dr. Seuleiman Hussein, Director Soba Teaching Hospital, Khartoum, Sudan for the laboratory facilities. We are also grateful to all study participants and field workers from KEMRI who efficiently mobilised the participants.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Anderson K, Gasim S & Elhassan AM (1994) Diagnosis of visceral leishmaniasis by polymerase chain reaction using blood, bone marrow and lymph node samples from patients from the Sudan. Tropical Medicine and International Health 2, 440444.
  • Antinori S, Calattini S, Longhi E et al. (2007) Clinical use of polymerase chain reaction performed on peripheral blood and bone marrow samples for the diagnosis and monitoring of visceral leishmaniasis in HIV-infected and HIV-uninfected patients: a single-center, 8-year experience in Italy and review of the literature. Clinical Infectious Diseases 44, 16021610.
  • Bhattarai NR, Van der Auwera G, Khanal B et al. (2009) PCR and direct agglutination as Leishmania infection markers among healthy Nepalese subjects living in areas endemic for Kala-Azar. Tropical Medicine and International Health 14, 404411.
  • Boelaert M, El Safi S, Jacquet D, De Muynck A, Van der Stuyft P & Le Ray D (1999) Operational validation of the direct agglutination test for diagnosis of visceral leishmaniasis. American Journal of Tropical Medicine and Hygiene 60, 129134.
  • Boelaert M, Bhattacharya S, Chappuis F et al. (2007) Evaluation of rapid diagnostic tests: visceral leishmaniasis. Nature Reviews. Microbiology 5, S30S39.
  • Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-van Dillen PME & Van Der Noordaa J (1990) Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28, 495503.
  • Chappuis F, Mueller Y, Nguimfack A et al. (2005) Diagnostic accuracy of two rK39 antigen-based dipsticks and the formol gel test for rapid diagnosis of visceral leishmaniasis in northeastern Uganda. Journal of Clinical Microbiology 43, 59735977.
  • Deborggraeve S, Laurent T, Espinosa D et al. (2008a) A simplified and standardized polymerase chain reaction format for the diagnosis of leishmaniasis. Journal of Infectious Diseases 198, 15651572.
  • Deborggraeve S, Boelaert M, Rijal S et al. (2008b) Diagnostic accuracy of a new Leishmania PCR for clinical visceral leishmaniasis in Nepal and its role in diagnosis of disease. Tropical Medicine and International Health 13, 13781383.
  • Hailu A (1990) Pre- and post-treatment antibody levels in visceral leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 84, 673675.
  • Harith AE, Kolk AH, Leewenburg J et al. (1988) Improvement of a direct agglutination test for field studies of visceral leishmaniasis. Journal of Clinical Microbiology 26, 13211325.
  • Hunt P, Steward R, Bueno de Mesuita J & Oldring L (2007) Neglected Diseases: A Human Rights Analysis. WHO, Geneva.
  • Maurya R, Singh RK, Kumar B, Salotra P, Rai M & Sundar S (2005) Evaluation of PCR for diagnosis of Indian kala-azar and assessment of cure. Journal of Clinical Microbiology 43, 30383041.
  • Mugasa C, Deborggraeve S, Schoone G et al. (2010a) Accordance and concordance of PCR and NASBA followed by oligochromatography for the molecular diagnosis of Trypanosoma brucei and Leishmania. Tropical Medicine and International Health in press.
  • Mugasa C, Laurent T, Schoone G et al. (2010b) Simplified molecular detection of Leishmania parasites in various clinical samples from patients with leishmaniasis. Parasites and Vectors, 3, 1318.
  • Prina E, Roux E, Mattei D & Milon G (2007) Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR. Microbes and Infection 9, 13071315.
  • Reithinger R & Dujardin JC (2007) Molecular diagnosis of leishmaniasis: current status and future applications. Journal of Clinical Microbiology 45, 2125.
  • Ritmeijer K, Melaku Y, Mueller M, Kipngetich S, O’keeffe C & Davidson RN (2006) Evaluation of a new recombinant K38 rapid diagnostic test for Sudanese visceral leishmaniasis. American Journal of Tropical Medicine and Hygiene 74, 7680.
  • Salotra P, Screenivas G & Pouge GP (2001) Development of a special species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post kala-azar dermal leishmaniasis. Journal of Clinical Microbiology 39, 357361.
  • Ter Horst R, Tefera T, Assefa G, Ebrahim AZ, Davidson RN & Ritmeijer K (2009) Field evaluation of rK39 test and direct agglutination test for diagnosis of visceral leishmaniasis in a population with high prevalence of human immunodeficiency virus in Ethiopia. American Journal of Tropical Medicine and Hygiene 80, 929934.
  • WHO (1996) Manual on Visceral Leishmaniasis. WHO, Geneva, Switzerland, pp. 179.