Hybridisations in UroGenital Schistosomiasis (HUGS):

Initial findings upon parasitological surveys in Mangochi and Nsanje Districts, Malawi

John Archer, Liverpool School of Tropical Medicine, UK

S. haematobium group introgressive hybridisation

• Mitochondrial genome inherited maternally
• Nuclear genome inherited from both parents

​

• Hybrid will therefore possess mitochondrial markers of a different species to those of nuclear markers

​

​

​

To identify recent hybridisation event:

​

• Target and genotype mitochondrial region (such as cox1) and nuclear ITS region

​

• If one region derived from one species (e.g., S. haematobium) and another region derived from a different species (e.g., S. mattheei), a recent hybridisation event has occurred

Whole mitochondrial genome (S. bovis)

Ribosomal RNA ITS1+2 region (S. haematobium & S. bovis)

S. haematobium? S. bovis? S. mattheei? etc…

S. haematobium? S. bovis? S. mattheei? etc…

Hybridisation: Impact on disease transmission and pathology? Some examples…

Diseases transmission

​

• Can viable hybrids be transmitted zoonotically? e.g., between humans and ruminants?

​

• Are hybrids able to infect a wider rage of snail intermediate host species?

​

• Are hybrids more (or less) resistant to treatment with praziquantel?

​

​

Disease pathology

​

• Do female hybrids produce more (or less) eggs?

​

• Are hybrid eggs more (or less) pathogenic? (can cause unusual egg morphology)

​

• Role of hybrids in female & male genital schistosomiasis

​

​

​

• 80% of 17M Malawians at-risk

​

​

• Affects all 29 districts within country

​

​

• Urogenital schistosomiasis highly prevalent along shoreline of Lake Malawi (30,000 Km²) and along Shire River (402 Km in length)

​

​

• Veterinary schistosomiasis overlooked; little known about non-human-infecting schistosomes throughout Malawi

Schistosomiasis in Malawi

• Recent parasitological surveillance (2018) found unusual egg morphologies (school-aged children) in Mangochi and Nsanje Districts

​

​

• Genotyping found S. mattheei – S. haematobium hybrids (Mangochi) and S. bovis – S. haematobium hybrids (Nsanje)

​

​

• Mangochi and Nsanje Districts selected as areas for further and more detailed investigation

S. haematobium group hybrids: Malawi

Hybridisations in UroGenital Schistosomiasis: HUGS

• Four-year Wellcome Trust funded Joint Investigator award

​

• Collaboration between Malawi-Liverpool-Wellcome Trust Clinical Research Programme (MLW), University of Malawi College of Medicine and the Liverpool School of Tropical Medicine (LSTM)

​

Aims to

​

• Ascertain if any altered pathological impact of infection with hybrids, as measured by point-of-contact assays and portable ultrasonography

​

• Reveal hybrid environmental transmission upon malacological and livestock (cattle and goat) surveillance

​

• Verify if spatial patterns of hybrid coinfection holds or alters after praziquantel treatment in these communities through one- and two-year longitudinal follow up studies

RBC counts

Haematuria

Mangochi District:

Nsanje District:

Pilot study: November 2021

• Parasitological surveys across four primary schools
• 363 school-aged children
• 38% mean prevalence of infection (~20% 50+ eggs p/10 mL urine)
• Malacological surveys: Lake Malawi southern shoreline

• Parasitological surveys across four primary schools
• 94 school-aged children
• 35% mean prevalence of infection (~14% 50+ eggs p/10 mL urine)
• Malacological surveys: Shire River

Mangochi District: Livestock GPS datalogging

• GPS dataloggers attached to eight cattle via collar

​

• Movements tracked May – July 2022

​

• Established water-contact points and water-contact behaviours (contact time, duration, purpose, etc)

​

• Frequent human water-contact also observed at these sites

​

• All eight infected (S. mattheei)

​

• One (calf) expelling S. haematobium – S. mattheei hybrid eggs

​

• Repeated using goats July – October 2022

Schistosome ‘needles in haystack’: High-throughput HRM screening

• Eggs, miracidia, cercariae, adult worm tissue, snail hosts (molecular xenomonitoring)… A lot of material to process!

​

​

• High-resolution melt qPCR assay targeting both mitochondrial (ND4, ND6 and tRNA-Lys) and nuclear (ITS2) regions to determine the presence of mixed-species ancestry

​

​

• Mitochondrial assay is species-specific multiplex (6 species) assay whereas nuclear assay is singleplex genus specific assay.

​

​

• Both assays produce amplicon products with melt temperatures (Tm) specific to each species

i: S. mattheei

ii: S. curassoni

iii: S. bovis

iv: S. haematobium

v: S. mansoni

vi: S. margrebowiei

Mitochondrial

Nuclear

Dr Lucas Cunningham, PDRA, LSTM

i: S. mansoni

ii: S. margrebowiei

iii: S. mattheei

iv: S. bovis

v: S. curassoni

vi: S. haematobium

Schistosome ‘needles in haystack’: High-throughput HRM screening

• When melt temperatures derived from both assays are plotted onto a scattergraph, it is possible to distinguish between different species based on where they cluster

​

• Can differentiate between hybrid and non-hybrid samples

​

• To validate and calibrate this approach, follow-up Sanger sequencing of samples to check for double chromatogram peaks at polymorphic positions

​

​

• Hybrids so far found in humans and cattle (both S. haematobium – S. mattheei)

​

• Cercariae to be screened soon…

Dr Lucas Cunningham, PDRA, LSTM

Hybrid

S. mattheei (A)

S. haematobium (G)

Double peak (G+A)

S. haematobium

S. mattheei

S. mansoni

S. margrebowiei

Mangochi: Samama region

• 382 households
• 1,200+ participants (stratified across age categories)

​

Nsanje: Mthawira region

• 405 households
• 1,200+ participants (stratified across age categories)

​

​

• Thousands of eggs and miracidia collected

​

• Pathology data collected (degree of haematuria and protein in urine, immunological data, questionnaire data)

​

• Livestock and snail work ongoing in these regions…

Human baseline survey: July 2022

Mangochi District

Nsanje District

Follow up survey: July 2023

Mangochi District

Nsanje District

• Individuals from both districts re-screened during 12-month follow-up survey (post-mass treatment with PZQ)

​

• Primary focus on those harbouring hybrid schistosome infections

​

​

• Any indication of pathological impact of infection with hybrids?

​

• Are hybrids more (or less) resistant to treatment with PZQ?

​

• Investigate any pathological associations between hybrid schistosome infections and female genital schistosomiasis (FGS) and male genital schistosomiasis (MGS)

Russell Stothard (co-PI)

Alexandra Juhàsz

Lucas Cunningham

Mohammad Al-harbi

Sam Jones

Sarah Rollason

Amber Read

James LaCourse

Janelisa Musaya (co-PI)

Seke Kayuni

Peter Makaula

Gladys Namacha

Donalles Kapira

Priscilla Chammudzi

David Lally

All health service assistance (HSAs) in Mangochi and Nsanje Districts

@HUGS_LSTM

Funders

john.archer@lstmed.ac.uk