1. Low prevalence of Leishmania donovani infection among the blood donors in kala-azar endemic areas of Bangladesh M. Mamun Huda, Shikha Rudra, Debashis Ghosh, Khondaker Rifat Hasan Bhaskar, Rajib Chowdhury, A.P. Dash, Sujit K. Bhattacharya, Rashidul Haque, Dinesh Mondal

2. Background Visceral leishmaniasis (VL) is a public health problem in Bangladesh with the highest burden in the Mymensingh district. In 2005, Health Ministers from Bangladesh, India and Nepal signed a MoU to eliminate VL from the sub-continent by 2015. The goal of the elimination program was to reduce VL cases below 1 per 10,000 people through: −ACD and proper case management, −Interruption of disease transmission through IVM strategies, and −Social mobilization. The disease is transmitted by a sand fly bite but recent literature suggested that it could be transmitted through blood transfusion if the donor blood had active infection with the leishmania parasite. But no information is available about the prevalence of Leishmania infection among blood donors in Bangladesh

3. Background (cont.) The blood transfusion unit of the Mymensingh Medical Collage (MMC) Hospital is the only public blood transfusion facility in the most VL endemic district, Mymensingh. Usually the blood donors attending this centre are relatives of patients who require blood transfusion. Since VL is clustered at the household and village level, theoretically there is a risk of transmission of VL through blood transfusion if the prevalence of active Leishmania infection is found high among the blood donors. This may necessitate introduction of routine screening for Leishmania infection among blood donors by the National VL Elimination Program. We have therefore undertaken this study to fill this knowledge gap in Bangladesh to support the National VL Elimination Program.

4. Objectives ♦ To investigate the LD infection prevalence among blood donor without past VL defined by the presence of anti- leishmania anti-body in their blood ♦ To determine the rate of active infection with Leishmania donovani among anti-leishmania anti-body positive blood donor defined by the presence of Leishmania DNA in their blood ♦ To develop policy recommendations for the national VL elimination programme in Bangladesh.

5. Methods: Ethical statement  The blood donors were enrolled in the study after obtaining written consent.  All rK-39 positive blood donors were told not to donate blood for the next six months. We then actively followed-up all the rK-39 positive blood donors for the next six months.  The study was approved by the Ethical Review Committee of icddr,b as well as Directorate General of Health Services, Government of Bangladesh.

6. Study site and population  The study was carried out in the Blood Transfusion Unit of the MMC Hospital, Mymensingh from August,2010 to April, 2011.  During the study period, voluntary blood donors attending the unit who resides in Mymensingh district were the study population

7. Methods: Sample size calculation About 6% sero prevalence by the rK39 ICT test among individuals without past VL infection and 3% ( i.e., fifty percent of them) were confirmed with active leishmania parasite infection (unpublished data). Thus to demonstrate the active infection rate of 3% among the blood donors with 1% precision and 95% confidence interval (CI), a minimum number of 1,117 blood donors was required.

8. Methods: Study design and sampling  The study was cross-section survey with 6 months follow up.  During the study period, voluntary blood donors attending the Blood Transfusion unit were invited to participate in the study.  A trained field research assistant (FRA) performed the consenting.  The medical officer performed clinical assessment and then enrolled the blood donor after checking inclusion and exclusion criteria of the study.  On each working day, up to 10 blood donors were enrolled.

9. Methods (cont.) Inclusion criteria:  Aged ≥18 to <60 years old;  Either sex;  Clinically healthy;  No history of VL in the past;  Resident of Mymensingh;  Voluntary consent to participate in the study. × Exclusion criteria:  Aged 60 years old;  Clinical evidence of acute or chronic illness;  Past history of VL or PKDL;  Non-resident of Mymensingh;  Non-consenting.

10. Methods (cont.) NOT metVoluntary blood donor Examined Inclusion & Exclusion criteria NOT enrolled into the study Enrolled into the study MET Perform rK39 test by Lab technician Collection of socio- demographic information by FRA Next six month follow up by FRA to see eventual development of VL using mobile phone contact or home visit (when possible) If the test is POSITIVE 1.Regarded as not eligible for donation of blood. 2.Collected a 3 ml venous blood in a tube with EDTA 3.Sample transported to the Parasitology Laboratory, icddr,b maintaining cold chain for Buffy coat preparation, DNA isolation and Ln-PCR analysis

11. Methods: Diagnostic Tools ☼Anti-leishmania anti-body detection: The test was done using commercially available kits (rK-39 ICT, Kala-azar DetectR, In Bios International, Seattle, USA) recommended by the WHO as per manufacturer’s instructions, but using two drops of blood drawn from the participant’s finger, instead of the serum sample. ☼Active Leishmania infection detection: The Ln-PCR test was carried out in Parasitology Laboratory by the expert Research Officer as per protocol described in a previous study done by our colleagues (Salam et al. 2009).

12. Methods: Data Analysis We entered all the data into the computer using EPI Info 3.5.1 Software We explored descriptive statistics to interrogate the nature of data. The 95% CI was calculated using Normal as well as Poisson distributional approach where applicable. We analyzed all the data using STATA 10

13. Result: Blood donor characteristics Characteristics; N=1195n (%) Male985 (82.4) Mean Age (SD)27.66 (7.95) Literate1115 (93.3) Occupation  Agriculture  Business  Job holder  Student  Others 77 (6.4) 256 (21.4) 246 (20.6) 432 (36.2) 148(15.4) Donate blood at least once in a year594 (49.7) Reason for blood donation  Relative or friend patient  Registered blood donor 1187 (99.4) 7 (0.6) During the study period, a total of 14367 blood donors attended at the blood transfusion unit of the MMC Hospital. Among them a total of 1195 blood donors were enrolled into the study.

14. Result: Blood donor characteristics (cont.) Characteristics; N=1195n (%) Heard about Kala-azar1128 (94.4) House type of blood donor  Precarious  Non-precarious 62 (5.2) 1133 (94.8) Mean no. of living room (SD) in the household3.53 (1.61) Average family size (SD)5.7 (2.3) Average bed net (SD) in the household3.31 (1.41) Use bed net at last night997 (83.4) Past KA patients in the household31 (2.6)

15. Result: Prevalence of Leishmania infection Serological testn (%) rK39 test results; N=1195  Positive  Negative 3 (0.3)* 1192 (99.7) Positivity grade; N=3  Grade I  Grade II  Grade III  Grade IV 0 (0.0) 3 (100.0) PCR test done; N=33 (100.0) PCR results; N=3  Positive  Negative 0 (0.0) 3 (100.0) Serological analysis of blood from the blood donors * 95% Confidence Interval, 0.05% -0.73%

16. © The risk for transmission of VL through donated blood from healthy blood donor volunteers in Mymensingh is very low © There is no strong justification for routine screening for Leishmania infection among blood donors in Bangladesh. Conclusion

17. Recommendation There is no need for introduction of routine screening to test for Leishmania infection among blood donors in VL endemic district of Bangladesh

18. Acknowledgement WHO Regional Office for South East-Asia (SEARO). Directorate General of Health Services, Government of Bangladesh. Blood Transfusion Department, Mymensingh Medical College, Mymensingh All blood donars who consented to participate in the study Dorothy Southern, Head, Training Support Group, Center for Communicable Diseases, icddr,b

19. Thank You