how Microbiopsy is helping to better understand leishmania major infection

Cutaneous leishmaniasis (CL) is a skin disease which disfigures millions of patients per year, sadly the World health organisation estimate that there are 600,000 to 1 million new cases occur globally per annum [i]. 

The parasite derived infection is commonly found in North and East Africa, the Middle East, Central and SW Asia.  Organisms responsible for CL are bites from sandfly (the vector) infected with a species of Leishmania called Leishmania major (L major).  

Symptoms 

The main symptoms of the disease are skin lesions, which begin as small painless papules or nodules which then often develop into painless open sores or ulcers  [ii].  Although the resultant infection impacts so many people per year, it is often a neglected disease. 

This is due to two main reasons; its epidemiological locations pointing to some of the poorest populations and furthermore, the disease is not deadly to most patients. 

However, left untreated the condition can cause severe cosmetic issues leading to psychological distress as well as social stigma.  Moreover, the resultant open wounds and ulcers are also targets for opportunistic secondary infections leading to further complications.

Mode of Infection 

To understand the mode of infection and how to both diagnose and treat the disease, it is important to understand the lifecycle of L Major.  L Major is a protozoan parasite which lives and replicates for most of its life in its immobile form (amastigotes) within host macrophages found in skin tissue. 

The main host for is a species of rodent called Meriones shawi (M shawi), which in turn then infect Sandflies (Phlebotomus papatasi) when the unsuspecting fly takes a blood meal containing infected macrophages. 

Upon infection, the blood meal is transferred to the midgut where the amastigotes are released and then rapidly transformed to a motile flagellated form (procyclic promastigotes) where they attach to the gut epithelium and rapidly replicate.

After 4-10 days, the promastigotes migrate from the midgut to both the foregut and pharynx of the sandfly, where they transform into highly infectious metacyclic promastigotes.  When the sandfly takes its next blood meal (which could then be instead from a human host) it regurgitates the metacyclic promastigotes, which are then engulfed by the new hosts phagocytes and eventually transferred to macrophages located in the hosts skin tissue. 

Within the macrophages, the metacyclic promastigotes  transformed back to amastigotes where they rapidly replicate, rupturing microphages and in doing so, go on to infect neighbouring macrophages, where the cycle repeats.  This then leads to lesion formation associated with CL [iii,iv]. 

Understanding Infectiousness of L. major to Sandflies  

In June 2025 an international  team of researchers at institutions in the Czech Republic, France and Algeria, published an article in PLoS Neglected Tropical Diseases entitled “Infectiousness of Leishmania major to Phlebotomus papatasi: differences between natural reservoir host Meriones shawi and laboratory model BALB/c mice” [v]. 

Using a variety of techniques such as microbiopsy, fluorescence imaging and qPCR, the team lead by Jovana Sádlová, demonstrated a better understanding pertaining the  degree of infectiousness of L. major to Sandflies. 

Furthermore the team also showed the heterogeneity of infection efficacy between the parasite’s natural host (M shawi) and a laboratory model rodent species (BALB/c mice). 

Findings 

• Previous field studies showed that rodent ear pinnae are preferred bite sites for the sandflies and therefore the group focussed on this tissue, for sampling potentially infected rodents. 

• The group found, that in agreement to previous studies, only 11% of sandflies became infected from feeding from the pinnae of infected M. shawi rodents.  This further suggested that the distribution parasites on the skin was patchy. 

• The group divided the ears into zones, symptomatic comprising of lesion centres and margins and then asymptomatic surrounding skin.  

• The findings were that the parasite burden was significantly higher in the symptomatic skin 

• Interestingly for M. shawi, sandflies only became infected when feeding on the margins not in the centre of the infected skin of the rodents.  However, this phenomena was not observed for the lab mice cohort (BALB/c) which showed that bite location on the infected tissue was similar at the centre and margins.  The group theorised that this was probably due to differences in immune response between the two species. 

• By using Harpera microbiopsy devices to collect tiny skin samples at precise areas of the skin and then image the samples using fluorescence microscopy, they were able to observe that less than 10 amastigotes from the margins of M. shawi, were sufficient to infect the sandfly vectors.   

• NB: The reason why the Harpera devices were ideal for this application was because they are firstly minimally invasive thus allowing for multiple sampling events from the same tissue.  However serendipitously, the Harpera devices sample at a depth of 260 µm, which is coincidentally the same bite depth as the sandfly proposes.  Furthermore, comparing DNA collected, it was confirmed that the microbiopsy device also removes a similar amount of tissue compared to the sandfly.     

• To confirm the surprising results from the microbiopsy experiment, the group allowed sandflies to feed on blood meal (with reducing concentrations of  amastigotes) through a chick-skin membrane. Following this to measure infection rates, the researchers examined amastigote numbers the midgut of the flies.  The results aligned with the groups previous results, where only 8-10% of the sandflies were infected with blood dosed with 1-10 amastigotes.  This lead to low or moderate infections after 8 days, where 100-1000 promastigotes were found in the sandfly guts. 

• Finally the group showed another key difference between the natural host (M. shawi) and the standard lab rodent model (BALB/c mice).   

• As mentioned above, not only was the bite location on the infected tissue similar at the centre and margins, in the BALB/c mice infected with the parasite, no infection was observed in sandflies when less than 1000 amastigotes were present in the blood.  This contrasted to as little as 1-10 amastigotes for the natural host. 

Conclusions 

The study demonstrated, when trying to elucidate the infectiousness of Leishmania major to sandflies, it is crucial to use the right host organism, be it the natural host or ana appropriate surrogate. 

In the study presented by  Jovana Sádlová et al, the laboratory mice used in the study (BALB/c mic) were less efficacious in infecting  sandflies compared to M. shawi. 

Moreover, no heterogeneity in infected tissue bite site location was observed. However this contrasted vastly to the host animal, where the periphery of infected tissue infected many more sandflies than the centre of the wound.   

One of the key tools used in the study was the Harpera microbiopsy Punch.  Although, the Harpera device was not specifically designed to mimic sandfly probosces, it serendipitously imitated a sandfly bite with unparalleled precision. 

This allowed researchers to garner great insight into rates of infection from host to vector.  Although use of Harpera has helped in further understand how cutaneous leishmaniasis develops, which is important in its own right, the study also demonstrates the wider utility of the device when sampling skin. 

Indeed, groups worldwide are using it for an increasing  number of applications such as in skin cancer and omics research . This means that in the future the Harpera punch could be used as a replacement of more invasive and painful biopsy techniques. 

Not only is this more comfortable for subjects but scientifically allows a potentially greater resolution in sampling geography and sampling cadence.