Study site and population
The study was carried out in the Blood Transfusion Department, Mymensingh Medical College Hospital, Mymensingh District from August 2010 to April 2011. During the study period, voluntary blood donors attending in the department were invited to participate in the study. On each working day, up to 10 voluntary blood donors were enrolled in the study after obtaining written informed consent and passing screening based on study inclusion and exclusion criteria. The study inclusion criteria were: age between 18–60 years old; either sex; clinically healthy; no history of VL in the past; permanent resident of Mymensingh District; and voluntary consent to participate in the study. Exclusion criteria were: age less than 18 years old or more than 60 years old; clinical evidence of acute or chronic illness; past history of Kala-azar or PKDL; presence of PKDL-like skin lesions; and refusal to consent to study participation.
Study design and sampling
The study design was a cross-sectional survey with 6 months follow up. A trained field research assistant (FRA) consented prospective subjects and collected socio-demographic information using a structured questionnaire, then the medical officer of the transfusion unit performed a clinical assessment and enrolled study participants after checking inclusion and exclusion criteria. The medical laboratory technician of the transfusion unit performed the rK39 strip test (rK39 ICT, Kala-azar DetectR, In Bios International, Seattle, USA) using finger prick blood from the participants, as per the manufacturer’s instructions. If a study participant tested positive for Leishmania infection by the rK39 strip test, then a lab technician collected 3 ml venous blood in an EDTA tube and this was transported to the Parasitology Laboratory, icddr,b maintaining cold chain for buffy coat preparation, DNA isolation and Ln-PCR analysis (see laboratory methods). All rK39 positive blood donors were regarded as not eligible for blood donation. Since the median incubation period for developing VL is approximately four months [2], all study participants, including those with positive rK39 strip tests, were followed for six months for eventual development of VL using mobile phone contact or home visits (when possible). All participants were instructed to contact study staff if they developed a fever of more than two weeks, or experienced weight loss, skin darkening and/or abdominal enlargement within six months of enrollment.
Laboratory methods
Preparation of buffy coat
Upon receipt at the laboratory, blood samples were centrifuged at 8000 rpm for 10 minutes at room temperature, and 500 μL of buffy coat was collected from the middle layer of the tube containing concentrated leukocytes. The buffy coat was kept in a 1.5-mL sterile microcentrifuge tube and preserved at −20°C for DNA extraction and PCR amplification.
DNA extraction from buffy coat
Buffy coat DNA was extracted for PCR using a QIAamp DNA blood mini kit (Qiagen, Hilden, Germany, Cat. no. 51106), as per the manufacturer’s instructions. The DNA was eluted in 0.2 mL of AE buffer (supplied with the Qiagen kit) after extraction. The purity of the DNA was satisfactory since the OD ratio at A260/A280 was within 1.7-1.9 for all DNA samples. Molecular-grade water was used instead of blood as an extraction control for checking for carry-over contamination in every run of DNA extraction and PCR amplification.
PCR analysis
Leishmania-specific nested PCR (Ln-PCR) was performed to detect parasite DNA in peripheral buffy coat using 2 μL of extracted DNA by the method described previously [14], with primers targeting the parasite’s SSU-rRNA region [15]. For the first PCR run, we used primers R221 5′-GGTTCCTTTCCTGATTTACG-3′ and R332 5′- GGCCGGTAAAGGCCGAATAG-3′. For the second PCR, 1 μL of 1:50 dilution of the first PCR product was used as a template in the presence of 0.15 μmol/L of the Leishmania-specific primers R223 and R333.
Sample size calculation
Seroprevalence by the rK39 strip test among individuals without past VL infection is 6% in the highly endemic areas of Mymensingh District, with 3% of seropositive individuals having confirmed active leishmania parasite infection by polymerase chain reaction (PCR) analysis (unpublished data). Thus to demonstrate an active infection rate of 3% among blood donors with 1% precision and 95% confidence interval (CI), a minimum sample size of 1,117 blood donors was required.
Data analysis
Socio-demographic data and laboratory results were entered into EPI Info 3.5.1 software (CDC, Atlanta, GA) for windows. After cleaning the data and checking for duplicates we explored the descriptive statistics to interrogate the nature of the data. A 95%CI was calculated using Normal as well as Poisson distributional approaches, where applicable. We analyzed all data using the statistical software STATA 10.1 (Stata Corporation, College Station, Texas).
Ethical statement
Blood donors were enrolled in the study after obtaining written consent. All rK39 positive blood donors were informed of their status and advised not to donate blood for the next six months. All rK39 positive blood donors were then followed for six months to see if they developed VL. The study was approved by the Research Review Committee and Ethical Review Committee of icddr,b, Dhaka, Bangladesh; and the WHO Regional Office for South-East Asia, New Delhi, Inida. The study was also approved by the Directorate General of Health Services, Government of Bangladesh.
