Identification of Trichomonas vaginalis 5-Nitroimidazole Resistance Targets.

Trichomonas vaginalis is the most common non-viral sexually transmitted infection. 5-nitroimidazoles are the only FDA-approved medications for T. vaginalis treatment. However, 5-nitroimidazole resistance has been increasingly recognized and may occur in up to 10% of infections. We aimed to delineate mechanisms of T. vaginalis resistance using transcriptome profiling of metronidazole (MTZ)-resistant and sensitive T. vaginalis clinical isolates. In vitro, 5-nitroimidazole susceptibility testing was performed to determine minimum lethal concentrations (MLCs) for T. vaginalis isolates obtained from women who had failed treatment (n = 4) or were successfully cured (n = 4). RNA sequencing, bioinformatics, and biostatistical analyses were performed to identify differentially expressed genes (DEGs) in the MTZ-resistant vs. sensitive T. vaginalis isolates. RNA sequencing identified 304 DEGs, 134 upregulated genes and 170 downregulated genes in the resistant isolates. Future studies with more T. vaginalis isolates with a broad range of MLCs are needed to determine which genes may represent the best alternative targets in drug-resistant strains.

Circulating Anodic Antigen (CAA): A Highly Sensitive Diagnostic Biomarker to Detect Active Schistosoma Infections-Improvement and Use during SCORE.

The Schistosomiasis Consortium for Operational Research and Evaluation (SCORE) was funded in 2008 to conduct research that would support country schistosomiasis control programs. As schistosomiasis prevalence decreases in many places and elimination is increasingly within reach, a sensitive and specific test to detect infection with Schistosoma mansoni and Schistosoma haematobium has become a pressing need. After obtaining broad input, SCORE supported Leiden University Medical Center (LUMC) to modify the serum-based antigen assay for use with urine, simplify the assay, and improve its sensitivity. The urine assay eventually contributed to several of the larger SCORE studies. For example, in Zanzibar, we demonstrated that urine filtration, the standard parasite egg detection diagnostic test for S. haematobium, greatly underestimated prevalence in low-prevalence settings. In Burundi and Rwanda, the circulating anodic antigen (CAA) assay provided critical information about the limitations of the stool-based Kato-Katz parasite egg-detection assay for S. mansoni in low-prevalence settings. Other SCORE-supported CAA work demonstrated that frozen, banked urine specimens yielded similar results to fresh ones; pooling of specimens may be a useful, cost-effective approach for surveillance in some settings; and the assay can be performed in local laboratories equipped with adequate centrifuge capacity. These improvements in the assay continue to be of use to researchers around the world. However, additional work will be needed if widespread dissemination of the CAA assay is to occur, for example, by building capacity in places besides LUMC and commercialization of the assay. Here, we review the evolution of the CAA assay format during the SCORE period with emphasis on urine-based applications.

Evaluation, Validation, and Recognition of the Point-of-Care Circulating Cathodic Antigen, Urine-Based Assay for Mapping Schistosoma mansoni Infections.

Efforts to control Schistosoma mansoni infection depend on the ability of programs to effectively detect and quantify infection levels and adjust programmatic approaches based on these levels and program goals. One of the three major objectives of the Schistosomiasis Consortium for Operational Research and Evaluation (SCORE) has been to develop and/or evaluate tools that would assist Neglected Tropical Disease program managers in accomplishing this fundamental task. The advent of a widely available point-of-care (POC) assay to detect schistosome circulating cathodic antigen (CCA) in urine with a rapid diagnostic test (the POC-CCA) in 2008 led SCORE and others to conduct multiple evaluations of this assay, comparing it with the Kato-Katz (KK) stool microscopy assay-the standard used for more than 45 years. This article describes multiple SCORE-funded studies comparing the POC-CCA and KK assays, the pros and cons of these assays, the use of the POC-CCA assay for mapping of S. mansoni infections in areas across the spectrum of prevalence levels, and the validation and recognition that the POC-CCA, although not infallible, is a highly useful tool to detect low-intensity infections in low-to-moderate prevalence areas. Such an assay is critical, as control programs succeed in driving down prevalence and intensity and seek to either maintain control or move to elimination of transmission of S. mansoni.

SCORE Studies on the Impact of Drug Treatment on Morbidity due to Schistosoma mansoni and Schistosoma haematobium Infection.

The Schistosomiasis Consortium for Operational Research (SCORE) was funded in 2008 to improve the evidence base for control and elimination of schistosomiasis-better understanding of the systemic morbidities experienced by children in schistosomiasis-endemic areas and the response of these morbidities to treatment, being essential for updating WHO guidelines for mass drug administration (MDA) in endemic areas. This article summarizes the SCORE studies that aimed to gauge the impact of MDA-based treatment on schistosomiasis-related morbidities. Morbidity cohort studies were embedded in the SCORE's larger field studies of gaining control of schistosomiasis in Kenya and Tanzania. Following MDA, cohort children had less undernutrition, less portal vein dilation, and increased quality of life in Year 5 compared with baseline. We also conducted a pilot study of the Behavioral Assessment System for Children (BASC-2) in conjunction with the Kenya gaining control study, which demonstrated beneficial effects of treatment on classroom behavior. In addition, the SCORE's Rapid Answers Project performed systematic reviews of previously available data, providing two meta-analyses related to morbidity. The first documented children's infection-related deficits in school attendance and achievement and in formal tests of learning and memory. The second showed that greater reductions in egg output following drug treatment correlates significantly with reduced odds of most morbidities. Overall, these SCORE morbidity studies provided convincing evidence to support the use of MDA to improve the health of school-aged children in endemic areas. However, study findings also support the need to use enhanced metrics to fully assess and better control schistosomiasis-associated morbidity.

Lessons Learned in Conducting Mass Drug Administration for Schistosomiasis Control and Measuring Coverage in an Operational Research Setting.

The Schistosomiasis Consortium for Operational Research and Evaluation (SCORE) was created to conduct research that could inform programmatic decision-making related to schistosomiasis. SCORE included several large cluster randomized field studies involving mass drug administration (MDA) with praziquantel. The largest of these were studies of gaining or sustaining control of schistosomiasis, which were conducted in five African countries. To enhance relevance for routine practice, the MDA in these studies was coordinated by or closely aligned with national neglected tropical disease (NTD) control programs. The study protocol set minimum targets of at least 90% for coverage among children enrolled in schools and 75% for all school-age children. Over the 4 years of intervention, an estimated 3.5 million treatments were administered to study communities. By year 4, the median village coverage was at or above targets in all studies except that in Mozambique. However, there was often a wide variation behind these summary statistics, and all studies had several villages with very low or high coverage. In studies where coverage was estimated by comparing the number of people treated with the number eligible for treatment, denominator estimation was often problematic. The SCORE experiences in conducting these studies provide lessons for future efforts that attempt to implement strong research designs in real-world contexts. They also have potential applicability to country MDA campaigns against schistosomiasis and other NTDs, most of which are conducted with less logistical and financial support than was available for the SCORE study efforts.

Impact of Different Mass Drug Administration Strategies for Gaining and Sustaining Control of Schistosoma mansoni and Schistosoma haematobium Infection in Africa.

This report summarizes the design and outcomes of randomized controlled operational research trials performed by the Bill & Melinda Gates Foundation-funded Schistosomiasis Consortium for Operational Research and Evaluation (SCORE) from 2009 to 2019. Their goal was to define the effectiveness and test the limitations of current WHO-recommended schistosomiasis control protocols by performing large-scale pragmatic trials to compare the impact of different schedules and coverage regimens of praziquantel mass drug administration (MDA). Although there were limitations to study designs and performance, analysis of their primary outcomes confirmed that all tested regimens of praziquantel MDA significantly reduced local Schistosoma infection prevalence and intensity among school-age children. Secondary analysis suggested that outcomes in locations receiving four annual rounds of MDA were better than those in communities that had treatment holiday years, in which no praziquantel MDA was given. Statistical significance of differences was obscured by a wider-than-expected variation in community-level responses to MDA, defining a persistent hot spot obstacle to MDA success. No MDA schedule led to elimination of infection, even in those communities that started at low prevalence of infection, and it is likely that programs aiming for elimination of transmission will need to add supplemental interventions (e.g., snail control, improvement in water, sanitation and hygiene, and behavior change interventions) to achieve that next stage of control. Recommendations for future implementation research, including exploration of the value of earlier program impact assessment combined with intensification of intervention in hot spot locations, are discussed.

Five-Year Impact of Different Multi-Year Mass Drug Administration Strategies on Childhood Schistosoma mansoni-Associated Morbidity: A Combined Analysis from the Schistosomiasis Consortium for Operational Research and Evaluation Cohort Studies in the Lake Victoria Regions of Kenya and Tanzania.

The WHO recommends mass treatment with praziquantel as the primary approach for Schistosoma mansoni-related morbidity control in endemic populations. The Schistosomiasis Consortium for Operational Research and Evaluation implemented multi-country, cluster-randomized trials to compare effectiveness of community-wide and school-based treatment (SBT) regimens on prevalence and intensity of schistosomiasis. To assess the impact of two different treatment schedules on S. mansoni-associated morbidity in children, cohort studies were nested within the randomized trials conducted in villages in Kenya and Tanzania having baseline prevalence ≥ 25%. Children aged 7-8 years were enrolled at baseline and followed to ages 11-12 years. Infection intensity and odds of infection were reduced both in villages receiving four years of annual community-wide treatment (CWT) and those who received biennial SBT over 4 years. These regimens were also associated with reduced odds of undernutrition and reduced odds of portal vein dilation at follow-up. However, neither hemoglobin levels nor the prevalence of the rare abnormal pattern C liver scores on ultrasound improved. For the combined cohorts, growth stunting worsened in the areas receiving biennial SBT, and maximal oxygen uptake as estimated by fitness testing scores declined under both regimens. After adjusting for imbalance in starting prevalence between study arms, children in villages receiving annual CWT had significantly greater decreases in infection prevalence and intensity than those villages receiving biennial SBT. Although health-related quality-of-life scores improved in both study arms, children in the CWT villages gained significantly more. We conclude that programs using annual CWT are likely to achieve better overall S. mansoni morbidity control than those implementing only biennial SBT.

Impact of Mothers' Schistosomiasis Status During Gestation on Children's IgG Antibody Responses to Routine Vaccines 2 Years Later and Anti-Schistosome and Anti-Malarial Responses by Neonates in Western Kenya.

The potential consequences of parasitic infections on a person's immune responsiveness to unrelated antigens are often conjectured upon in relationship to allergic responses and autoimmune diseases. These considerations sometimes extend to whether parasitic infection of pregnant women can influence the outcomes of responses by their offspring to the immunizations administered during national Expanded Programs of Immunization. To provide additional data to these discussions, we have enrolled 99 close-to-term pregnant women in western Kenya and determined their Schistosoma mansoni and Plasmodium falciparum infection status. At 2 years of age, when the initial immunization schedule was complete, we determined their children's IgG antibody levels to tetanus toxoid, diphtheria toxoid, and measles nucleoprotein (N-protein) antigens using a multiplex assay. We also monitored antibody responses during the children's first 2 years of life to P. falciparum MSP119 (PfMSP119), S. mansoni Soluble Egg Antigen (SEA), Ascaris suum hemoglobin (AsHb), and Strongyloides stercoralis (SsNIE). Mothers' infections with either P. falciparum or S. mansoni had no impact on the level of antibody responses of their offspring or the proportion of offspring that developed protective levels of antibodies to either tetanus or diphtheria antigens at 2 years of age. However, children born of S. mansoni-positive mothers and immunized for measles at 9 months of age had significantly lower levels of anti-measles N-protein antibodies when they were 2 years old (p = 0.007) and a lower proportion of these children (62.5 vs. 90.2%, OR = 0.18, 95% CI = 0.04-0.68, p = 0.011) were considered positive for measles N-protein antibodies. Decreased levels of measles antibodies may render these children more susceptible to measles infection than children whose mothers did not have schistosomiasis. None of the children demonstrated responses to AsHb or SsNIE during the study period. Anti-SEA and anti-PfMSP119 responses suggested that 6 and 70% of the children acquired schistosomes and falciparum malaria, respectively, during the first 2 years of life.

Osteopontin Is Upregulated in Human and Murine Acute Schistosomiasis Mansoni.

BACKGROUND: Symptomatic acute schistosomiasis mansoni is a systemic hypersensitivity reaction against the migrating schistosomula and mature eggs after a primary infection. The mechanisms involved in the pathogenesis of acute schistosomiasis are not fully elucidated. Osteopontin has been implicated in granulomatous reactions and in acute hepatic injury. Our aims were to evaluate if osteopontin plays a role in acute Schistosoma mansoni infection in both human and experimentally infected mice and if circulating OPN levels could be a novel biomarker of this infection. METHODOLOGY/PRINCIPAL FINDINGS: Serum/plasma osteopontin levels were measured by ELISA in patients with acute (n = 28), hepatointestinal (n = 26), hepatosplenic (n = 39) schistosomiasis and in uninfected controls (n = 21). Liver osteopontin was assessed by immunohistochemistry in needle biopsies of 5 patients. Sera and hepatic osteopontin were quantified in the murine model of schistosomiasis mansoni during acute (7 and 8 weeks post infection, n = 10) and chronic (30 weeks post infection, n = 8) phase. Circulating osteopontin levels are increased in patients with acute schistosomiasis (p = 0.0001). The highest levels of OPN were observed during the peak of clinical symptoms (7-11 weeks post infection), returning to baseline level once the granulomas were modulated (>12 weeks post infection). The plasma levels in acute schistosomiasis were even higher than in hepatosplenic patients. The murine model mirrored the human disease. Macrophages were the major source of OPN in human and murine acute schistosomiasis, while the ductular reaction maintains OPN production in hepatosplenic disease. Soluble egg antigens from S. mansoni induced OPN expression in primary human kupffer cells. CONCLUSIONS/SIGNIFICANCE: S. mansoni egg antigens induce the production of OPN by macrophages in the necrotic-exudative granulomas characteristic of acute schistosomiasis mansoni. Circulating OPN levels are upregulated in human and murine acute schistosomiasis and could be a non-invasive biomarker of this form of disease.

Serum osteopontin is a biomarker of severe fibrosis and portal hypertension in human and murine schistosomiasis mansoni.

Schistosomiasis is a major cause of fibrosis and portal hypertension. The reason 4-10% of infected subjects develops hepatosplenic schistosomiasis remains unclear. Chronically infected male CBA/J mice reproduce the dichotomic forms of human schistosomiasis. Most mice (80%) develop moderate splenomegaly syndrome (similar to hepatointestinal disease in humans) and 20% present severe hypersplenomegaly syndrome (analogous to human hepatosplenic disease). We demonstrated that the profibrogenic molecule osteopontin discriminates between mice with severe and mild disease and could be a novel morbidity biomarker in murine and human schistosomiasis. Failure to downregulate osteopontin during the chronic phase may explain why hepatosplenic subjects develop severe fibrosis.

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

BACKGROUND: Intestinal 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. METHODS: This 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. RESULTS: Overall, 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. CONCLUSIONS: The 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.

Identification of cytokeratin 18 as a biomarker of mouse and human hepatosplenic schistosomiasis.

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.

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.