Mohammad H. Alharbi

Background and Aim: Toxoplasma gondii relies predominantly on purine salvage pathways for intracellular survival due to the absence of complete de novo purine biosynthesis. Targeting parasite purine-associated metabolism, therefore, represents a promising therapeutic strategy for toxoplasmosis.,Methods: An integrated computational and experimental workflow was employed to identify candidate inhibitors targeting T. gondii adenosine kinase (AK). High-throughput virtual screening of the ChemBridge library was performed, followed by molecular docking, 100 ns molecular dynamics simulations, and MM/PBSA binding free energy analysis. Biological evaluation included RH-2F β-galactosidase proliferation assays, purine rescue experiments, intracellular ATP depletion assays, DCFDA-based ROS quantification, and JC-1 mitochondrial membrane potential analysis in infected HFF monolayers.,Results: TAK0104 demonstrated favorable binding within the ACP-defined catalytic cavity of adenosine kinase with a docking score of -8.73 kcal/mol and stable ligand retention during a 100 ns molecular dynamics simulation. Biologically, TAK0104 inhibited intracellular tachyzoite proliferation with an IC50 of 322.6 nM and exhibited comparatively low host-cell toxicity (TD50 = 10,701 nM; therapeutic index = 33.2). Purine rescue experiments demonstrated that TAK0104-mediated growth inhibition reduced parasite proliferation to 23.4 ± 4.09% relative to untreated infected controls, whereas hypoxanthine supplementation restored proliferation to 80.8 ± 8.50%. TAK0104 further induced concentration-dependent ATP depletion, intracellular ROS accumulation, and mitochondrial membrane depolarization in infected host-cell cultures, indicating substantial metabolic stress associated with treatment.,Conclusions: TAK0104 demonstrated potent anti-Toxoplasma activity associated with disruption of parasite purine metabolism, accompanied by energetic, oxidative, and mitochondrial stress responses.

In November 2017, Biomphalaria pfeifferi, the key intermediate host for Schistosoma mansoni in Africa, was first reported in Lake Malawi, Mangochi District. Two subsequent malacological surveys in 2018 and 2019 confirmed its lacustrine presence, as well as its presence along the Upper Shire River. These surveys provided sufficient specimens for analyses of the genetic structure and a transmission assessment for intestinal schistosomiasis. A total of 76 collected snails were characterized by a DNA sequence analysis of a 650 bp fragment of the mitochondrial cytochrome oxidase subunit 1 (cox1); by size fractionation of six fluorescently labelled microsatellite loci (Bgμl16, Bgμl, Bpf8, rg6, U-7, and rg9);by denaturing PAGE; and by detection of pre-patent Schistosoma infection by real-time PCR with a TaqMan^® probe. Five closely related cox1 haplotypes were identified, all present within a single location, with only one haplotype common across all the other locations sampled. No allelic size variation was detected with the microsatellites and all loci were monomorphic. Overall, the pre-patent prevalence of Schistosoma spp. was 31%, with infected snails found at several sampling locations. In this part of Lake Malawi, Bi. pfeifferi exhibits low genetic diversity and is clearly being exposed to the miracidia of S. mansoni, which is likely facilitating the autochthonous transmission of this parasite.

The freshwater snail genus Bulinus plays a vital role in transmitting parasites of the Schistosoma haematobium group. A hybrid schistosome between S. haematobium and S. mattheei has been recently detected using DNA-based identification methods in school children along the Lake Malawi shoreline in Mangochi District. This finding raised the need for contemporary revaluation of local interactions between schistosomes and snails, with a particular focus on snail species within the Bulinus africanus group. In 2017 and 2018, malacological surveys sampled several freshwater sites in Mangochi District. Collected snails (n = 250) were characterised using cytochrome oxidase subunit 1 gene (cox1), with DNA barcoding of the ‘Folmer’ region and a rapid PCR-RFLP typing assay with double digestion with HaeIII and SacI restriction enzymes. DNA cox1 sequence analysis, with phylogenetic tree construction, suggested the presence of at least three B. africanus group taxa in Lake Malawi, B. globosus, alongside first reports of B. africanus and B. angolensis, which can be differentiated by PCR-RFLP methods. In addition, a total of 30 of the 106 B. africanus group snails (28.30%) were positive to the Schistosoma-specific screen using real-time PCR methods. This study provides new insight into the recent changes in the epidemiology of urogenital schistosomiasis as likely driven by a new diversity of B. africanus group snails within the Lake.

Schistosome eggs cause granulomata and pathological abnormalities, detectable with non-invasive radiological techniques such as ultrasonography which could be useful in male genital schistosomiasis (MGS). As part of our novel MGS study among fishermen along Lake Malawi, we describe pathologies observed on ultrasonography and praziquantel (PZQ) treatment over time. Fishermen aged 18+ years were recruited, submitted urine and semen for parasitological and molecular testing, and thereafter, transabdominal pelvic and scrotal ultrasonography, assessing pathologies in the prostate, seminal vesicles, epididymis and testes. Standard PZQ treatment and follow-up invitation at 1-, 3-, 6- and 12-months’ time-points were offered. A total of 130 recruited fishermen underwent ultrasonography at baseline (median age: 32.0 years); 27 (20.9%, n = 129) had S. haematobium eggs in urine (median: 1.0 egg/10 mL), 10 (12.3%, n = 81) in semen (defined as MGS, median: 2.9 eggs/mL ejaculate) and 16 (28.1%, n = 57) had a positive seminal Schistosoma real-time PCR. At baseline, 9 fishermen (6.9%, n = 130) had abnormalities, with 2 positive MGS having prostatic and testicular nodules. Fewer abnormalities were observed on follow-up. In conclusion, pathologies detected in male genitalia by ultrasonography can describe MGS morbidity in those with positive parasitological and molecular findings. Ultrasonography advances and accessibility in endemic areas can support monitoring of pathologies’ resolution after treatment.

The freshwater snail genus Bulinus plays a vital role in transmitting parasites of the Schistosoma haematobium group. A hybrid schistosome between S. haematobium and S. mattheei has been recently detected using DNA-based identification methods in school children along the Lake Malawi shoreline in Mangochi District. This finding raised the need for contemporary revaluation of local interactions between schistosomes and snails, with a particular focus on snail species within the Bulinus africanus group. In 2017 and 2018, malacological surveys sampled several freshwater sites in Mangochi District. Collected snails (n= 250) were characterised using cytochrome oxidase subunit 1 gene (cox1), with DNA barcoding of the ‘Folmer’ region and a rapid PCR-RFLP typing assay with double digestion with HaeIII and SacI restriction enzymes. DNA cox1 sequence analysis, with phylogenetic tree construction, suggested the presence of at least three Bu. africanus group taxa in Lake Malawi, Bu. globosus, alongside first reports of Bu. africanus and Bu. angolensis, which can be differentiated by PCR-RFLP methods. In addition, A total of 30 of the 106 Bu. africanus group snails (28.30%) were positive to the Schistosoma-specific screen using real-time PCR methods. This study provides new insight into the recent changes in the epidemiology of urogenital schistosomiasis as likely driven by a new diversity of Bu. africanus group snails within the lake

Urogenital schistosomiasis causes morbidity within the genitalia but is underreported and infrequently examined in men. To draw attention to male genital schistosomiasis (MGS), a longitudinal cohort study was conducted among fishermen along the southwestern shoreline of Lake Malawi. A case series of five participants is presented inclusive of questionnaire interviews, parasitological examinations, ultrasonography, and provision of a standard dose (40 mg/kg) of praziquantel (PZQ) treatment at baseline, 1-, 3-, 6-, and 12-month follow-up time points. Eggs of Schistosoma haematobium were observed in urine or semen across all time points; parasitological diagnostics were bolstered by real-time PCR for Schistosoma DNA in semen and by portable ultrasonography to document putative MGS-associated morbidity. We highlight the importance of developing standard diagnostic tests for MGS and increasing the accessibility of PZQ treatment to men, especially those in at-risk endemic areas.