Ibrahim N. Mwangi

Reduced Susceptibility to Praziquantel among Naturally Occurring Kenyan Isolates of Schistosoma mansoni

Background The near exclusive use of praziquantel (PZQ) for treatment of human schistosomiasis has raised concerns about the possible emergence of drug-resistant schistosomes. Methodology/Principal Findings We measured susceptibility to PZQ of isolates of Schistosoma mansoni obtained from patients from Kisumu, Kenya continuously exposed to infection as a consequence of their occupations as car washers or sand harvesters. We used a) an in vitro assay with miracidia, b) an in vivo assay targeting adult worms in mice and c) an in vitro assay targeting adult schistosomes perfused from mice. In the miracidia assay, in which miracidia from human patients were exposed to PZQ in vitro, reduced susceptibility was associated with previous treatment of the patient with PZQ. One isolate (“KCW”) that was less susceptible to PZQ and had been derived from a patient who had never fully cured despite multiple treatments was studied further. In an in vivo assay of adult worms, the KCW isolate was significantly less susceptible to PZQ than two other isolates from natural infections in Kenya and two lab-reared strains of S. mansoni. The in vitro adult assay, based on measuring length changes of adults following exposure to and recovery from PZQ, confirmed that the KCW isolate was less susceptible to PZQ than the other isolates tested. A sub-isolate of KCW maintained separately and tested after three years was susceptible to PZQ, indicative that the trait of reduced sensitivity could be lost if selection was not maintained. Conclusions/Significance Isolates of S. mansoni from some patients in Kisumu have lower susceptibility to PZQ, including one from a patient who was never fully cured after repeated rounds of treatment administered over several years. As use of PZQ continues, continued selection for worms with diminished susceptibility is possible, and the probability of emergence of resistance will increase as large reservoirs of untreated worms diminish. The potential for rapid emergence of resistance should be an important consideration of treatment programs.

Introgressive hybridization of human and rodent schistosome parasites in western Kenya

Hybridization and introgression can have important consequences for the evolution, ecology and epidemiology of pathogenic organisms. We examined the dynamics of hybridization between a trematode parasite of humans, Schistosoma mansoni, and its sister species, S. rodhaini, a rodent parasite, in a natural hybrid zone in western Kenya. Using microsatellite markers, rDNA and mtDNA, we showed that hybrids between the two species occur in nature, are fertile and produce viable offspring through backcrosses with S. mansoni. Averaged across collection sites, individuals of hybrid ancestry comprised 7.2% of all schistosomes collected, which is a large proportion given that one of the parental species, S. rodhaini, comprised only 9.1% of the specimens. No F1 individuals were collected and all hybrids represented backcrosses with S. mansoni that were of the first or successive generations. The direction of introgression appears highly asymmetric, causing unidirectional gene flow from the rodent parasite, S. rodhaini, to the human parasite, S. mansoni. Hybrid occurrence was seasonal and most hybrids were collected during the month of September over a 2‐year period, a time when S. rodhaini was also abundant. We also examined the sex ratios and phenotypic differences between the hybrids and parental species, including the number of infective stages produced in the snail host and the time of day the infective stages emerge. No statistical differences were found in any of these characteristics, and most of the hybrids showed an emergence pattern similar to that of S. mansoni. One individual, however, showed a bimodal emergence pattern that was characteristic of both parental species. In conclusion, these species maintain their identity despite hybridization, although introgression may cause important alterations of the biology and epidemiology of schistosomiasis in this region.

Interactions between Natural Populations of Human and Rodent Schistosomes in the Lake Victoria Region of Kenya: A Molecular Epidemiological Approach

Background Schistosoma mansoni exists in a complex environmental milieu that may select for significant evolutionary changes in this species. In Kenya, the sympatric distribution of S. mansoni with S. rodhaini potentially influences the epidemiology, ecology, and evolutionary biology of both species, because they infect the same species of snail and mammalian hosts and are capable of hybridization. Methodology/Principal Findings Over a 2-year period, using a molecular epidemiological approach, we examined spatial and temporal distributions, and the overlap of these schistosomes within snails, in natural settings in Kenya. Both species had spatially and temporally patchy distributions, although S. mansoni was eight times more common than S. rodhaini. Both species were overdispersed within snails, and most snails (85.2% for S. mansoni and 91.7% for S. rodhaini) only harbored one schistosome genotype. Over time, half of snails infected with multiple genotypes showed a replacement pattern in which an initially dominant genotype was less represented in later replicates. The other half showed a consistent pattern over time; however, the ratio of each genotype was skewed. Profiles of circadian emergence of cercariae revealed that S. rodhaini emerges throughout the 24-hour cycle, with peak emergence before sunrise and sometimes immediately after sunset, which differs from previous reports of a single nocturnal peak immediately after sunset. Peak emergence for S. mansoni cercariae occurred as light became most intense and overlapped temporally with S. rodhaini. Comparison of schistosome communities within snails against a null model indicated that the community was structured and that coinfections were more common than expected by chance. In mixed infections, cercarial emergence over 24 hours remained similar to single species infections, again with S. rodhaini and S. mansoni cercarial emergence profiles overlapping substantially. Conclusions/Significance The data from this study indicate a lack of obvious spatial or temporal isolating mechanisms to prevent hybridization, raising the intriguing question of how the two species retain their separate identities.

No Apparent Reduction in Schistosome Burden or Genetic Diversity Following Four Years of School-Based Mass Drug Administration in Mwea, Central Kenya, a Heavy Transmission Area

Background Schistosomiasis is a debilitating neglected tropical disease that infects over 200 million people worldwide. To combat this disease, in 2012, the World Health Organization announced a goal of reducing and eliminating transmission of schistosomes. Current control focuses primarily on mass drug administration (MDA). Therefore, we monitored transmission of Schistosoma mansoni via fecal egg counts and genetic markers in a typical school based MDA setting to ascertain the actual impacts of MDA on the targeted schistosome population. Methods For 4 years, we followed 67 children enrolled in a MDA program in Kenya. Infection status and egg counts were measured each year prior to treatment. For 15 of these children, for which there was no evidence of acquired resistance, meaning they became re-infected following each treatment, we collected microsatellite genotype data from schistosomes passed in fecal samples as a representation of the force of transmission between drug treatments. We genotyped a total of 4938 parasites from these children, with an average of 329.2 parasites per child for the entire study, and an average of 82.3 parasites per child per annual examination. We compared prevalence, egg counts, and genetic measures including allelic richness, gene diversity (expected heterozygosity), adult worm burdens and effective number of breeders among time points to search for evidence for a change in transmission or schistosome populations during the MDA program. Findings We found no evidence of reduced transmission or schistosome population decline over the course of the program. Although prevalence declined in the 67 children as it did in the overall program, reinfection rates were high, and for the 15 children studied in detail, schistosome egg counts and estimated adult worm burdens did not decline between years 1 and 4, and genetic diversity increased over the course of drug treatment. Interpretation School based control programs undoubtedly improve the health of individuals; however, our data show that in an endemic area, such a program has had no obvious effect on reducing transmission or of significantly impacting the schistosome population as sampled by the children we studied in depth. Results like these, in combination with other sources of information, suggest more integrated approaches for interrupting transmission and significantly diminishing schistosome populations will be required to achieve sustainable control.

Praziquantel sensitivity of Kenyan Schistosoma mansoni isolates and the generation of a laboratory strain with reduced susceptibility to the drug

Schistosomiasis is a neglected tropical disease caused by blood-dwelling flukes of the genus Schistosoma. While the disease may affect as many as 249 million people, treatment largely relies on a single drug, praziquantel. The near exclusive use of this drug for such a prevalent disease has led to concerns regarding the potential for drug resistance to arise and the effect this would have on affected populations. In this study, we use an in vitro assay of drug sensitivity to test the effect of praziquantel on miracidia hatched from eggs obtained from fecal samples of Kenyan adult car washers and sand harvesters as well as school children. Whereas in a previous study we found the car washers and sand harvesters to harbor Schistosoma mansoni with reduced praziquantel sensitivity, we found no evidence for the presence of such strains in any of the groups tested here. Using miracidia derived from seven car washers to infect snails, we used the shed cercariae to establish a strain of S. mansoni with significantly reduced praziquantel sensitivity in mice. This was achieved within 5 generations by administering increasing doses of praziquantel to the infected mice until the parasites could withstand a normally lethal dose. This result indicates that while the threat of praziquantel resistance may have diminished in the Kenyan populations tested here, there is a strong likelihood it could return if sufficient praziquantel pressure is applied.

Schistosomes of small mammals from the Lake Victoria Basin, Kenya: new species, familiar species, and implications for schistosomiasis control.

Recent schistosomiasis control efforts in sub-Saharan Africa have focused nearly exclusively on treatment of humans with praziquantel. However, the extent to which wild mammals act as reservoirs for Schistosoma mansoni and therefore as sources of renewed transmission following control efforts is poorly understood. With the objective to study the role of small mammals as reservoir hosts, 480 animals belonging to 9 rodent and 1 insectivore species were examined for infection with schistosomes in Kisumu, in the Lake Victoria Basin, Kenya. Animals were collected from 2 sites: near the lakeshore and from Nyabera Marsh draining into the lake. A total of 6.0% of the animals captured, including 5 murid rodent species and 1 species of shrew (Crocidura olivieri) were infected with schistosomes. Four schistosome species were recovered and identified using cox1 DNA barcoding: S. mansoni, S. bovis, S. rodhaini and S. kisumuensis, the latter of which was recently described from Nyabera Marsh. Schistosoma mansoni and S. rodhaini were found infecting the same host individual (Lophuromys flavopunctatus), suggesting that this host species could be responsible for the production of hybrid schistosomes found in the area. Although the prevalence of S. mansoni infection in these reservoir populations was low (1.5%), given their potentially vast population size, their impact on transmission needs further study. Reservoir hosts could perpetuate snail infections and favour renewed transmission to humans once control programmes have ceased.

Schistosoma kisumuensis n. sp. (Digenea: Schistosomatidae) from murid rodents in the Lake Victoria Basin, Kenya and its phylogenetic position within the S. haematobium species group.

Schistosoma kisumuensis n. sp. is described based on 6 adult males and 2 adult females collected from the circulatory system of 3 murid rodent species, Pelomys isseli, Mastomys natalensis, and Dasymys incomtus. Specimens were collected from a single location, Nyabera Swamp, in Kisumu, Kenya in the Lake Victoria Basin. This new species is morphologically similar to members of the S. haematobium group, currently represented by 8 species parasitizing artiodactyls and primates, including humans. Schistosoma kisumuensis differs from these species by producing relatively small Schistosoma intercalatum-like eggs (135.2 x 52.9 microm) with a relatively small length to width ratio (2.55). Comparison of approximately 3000-base-pair sequences of nuclear rDNA (partial 28S) and mtDNA (partial cox1, nad6, 12S) strongly supports the status of S. kisumuensis as a new species and as a sister species of S. intercalatum. The cox1 genetic distance between these two species (6.3%) is comparable to other pairwise comparisons within the S. haematobium group. Separation of the Congo River and Lake Victoria drainage basins is discussed as a possible factor favoring the origin of this species.

Polymorphism associated with the Schistosoma mansoni tetraspanin-2 gene

A vaccine against schistosomiasis would contribute significantly to reducing the 3-70 million disability-adjusted life years lost annually to the disease. Towards this end, inoculation with the large extracellular loop (EC-2) of Schistosoma mansoni tetraspanin-2 protein (Sm-TSP-2) has proved effective in reducing worm and egg burdens in S. mansoni-infected mice. The EC-2 loop of Schistosoma japonicum TSP-2, however, has been found to be highly polymorphic, perhaps diminishing the likelihood that this antigen can be used for vaccination against this species. Here, we examine polymorphism of the EC-2 of Sm-TSP-2 in genetically unique worms derived from six individuals from Kisumu, Kenya.

Non-invasive sampling of schistosomes from humans requires correcting for family structure.

For ethical and logistical reasons, population-genetic studies of parasites often rely on the non-invasive sampling of offspring shed from their definitive hosts. However, if the sampled offspring are naturally derived from a small number of parents, then the strong family structure can result in biased population-level estimates of genetic parameters, particularly if reproductive output is skewed. Here, we document and correct for the strong family structure present within schistosome offspring (miracidia) that were collected non-invasively from humans in western Kenya. By genotyping 2,424 miracidia from 12 patients at 12 microsatellite loci and using a sibship clustering program, we found that the samples contained large numbers of siblings. Furthermore, reproductive success of the breeding schistosomes was skewed, creating differential representation of each family in the offspring pool. After removing the family structure with an iterative jacknifing procedure, we demonstrated that the presence of relatives led to inflated estimates of genetic differentiation and linkage disequilibrium, and downwardly-biased estimates of inbreeding coefficients (FIS). For example, correcting for family structure yielded estimates of FST among patients that were 27 times lower than estimates from the uncorrected samples. These biased estimates would cause one to draw false conclusions regarding these parameters in the adult population. We also found from our analyses that estimates of the number of full sibling families and other genetic parameters of samples of miracidia were highly intercorrelated but are not correlated with estimates of worm burden obtained via egg counting (Kato-Katz). Whether genetic methods or the traditional Kato-Katz estimator provide a better estimate of actual number of adult worms remains to be seen. This study illustrates that family structure must be explicitly accounted for when using offspring samples to estimate the genetic parameters of adult parasite populations.

Development of a Loop Mediated Isothermal Amplification for Diagnosis of Ascaris lumbricoides in Fecal Samples

Ascaris lumbricoides is a nematode parasite that causes the common tropical infection ascariasis in humans. It is also considered among the neglected tropical diseases. Diagnosis relies mainly on microscopy-based methods which are laborious, are limited by low sensitivity, and require high expertise. We have developed a loop mediated isothermal amplification (LAMP) for diagnosis of ascariasis in fecal samples, based on the first internal transcribed (ITS-1) spacer region of the ribosomal DNA. We used Primer Explorer V4 software to design primers. Ascaris adult and ova were obtained from naturally infected school children, whose parents/guardians gave consent for their participation in the study. Genomic DNA was extracted using alkaline lysis method and amplified by LAMP at 63°C for 45 minutes. LAMP products were visualized by naked eyes after adding SYBR Green dye and also on agarose gel. LAMP successfully and reliably detected Ascaris DNA from a single egg and in fecal samples. The assay specifically detected Ascaris DNA without amplifying DNA from ova of other parasites which commonly coexist with A. lumbricoides in feces. The developed LAMP assay has great potential for use in ascariasis diagnosis at the point of care and in low infection intensity situation that characterize control and elimination campaigns.