Comparison of collection methods for Phlebotomus argentipes sand flies to use in a molecular xenomonitoring system for the surveillance of visceral leishmaniasis.

Background: The kala-azar elimination programme has resulted in a significant reduction in visceral leishmaniasis (VL) cases across the Indian Subcontinent. To detect any resurgence of transmission, a sensitive cost-effective surveillance system is required. Molecular xenomonitoring (MX), detection of pathogen DNA/RNA in vectors, provides a proxy of human infection in the lymphatic filariasis elimination programme. To determine whether MX can be used for VL surveillance in a low transmission setting, large numbers of the sand fly vector Phlebotomus argentipes are required. This study will determine the best method for capturing P. argentipes females for MX. Methodology/Principal findings: The field study was performed in two programmatic and two non-programmatic villages in Bihar, India. A total of 48 households (12/village) were recruited. Centers for Disease Control and Prevention light traps (CDC-LTs) were compared with Improved Prokopack (PKP) and mechanical vacuum aspirators (MVA) using standardised methods. Four 12x12 Latin squares, 576 collections, were attempted (12/house, 144/village,192/method). Molecular analyses of collections were conducted to confirm identification of P. argentipes and to detect human and Leishmania DNA. Operational factors, such as time burden, acceptance to householders and RNA preservation, were also considered. A total of 562 collections (97.7%) were completed with 6,809 sand flies captured. Females comprised 49.0% of captures, of which 1,934 (57.9%) were identified as P. argentipes. CDC-LTs collected 4.04 times more P. argentipes females than MVA and 3.62 times more than PKP (p<0.0001 for each). Of 21,735 mosquitoes in the same collections, no significant differences between collection methods were observed. CDC-LTs took less time to install and collect than to perform aspirations and their greater yield compensated for increased sorting time. No significant differences in Leishmania RNA detection and quantitation between methods were observed in experimentally infected sand flies maintained in conditions simulating field conditions. CDC-LTs were favoured by householders. Conclusions/Significance: CDC-LTs are the most useful collection tool of those tested for MX surveillance since they collected higher numbers of P. argentipes females without compromising mosquito captures or the preservation of RNA. However, capture rates are still low. Author summary: Molecular xenomonitoring, screening insects for pathogen DNA/RNA, may be used for surveillance of diseases transmitted by insects. Since the proportion of insects infected with pathogens is very low in areas targeted for disease elimination, large numbers of females need to be screened. We compared three different methods for collecting Phlebotomus argentipes sand fly females, the vector of parasites causing the disease visceral leishmaniasis in the Indian subcontinent, to determine which collected the largest number of females. Other factors that may also influence selection of a particular method of collection by a disease control programme, such as the time it takes to collect and sort samples, the acceptance of householders for a particular collection method and whether RNA degradation in insect samples differed between collection methods, were also considered. Centers for Disease Control and Prevention light traps (CDC-LTs) proved to be more useful than two types of aspiration methods for collecting higher numbers of sand fly females and RNA preservation was retained. Furthermore, they took less time to install than to perform aspirations and were favoured by householders. Therefore, CDC-LTs were considered to be the most suitable collection method for molecular xenomonitoring of sand flies in India. [ABSTRACT FROM AUTHOR]