A Novel Noninvasive Method for Diagnosis of Visceral Leishmaniasis by. rk39 Test in Sputum Samples

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1 JCM Accepts, published online ahead of print on 0 June 00 J. Clin. Microbiol. doi:0./jcm.00-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 0 0 A Novel Noninvasive Method for Diagnosis of Visceral Leishmaniasis by rk Test in Sputum Samples Visceral Leishmaniasis (VL) is a major public health problem of the eastern states in India namely Bihar, West Bengal, Jharkhand, and Eastern Uttar Pradesh. Bihar alone accounts for >0% of the total cases of kala-azar reported from India. The disease is reported to be predominant in the poor and malnourished people (). Development of a simple, noninvasive, cheap, and reliable diagnostic tool has been suggested as a pre-requisite (-). Ideally, diagnosis of kala-azar is done by direct demonstration of the parasite under the microscope in splenic or bone marrow aspirates (-). Detection of parasite in splenic aspirate (SA) is sensitive but is invasive, painful, carries risk of serious/fatal hemorrhage whereas bone marrow aspirates are safer and relatively easy to perform but are less sensitive (0 %) and demand technical support. In vitro cultivation of parasites is expensive, time consuming and requires expertise thus severely restricting its use in routine diagnosis. Serological tests though good but have their own limitations viz: cannot differentiate between present and past infection, asymptomatic vs. clinical cure etc. Sensitivity and specificity also varies from test to test and some serological tests are not user-friendly and not suitable for field conditions (). In the last few years, a serum IgG based detection system using rk- antigen has been found useful in diagnosis of VL. The test was found highly sensitive, specific, simple, reproducible and results can be obtained within 0- min (). To the best of our knowledge, use of rk in detection of VL infection in sputum samples has not been reported. This is perhaps the first early report for use of rk strips (InBios, Seattle, USA) in the detection of active VL cases in sputum samples. The assay was performed according to the manufacturer s protocol. Briefly, bottom of the absorbent pad of rk strip was dipped in freshly collected sputum samples for Downloaded from on February, 0 by guest

2 0 0 minutes. Two drops of the chase buffer provided with the kit was added to the pad, and was allowed to migrate up to the strip by capillary action. The results were read after 0 minutes. Appearance of a red upper (control) band indicated the proper functioning of the test and that of a lower red (test) band suggested the presence of anti-k IgG in the sputum. The test was considered positive if two bands viz; upper and lower were present, where as the test was considered negative if only the upper control band appeared. A total of 0 sputum samples from confirmed VL patients and controls were screened by rk- strip test. Of the microscopically confirmed VL cases, (.%) were found positive by rk- in the sputum and all in blood samples (00%) (Table ). However, in endemic and non-endemic controls, the rk- test in sputum was found more specific than blood because sputum did not show any positive reaction, whereas.% positivity in endemic control and no positivity in non endemic control were observed in blood sample. This test was also found more specific in the other disease group because none of the sputum samples cross reacted with rk- strip, whereas blood of (.%) patients showed positive reaction. The.% &.% positivity in healthy endemic controls and other diseases groups respectively, in blood, by rk strip test were false positive because the positive cases of both the groups did not show LD positive reaction by PCR (). Moreover, these subjects didn t exhibit clinical symptoms of visceral leishmaniasis like fever, pancytopenia, and hepatosplenomegaly. Even the follow up for more than months did not show conversion of any of these cases to symptomatic kala-azar cases. Furthermore, the PCR () of sputum in both these groups were also for negative for LD infection. Lastly, it was not possible ethically to subject these cases to splenic / bone marrow aspiration. The present data clearly demonstrates that though the newly developed sputum based rk- test is only 0.0% less sensitive, it is much more specific than the rk- test for diagnosis of VL in blood. So this test can be easily adapted for routine diagnosis of VL using Downloaded from on February, 0 by guest

3 0 sputum samples. This will be more beneficial for diagnosis of VL in difficult field conditions as it is purely non invasive, easily collectable, needs no special equipment or sophisticated technology and the result can be read visually. Finally this diagnostic tool can be useful for the Kala-Azar elimination programme which is scheduled to end by 0 in the Indian subcontinent (). Table. Comparison of sensitivity and specificity of rk strip test in diagnosis of Kala azar in Sputum and Serum, of LD body (Microscopy) positive VL patients and controls. Patients category n LD body (Microscopy) (SA or BM) Positive no. (%) rk strip test (Serum) Positive no. (%) rk strip test (Sputum) Positive no. (%) Confirmed VL (00) (00) (.) patients Healthy endemic NA (.) 0 (0) controls Healthy non-endemic 0 NA 0 (0) 0 (0) controls * Other diseases NA (.) 0 (0) Total no. of samples 0 NA, not applicable; LD, Leishmania donovani; VL, visceral leishmaniasis; AFB, acid fast bacilli; SA, splenic aspirate; BM, bone marrow aspirate. * Other diseases control patients of tuberculosis (n =), Leprosy (n =) and Malaria (n =). REFERENCES. World Health Organization. 00. The World Health Report. World Health Organization, Geneva, Switzerland.. Desjeux, P. Leishmaniasis: 00. Current situation and new perspectives. Comp. Immunol. Microbiol. Infect. Dis. :0.. Guerin PJ, Olliaro P, Sundar S. 00. Visceral leishmaniasis: current status of control, diagnosis, and treatment, and a proposed research and development agenda. The Lancet Infectious Diseases. : 0. Downloaded from on February, 0 by guest

4 0 0. Pearson RD, de Queiroz SA.. Clinical spectrum of leishmaniasis. Clin Infect Dis. :.. Berman, J. 00. Recent developments in leishmaniasis: epidemiology, diagnosis, and treatment. Current Infectious Disease Reports. :-.. Boelaert M, El-Safi S, Hailu A, Mukhtar M, Rijal S, Sundar S, Wasunna M, Aseffa A, Mbui J, Menten J, Desjeux P, Peeling RW. 00. Diagnostic tests for kala-azar: a multi-centre study of the freeze-dried DAT, rk strip test and Katex in East Africa and the Indian subcontinent. Trans R Soc Trop Med Hyg. 0:-0.. Sundar S, Reed SG, Singh VP, Kumar PC, Murray HW.. Rapid accurate field diagnosis of Indian visceral leishmaniasis. Lancet. :.. Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, Negi NS. 00. Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol. :-.. TDR. Press release: 00. Elimination of kala-azar from endemic countries in the south-east Asia region. diseases/leish/press_release.htm. Potential conflicts of interest All authors: no conflicts. List of Authors D. Singh Agamkuan, Patna, India K. Pandey Downloaded from on February, 0 by guest

5 Department of Clinical Medicine Rajendra Memorial Research Institute of Medical Sciences (ICMR) Agamkuan, Patna India V. N. R. Das Department of Clinical Medicine, Agamkuan, Patna India S. Das Agamkuan, Patna India S. Kumar Rajendra Memorial Research Institute of Medical Sciences(ICMR), Agamkuan, Patna R. K. Topno Department of Epidemiology, Agamkuan, Patna and P. Das* Downloaded from on February, 0 by guest

6 Agamkuan, Patna India *Tel: +-- Fax: drpradeep.das@gmail.com Downloaded from on February, 0 by guest