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Keywords:

  • Sudan;
  • leishmaniasis;
  • diagnosis;
  • direct agglutination test

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Study area and population
  6. Microscopy
  7. Direct agglutination test
  8. PCR
  9. Statistical analysis
  10. Ethical clearance
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

The direct agglutination test (DAT) based on freeze-dried (FD) Leishmania donovani antigen was evaluated for the serodiagnosis of kala-azar in a rural setting in eastern Sudan. The performance of the FD-DAT was compared with standard liquid antigen (LQ) by testing serum samples and blood samples collected on filter paper of microscopically and PCR-confirmed VL patients, apparently healthy endemic controls and patients with other relevant infectious diseases for the region. In the present study, the FD-DAT had a sensitivity of 96.8% and a specificity of 96.2%. The LQ-DAT had a sensitivity of 91.0% and a specificity of 96.6%. A high degree of agreement (97.3%; r-value 0.94) was observed between the FD-DAT and the LQ-DAT, as well as between the FD-DAT performed on serum samples and corresponding blood samples collected on filter paper (agreement 97.8%; r-value 0.79). The FD-DAT is very suitable as diagnostic test for kala-azar in remote rural conditions as it is sensitive, specific and stable. The antigen is affordable, reproducible and available, which contributes to the sustainability of the DAT as a diagnostic test for VL.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Study area and population
  6. Microscopy
  7. Direct agglutination test
  8. PCR
  9. Statistical analysis
  10. Ethical clearance
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

Visceral leishmaniasis (VL) or kala-azar is one of the most important parasitic tropical diseases in Sudan, which is one of the most important foci in the world (Osman et al. 2000). The disease was first described in Sudan by Neave (1904), and the first epidemics in 1936–38 were reported by Stephenson (1940) in the Upper Nile Province and Fung area (Osman et al. 2000; Zijlstra & El-Hassan 2001). Untreated, VL is fatal; hence early and accurate diagnosis is essential. In Sudan, two methods are commonly used in the routine diagnosis of the disease: parasitology and serology.

Parasitological testing comprises microscopical examination of tissue smears and parasite culture. The latter method may be more sensitive and reliable than microscopy in diagnosis of VL, especially when there are few parasites present [World Health Organization (WHO) 1996]. Culture may give information on the viability of the parasite, which can be important in evaluating VL chemotherapy (Lightner et al. 1983). The parasitological methods are highly specific, but their sensitivity can be as low as 50–60% (Siddig et al. 1988; Zijlstra et al. 1992) depending on the type of tissue and method employed (reviewed by Osman et al. 2000).

Molecular methods, particularly polymerase chain reaction (PCR), may provide a sensitive and specific alternative for the diagnosis of leishmaniasis (Schallig & Oskam 2002). The performance of PCR and its value for the diagnosis of VL in Sudan has been extensively evaluated (Osman 1998). These studies demonstrated that PCR is more sensitive than traditional microscopy (Osman et al. 1997a), can be used as a test of cure (Osman et al. 1997b) and to assess the success of treatment (Osman et al. 1998b). In addition, PCR is very useful for the diagnosis of post-kala-azar dermal leishmaniasis (Osman et al. 1998a).

Although PCR is a valuable diagnostic tool, its application in rural conditions is hampered due to the necessity of having electricity and sophisticated equipment available. Therefore, serology is often used in the diagnosis of VL. The direct agglutination test (DAT) remains the first-line diagnostic tool in Sudan as it appears to be a simple and economical test with high sensitivity and specificity (El-Harrith et al. 1988). The test is very suitable for use in the field as demonstrated by Zijlstra et al. (1991). The development of a freeze-dried (FD) antigen increased the applicability of the test under rural conditions as, in contrast to liquid antigen, a cold chain for preserving the antigen is not necessary (Meredith et al. 1995).

In the present study, we have evaluated the use of the DAT based on FD antigen for the diagnosis of VL in a rural setting in Sudan. Serum and blood samples collected on filter paper of confirmed kala-azar patients (confirmation by PCR and microscopy) were tested in DAT based on FD or liquid antigen (standard test; Abdel-Hameed et al. 1989). Samples obtained from apparently healthy endemic controls and patients with other relevant infectious diseases for the region were used to assess the specificity of the test.

Study area and population

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Study area and population
  6. Microscopy
  7. Direct agglutination test
  8. PCR
  9. Statistical analysis
  10. Ethical clearance
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

The study was conducted in an established VL endemic area in east Sudan, in Kassab rural hospital, Médecins sans Frontières, MSF kala-azar centre (Gaderif state) in 2003. Cases (n = 61) were selected from patients with microscopically confirmed VL admitted to hospital. The following diagnostic samples were collected from the selected patients:

  • 61 serum samples
  • 46 corresponding whole blood samples on filter paper
  • 61 lymph node aspirations (collected in 250 μl lysis buffer, comprising of 50 mm NaCl, 50 mm Tris, 10 mm EDTA (pH 8), 1% Triton X-100 and 50 μg proteinase K, for PCR analysis)
  • 61 lymph node smears (for microscopical examination).

The following additional serum samples, collected from individuals living in the same area, were used for serological evaluation:

  • 102 samples from apparently healthy individuals (i.e. people without any current symptoms of visceral leishmaniasis)
  • Eight patients with confirmed tuberculosis
  • Eight patients with confirmed malaria
  • Eight patients with confirmed schistosomiasis.

Microscopy

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Study area and population
  6. Microscopy
  7. Direct agglutination test
  8. PCR
  9. Statistical analysis
  10. Ethical clearance
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

Lymph node smears were fixed with absolute methanol and stained with Giemsa according to WHO protocol (WHO 1996). Stained slides were examined using a 100× oil immersion objective.

Direct agglutination test

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Study area and population
  6. Microscopy
  7. Direct agglutination test
  8. PCR
  9. Statistical analysis
  10. Ethical clearance
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

The DAT was performed as described by Meredith et al. (1995). In brief, serum or blood samples (after overnight elution from filter paper) were diluted in physiological saline (0.9% NaCl) containing 0.8%β-mercaptoethanol. Twofold dilution series of the samples were made, starting at a dilution of 1:100 and going up to a maximum serum dilution of 1:102 400 in the case of serum or 1:51 200 in the case of blood eluted from filter paper.

Freeze-dried DAT antigen (Leishmania donovani promastigotes) produced by KIT Biomedical Research (Batch 0312, expiration date June 2005) was reconstituted with 5 ml physiological saline. Liquid DAT antigen was a gift from Dr A. El-Harith (El Ahfad University College, Omdurman, Sudan).

Fifty microlitres of DAT antigen solutions (concentration of 5 × 107 parasites/ml) was added to each well containing 50 μl diluted sample and the results were read after 18 h of incubation at room temperature. A sample is considered positive if it has a titre ≥1:1600, the cut-off value of the DAT (El-Harrith et al. 1988). Both DAT tests were performed by (K.A.) on the collected stored sera at the BNRTI and confirmed at KIT in Amsterdam.

PCR

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Study area and population
  6. Microscopy
  7. Direct agglutination test
  8. PCR
  9. Statistical analysis
  10. Ethical clearance
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

DNA was extracted from lymph node samples collected in lysis buffer by using the chloroform–phenol method (Osman et al. 1997a). Mini circle DNA was amplified using L. donovani-specific DB8 (5′-CCAGTTTCCCGCCCCG-3′) and AJS3 (5′-GGGGGTTGGTGTAGGGC-3′) synthetic oligonucleotides and amplification protocol as described by Smyth et al. (1992). PCR products were stored at 4 °C before analysis on a 1% agarose gel.

Statistical analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Study area and population
  6. Microscopy
  7. Direct agglutination test
  8. PCR
  9. Statistical analysis
  10. Ethical clearance
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

The sensitivity (i.e. the probability that the assay will be positive when the infection is present) and the specificity (i.e. the probability that the assay will be negative when the infection is absent) were calculated using the formulas:

  • image

and

  • image

where TN represents true negative, TP true positive, FN false negative and FP false positive. The sensitivity of the two tests, FD-DAT and LQ-DAT, was assessed with sera from confirmed VL patients (n = 61). Sera of healthy controls (n = 102) and sera of patients with confirmed other diseases (n = 24) were used to determine the specificity of DAT.

Furthermore, the degree of agreement between the evaluated tests was determined. The agreement between the tests was determined by calculating κ values with 95% confidence intervals using Epi-info version 6. Kappa values express the agreement beyond chance and a κ-value of 0.60–0.80 represents a substantial agreement beyond chance, whereas a κ-value of >0.80 represents almost perfect agreement beyond chance (Altman 1991).

Ethical clearance

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Study area and population
  6. Microscopy
  7. Direct agglutination test
  8. PCR
  9. Statistical analysis
  10. Ethical clearance
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

Ethical approval for this study was obtained from the ethical committee of the Blue Nile Research and Training Institute/University of Gezira (Wad Medani, Sudan) and from the State Health Authority in Gedarif. Patients were only included in the study after appropriate informed consent was obtained.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Study area and population
  6. Microscopy
  7. Direct agglutination test
  8. PCR
  9. Statistical analysis
  10. Ethical clearance
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

Sixty-one patients tested positive by both PCR and microscopical analysis, confirming their parasitological status. The results of testing positive and negative controls and of the serum samples of patients with other confirmed infectious diseases with FD-DAT and LQ-DAT are summarized in Table 1. Calculation of the sensitivity of the assays revealed that the FD-DAT had a sensitivity of 96.8% (two false-negative results) and the LQ-DAT had a sensitivity of 91.0% (six false-negative results). The two false FD-DAT negative patients were also tested negative with LQ-DAT.

Table 1.  Comparison between direct agglutination test (DAT) based on freeze-dried antigen (FD-DAT) and liquid antigen (LQ-DAT) using serum samples confirmed visceral leishmaniasis (VL) cases, healthy endemic controls and samples from patients with confirmed other diseases. Values represent the number of samples found positive by the test/total number of samples tested
Patient categoryFD-DATLQ-DAT
Confirmed VL (n = 61)59/6155/61
Healthy endemic controls (n = 102)5/1024/102
Schistosomiais (n = 8)0/80/8
Malaria (n = 8)0/80/8
Tuberculosis (n = 8)0/80/8

None of the serum samples from patients with other confirmed infectious diseases tested positive with FD-DAT or LQ-DAT. Four endemic negative controls tested positive with LQ-DAT and one additional patient tested positive with FD-DAT. The DAT titres of these false-positive patients were at or one step above the cut-off titre (1:1600) of the DAT test. The specificity of the FD-DAT was 96.2% and of the LQ-DAT 96.6%.

There was a high degree of agreement (97.3%) between the FD-DAT and the LQ-DAT (Table 2). The agreement beyond chance (κ-value) was 0.94. A high degree of agreement was also observed between the FD-DAT performed on serum samples and corresponding blood samples collected on filter paper (Table 3; agreement 97.8%; κ-value 0.79).

Table 2.  Comparison between FD-DAT and LQ-DAT for the detection of Leishmania antibodies in human serum samples
FD-DATLQ-DAT
+ (n)− (n)Total
  1. Agreement = 97.3%; κ-value = 0.94.

+ (n)59564
− (n)0123123
Total59128187
Table 3.  Comparison between FD-DAT performed on serum samples and blood samples collected on filter paper for the detection of Leishmania antibodies in confirmed human samples
Serum samplesFilter paper samples
+ (n)− (n)Total
  1. Agreement = 97.8%; κ-value = 0.79.

+ (n)43144
− (n)022
Total43346

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Study area and population
  6. Microscopy
  7. Direct agglutination test
  8. PCR
  9. Statistical analysis
  10. Ethical clearance
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

People affected by visceral leishmaniasis (VL) often live in poor rural areas with limited health care resources (Desjeux 1996). Therefore, practical, sensitive and specific diagnostic tools that can be employed under harsh conditions are essential. The DAT is a well-validated test for the serodiagnosis of VL and has been in use in Sudan for the last 15 years (Abdel-Hameed et al. 1989). The availability of an FD antigen for this test should increase the applicability of the DAT in peripheral health structures as, in contrast to liquid antigen, a cold chain is not needed and problems with transportation can be circumvented (Meredith et al. 1995). In the present study, we have evaluated and compared the performance of the FD antigen with liquid antigen, which is currently being used as the standard test.

The sensitivity and specificity of both antigens were concordant. The sensitivity of the DAT is comparable with previous reports (El-Harrith et al. 1988; Abdel-Hameed et al. 1989; Zijlstra et al. 1991; Meredith et al. 1995; Oskam et al. 1999). Two patients were missed with both tests, and antibody titres could not be detected with DAT. These patients may have been co-infected with HIV (status not assessed in this study), which would have affected their ability to produce antibodies and subsequently are tested negative with DAT (Hailu & Berhe 2002). This was pointed out by WHO (1996), reporting that 20–40% of HIV patients may be serologically negative and diagnosis must therefore rely on the parasitological detection of leishmania. Serology has low sensitivity in HIV patients due to their compromised immune response. This contrasts with the results of Hailu and Berhe from Ethiopia with good DAT performance in HIV/VL co-infected patients.

Low specificity values of the DAT have been reported in several studies (Zijlstra et al. 1991; Boelaert et al. 1999), although they used a control series of unical suspects rather than healthy endemic controls. However, in the present study a relatively high specificity (>90%) of both DAT tests is found, which is comparable with previous studies in which the FD antigen was used (Meredith et al. 1995; Oskam et al. 1999; Schoone et al. 2001). Several studies demonstrated that the specificity of the DAT depends on the cut-off titre chosen. A high cut-off titre generally yields higher specificity (Oskam et al. 1999; Chappuis et al. 2003). But our cut-off titre is low compared with other studies (Chappuis et al. 2003) and still a very good specificity of the test was achieved. It is also suggested that the specificity of the DAT depends to a certain extent on the choice of the control group (Chappuis et al. 2003). When control groups are composed of non-symptomatic people or patients with confirmed other diseases, as is the case here, the specificity of the test tends to be higher, than when only clinical suspects are studied. Dipstick tests using rk39 may show lower sensitivity and specificity (Zijlstra & El-Hassan 2001; Veeken 2003).

Some apparently healthy controls were found DAT positive, albeit at or one step above the cut-off titre of the test. These subjects were not parasitologically examined or followed-up. These supposed ‘false-positive’ case may have been individuals with an early Leishmania infection, or they may have had and been treated for leishmaniasis in the past. The specificity of the DAT, as any serological test, is affected by the persistence of antibodies after treatment of leishmaniasis (Hailu 1990; Zijlstra et al. 2001).

DAT performed on matching blood spotted on filter paper and serum samples gave highly concordant results. This increases the applicability of DAT under field conditions. The combination of DAT and blood spotted on filter paper is, for example, very useful when large numbers of diagnostic samples have to be collected at the same time in a remote rural area where there are no laboratory facilities. The samples can easily be collected, taken to a laboratory and subsequently analysed.

In conclusion, our results show that in a rural setting in eastern Sudan, the DAT based on FD antigen is a suitable tool for the diagnosis of kala-azar. The test is sensitive and specific and problems with transportation and storage of the antigen are circumvented. Furthermore, the low cost of the FD antigen (around €1.85 per test) compared with the reported high cost of the liquid antigen (US$ 4.50 per test, Chappuis et al. 2003) contributes to the affordability of the test. Local production should be encouraged to ensure sustainability. Although the local cost of the LQ.DAT test is only 0.5$, storage and transport of the FD.DAT are much cheaper and safer, especially in rural settings. Moreover, the FD antigen can be produced in relatively large batches, which increases the reproducibility of the test, a problem encountered with liquid antigen of which only small batches are produced. Finally, the availability of a dedicated production facility for the FD antigen increases the sustainability the DAT as a diagnostic test for VL.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Study area and population
  6. Microscopy
  7. Direct agglutination test
  8. PCR
  9. Statistical analysis
  10. Ethical clearance
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References

Work on the evaluation of the DAT as a diagnostic test for visceral leishmaniasis was partly supported by a grant from the Hubrecht-Jansen Foundation (Koninklijk Instituut voor de Tropen/Royal Tropical Institute, Amsterdam, The Netherlands). Bakri Y. M. Nour is supported by a grant (WB93-388) from The Netherlands Foundation for the Advancement of Tropical research.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Study area and population
  6. Microscopy
  7. Direct agglutination test
  8. PCR
  9. Statistical analysis
  10. Ethical clearance
  11. Results
  12. Discussion
  13. Acknowledgements
  14. References
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