Vector incrimination 

Dr Matthew Rogers: In this section, we will introduce you to the various steps required to incriminate a sand fly species as a vector for leishmaniasis. We will outline the different approaches and their relative merits for addressing the incrimination criteria. And secondly, explain the methods available for quantifying vectorial roles. This section is intended to build on your knowledge of sand fly and parasite biology. Globally, there are over 700 species of phlebotomine sand flies, although only 50 so far have been proven to transmit disease to humans. These are the four main criteria by which all insect vectors of disease are incriminated. They are– 1, to demonstrate that the vector species in question bites humans.

2, to demonstrate that the species is able to transmit the parasite. 3, to demonstrate that the distribution of the species coincides in space and time with the distribution of infection in humans. 4, to demonstrate natural infections of the species with parasites which are indistinguishable from the parasites isolated from human patients in the same region. To begin to incriminate a sand fly species as a leishmaniasis vector, you need to find those sand flies that bite humans. The most conclusive evidence for this would be to catch sand flies in the act of biting on a human volunteer. However, for leishmaniasis, this is unethical and not allowed, as there is no safe and effective drug able to protect the volunteer from infection.

Similarly, human landing catches are not allowed because of the same infection risk, even though the intention is to trap the sand fly before it bites. These two methods have the advantage that they directly record the human biting behaviour of the fly, thus making a stronger case for the incrimination of that species. An alternative would be to use the human volunteers to rest inside a fly-proof netting as the bait for a trap, which catches sand flies seeking a blood meal. Exit traps positioned in the windows or eaves of houses may also be used to catch sand flies leaving human dwellings to digest their blood meal.

More commonly, however, collecting sand flies from areas in which they like to rest to digest their blood meals is used to catch flies close to or within human dwellings. A variant of this approach are spray catches, which use a knock-down dose of insecticide to catch sand flies in human dwellings. Alternatively, sand flies may be passively trapped inside or outside houses by setting traps to catch sand flies in an area of disease transmission.

Traps that have been used to sample sand flies are either interceptive– for example, sticky traps, which catch sand flies as they travel between a human habitation and a potential resting or egg laying site– and/or attractive– such as CDC, Shannon, or Disney traps, which use various combinations of light and/or synthetic host odours, such as carbon dioxide to mimic human breath, to attract insects from a particular area of disease. After trapping sand flies, the next task is to identify if they took a blood meal from humans. There are two main approaches to do this, immunological or molecular. Immunological methods rely on visualising or measuring the antigen-antibody reaction between the sand fly blood meal, the antigen, and a panel of host-specific antibodies.

This could be done by eye, such as the precipitin test, or using a spectrophotometer after an enzyme-linked immunosorbent assay, ELISA. Molecular methods use the Polymerase Chain Reaction, PCR, to specifically amplify host DNA from the sand fly blood meal using species-specific primers. This can be visualised by UV illumination after separation on an agarose gel, or measured through emitted light intensity using a light-cycler during the PCR reaction. Sand flies found to blood feed on humans should then be identified to species level using molecular techniques.

From experimental studies, it is known that most Leishmania reach maturity in sand flies one to weeks after infection. Critically, Leishmania need to survive the digestion and defecation of the blood meal to successfully establish a transmissible infection. All Leishmania pathogenic to humans multiply and complete their development in the anterior part of the midgut, near the sand fly’s head. Characteristically, mature infected flies bear infections on the stomodeal valve, which are embedded within a gel-like blockage of promastigote secretory gel, PSG. Leishmania transmission is by regurgitation of parasites from this region of the sand fly gut.

Following the identification of sand fly species in an area of disease which bite humans, attention is next paid to proving the ability of that sand fly species to host and transmit Leishmania parasites recovered from the human infections. Ideally, sand flies have been colonised– or, failing this, collected fresh from the field– are allowed to blood feed on animals experimentally infected with parasites recovered from recent human cases, or through a membrane feeding device. Sand flies should be sampled after the blood meal is digested and defecated, from day 4 onwards, to assess the survivorship and growth of the parasites.

Sampling over the course of the infection will tell you if the vector is susceptible to infection, permits the multiplication of parasites, and allows them to mature into the infectious metacyclic promastigote form– i.e. undergo metacyclogenesis. Additionally, if the midgut is carefully dissected, the intensity of the infection in different regions of the gut can also be assessed. This can further inform you of the vectorial competency of that sand fly species and the likelihood of transmission, especially if the infection colonises the anterior portion of the midgut in later-stage infections by attaching to the stomodeal valve and/or blocking it with promastigote secretory gel.

If it is possible, recovering the infected metacyclic promastigotes from sand flies force-fed on capillaries or fed on a membrane further demonstrates the vectorial competence of the sand fly species in question. This is definitively proven if the infected sand flies can then transmit their parasites and generate infections in susceptible, uninfected lab animals, thus reproducing those infections found in humans.

Next, entomological surveys combined with case detection will be required to demonstrate that the suspected sand fly species occurs at the same time and same space as infections in the human population. Sand fly infection rates in the field are notoriously low– less than 1%. However, demonstrating the same parasite species from wild caught flies as the same as those recovered and identified from local patients is the final layer of proof required for vector incrimination.

To do this, a variety of approaches can be used to identify the species of parasites dissected from wild caught sand flies through dissection, parasite culture, and biochemical or molecular identification; or by screening flies for the presence of parasite DNA by PCR, or parasite antigens by ELISA or immunofluorescence.