William Evan Secor

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.

Extensive Genetic Diversity, Unique Population Structure and Evidence of Genetic Exchange in the Sexually Transmitted Parasite Trichomonas vaginalis

Background Trichomonas vaginalis is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted infection world-wide. Despite its prevalence, little is known about the genetic diversity and population structure of this haploid parasite due to the lack of appropriate tools. The development of a panel of microsatellite makers and SNPs from mining the parasites genome sequence has paved the way to a global analysis of the genetic structure of the pathogen and association with clinical phenotypes. Methodology/Principal Findings Here we utilize a panel of T. vaginalis-specific genetic markers to genotype 235 isolates from Mexico, Chile, India, Australia, Papua New Guinea, Italy, Africa and the United States, including 19 clinical isolates recently collected from 270 women attending New York City sexually transmitted disease clinics. Using population genetic analysis, we show that T. vaginalis is a genetically diverse parasite with a unique population structure consisting of two types present in equal proportions world-wide. Parasites belonging to the two types (type 1 and type 2) differ significantly in the rate at which they harbor the T. vaginalis virus, a dsRNA virus implicated in parasite pathogenesis, and in their sensitivity to the widely-used drug, metronidazole. We also uncover evidence of genetic exchange, indicating a sexual life-cycle of the parasite despite an absence of morphologically-distinct sexual stages. Conclusions/Significance Our study represents the first robust and comprehensive evaluation of global T. vaginalis genetic diversity and population structure. Our identification of a unique two-type structure, and the clinically relevant phenotypes associated with them, provides a new dimension for understanding T. vaginalis pathogenesis. In addition, our demonstration of the possibility of genetic exchange in the parasite has important implications for genetic research and control of the disease.

Macrophage‐derived hedgehog ligands promotes fibrogenic and angiogenic responses in human schistosomiasis mansoni

Schistosomiasis mansoni is a major cause of portal fibrosis and portal hypertension. The Hedgehog pathway regulates fibrogenic repair in some types of liver injury.

High prevalence of schistosomiasis in Mbita and its adjacent islands of Lake Victoria, western Kenya

BackgroundIntestinal schistosomiasis continues to be a significant cause of morbidity among communities located around Lake Victoria and on its islands. Although epidemiological surveys have been conducted in other areas bordering the lake in western Kenya, Mbita district and its adjacent islands have never been surveyed, largely due to logistical challenges in accessing these areas. Consequently, there is a paucity of data on prevalence of schistosomiasis and soil-transmitted helminth (STH) infections that are endemic in this region.MethodsThis cross-sectional study determined the prevalence, intensity of infection and geographical distribution of schistosome and STH infections among 4,065 children aged 5–19 years in 84 primary schools in Mbita and nearby islands of Lake Victoria (Mfangano, Ringiti, Rusinga and Takawiri), in western Kenya. Single stool samples were collected and examined for eggs of Schistosoma mansoni and STHs (Hookworms, Ascaris lumbricoides and Trichuris trichiura) using the Kato-Katz technique. Primary schools were mapped using geographical information system data on PDAs and prevalence maps generated using ArcView GIS software.ResultsOverall, 65.6% (95% CI = 64.2-67.1%) of children were infected with one or more helminth species; 12.4% (95% CI = 11.4-13.4%) of children were infected with one or more STH species. Mean school prevalence of S. mansoni infection was 60.5% (95% CI = 59.0-62.0%), hookworms 8.4% (95% CI = 7.6-9.3%), A. lumbricoides 3.3% (95% CI = 2.7-3.8%), and T. trichiura 1.6% (95% CI = 1.2-2.0%). Interestingly, the mean S. mansoni prevalence was 2-fold higher on the islands (82%) compared to the mainland (41%) (z = 5.8755, P < 0.0001). Similarly, intensity of infection was 54% higher on the islands (217.2 ± 99.3) compared to the mainland (141.3 ± 123.7) (z = 3.9374, P < 0.0001). Schools in closest proximity to Lake Victoria had the highest S. mansoni prevalence while prevalence of STHs was more homogenously distributed.ConclusionsThe very high prevalence of schistosomiasis in Mbita and the 4 islands is quite alarming, and indicates an urgent and critical need for control interventions. Findings from this survey indicate the need to implement treatment in remote areas not previously covered by mass drug administration programs.

Trichomonas vaginalis Metronidazole Resistance Is Associated with Single Nucleotide Polymorphisms in the Nitroreductase Genes ntr4Tv and ntr6Tv

ABSTRACT Metronidazole resistance in the sexually transmitted parasite Trichomonas vaginalis is a problematic public health issue. We have identified single nucleotide polymorphisms (SNPs) in two nitroreductase genes (ntr4Tv and ntr6Tv) associated with resistance. These SNPs were associated with one of two distinct T. vaginalis populations identified by multilocus sequence typing, yet one SNP (ntr6Tv A238T), which results in a premature stop codon, was associated with resistance independent of population structure and may be of diagnostic value.

Identification of Cytokeratin 18 as a Biomarker of Mouse and Human Hepatosplenic Schistosomiasis

ABSTRACT Previously, we demonstrated unique protein expression patterns in 20-week-Schistosoma mansoni-infected CBA/J mice with moderate splenomegaly syndrome (MSS) or hypersplemomegaly syndrome (HSS). To better understand the development of severe pathology, we compared the two-dimensional differential in-gel electrophoresis (2D-DIGE) proteomic signatures of livers from uninfected mice and mice infected for 6, 8, 12, or 20 weeks and found significant changes in collagen isoforms, interleukin-2 (IL-2), cytokeratin 18, hydroxyproline, S. mansoni phosphoenolpyruvate carboxykinase, major urinary protein isoforms, and peroxiredoxin 6. Cytokeratin 18, hydroxyproline, and connective tissue growth factor (CTGF) were chosen for analysis in mouse and human sera using targeted biochemical assays. Consistent with the liver analysis, cytokeratin 18, CTGF, and hydroxyproline were significantly elevated in sera from mice with HSS compared to those from uninfected mice or mice with MSS. Moreover, cytokeratin 18 and CTGF were found to be markers for subjects with hepatosplenic and intestinal schistosomiasis, respectively, while serum hydroxyproline was a strong indicator of fibrosis for severe HS. These findings indicate that schistosome-associated changes to the liver can be detected in the serum and reveal the potential for cytokeratin 18 to be used as a diagnostic marker for early detection of hepatosplenic schistosomiasis.

Differential Patterns of Liver Proteins in Experimental Murine Hepatosplenic Schistosomiasis

ABSTRACT Schistosoma mansoni eggs produced by adult worms in the mesenteric vasculature become trapped in the liver, where they induce granulomatous lesions and strong immune responses. Infected individuals suffer from intestinal schistosomiasis (INT) in 90% of cases, whereas the remaining 10% present with severe hepatosplenic schistosomiasis (HS). The CBA/J mouse model mimics human disease, with 20% of infected mice developing hypersplenomegaly syndrome (HSS) that resembles HS and 80% developing moderate splenomegaly syndrome (MSS) similar to INT. We studied differential patterns of protein expression in livers of 20-week-infected CBA/J mice with MSS or HSS to understand the molecular changes that underlie these two disease forms. Using differential in-gel electrophoresis to identify differentially expressed protein spots, we found 80 protein spots significantly changed with infection and 35 changes specific to severe disease. In particular, the abundances of prohibitin 2, transferrin isoforms, and major urinary protein isoforms were significantly altered in HSS mice. Furthermore, annexin 5, glutathione S-transferase pi class, and S. mansoni phosphoenolpyruvate carboxykinase expression levels changed significantly with schistosome infection. Additionally, levels of major urinary protein decreased and levels of transferrin increased significantly in the sera of HSS mice compared to levels in sera of MSS or control mice, and these differences correlated to the degree of splenomegaly. These findings indicate that the liver protein abundances differ between MSS and HSS mice and may be used for the development of diagnostic markers for the early detection of hepatosplenic schistosomiasis.

Genetic Indicators of Drug Resistance in the Highly Repetitive Genome of Trichomonas vaginalis

Abstract Trichomonas vaginalis, the most common nonviral sexually transmitted parasite, causes ∼283 million trichomoniasis infections annually and is associated with pregnancy complications and increased risk of HIV-1 acquisition. The antimicrobial drug metronidazole is used for treatment, but in a fraction of clinical cases, the parasites can become resistant to this drug. We undertook sequencing of multiple clinical isolates and lab derived lines to identify genetic markers and mechanisms of metronidazole resistance. Reduced representation genome sequencing of ∼100 T. vaginalis clinical isolates identified 3,923 SNP markers and presence of a bipartite population structure. Linkage disequilibrium was found to decay rapidly, suggesting genome-wide recombination and the feasibility of genetic association studies in the parasite. We identified 72 SNPs associated with metronidazole resistance, and a comparison of SNPs within several lab-derived resistant lines revealed an overlap with the clinically resistant isolates. We identified SNPs in genes for which no function has yet been assigned, as well as in functionally-characterized genes relevant to drug resistance (e.g., pyruvate:ferredoxin oxidoreductase). Transcription profiles of resistant strains showed common changes in genes involved in drug activation (e.g., flavin reductase), accumulation (e.g., multidrug resistance pump), and detoxification (e.g., nitroreductase). Finally, we identified convergent genetic changes in lab-derived resistant lines of Tritrichomonas foetus, a distantly related species that causes venereal disease in cattle. Shared genetic changes within and between T. vaginalis and Tr. foetus parasites suggest conservation of the pathways through which adaptation has occurred. These findings extend our knowledge of drug resistance in the parasite, providing a panel of markers that can be used as a diagnostic tool.

Schistosome-induced cholangiocyte proliferation and osteopontin secretion correlate with fibrosis and portal hypertension in human and murine schistosomiasis mansoni

Schistosomal egg antigens induce host bile ductular cells to proliferate and produce osteopontin (OPN), a pro-fibrogenic factor that stimulates hepatic stellate cells to become myofibroblasts. The numbers of OPN-producing bile ductules correlate with fibrogenesis and portal hypertension in humans and mice.

An ELISA Test to Detect Human Serum Antibodies Reactive with Encephalitozoon intestinalis

Microsporidia are a group of obligate intracellular protistan parasites that have recently gained recognition as opportunistic infections in persons with AIDS or in those who are otherwise immunocompromised. To date, eight genera have been identified as human pathogens, Encephalitozoon, Enterocytozoon, Nosema, Virtaformu. Pleistophora, Trachipleistophora, Brachiola. and the collective genus Microsporidiwn [3]. A frequent causative agent of microsporidial diarrhea and systemic infection among AIDS patients is Encephulitozoon intestinalis [8]. This parasite can also cause chmnic diarrhea in immunounnptent persons [7]. However. many epidemiologic aspects of this infection remain poorly understood as the diagnostic tools for this parasite are not amenable to widespread use. In an attempt to improve this situation, we have developed an ELISA test to detect human serum antibodies reactive with Encephulitozoon intestinulis.