Evaluation of a simple,rapid and field-adapted diagnostic assay for enterotoxigenic E. coli and Shigella

Understanding the global burden of enterotoxigenic E. coli (ETEC) and Shigella diarrhea as well as estimating the cost effectiveness of vaccines to control these two significant pathogens have been hindered by the lack of a diagnostic test that is rapid, simple, sensitive, and can be applied to the endemic countries. We previously developed a simple and rapid assay, Rapid Loop mediated isothermal amplification based Diagnostic Test (RLDT) for the detection of ETEC and Shigella spp. (Shigella). In this study, the RLDT assay was evaluated in comparison with quantitative PCR (qPCR), culture and conventional PCR for the detection of ETEC and Shigella. This validation was performed using previously collected stool samples from endemic countries, from the travelers to the endemic countries, as well as samples from a controlled human infection model study of ETEC. The performance of RLDT from dried stool spots was also validated. RLDT resulted in excellent sensitivity and specificity compared to qPCR (99% and 99.2% respectively) ranging from 92.3 to 100% for the individual toxin genes of ETEC and 100% for Shigella. Culture was less sensitive compared to RLDT. No significant differences were noted in the performance of RLDT using samples from various sources or stool samples from moderate to severe diarrhea or asymptomatic infections. RLDT performed equally well in detection of ETEC and Shigella from the dried stool samples on filter papers. This study established that RLDT is sufficiently sensitive and specific to be used as a simple and rapid diagnostic assay to detect ETEC and Shigella in endemic countries to determine disease burden of these pathogens in the national and subnational levels. This information will be important to guide public health and policy makers to prioritize resources for accelerating the development and introduction of effective preventative and/or treatment interventions against these enteric infections.     

Development of a simple,rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries

Enterotoxigenic E. coli (ETEC) and Shigella spp (Shigella) are complex pathogens. The diagnostic assays currently used to detect these pathogens are elaborate or complicated, which make them difficult to apply in resource poor settings where these diseases are endemic. The culture methods used to detect Shigella are not sensitive, and the methods used to detect ETEC are only available in a few research labs. To address this gap, we developed a rapid and simple diagnostic assay–"Rapid LAMP based Diagnostic Test (RLDT)." The six minutes sample preparation method directly from the fecal samples with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, ETEC [heat labile toxin (LT) and heat stable toxins (STh, and STp) genes] and Shigella (ipaH gene) detection was made simple, rapid (<50 minutes), and inexpensive. This assay is cold chain and electricity free. Moreover, RLDT requires minimal equipment. To avoid any end user’s bias, a battery-operated, handheld reader was used to read the RLDT results. The results can be read as positive/negative or as real time amplification depending on the end user’s need. The performance specifications of the RLDT assay, including analytical sensitivity and specificity, were evaluated using fecal samples spiked with ETEC and Shigella strains. The limit of detection was ~10⁵ CFU/gm of stool for LT, STh, and STp and ~10⁴ CFU/gm of stool for the ipaH gene, which corresponds to about 23 CFU and 1 CFU respectively for ETEC and Shigella per 25uL reaction within 40 minutes. The RLDT assay from stool collection to result is simple, and rapid and at the same time sufficiently sensitive. RLDT has the potential to be applied in resource poor endemic settings for the rapid diagnosis of ETEC and Shigella.     

Toxicity and Immunogenicity of Enterotoxigenic Escherichia coli Heat-Labile and Heat-Stable Toxoid Fusion 3xSTaA14Q-LTS63K/R192G/L211A in a Murine Model

Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC) are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT) and heat-stable type Ib toxin (STa) disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STa_P13F) fused at the N- or C-terminus, or inside the A subunit of LT_R192G elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011). In this study, we generated a different STa toxoid (STa_A14Q) and a triple-mutant LT toxoid (LT_{S63K/R192G/L211A}, tmLT), constructed a toxoid fusion (3xSTa_A14Q-tmLT) that carried 3 copies of STa_A14Q for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTa_A14Q-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea.     

Exopolysaccharides Synthesized by Lactobacillus reuteri Protect against Enterotoxigenic Escherichia coli in Piglets

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in piglets; ETEC cells colonize the intestinal mucosa with adhesins and deliver toxins that cause fluid loss. This study determined the antiadhesive properties of bacterial exopolysaccharides (reuteran and levan) and related glycans (dextran and inulin) in a small intestinal segment perfusion (SISP) model. The SISP model used 10 jejunal segments from 5-week-old piglets. Five segments were infected with ETEC expressing K88 fimbriae (ETEC K88), while five segments were treated with saline. Every two segments (ETEC and non-ETEC infected) were infused with 65 ml of 10 g liter⁻¹ of glycans or saline (control) for 8 h. High-resolution melting-curve (HRM) quantitative PCR (qPCR) indicated that E. coli is the dominant bacterium in infected segments, while other bacteria were predominant in noninfected segments. Infection by ETEC K88 was also verified by qPCR; gene copy numbers of K88 fimbriae and the heat-labile toxin (LT) in mucosal scrapings and outflow fluid of infected segments were significantly higher than those in noninfected segments. Genes coding for K88 fimbriae and LT were also detected in noninfected segments. LT amplicons from infected and noninfected segments were 99% identical over 481 bp, demonstrating the presence of autochthonous ETEC K88. All glycans reduced fluid loss caused by ETEC K88 infection. Reuteran tended (P = 0.06) to decrease ETEC K88 levels in mucosal scraping sample, as judged by qPCR. Fluorescent in situ hybridization analysis demonstrated that reuteran significantly (P = 0.012) decreased levels of adherent ETEC K88. Overall, reuteran may prevent piglet diarrhea by reducing adhesion of ETEC K88.     

Glucose Significantly Enhances Enterotoxigenic Escherichia coli Adherence to Intestinal Epithelial Cells through Its Effects on Heat-Labile Enterotoxin Production

The present study tested whether exposure of enterotoxigenic Escherichia coli (ETEC) to glucose at different concentrations in the media results in increased bacterial adherence to host cells through increased heat-labile enterotoxin (LT) production, thereby suggesting the effects are physiological. Porcine-origin ETEC strains grown in Casamino acid yeast extract medium containing different concentrations of glucose were washed and inoculated onto IPEC-J2 porcine intestinal epithelial cells to test for effects on adherence and host cell cAMP concentrations. Consistent with previous studies, all LT⁺ strains had higher ETEC adherence to IPEC-J2 cells than did LT⁻ strains. Adherence of the LT⁻ but not the LT⁺ strains was increased by pre-incubating the IPEC-J2 cells with LT and decreased by co-incubation with GM1 ganglioside in a dose-dependent manner (P<0.05). To determine whether the glucose concentration of the cell culture media has an effect on adherence, IPEC-J2 cells were inoculated with LT⁺ or LT⁻ strains in cell culture media containing a final glucose concentration of 0, 0.25, 0.5, 1.0 or 2.0%, and incubated for 4 h. Only media containing 0.25% glucose resulted in increased adherence and cAMP levels, and this was limited to IPEC-J2 cells inoculated with LT⁺ strains. This study supports the hypothesis that glucose, at a concentration optimal for LT expression, enhances bacterial adherence through the promotion of LT production. Hence, these results establish the physiological relevance of the effects of glucose on LT production and provide a basis for how glucose intake may influence the severity of ETEC infection.     

Prevalence,risk factors and health consequences of soil-transmitted helminth infection on the Bijagos Islands,Guinea Bissau: A community-wide cross-sectional study

Soil-transmitted helminths (STH) are endemic and widespread across Sub-Saharan Africa. A community wide soil-transmitted helminth (STH) prevalence survey was performed on the island of Bubaque in Guinea-Bissau using both Kato-katz microscopy and qPCR methodology. Predictors of infection and morbidity indicators were identified using multivariable logistic regression, and diagnostic methods were compared using k statistics. Among 396 participants, prevalence of STH by microscopy was 23.2%, hookworm was the only species identified by this method and the mean infection intensity was 312 eggs per gram. qPCR analysis revealed an overall prevalence of any STH infection of 47.3%, with the majority A. duodenale (32.3%), followed by N. americanus (15.01%) and S. stercoralis (13.2%). A. lumbricoides, and T. trichiura infections were negligible, with a prevalence of 0.25% each. Agreement between diagnostic tests was k = 0.22, interpreted as fair agreement, and infection intensity measured by both methods was only minimally correlated (R_s = -0.03). STH infection overall was more common in females and adults aged 31–40. STH infection was associated with open defaecation, low socio-economic status and further distance to a water-source. The prevalence of anaemia (defined as a binary outcome by the WHO standards for age and sex) was 69.1%, and 44.2% of children were malnourished according to WHO child growth standards. Hookworm infection intensity by faecal egg count showed no statistically significant association with age (R_s 0.06) but S. Stercoralis infection intensity by qPCR cycle threshold was higher in pre-school aged children (R_s = 0.30, p-value 0.03) There was no statistically significant association between STH infection and anaemia (OR 1.0 p = 0.8), stunting (OR 1.9, p-value 0.5) and wasting (OR 2.0, p-value 0.2) in children. This study reveals a persistent reservoir of STH infection across the community, with high rates of anaemia and malnutrition, despite high-coverage of mebendazole mass-drug administration in pre-school children. This reflects the need for a new strategy to soil-transmitted helminth control, to reduce infections and ultimately eliminate transmission.     

Comparison of the egg recovery rates and limit of detection for soil-transmitted helminths using the Kato-Katz thick smear,faecal flotation and quantitative real-time PCR in human stool

BackgroundMonitoring the success of soil-transmitted helminth (STH) control programs relies on accurate diagnosis and quantitative assessment of infection prevalence and intensity. As preventative chemotherapeutic program coverage for STH expands, the necessity of gaining insights into the relative or comparative sensitivities, in terms of limits of detection (LOD) and egg-recovery-rates (ERR) for microscopy and quantitative polymerase chain reaction qPCR-based diagnostic techniques becomes imperative to inform suitability for their intended use for large scale STH monitoring and treatment efficacy studies.Methodology/Principal findingsThe diagnostic performance in terms of ERR and LOD of the Kato-Katz (KK) thick smear technique, sodium nitrate (NaNO₃) faecal floatation (FF) and qPCR for the accurate detection and enumeration of STH eggs were calculated and expressed in eggs per gram (EPG), by experimentally seeding parasite-free human faeces with Ascaris spp., Trichuris spp. and Necator americanus eggs representing low, medium and high intensity infections. The efficiency of NaNO₃ flotation was also calculated over a range of specific gravities (SpGr) for the optimum recovery of STH eggs. FF of SpGr 1.30 recovered 62.7%, 11% and 8.7% more Trichuris spp., Necator americanus and Ascaris spp. eggs respectively, than the recommended SpGr of 1.20. All diagnostic methods demonstrated strong direct correlation to the intensity of seeded EPG. KK and FF (SpGr 1.30) resulted in significant lower ERRs compared to qPCR (p <0.05). qPCR demonstrated significantly (p <0.05) greater sensitivity with an ability to detect as little as 5 EPG for all three STH, compared to 50 EPG by KK and FF (SpGr 1.30).Conclusions/SignificanceThis study compares the diagnostic parameters in terms of LOD and ERRs of STHs for the KK, FF and qPCR. These results indicate that the diagnostic performance of qPCR assays should be considered by control programs in the phase that aims to seek confirmation of transmission break and cessation of preventive chemotherapy in low-transmission settings, in line with the control targets of the WHO neglected tropical diseases 2030 Roadmap.     

Assessment of environmental contamination with soil-transmitted helminths life stages at school compounds,households and open markets in Jimma Town,Ethiopia

BackgroundIt remains largely unknown where and how infections with soil-transmitted helminths (STHs; Ascaris, Trichuris, Necator and Ancylostoma) occur. We therefore aimed to identify possible sources of infection by assessing the environmental contamination in an STH-endemic area.MethodsWe first performed a series of laboratory experiments designed to optimize a soil straining-flotation method to detect and quantify Ascaris and Trichuris eggs in soil, and to validate the diagnostic performance of the optimized method when followed by microscopy and qPCR. In a second phase, we applied this method to assess the level of STH contamination in 399 environmental samples collected from 10 school compounds, 50 households and 9 open markets in Jimma Town (Ethiopia). Subsequently, we explored associations between the environmental contamination and both the corresponding STH epidemiology at the level of the schools and the household characteristics. Finally, we assessed the knowledge, attitude and practice (KAP) towards STHs in school children.Principal findingsOur soil straining-flotation method has an analytical sensitivity of 50 eggs per 100 grams of soil and egg recovery rate of 36.0% (Ascaris) and 8.0% (Trichuris). The analysis of field samples with both microscopy and qPCR revealed the presence of 8 different helminth species of medical importance, including but not limited to the human STHs. There was a significant association between the environmental contamination and prevalence of any STH infections at the school level only. The KAP indicated a lack of knowledge and awareness of STHs.Conclusions/SignificanceOur optimized straining-flotation method has a moderate diagnostic performance and revealed that life stages of helminths are ubiquitous in the environment, which might be due to the poor sanitary facilities at both the schools and the households, and a poor level of KAP towards STHs. Further research is required to gain more insights into the contribution of these life stages to transmission.     

Diagnosis,Treatment and Risk Factors of Strongyloides stercoralis in Schoolchildren in Cambodia

Background

Worldwide, an estimated 30 to 100 million people are infected with Strongyloides stercoralis, a soil-transmitted helminth. Information on the parasite is scarce in most settings. In semi-rural Cambodia, we determined infection rates and risk factors; compared two diagnostic methods (Koga agar plate [KAP] culture and Baermann technique) for detecting S. stercoralis infections, using a multiple stool examination approach; and assessed efficacy of ivermectin treatment.

Methods/Principal Findings

We performed a cross-sectional study in 458 children from four primary schools in semi-rural villages in Kandal province, using three diagnostic procedures (Kato-Katz, KAP culture and Baermann technique) on three stool samples. Infected children were treated with ivermectin (100 µg/kg/day for two days) and re-examined three weeks after treatment. Hookworm, S. stercoralis, Trichuris trichiura, and small trematode eggs were most prevalent, with 24.4% of children being infected with S. stercoralis. The sensitivity of KAP culture and Baermann technique was 88.4% and 75.0%, respectively and their negative predictive values were 96.4% and 92.5%, respectively. The cumulative prevalence of S. stercoralis increased from 18.6% to 24.4%, after analyzing three stool samples, which was close to the modeled ‘true’ prevalence of 24.8%. Children who reported defecating in latrines were significantly less infected with S. stercoralis than those who did not use latrines (p<0.001). Itchy skin and diarrhea were significantly associated with S. stercoralis infection. The cure rate of ivermectin was 98.3%.

Conclusions/Significance

S. stercoralis infection is highly prevalent among semi-rural Cambodian schoolchildren. The sensitivity of KAP culture is higher than that of the Baermann technique. In the absence of a “gold standard”, analysis of multiple stool samples by different diagnostic methods is required to achieve a satisfactory level of sensitivity. Almost three-quarters of the infections could have been avoided by proper sanitation. Ivermectin is highly efficacious against S. stercoralis but prohibitive costs render the drug inaccessible to most Cambodians.     

Detection of toxoplasmic encephalitis in HIV positive patients in urine with hydrogel nanoparticles

BackgroundDiagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circumstances. The poor clinical sensitivity of quantitative polymerase chain reaction (qPCR) for Toxoplasma in blood and CSF and the limited availability of molecular diagnostics and imaging technology leaves clinicians in resource-limited settings with few options other than empiric treatment.Methology/principle findingsHere we describe proof of concept for a novel urine diagnostics for TE using Poly-N-Isopropylacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substantially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml of T. gondii antigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurological symptoms compatible with TE were tested for 1) T. gondii serology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected, T. gondii seropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitoring (PRM) for the presence of T. gondii antigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic.Conclusion/significancesOur results demonstrate nanoparticle technology’s potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.     

Prevalence of diarrhoeal pathogens among children under five years of age with and without diarrhoea in Guinea-Bissau

BackgroundChildhood diarrhoea, a major cause of morbidity and mortality in low-income regions, remains scarcely studied in many countries, such as Guinea-Bissau. Stool sample drying enables later qPCR analyses of pathogens without concern about electricity shortages.MethodsDried stool samples of children under five years treated at the Bandim Health Centre in Bissau, Guinea-Bissau were screened by qPCR for nine enteric bacteria, five viruses, and four parasites. The findings of children having and not having diarrhoea were compared in age groups 0–11 and 12–59 months.ResultsOf the 429 children– 228 with and 201 without diarrhoea– 96.9% and 93.5% had bacterial, 62.7% and 44.3% viral, and 52.6% and 48.3% parasitic pathogen findings, respectively. Enteroaggregarive Escherichia coli (EAEC; 60.5% versus 66.7%), enteropathogenic E. coli (EPEC; 61.4% versus 62.7%), Campylobacter (53.2% versus 51.8%), and enterotoxigenic E. coli (ETEC; 54.4% versus 44.3%) were the most common bacterial pathogens. Diarrhoea was associated with enteroinvasive E. coli (EIEC)/Shigella (63.3%), ETEC (54.4%), astrovirus (75.0%), norovirus GII (72.6%) and Cryptosporidium (71.2%). The only pathogen associated with severe diarrhoea was EIEC/Shigella (p<0.001). EAEC was found more frequent among the infants, and EIEC/Shigella, Giardia duodenalis and Dientamoeba fragilis among the older children.ConclusionsStool pathogens proved common among all the children regardless of them having diarrhoea or not.     

Diarrheagenic Escherichia coli Carrying Supplementary Virulence Genes Are an Important Cause of Moderate to Severe Diarrhoeal Disease in Mexico

Diarrheagenic Escherichia coli (DEC) cause acute and persistent diarrhoea worldwide, but little is known about their epidemiology in Mexico. We determined the prevalence of bacterial enteropathogens in 831 children with acute diarrhoea over a four-year period in Yucatan, Mexico. Six DEC supplementary virulence genes (SVG), mainly associated with enteroaggregative E. coli (EAEC), were sought in 3100 E. coli isolates. DEC was the most common bacterial enteropathogen (28%), surpassing Salmonella (12%) and Shigella (9%). Predominant DEC groups were diffusely adherent E. coli (DAEC) (35%), EAEC (24%), and enteropathogenic E. coli (EPEC) (19%). Among children with DEC infections, 14% had severe illness mainly caused by EPEC (26%) and DAEC (18%); 30% had moderate diarrhoea mainly caused by DAEC (36%), mixed DEC infections (33%) and EAEC (32%). DAEC was most prevalent during spring, while ETEC, EAEC and EPEC predominated in summer. EAEC was more frequent in children 6–24 months old than in those younger than 6 months of age (P = 0.008, OR = 4.2, 95% CI, 1.3–13.9). The presence of SVG dispersin, (aatA), dispersin-translocator (aatA), enteroaggregative heat-stable toxin 1 (astA), plasmid encoded toxin (pet), cytolethal distending toxin (cdt) was higher in DEC than non-DEC strains, (36% vs 26%, P <0.0001, OR = 1.5, 95% CI, 1.3–1.8). 98% of EAEC-infected children harboured strains with SVG; 85% carried the aap-aatA gene combination, and 33% of these also carried astA. 28% of both EPEC and ETEC, and 6% of DAEC patients had strains with SVG. 54% of EPEC patients carried pet-positive strains alone or in combination with astA; only this DEC group harboured cdt-positive isolates. All ETEC patients carried astA- or astA-aap-positive strains. astA and aap were the most common SVG in DAEC (3% and 2%) and non-DEC strains (21% and 13%). DEC carrying SVG are an important cause of moderate to severe bacterial diarrhoea in Mexican children.     

Community Rates of IgG4 Antibodies to Ascaris Haemoglobin Reflect Changes in Community Egg Loads Following Mass Drug Administration

BackgroundConventional diagnostic methods for human ascariasis are based on the detection of Ascaris lumbricoides eggs in stool samples. However, studies of ascariasis in pigs have shown that the prevalence and the number of eggs detected in the stool do not correlate well with exposure of the herd to the parasite. On the other hand, an ELISA test measuring antibodies to Ascaris suum haemoglobin (AsHb) has been shown to be useful for estimating transmission intensity on pig farms. In this study, we further characterized the AsHb antigen and screened samples from a population-based study conducted in an area that is endemic for Ascaris lumbricoides in Indonesia to assess changes in AsHb antibody rates and levels in humans following mass drug administration (MDA).Conclusion/SignificanceIgG4 antibody levels to AsHb appear to reflect recent exposure to Ascaris. The antibody prevalence rate may be a useful indicator for Ascaris transmission intensity in communities that can be used to assess the impact of control measures on the force of transmission.     

Diagnostic test accuracy for detecting Schistosoma japonicum and S. mekongi in humans: A systematic review and meta-analysis

BackgroundMost of national schistosomiasis elimination programmes in Asia are relying on stool examination, particularly Kato Katz stool examination technique for regular transmission monitoring. However, the Kato-Katz technique has shown low sensitivity for the detection of light-intensity infections, and therefore highly sensitive diagnostic tools are urgently required to monitor prevalence of infection in low transmission settings. The objective of this systematic review was to evaluate and synthesize the performance of diagnostic tests for detecting Schistosoma japonicum and S. mekongi infection in people living in endemic areas.Methodology/Principal findingsWe comprehensively searched these nine electronic databases and other resources until July 2019, with no language or publication limits: PubMed, EMBASE, MEDLINE, Web of Science, BIOSIS Citation Index, HTA, CINAHL PLUS, The Cochrane Library, and PsycINFO. We included original studies that assessed diagnostic performance using antibody, antigen, and molecular tests with stool examination test as a reference standard. Two reviewers independently extracted a standard set of data and assessed study quality. We estimated the pooled estimates of sensitivity and specificity for each index test. We used diagnostic odds ratio to determine the overall accuracy and hierarchical summary receiver operating characteristics (HSROC) curve to assess the index tests performance.Fifteen studies (S. japonicum [n = 13] and S. mekongi [n = 2]) testing 15,303 participants were included in the review. Five studies reported performance of enzyme-linked immunosorbent assay (ELISA), seven studies reported indirect hemagglutination assay (IHA), and four studies reported polymerase chain reaction (PCR) for detecting S. japonicum. The pooled sensitivity and specificity were 0.93 (95% CI: 0.84–0.98) and 0.40 (95% CI: 0.29–0.53) for ELISA, 0.97 (95% CI: 0.90–0.99) and 0.66 (95% CI: 0.58–0.73) for IHA, and 0.89 (95% CI: 0.71–0.96) and 0.49 (95% CI: 0.29–0.69) for PCR respectively. A global summary indicated the best performance for IHA, closely followed by ELISA. We were unable to perform meta-analysis for S. mekongi due to insufficient number of studies.Conclusions/SignificanceIHA showed the highest detection accuracy for S. japonicum. Further studies are needed to determine the suitable diagnostic methods to verify the absence of transmission of S. mekongi and also to compare detection accuracy against more sensitive reference standards such as PCR.     

Escherichia coli Expressing EAST1 Toxin Did Not Cause an Increase of cAMP or cGMP Levels in Cells, and No Diarrhea in 5-Day Old Gnotobiotic Pigs

Background

Enterotoxigenic Escherichia coli (ETEC) strains are the leading bacterial cause of diarrhea to humans and farm animals. These ETEC strains produce heat-labile toxin (LT) and/or heat-stable toxins that include type I (STa), type II (STb), and enteroaggregative heat-stable toxin 1 (EAST1). LT, STa, and STb (in pigs) are proven the virulence determinants in ETEC diarrhea. However, significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains.

Methodology/Principal Findings

In this study, we constructed E. coli strains to express EAST1 toxin as the only toxin and studied them in cell lines and five-day old gnotobiotic piglets to determine significance of EAST1 toxin. Data from in vitro studies indicated that EAST1 did not stimulate an increase of intracellular cyclic AMP or GMP levels in T-84 cells or porcine cell line IPEC-J2, nor did it enhance LT or STa toxin of ETEC strains in stimulation of cAMP or cGMP in T-84 cells. In addition, 5-day old gnotobiotic pigs challenged with E. coli strains expressing EAST1 as the only toxin did not developed diarrhea or signs of clinical disease during 72 h post-inoculation.

Conclusion/Significance

Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea.     

Real-time PCR Demonstrates High Prevalence of Schistosoma japonicum in the Philippines: Implications for Surveillance and Control

Background

The Philippines has a population of approximately 103 million people, of which 6.7 million live in schistosomiasis-endemic areas with 1.8 million people being at risk of infection with Schistosoma japonicum. Although the country-wide prevalence of schistosomiasis japonica in the Philippines is relatively low, the prevalence of schistosomiasis can be high, approaching 65% in some endemic areas. Of the currently available microscopy-based diagnostic techniques for detecting schistosome infections in the Philippines and elsewhere, most exhibit varying diagnostic performances, with the Kato-Katz (KK) method having particularly poor sensitivity for detecting low intensity infections. This suggests that the actual prevalence of schistosomiasis japonica may be much higher than previous reports have indicated.

Methodology/Principal Findings

Six barangay (villages) were selected to determine the prevalence of S. japonicum in humans in the municipality of Palapag, Northern Samar. Fecal samples were collected from 560 humans and examined by the KK method and a validated real-time PCR (qPCR) assay. A high S. japonicum prevalence (90.2%) was revealed using qPCR whereas the KK method indicated a lower prevalence (22.9%). The geometric mean eggs per gram (GMEPG) determined by the qPCR was 36.5 and 11.5 by the KK. These results, particularly those obtained by the qPCR, indicate that the prevalence of schistosomiasis in this region of the Philippines is much higher than historically reported.

Conclusions/Significance

Despite being more expensive, qPCR can complement the KK procedure, particularly for surveillance and monitoring of areas where extensive schistosomiasis control has led to low prevalence and intensity infections and where schistosomiasis elimination is on the horizon, as for example in southern China.     

Tick-borne relapsing fever Borreliosis,a major public health problem overlooked in Senegal

BackgroundTick-borne relapsing fever (TBRF) is the most common vector-borne bacterial disease in humans in West Africa. It is frequently clinically confused with malaria. Our study aims to determine, on a micro-geographic scale, the conditions for the maintenance and spread of TBRF in the Niakhar district of Senegal.Methodology/Principal findingsWe conducted clinical, entomological and animal reservoir investigations. Field surveys were carried out in order to investigate the presence of Ornithodoros sonrai vector ticks and to detect Borrelia spp. by qPCR using the 16S rRNA and glpQ genes, respectively. Micromammal trapping series were carried out inside homes and Borrelia infection was detected using brain tissue qPCR. Capillary blood samples from febrile patients were also tested for Borrelia using qPCR. More than 97% (40/41) of the villages surveyed were infested with O. sonrai ticks. The prevalence of Borrelia spp. infections in ticks was 13% (116/910), and over 73% (85/116) were positively confirmed as being Borrelia crocidurae. Borreliosis cases accounted for 12% (94/800) of episodes of fever and all age groups were infected, with children and young people between the ages of 8–14 and 22–28 being the most infected by the disease (16% and 18.4%). TBRF cases occurred in all seasons, with a peak in August. In two species of small rodents that were found to be infected (Arvicanthis niloticus, Mus musculus), the proportion of Borrelia infection was 17.5% (10/57), and the highest prevalence of infection (40.9%, 9/22) was observed in A. niloticus.Conclusion/SignificanceOur study indicates that TBRF is an endemic disease in the Niakhar district, where children and young people are the most infected. Arvicanthis niloticus and O. sonrai ticks are massively present and appear to be the main epidemiological reservoirs causing its extensive spread to humans.     

Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

IntroductionThe clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing.Material and methodsIndividual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes.ResultsThe prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients.Conclusion and recommendationOur findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.     

Prevalence of soil-transmitted helminth infections,schistosomiasis, and lymphatic filariasis before and after preventive chemotherapy initiation in the Philippines: A systematic review and meta-analysis

ObjectiveTo estimate the impact of preventive chemotherapy on the prevalence and intensity of soil-transmitted helminth (STH) infections, schistosomiasis, and lymphatic filariasis in the Philippines, using systematic review and meta-analysis.MethodsWe included reports reporting prevalence of STH infections, schistosomiasis, or lymphatic filariasis in the Philippines published until 31 March 2021. Peer-reviewed studies were identified in electronic databases. Grey literature reports by the University of the Philippines and the Department of Health were also included. Pooled infection prevalence, before and after the initiation of preventive chemotherapy, stratified by age group, was calculated using the inverse variance heterogeneity model.FindingsA total of 109 reports were included in the review and meta-analysis. Overall prevalence of moderate-heavy intensity Ascaris lumbricoides (6.6%) and Trichuris trichiura (2.7%) infection after initiation of preventive chemotherapy were significantly lower than the prevalence prior to initiation (23.6% for A. lumbricoides and 12.2% for T. trichiura). Prevalence reductions were also found in school and preschool-age children for A. lumbricoides and T. trichiura. Studies conducted after preventive chemotherapy initiation had significantly lower overall prevalence of moderate-heavy intensity schistosomiasis (3.1% vs 0.2%) and of schistosomiasis in school-age children (30.5% vs 1%). Pooled prevalence of lymphatic filariasis prior to preventive chemotherapy initiation was 3.2% across 12 provinces, while currently only two provinces still have prevalence of more than 1%. There were no published studies reporting prevalence of lymphatic filariasis after initiation of preventive chemotherapy. Heterogeneity was high with I² mostly above 90%.ConclusionThe burden of STH infections and schistosomiasis in children were significantly lower in studies conducted following the initiation of preventive chemotherapy. Eliminating morbidity and interrupting transmission, however, may require expanded control initiatives including community-wide treatment, and improved water, sanitation, and hygiene. Lymphatic filariasis burden has decreased since the implementation of preventive chemotherapy, with all but two provinces having reached the elimination of lymphatic filariasis as a public health problem.     

Evaluation of Circulating Cathodic Antigen (CCA) Urine-Tests for Diagnosis of Schistosoma mansoni Infection in Cameroon

Background

The Kato-Katz is the most common diagnostic method for Schistosoma mansoni infection. However, the day-to-day variability in host egg-excretion and its low detection sensitivity are major limits for its use in low transmission zones and after widespread chemotherapy. We evaluated the accuracy of circulating cathodic antigen (CCA) urine-assay as a diagnostic tool of S. mansoni. In comparison, a low sensitive CCA test (CCA-L) was assessed.

Methodology

The study was conducted in three settings: two foci with single S. mansoni infections (settings A and B), and one mixed S. mansoni – S. haematobium focus (setting C). Stool and urine samples were collected from school-children on three consecutive days. Triplicate Kato-Katz readings were performed per stool sample. Each urine sample was tested with one CCA and only the first urine sample was subjected to CCA-L. Urine samples were also examined for S. haematobium eggs using the filtration method and for microhaematuria using urine reagent strips. Overall, 625 children provided three stool and three urine samples.

Principal Findings

Considering nine Kato-Katz thick smears as ‘reference’ diagnostic test, the prevalence of S. mansoni was 36.2%, 71.8% and 64.0% in settings A, B and C, respectively. The prevalence of S. haematobium in setting C was 12.0%. The sensitivities of single Kato-Katz, CCA and CCA-L from the first stool or urine samples were 58%, 82% and 46% in setting A, 56.8%, 82.4% and 68.8% in setting B, and 49.0%, 87.7% and 55.5% in setting C. The respective specificities were 100%, 64.7% and 100%; 100%, 62.3% and 91.3%; and 100%, 42.5% and 92.0%. Mixed infection with S. haematobium did not influence the CCA test results for S. mansoni diagnosis.

Conclusions/Significance

Urine CCA revealed higher sensitivity than CCA-L and triplicate Kato-Katz, and produced similar prevalence as nine Kato-Katz. It seems an attractive method for S. mansoni diagnosis.