|Year : 2012 | Volume
| Issue : 2 | Page : 88-90
Comparison of an indirect hemagglutination test and bone marrow aspiration for the diagnosis of visceral leishmaniasis in Aseer area, Saudi Arabia
Abdul-moneim Jamil1, Fakhreldin Mohamed Omer2, Salah E.A. Abdalla2, Nadia El-Menshawy3, Ali M. Al-Bin-Ali4
1 Clinical Pathology & Laboratory Haematology, Department of Pathology, College of Medicine, King Khalid University, Abha, Saudi Arabia
2 Department of Microbiology, College of Medicine, King Khalid University, Abha, Saudi Arabia
3 Clinical Pathology & Laboratory Haematology, Haematology Unit, Mansoura University, Mansoura, Egypt
4 Department of Pediatrics, College of Medicine, King Khalid University, Abha, Saudi Arabia
|Date of Submission||05-Dec-2011|
|Date of Acceptance||17-Jan-2012|
|Date of Web Publication||23-Jun-2014|
Clinical Pathology & Laboratory Haematology, Department of Pathology, College of Medicine, King Khalid University, P.O. Box 641, 61421 Abha
Source of Support: None, Conflict of Interest: None
In the Aseer area, in the southern western region of Saudi Arabia, visceral leishmaniasis or Kala-azar is an endemic and serious disease that mainly affects children.
In this study, an anti-leishmania antibody diagnostic test, the indirect hemagglutination (IHA) test, was compared with the gold standard microscopic detection of Leishmania donovani bodies in bone marrow aspiration (LDB/BMA) smears. Factors such as sensitivity, specificity as well as reliability will govern whether health authorities should continue the use of the IHA assay or replace it with more specific and sensitive as well as reliable techniques.
Patients and methods
A total of 582 patient samples were included in this study. All were tested by IHA and by microscopic examination for LDB/BMA smears. Results of the tests are statistically analyzed and tabulated.
The comparison showed a poor correlation between the microscopic detection of parasite forms in the gold standard bone marrow smear examination (LDB/BMA) and the serological antibody screening of patient samples by IHA (P⩽0.05). This type of results and correlation poses serious questions relating to the sensitivity, specificity, and ultimately, the reliability of the IHA test in the diagnosis of visceral leishmaniasis.
A better alternative for IHA test might be the introduction of methods that detect the parasite it self or its antigens that can be used in conjunction with microscopic examination for LDB/BMA. In this respect, PCR–ELISA and KATEX agglutination tests represent obvious candidates.
Keywords: Aseer area, bone marrow aspiration, indirect hemagglutination, Kala-azar, leishmania donovani, visceral leishmaniasis
|How to cite this article:|
Jamil Am, Mohamed Omer F, Abdalla SE, El-Menshawy N, Al-Bin-Ali AM. Comparison of an indirect hemagglutination test and bone marrow aspiration for the diagnosis of visceral leishmaniasis in Aseer area, Saudi Arabia. Egypt J Haematol 2012;37:88-90
|How to cite this URL:|
Jamil Am, Mohamed Omer F, Abdalla SE, El-Menshawy N, Al-Bin-Ali AM. Comparison of an indirect hemagglutination test and bone marrow aspiration for the diagnosis of visceral leishmaniasis in Aseer area, Saudi Arabia. Egypt J Haematol [serial online] 2012 [cited 2022 Aug 13];37:88-90. Available from: http://www.ehj.eg.net/text.asp?2012/37/2/88/135060
| Introduction|| |
In the Aseer area, in the southern western region of Saudi Arabia, visceral leishmaniasis (VL) or Kala-azar is an endemic and serious disease that mainly affects children 1. A definite diagnosis of Kala-azar has traditionally been made on the basis of the presence of Leishmania donovani amastigotes in biopsy material from the spleen 2 or in bone marrow or lymph node aspirates. Besides being invasive, such techniques are often less sensitive 3. In addition, these techniques are cumbersome and time consuming and are not suitable for field conditions 4. Other noninvasive tests that detect antigens in body fluids are available and they include direct agglutination tests for antigen detection in urine samples 5. The antigen levels detected by these methods correlate broadly with parasite loads and the techniques are applicable under field conditions 6.
In the southern part of Saudi Arabia, VL is endemic and there are few studies on the epidemiology, clinical aspects, and laboratory diagnosis of VL. In the Aseer area, the diagnosis of VL is made mainly on the basis of microscopic detection of Leishmania donovani bodies in routinely stained bone marrow aspiration (LDB/BMA) smears and the anti-leishmania antibody screening test, the indirect hemagglutination (IHA) assay.
The aim of the present study is to compare the serology-based test, namely, the IHA assay, with the standard microscopic observation of amastigote parasite stages in bone marrow aspirates (LDB/BMA).
| Patients and methods|| |
This retrospective study was carried out at Aseer Central Hospital and the reference laboratories of King Khalid University, College of Medicine, Microbiology, and Pathology Departments. Both the hospital and the university are located at the south west of Saudi Arabia, Aseer area, Abha.
Samples were collected from 582 patients suspected of having VL during the period from June 1985 to August 2008. The diagnosis of VL was suspected on the basis of the detection of anti-leishmania antibodies by the IHA assay and was confirmed by a direct microscopic observation of leishmania amastigotes in bone marrow smears.
The BMA were collected from all patients by a posterior superior iliac spine puncture. Smears prepared from the aspirates were stained by Romanowsky stains, mostly May-Grünwald–Giemsa or Giemsa alone. On microscopic examination, amastigotes appeared as rounded or oval bodies, measuring 2–3 µm in length, and were found extracellular or intracellular in the monocyte/macrophage. In some cases, a more sharply demarcated and characteristic ‘double spot’ appearance of LDB was observed. In instances where parasitemia was scarce, bone marrow smears were confirmed as negative for LDB only after four members of our staff had examined the marrow slides for a minimum of 15 min each 7.
The Cellognost-Leishmaniasis indirect erythrocyte agglutination micromethod was used for the qualitative detection and the quantitative determination of specific antibodies to Leishmania donovani in human serum. The assay was performed according to the manufacturer’s instructions (Dade Behring GmbH, Marburg, Germany) 8. The performance of each lot of the test was tested with positive and negative control sera. Any titer greater than 1/64 was considered positive. A positivity titer that ranged between 1/64 and 1/256 was considered as mild, 1/512 and 1/1024 as moderate, and 1/2048 and 1/4096 as severe.
Fisher’s exact test and χ2-test were used to compare proportions. Statistical significance was defined as P value of 0.05 or less.
| Results|| |
During the study period, 582 samples were received and examined by both BMA/LDB and the IHA assay.
Out of the 582 samples, only 140 were positive for BMA/LDB, whereas 442 were negative. Out of these 140 BMA/LDB-positive samples, only 21 were simultaneously positive for IHA assay whereas 119 were negative. Out of the 442 BMA/LDB-negative samples, only 23 were IHA positive whereas 419 were negative. Hence, out of the 582 samples examined, only 44 were IHA positive, whereas 538 samples were negative. These results are summarized in [Table 1].
|Table 1: Comparison of indirect hemagglutination test and microscopic identification of Leishmania donovani bodies in bone marrow aspiration|
Click here to view
The most important observation of this retrospective study is the poor correlation between the microscopic detection of parasite forms in the gold standard bone marrow smear examination (LDB/BMA) and the serological antibody screening of patient samples by the IHA assay (P⩽0.05).
To determine the validity of the IHA assay, positivity association between LDB/BMA and IHA results was assessed. Out of the 140 positive samples in LDB/BMA, only 21 were simultaneously positive in the IHA assay (n=21/140), that is 15% [Table 2].
| Discussion|| |
The diagnosis of VL is not always easy, considering the level of overlap in the clinical features between VL and a host of other commonly occurring infections including malaria, typhoid, tuberculosis, and HIV, some even existing as coinfections with VL 9,10. An additional problem, not unique to VL, is the sequestration of parasitic stages in the spleen, bone marrow, and/or lymph nodes. Thus, there is a need for accurate diagnostic laboratory methods that should confirm or at least aid a final informed clinical decision making 11,12.
In the present study, we have compared the performance of a serological anti-lieshmania antibody screening IHA assay against the standard technique of detecting LDB/BMA. Our results showed a basic lack of correlation between serology and the microscopic detection of LDB in BMA smears. The reasons for this poor correlation may be multifold. Although the IHA assay is considered to be a sensitive and reliable indicator for the presence of anti-lieshmania antibodies in exposed individuals, it is, however, not a reliable indicator of the presence of parasites or parasite antigens.
In this respect, although BMA could be a sensitive diagnostic method 13, some have advocated the use of spleen aspirate for the microscopic detection of LDBs on the grounds that it is more sensitive (≥95%) than BMA 2 despite the fact that the technique may be risky 14.
Aspirates of lymph nodes could be a better alternative for BMA, being less invasive and cumbersome 15, 16, although in our case, this is unlikely as lymphadenopathy is lacking in most of the cases 7.
The present study confirms the poor performance of antibody-detecting methods by the IHA assay in comparison with the microscopic detection of LDB in BMA.
The low sensitivity of the IHA assay in our case in comparison with observation of LDB/BMA may be attributed to one or more of the following reasons. The first reason could be comorbidity, a feature that is significantly suspected in our cases and that highly influences the performance of serological tests including the IHA test for diagnosis of VL 9, 10, 17. Second, the poor nutritional status of many patients and its effect on the immunity and the immune response of the patients to parasitic infections could be another reliable explanation for the low IHA titer (i.e. <1/64) and may thus be considered falsely serologically negative 18.
The fact that the detection by such assays depends on elevated levels of the specific immunoglobulin highlights the factor of sensitivity. Another complicating factor is the polyclonal and nonspecific activation of B lymphocytes, a hallmark of VL infection, which produces large amounts of antibodies against irrelevant proteins and haptens 19. These vast amounts of activated B lymphocytes and the accompanying production of polyclonal antibodies have been advocated as the reasons for the increased false positivity in the IHA assay 20,21.
From the above, it can be safely assumed that a positive result from these antibody-detecting methods might not actually confirm the diagnosis of active VL. A better alternative will be serology that detects parasite antigens 22. Such tests are also useful in cases where the patient is immunocompromised with very poor antibody responses, for example end-stage AIDS patients 23,24. One such technique, the latex agglutination test, KATEX, which detects Leishmania antigens in urine, has been reported to have 100% specificity and about 68–100% sensitivity 5, 6, 25. This test was reported to work well irrespective of the geographical origin of the samples. Whether the test has the ability to detect asymptomatic cases of VL, as well as monitoring therapy, needs to be confirmed 4.
However, far superior testing may be provided by DNA hybridization methodology, including PCR techniques 26–30. A PCR–ELISA that transcends ‘zymodemal’ barriers has also been introduced 31. In general, PCR–ELISA and similar techniques can detect parasites in peripheral blood and aid the quantitative analysis of parasite burden 32. Sensitivity levels are usually higher than those of the indirect fluorescent antibody test, parasite culture, or microscopy. Detection thresholds of 0.1 promastigote per 1fg of genomic material are not unusual. Using PCR–ELISA, the detection rates were 83% in blood samples of clinically diagnosed cases of mucocutaneous leishmaniasis 33.
| Conclusion|| |
A better alternative for IHA test might be the introduction of methods that detect the parasite itself or its antigens that can be used in conjunction with microscopic examination for LDB/BMA. In this respect, PCR-ELISA and KATEX agglutination tests represent obvious candidates.
| Acknowledgements|| |
The authors would like to express their appreciation to Prof. Ahmad Mahfouz for his valuable input of statistical analysis of our results.
| References|| |
|1.||Peters W, Al Zahrani MA. The leishmaniasis? A public health problem in Saudi Arabia. Saudi Med J. 1987;8:333–343 |
|2.||Chulay JD, Bryceson ADM. Quantitation of amastigotes of Leishmania donovani in smears of splenic aspirates from patients with visceral leishmaniasis. Am J Trop Med Hyg. 1983;32:475–479 |
|3.||Bain BJ. Bone marrow aspiration. J Clin Pathol. 2001;54:657–663 |
|4.||Singh S, Sivakumar R. Recent advances in the diagnosis of leishmaniasis. J Postgrad Med. 2003;49:55–60 |
|5.||Attar ZJ, Chance ML, El Safi S, Carney J, Azazy A, El Hadi M, et al. Latex agglutination test for the detection of urinary antigens in visceral leishmaniasis. Acta Trop. 2001;78:11–16 |
|6.||Vilaplana C, Blanco S, Domínguez J, Giménez M, Ausina V, Tural C, et al. Noninvasive method for diagnosis of visceral leishmaniasis by a latex agglutination test for detection of antigens in urine samples. J Clin Microbiol. 2004;42:1853–1854 |
|7.||Sheikha A. Dyserythropoiesis in 105 patients with visceral leishmaniasis. Lab Hematol. 2004;10:206–211 |
|8.||Behring D Cellognost-leishmaniasis. 2004. Available at: www.dadebehring.com [Accessed 29 November 2011] |
|9.||Oren R, Schnur LF, Yehuda DB, Mayner V, Okon E, Rachmilewitz EA. Visceral leishmaniasis: a difficult diagnosis and unusual causative agent. J Infect Dis. 1991;164:746–749 |
|10.||Berhe N, Hailu A, Wolday D, Negesse Y, Cenini P, Fronunel D. Ethiopian visceral leishmaniasis patients co-infected with human immunodeficiency virus. Trans R Soc Trop Med Hyg. 1995;89:205–207 |
|11.||Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol. 2002;9:951–958 |
|12.||Murray HW, Berman JD, Davies CR, Saravia NG. Advances in leishmaniasis. Lancet. 2005;366:1561–1577 |
|13.||Da Silva MRB, Stewart JM, Costa CHN. Sensitivity of bone marrow aspirates in the diagnosis of visceral leishmaniasis. Am J Trop Med Hyg. 2005;72:811–814 |
|14.||Melby PCBehrman RE, Kliegman RM, Jenson HB. Leishmaniasis. Nelson textbook of paediatrics. 200017th ed. Philadelphia WB Saunders:1130–1133 |
|15.||Babiker ZOE, Davidson R, Mazinda C, Kipngetich S, Ritmeijer K. Utility of lymph node aspiration in the diagnosis of visceral leishmaniasis in Sudan. Am J Trop Med Hyg. 2007;76:689–693 |
|16.||Abass EM, Mansour D, El Harith A. Demonstration of agglutinating anti-Leishmania antibodies in lymph node aspirate for confirmation of kala-azar serodiagnosis. J Med Microbiol. 2007;56:1256–1258 |
|17.||Dakic ZD, Pelemis MR, Stevanovic GD, Poluga JL, Lavadinovic LS, Milosevic IS, et al. Epidemiology and diagnostics of visceral leishmaniasis in Serbia. Clin Microbiol Infect. 2009;15:1173–1176 |
|18.||Lima Maciel BL, Lacerda HG, Queiroz JW, Galvão J, Pontes NN, Dimenstein R, et al. Association of nutritional status with the response to infection with Leishmania chagasi. Am J Trop Med Hyg. 2008;79:591–598 |
|19.||Galvao Castro B, Sa Ferreira JA, Marzochi KF. Polyclonal B cell activation, circulating immune complexes and autoimmunity in human American visceral leishmaniasis. Clin Exp Immunol. 1984;56:58–66 |
|20.||Malik GM, Reshi SH, Abdalla SE. False positive serology for visceral leishmanias (kala-azar) using the indirect haemagglutination (IHA) method. J Trop Med Hyg. 1992;95:67–70 |
|21.||Malik GM, Abdalla RE, Al Knawy B, Abdalla SE. Lymphoma as a cause of false positive serology for visceral leishmaniasis (kala-azar) using the indirect haemagglutination (IHA) method. J Trop Med Hyg. 1995;98:285–286 |
|22.||Senaldi G, Xiao Su H, Hoessli DC, Bordier C. Serological diagnosis of visceral leishmaniasis by a dot-enzyme immunoassay for the detection of a Leishmania donovani-related circulating antigen. J Immunol Methods. 1996;193:9–15 |
|23.||Fernandez Guerrero ML, Aguado JM, Buzon L, Barros C, Montalban C, Martin T, et al. Visceral leishmaniasis in immunocompromised hosts. Am J Med. 1987;83:1098–1102 |
|24.||Martinez de Letona LJ, Vazquez CM, Maestu RP. Visceral leishmaniasis as an opportunistic infection. Lancet. 1986;1:1094 |
|25.||Sarkari B, Chance M, Hommel M. A capture ELISA for the diagnosis of visceral leishmaniasis using a monoclonal antibody against a leishmanial urinary antigen. Iran Biomed J. 2005;9:117–122 |
|26.||Lopez M, Montoya Y, Arana M, Cruzalegui F, Braga J, Llanos Cuentas A, et al. The use of nonradioactive DNA probes for the characterization of Leishmania isolates from Peru. Am J Trop Med Hyg. 1988;38:308–314 |
|27.||Cortes S, Rolão N, Ramada J, Campino L. PCR as a rapid and sensitive tool in the diagnosis of human and canine leishmaniasis using Leishmania donovani s.l. – specific kinetoplastid primers. Trans R Soc Trop Med Hyg. 2004;98:12–17 |
|28.||Osman OF, Oskam L, Zijlstra EE, Kroon NCM, Schoone GJ, Khalil ETAG, et al. Evaluation of PCR for diagnosis of visceral leishmaniasis. J Clin Microbiol. 1997;35:2454–2457 |
|29.||Osman OF, Oskam L, Kroon NCM, Schoone GJ, Khalil ETAG, El Hassan AM, et al. Use of PCR for diagnosis of post-kala-azar dermal leishmaniasis. J Clin Microbiol. 1998;36:1621–1624 |
|30.||Osman OF, Oskam L, Zijlstra EE, El Hassan AM, El Naeim DA, Kager PA. Use of the polymerase chain reaction to assess the success of visceral leishmaniasis treatment. Trans R Soc Trop Med Hyg. 1998;92:397–400 |
|31.||Martin Sanchez J, Lopez Lopez MC, Acedo Sanchez C, Castro Fajardo JJ, Pineda JA, Morillas Marquez F. Diagnosis of infections with Leishmania infantum using PCR-ELISA. Parasitology. 2001;122:607–615 |
|32.||Pizzuto M, Piazza M, Senese D, Scalamogna C, Calattini S, Corsico L, et al. Role of PCR in diagnosis and prognosis of visceral leishmaniasis in patients coinfected with human immunodeficiency virus type 1. J Clin Microbiol. 2001;39:357–361 |
|33.||Matsumoto T, Hashiguchi Y, Gomez EA, Calvopiña MH, Nonaka S, Saya H, et al. Comparison of PCR results using scrape/exudate, syringe-sucked fluid and biopsy samples for diagnosis of cutaneous leishmaniasis in Ecuador. Trans R Soc Trop Med Hyg. 1999;93:606–607 |
[Table 1], [Table 2]