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- Materials and methods
Objective To evaluate five kala-azar serological tests for field use.
Method Serological survey in Pandit Ka Purva village in Varanasi district, India, using Sia water test, aldehyde test, direct agglutination test (DAT), micro-enzyme-linked immunosorbent assay (ELISA) and dot-ELISA.
Results The total population of the village was 518, 67 of whom showed typical clinical and parasitological features of kala-azar, including seven who died. The age distribution of kala-azar cases showed significant differences, being highest among the 45–54-year age group. The disease was more prevalent among males. Serum samples were collected from 498 persons (96% of total population) including 67 kala-azar cases and 40 disease controls (malaria, TB, leprosy, typhoid). Ten 10 serum samples from healthy controls living in endemic area were also collected. The test sensitivities were: Sia water test, 85.0%; aldehyde test, 62.7%; DAT, 94.0%; micro-ELISA, 91.0% and dot-ELISA, 97.0%. The test specificities were: Sia water test 92.5%, aldehyde test, 93.2%, DAT, 96.7; micro-ELISA, 97.6% and dot-ELISA, 98.4%.
Conclusion The dot-ELISA is highly sensitive and specific, cheap, and easy to interpret with the naked eye, making it a powerful screening test for the surveillance and diagnosis of Indian kala-azar at field level.
Objectif: Evaluer 5 tests sérologiques du kala-azar pour leur utilisation sur le terrain.
Méthode: Suivi sérologique dans le village de Pandit Ka Purva dans le district de Varanasi en Inde, en utilisant les tests Sia water, Aldéhyde, DAT, micro-ELISA et dot-ELISA.
Résultats La population du village était de 518 habitants dont 67 avec des caractéristiques cliniques et parasitologiques typiques du kala-azar; 7 personnes parmi ces derniers sont décédées. Une différence significative a été observée dans la distribution d’âge des cas de kala-azar; le nombre étant plus élevée dans le groupe d’âge de 45–54 ans. La maladie était plus prévalente chez les hommes. Des échantillons de sérum ont été prélevés chez 498 personnes (96% de la population) dont 67 cas de kala-azar et 40 cas contrôles (malaria, TB, Lèpre, typhoïde). Dix (10) échantillons de sérum de contrôles sains vivant dans la zone endémique ont également été inclus. Les sensitivités des tests étaient les suivantes:
Sia water: 85.0%, Aldéhyde: 62.7%, DAT: 94.0%, micro-ELISA: 91.0% et dot-ELISA: 97.0%. Leurs spécificités étaient les suivantes: Sia water: 92.5%, Aldéhyde: 93.2%, DAT: 96.7%, micro-ELISA: 97.6% et dot-ELISA: 98.4%.
Conclusion: Le dot-ELISA a une sensitivité et une spécificité plus élevées, est moins coûteux et facile à interpréter à l’œil nu, ce qui le rend plus performant pour le criblage des échantillons pour la surveillance et le diagnostic sur le terrain du kala-azar indien.
Motsclés kala-azar, micro-ELISA, DAT, dot-ELISA, sérodiagnostic
Objetivo Evaluar 5 pruebas serológicas de kala-azar para su uso en el campo.
Método Encuesta serológica en el poblado de Pandit Ka Purva en el distrito Varanasi, India, utilizando las pruebas de Sia water, prueba de aldehídos, DAT, micro-ELISA y punto-ELISA.
Resultados La población total era de 518, de los cuales 67 mostraban signos clínicos típicos y características parasitológicas de kala-azar, incluyendo los 7 que murieron. La distribución por edad mostraba diferencias significativas, siendo más alta en el grupo de 45–54 años. La enfermedad era más prevalente en los hombres. Se recolectaron muestras de suero a 498 personas (96% de la población), incluyendo 67 casos de kala-azar y 40 enfermos control (malaria, Tuberculosis, lepra, tifoidea). También se tomaron diez muestras de suero a controles residentes en la zona endémica. Las sensibilidad de los test fueron: test de Sia water, 85.0%; test de aldehidos, 62.7%; DAT, 94.0%; micro-ELISA, 91.0% y punto-ELISA, 97.0%. La especificidad en cada caso fué: Test Sia water 92.5%, Test Aldehido test, 93.2%, DAT, 96.7; micro-ELISA, 97.6% y punto-ELISA, 98.4%.
Conclusión El punto-ELISA es altamente sensible y específico, barato y fácil de interpretar a simple vista, convirtiéndolo en una prueba de tamizaje muy poderosa para el seguimiento y el diagnóstico de kala-azar en el campo.
Palabras clave kala-azar, micro-ELISA, dot-ELISA, diagnóstico serológico
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- Materials and methods
Kala-azar or visceral leishmaniasis (VL) is a major public health problem in many parts of the world. Half a million cases of VL and 57 000 deaths of VL patients are reported worldwide annually (http://www.who.int/tdr/disease/leish/diseaseinfo.htm). The estimated annual incidence of VL in India is between 100 000 and 200 000 (World Health Organization 2000). The state of Bihar accounts for nearly 90% of the cases, followed by West Bengal and eastern Uttar Pradesh. Most of the affected population is very poor and illiterate and not aware of preventive measures. These patients cannot afford expensive diagnosis or treatment. Demonstration of parasites is the most reliable and conventional method of diagnosing VL. The parasite is commonly found in the splenic or bone marrow aspirate and in the buffy coat of peripheral blood [in human immunodeficiency virus (HIV) co-infection]. The sensitivity of the splenic aspirate smear is more than 95% and is regarded as the gold standard for the diagnosis of kala-azar. But splenic aspiration carries the risk of severe haemorrhage. The sensitivity of bone marrow smear, the other diagnostic option, is only about 60–85% and the procedure is quite painful. Although several serodiagnostic tests, such as the enzyme-linked immunosorbent assay (ELISA) and the indirect fluorescent antibody test (IFAT) have been developed, none are in wide use at the field level because of their high cost, need for electricity, complicated technical procedures and unsatisfactory specificity and sensitivity (Sundar & Rai 2002). The direct agglutination test (DAT) is a cheap, sensitive and specific test that is popular in Africa. But its drawbacks are batch to batch variation, instability of the antigen, need for incubation and a cumbersome procedure; thus, it is not popular in India (Sundar & Rai 2002). The aldehyde test, Sia water test, complement fixation test, indirect agglutination test, IFAT, DAT and ELISA all have advantages and disadvantages.
The rK39 strip test, a rapid immunochromatographic test based on a 39-amino acid antigen found in the kinesin region of amastigotes of Leishmania chagasi, is cheap, easy to use, requires no equipment, and has high sensitivity and specificity (Sundar et al. 1998). However, like other antibody-based tests, it remains positive for long periods after cure, and cannot be used for predicting cure or in relapses (Badaro et al. 1996; Zijlstra et al. 2001). Another drawback of this format is that an individual with a positive rK39 strip test result may suffer from illness(es) (malaria, typhoid fever, or tuberculosis) with clinical features similar to those of VL, yet be misdiagnosed as suffering from VL. Notwithstanding these limitations, the rK39 immunochromatographic strip test has proved to be versatile in predicting acute infection, and it is the only available format for diagnosis of VL with acceptable sensitivity and specificity levels, which is also inexpensive (US$1–1.5) and simple and can be performed even by paramedics in prevailing difficult field conditions. Subclinical VL with few or no symptoms and positive anti-leishmanial serology or documented seroconversion (Badaro et al. 1986, 1996), where direct organ aspirate smears are often negative for parasites (Ho et al. 1982; Marty et al. 1994) is common in several areas where the disease is endemic (Ho et al. 1982; Badaro et al. 1986, 1996; Marty et al. 1994) and may occur in India too (Sacks et al. 1987; Saran et al. 1992). At least some false-positive responses might be expected among patients with subclinical infections, especially those who do not eventually self-cure (Badaro et al. 1996). However, the frequency of subclinical infection in India has not been clearly established, as no clinical or laboratory markers have been described to identify the disease before it is manifested clinically.
Antigen detection is more specific than antibody-based immunodiagnostic tests. Recently, a new latex agglutination test (KATEX) has been developed to detect leishmanial antigen in the urine of patients with kala-azar (Attar et al. 2001). Evaluation of this test is currently underway in India, Sudan, Nepal and Brazil. DNA detection using polymerase chain reaction has also been developed by many laboratories, with a high level of accuracy. Unfortunately, none of these new tools are field-friendly and commercially available. There is therefore a need to develop a specific test that can minimize the chances of false positivity and can help in the early diagnosis of kala-azar. The present study was designed and conducted before the development of the rK39 rapid test to compare the sensitivity and specificity indices of the serological tests performed for the diagnosis of kala-azar in a village of Varanasi district, India.
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- Materials and methods
Reports at the Sir Sunder Lal Hospital, Banaras Hindu University, of a large number of kala-azar cases from one particular village in Varanasi district, Uttar Pradesh, led us to conduct an epidemiological study using standard procedures. The overall prevalence and case fatality of the disease were 12.9% and 10.5% respectively. A history of fever and hepatosplenomegaly was noted in all cases. The case definition was the presence of parasites in bone marrow or splenic aspirate smears. The disease was more prevalent among adults, but occurred among children also (Kumar et al. 1999). Hence it was of interest to compare various specific and non-specific tests available at that time for presence of anti-leishmanial antibodies. This study reports the sero-epidemiology of kala-azar of the village. Sixty-seven parasitologically confirmed VL cases, 481 controls including village residents and healthy endemic controls (relatives of VL patients from Muzaffarpur, Bihar), as well as other infection controls such as malaria, tuberculosis, leprosy, and typhoid cases were studied. The population of the village was 518 at the time, of which 67 (39 males and 28 females) showed typical features of kala-azar, including seven who died. Ninety-six percent including the 67 VL patients (498/518) of the inhabitants, participated in this study. The positivity of LD bodies in splenic aspirate and/or bone marrow smears (Leishman staining) were detemined in the suspected kala-azar cases (data not shown).
The age distribution of kala-azar cases showed significant differences. The disease occurred most frequently in the 45–54-year age groups, followed by those older than 55 years, and was least frequent in the age group below 5 years. Kala-azar was more prevalent among males than females (Table 1). All patients were treated with sodium stibogluconate (20 mg/kg/day i.v. for 30 days); 60 (89.6%) improved and seven (10.5%) died from concurrent infection or advanced disease (data not shown).
Table 1. Age and sex distribution of kala-azar cases in Pandit Ka Purva village, Varanasi district, India
| ||No. of subjects surveyed||n||No. of VL cases*||Z-value||P-value|
|Age group (years)†|
| <5||76||71||2 (2.6)|| || |
| 6–14||149||147||17 (11.4)|| || |
| 15–24||94||93||11 (11.7)|| || |
| 25–34||94||92||13 (13.8)|| || |
| 35–44||47||44||6 (12.8)|| || |
| 45–54||25||24||12 (48.0)|| || |
| >55||33||27||6 (18.2)|| || |
| Male||266|| ||39 (14.7)||1.21||>0.05|
| Female||252|| ||28 (11.1)|| || |
| Total||518||498||67 (12.9)|| || |
The nonimmunological tests such as the Sia water and aldehyde tests were also performed for the comparison with DAT, micro-ELISA and dot-ELISA. The positivity of the Sia water test was 85%, and that of the aldehyde test, 62.7% (Table 2). The result of aldehyde test was analysed on the basis of early and late stages of kala-azar cases. Sixty-seven serum samples of 19 early kala-azar patients (EKA; 1–3 months of fever) and 48 samples of late kala-azar patients (LKA; 4–12 months of fever) were studied. The aldehyde test was positive in 72.92% LKA cases and in 36.84% EKA cases (data not shown). Cross reactivity occurred with the serum samples of malaria, tuberculosis and leprosy cases. The DAT was positive in 94.0% patients with kala-azar at 1:800 dilution. Sixty-one of 67 kala-azar patients (91.0%) were positive with micro-ELISA and six had absorbance in the negative range. In one healthy control from endemic areas, the micro-ELISA was false-positive. Dot-ELISA showed a sensitivity of 97.0% and cross-reactivity with tuberculosis. Of the healthy inhabitants, 7.7% tested positive with the Sia water test, 6.5% with the aldehyde test, 3.5% with the DAT, 2.1% with micro-ELISA, and 1.6% with dot-ELISA (Table 2). The sensitivity and specificity of the various serological tests are shown in Table 3.
Table 2. Sia water test, aldehyde test, DAT, ELISA and dot-ELISA using sera of kala-azar cases and healthy individuals in Pandit Ka Purva village and Muzaffarpur, Bihar, Varanasi district and other infectious diseases
|Subjects (no.)||Sia water test positive (%)||Aldehyde test positive (%)||DAT positive (%)||Micro-ELISA positive (%)||Dot-ELISA positive (%)|
|Kala-azar (67)||57 (85.0)||42 (62.7)||63 (94.0)||61 (91.0)||65 (97.0)|
|Healthy control (431)||33 (7.65)||28 (6.49)||15 (3.48)||9 (2.08)||7 (1.62)|
|Healthy control (endemic) (10)||2 (20.0)||1 (10.0)||1 (10.0)||1 (10.0)||0 (0.0)|
|Malaria (10)||1 (10.0)||2 (20.0)||0 (0.0)||0 (0.0)||0 (0.0)|
|Tuberculosis (10)||2 (20.0)||3 (30.0)||1 (10.0)||2 (20.0)||1 (10.0)|
|Leprosy (10)||1 (10.0)||1 (10.0)||0 (0.0)||0 (0.0)||0 (0.0)|
|Typhoid (10)||0 (0.0)||0 (0.0)||0 (0.0)||0 (0.0)||0 (0.0)|
Table 3. The sensitivity and specificity of various serological tests in Pandit Ka Purva village, Varanasi district, India
|Sia water test||85.01||92.50|
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- Materials and methods
This study provides an evaluation of serological tests for sensitivity and specificity at the community level. At the Kala-azar Medical Research Centre, definitive diagnosis of VL is based on demonstration of parasites in biopsies or aspirates of infected spleen/bone marrow. In preparations stained with Giemsa or Leishman stain, the cytoplasm appears pale blue, with a relatively large nucleus that stains red. After identification, parasite density can be scored microscopically by means of a logarithmic scale ranging from 0 (no parasite per 1000 oil immersion fields) to 6 (100 parasites per field) (Chulay & Bryceson 1983). The sensitivity of the bone marrow smear is about 60–85%. Splenic aspirate, though associated with the risk of fatal haemorrhage in inexperienced hands, is one of the most valuable methods for diagnosis of kala-azar, with a sensitivity exceeding 95%. It requires no special equipment, from the patient's standpoint it is generally preferable to the more painful bone marrow aspirate, and it has proven to be safe and relatively easy to perform in experienced hands. For patients suspected to have VL, splenic aspirate can be performed even when the spleen is not palpable, after demarcating the area of splenic dullness by percussion. The only risk of splenic puncture is bleeding from a soft and enlarged spleen. At our treatment centre, fatal bleeding has occurred only twice in 9612 splenic aspirate procedures performed over the last 15 years. To minimize the risk of excessive blood loss, splenic puncture should be avoided in patients with a platelet count of <40 000 platelets/l and a prothrombin time of >5 s over the control. This approach suffers because of lack of adequate sensitivity when the load of organisms is low. However, in the state of Bihar, where VL is rampant, medical centres in the cities, primary health centres in the villages and private practitioners perform bone marrow aspiration. Now, the rK39 rapid test is also available at selected centres.
Non-immunological tests such as the Sia water and aldehyde tests confirm the hypergammaglobulineamic state characteristic of kala-azar. The Sia water test is positive mainly because of the presence of euglobulins. Hypergammaglobulineamia in other infectious diseases can also account for the cross-reaction (Manson-Bahr & Apted 1983). The aldehyde test does not become positive in the early weeks of the disease (Ghose et al. 1980). Aikat et al. (1979) reported 80% aldehyde test positivity in kala-azar patients when the disease had been present for about 3 months. In our study, the Sia water test had 85% positivity, and the aldehyde test, 62.7%. These tests are based on the level of IgG and positive results are also obtained with other causes of raised immunoglobulin levels, whether IgG or IgM classes. Therefore, they are merely screening tests which require further investigations if positive. The tests are positive in about 85% of patients with VL, and they do not become positive until about 3 months after infection. Hence these tests may detect only symptomatic cases and fail to detect early leishmaniasis (Gupta 1996).
Direct agglutination test was described by Harith et al. (1986) and later validated for its potential use in the serodiagnosis and seroepidemiology of VL (Harith et al. 1987). However, it is yet to become a serodiagnostic tool for the diagnosis of kala-azar in India. Several laboratories in India reported satisfactory sensitivity and specificity levels for this test (Vinayak et al. 1994; Sundar et al. 1996). Although the DAT showed a high degree of repeatability within the centres, its reproducibility across the centres was quite weak (Boelaert et al. 1999a,b). Moreover, difficult field conditions, the fragility of aqueous antigen, the lack of cold chain, and batch-to-batch variations in the antigen, along with non-standardization of test readings, have severely limited its widespread applicability in regions of endemicity. Freeze-dried antigens developed in Belgian and Dutch laboratories are likely to overcome some of these handicaps (Boelaert et al. 1999a,b; Oskam et al. 1999) However, unless this improved antigen is produced indigenously to make it affordable and DAT is made user-friendly with one-step dilution and reduced incubation time, its field use is unlikely in endemic countries like India. Like most antibody-based tests, DAT may yield positive results for a long time after complete cure and thus has not proved to be of much prognostic value (Harith et al. 1988).
The absence of trypanosomiasis on the Indian subcontinent makes ELISA test a powerful and relevant tool in the serodiagnosis of kala-azar. ELISA is an accurate, safe, economical and non-invasive technique compared with splenic and bone marrow aspiration for kala-azar diagnosis (Ho et al. 1983). It can be employed to determine the class of antibodies against the causative organism. In our study, micro-ELISA could detect anti-leishmanial antibodies in 91.0% of kala-azar cases. In parasitologically confirmed patients with kala-azar, Srivastava (1989) observed 100% sensitivity with ELISA, but with the sera of healthy controls they found 7.1% false-positivity. Cross reactions were evident among sera of patients suffering from tuberculosis and other infectious diseases as indicated by several studies (Xu et al. 1982; Pappas et al. 1983; Walton et al. 1986) when whole antigen of the Leishmania parasite was used. Our results showed that some of the healthy individuals were positive with all the serological tests. We could not rule out that these individuals were exposed to the infected sandfly and resolved the infection or that they later developed full-blown infection or due to false-positivity of the tests.
Jaffe and Zalis (1988) used affinity-purified parasite proteins to develop a dot-blot assay for VL and found a sensitivity of 90–100%. In our study, the specificity of dot-ELISA was 98.4% and no cross-reactions were observed except with one tuberculosis patient. Thus, the dot-ELISA used in our study has a unique advantage in serological diagnosis of kala-azar over the other tests if we use the electro-eluted potentially immunodominant antigens from polyacrylamide gels identified by Western blotting. If the high degree of sensitivity and specificity demonstrated in our experiments could be transferred to rapid testing formats, invasive and risky procedures like spleen or bone marrow aspiration could be eliminated for the majority of patients presenting for the first time with VL. A rapid dot-ELISA has been developed for the detection of anti-leishmania antibodies in the dog (Vercammen et al. 1998). The test procedure lasted only 30 min. Recently, a simple and rapid dot-ELISA (requiring only 5 min) was reported for the detection of Mycobacterium tuberculosis circulating antigen in serum (Attallah et al. 2003).
For community-based diagnosis, a simple and cheap test is necessary. As plate ELISA requires expensive equipment and other facilities, a cheaper modification, i.e. dot-ELISA was standardized. The conditions in endemic areas are most primitive and we desperately need a simple, cheap and definitive serodiagnostic tool. Dot-ELISA is more sensitive than ELISA, quick, inexpensive and does not require a high degree of technical skill. Dot-ELISA proved to be economical, regarding use of reagents and was very easy to perform. Interpretation was easy by visual inspection of reaction end points in the nitrocellulose discs, obviating the need for an ELISA reader. Therefore, the dot-ELISA appears a suitable tool for seroepidemiological surveys and for individual diagnosis of VL.