Xenodiagnosis to address key questions in visceral leishmaniasis control and elimination

Abstract

Visceral leishmaniasis (VL) remains an important public health issue worldwide causing substantial morbidity and mortality. The Indian subcontinent accounted for up to 90% of the global VL burden in the past but made significant progress during recent years and is now moving towards elimination. However, to achieve and sustain elimination of VL, knowledge gaps on infection reservoirs and transmission need to be addressed urgently. Xenodiagnosis is the most direct way for testing the infectiousness of hosts to the vectors and can be used to investigate the dynamics and epidemiology of Leishmania donovani transmission. There are, however, several logistic and ethical issues with xenodiagnosis that need to be addressed before its application on human subjects. In the current Review, we discuss the critical knowledge gaps in VL transmission and the role of xenodiagnosis in disease transmission dynamics along with its technical challenges. Establishment of state of the art xenodiagnosis facilities is essential for the generation of much needed evidence in the VL elimination initiative.

Fig 1. Dynamics of human infectious reservoirs for VL.

Human infection is initiated when an infected female P. argentipes injects L. donovani parasites during a blood meal. Most infections do not lead to clinical symptoms and are suppressed or eliminated by the innate or adaptive immunity. Only a fraction of infected, asymptomatic subjects progress to clinical VL disease. After treatment of clinical infection, most patients develop protective immunity against the disease. Approximatly 1% to 10% of former VL patients may develop a chronic cutaneous form called PKDL. PKDL without history of VL is rare. HIV positive individuals are more at risk of developing VL once infected and become important source of transmission due to their potentially high parasitic load. PKDL, post kala-azar dermal leishmaniasis; VL, visceral leishmaniasis.

Fig 2. The infectivity of the subject can be tested with direct xenodiagnosis using sand fly vector female P. argentipes.

(A) Pathogen free colony of sand fly. (B) Three-to-five day old unfed P. argentipes females removed from a colony cage. (C) An empty 2-inch diameter polycarbonate feeding chamber. (D) Loaded with sand flies by mouth aspirator through a small entry port. The feeding chamber has a screen-mesh bottom, through which the sand flies can feed, and vented top to prevent moisture condensation that might entrap sand flies inside the chamber. (E) The loaded feeding chamber (with 20 to 30 female P. argentipes) is then strapped with the screened side against the skin by a band for 30 minutes on the arm or leg (location can be decided during xenodiagnosis process based on logistic as well as patients comfort). This procedure usually takes place in the xenodiagnosis room, separate from the closed colony insectary room. (F) After 30 minutes of feeding, the feeding chamber is removed from the patient’s arm or leg and sand flies are released from the feeding chamber into a polycarbonate holding cage where they can be separated as fed and unfed. (G) Only blood-engorged females sand flies are transferred into a 1-pint holding container with screen-mesh top and held for at least 72 hours in an environmental cabinet at 28°C and 80% humidity with a 17:7 (Light: Dark) photoperiod. (H) All 3 to 5 days postfeeding surviving flies are then dissected and examined under the microscope for the presence or absence of Leishmania promastigotes. A subject will be considered positive for infectivity to sand flies if an infection is observed in at least one sand fly.