PCR In Stool Samples Research Articles

Detection of cytomegalovirus (CMV) by digital PCR in stool samples for the non-invasive diagnosis of CMV gastroenteritis

BackgroundCMV gastroenteritis is common in patients receiving allogeneic hematopoietic stem cell transplantation and it is difficult to distinguish from acute graft-versus-host disease (aGvHD), which has very similar symptoms but needs quite different treatment. CMV gastroenteritis is caused by local infection or reactivation of CMV in the gastrointestinal tract while aGvHD is due to immune rejection. The gold standard of diagnosis of CMV gastroenteritis and aGvHD is gastrointestinal biopsy under endoscopy, which is invasive and can potentially lead to severe side effects. Stool samples testing with quantitative polymerase chain reaction (qPCR) may be an alternative, while the application in trace level measurements and precision are not all satisfactory enough in reported research.MethodsIn this study, we designed a novel method that extracted the cell free DNA (cfDNA) from the fecal supernatant to perform digital PCR (dPCR) for the detection of CMV, analyzed the performance and compared it with the total DNA extracted by the current procedure.ResultsTwenty-two paired stool samples using two DNA extraction methods proved that the cfDNA extraction method had markedly higher DNA concentrations and control gene copy number, suggesting that cfDNA may be more informative and more useful for the detection of CMV DNA segment. The dPCR approach in detecting CMV DNA segment also exhibit good linearity (R2 = 0.997) and higher sensitivity (limit of detection at 50% was 3.534 copies/μL). Eighty-two stool samples from 44 immunocompromised patients were analyzed, CMV-positive rate was 28%, indicating that more than one-quarter of the gastrointestinal symptoms within these patients may be caused by CMV infection or reactivation.ConclusionThe combined results suggest that detection of CMV by dPCR in cfDNA of stool supernatant is a powerful method to identify CMV gastroenteritis and helps in clinical treatment decision making.

Should Dientamoeba fragillis be looked for in pediatric digestive pathology of an unknown cause? A proposed pilot case–control study

IntroductionDientamoeba (D.) fragilis is a common intestinal protozoan with an unresolved clinical significance. The association between D. fragilis and the etiology of gastrointestinal symptoms in children is unclear. Metronidazole is often used for treatment. The aims of this study are to clarify the clinical relevance of D. fragilis in children with gastrointestinal symptoms, and to determine the clinical and microbiological efficacy of metronidazole in D. fragilis-infected children with gastrointestinal complaints. MethodsA prospective case–control study was performed from October 2017 to February 2019. A total of 106 individuals aged 1–17 were included. Out of the 106; 59 showed gastrointestinal symptoms (case group), and 47 were without gastrointestinal symptoms (control group). We excluded 2 patients from the case group. D. fragilis was diagnosed by real-time PCR in stool samples. A 10-day course of oral Metronidazole was prescribed in D. fragilis positive children with GI symptoms. Clinical data before and after the treatment as well as peripheral eosinophilia in previous blood samples, were recorded. ResultsOf the 104 participants, D. fragilis was found in 17 (29.8%) children from the case group, whereas in the control group the parasite was detected in 11 patients (23.4%) with an odds ratio (OR) of 1.39 (IC 95% 0.53–3.75, p=0.46). The most prevalent clinical manifestation was abdominal pain (46/57, 80.7%). Seventeen cases with a positive PCR received anti-parasitic treatment according to the established protocol, although during the collection period we received only 11 stool samples to perform the post-treatment follow-up. The PCR of the D. fragilis remained positive in 3 patients (3/11, 27.27%). Despite achieving the eradication of the parasite, 4/8 patients (50%) continued with digestive symptoms. ConclusionsAccording to our study there were no differences between the D. fragilis infection in children with or without gastrointestinal symptoms. No relation was found between the clinical and microbiological responses after said D. fragilis treatment. Therefore, we conclude that it is not justified to look specifically for D fragilis in pediatric patients with abdominal symptoms.

Should Dientamoeba fragillis be looked for in pediatric digestive pathology of an unknown cause? A proposed pilot case–control study

IntroductionDientamoeba (D.) fragilis is a common intestinal protozoan with an unresolved clinical significance. The association between D. fragilis and the etiology of gastrointestinal symptoms in children is unclear. Metronidazole is often used for treatment. The aims of this study are to clarify the clinical relevance of D. fragilis in children with gastrointestinal symptoms, and to determine the clinical and microbiological efficacy of metronidazole in D. fragilis-infected children with gastrointestinal complaints. MethodsA prospective case–control study was performed from October 2017 to February 2019. A total of 106 individuals aged 1–17 were included. Out of the 106; 59 showed gastrointestinal symptoms (case group), and 47 were without gastrointestinal symptoms (control group). We excluded 2 patients from the case group. D. fragilis was diagnosed by real-time PCR in stool samples. A 10-day course of oral Metronidazole was prescribed in D. fragilis positive children with GI symptoms. Clinical data before and after the treatment as well as peripheral eosinophilia in previous blood samples, were recorded. ResultsOf the 104 participants, D. fragilis was found in 17 (29.8%) children from the case group, whereas in the control group the parasite was detected in 11 patients (23.4%) with an odds ratio (OR) of 1.39 (IC 95% 0.53–3.75, p=0.46). The most prevalent clinical manifestation was abdominal pain (46/57, 80.7%). Seventeen cases with a positive PCR received anti-parasitic treatment according to the established protocol, although during the collection period we received only 11 stool samples to perform the post-treatment follow-up. The PCR of the D. fragilis remained positive in 3 patients (3/11, 27.27%). Despite achieving the eradication of the parasite, 4/8 patients (50%) continued with digestive symptoms. ConclusionsAccording to our study there were no differences between the D. fragilis infection in children with or without gastrointestinal symptoms. No relation was found between the clinical and microbiological responses after said D. fragilis treatment. Therefore, we conclude that it is not justified to look specifically for D fragilis in pediatric patients with abdominal symptoms.

Effect of a Probiotic Mixture in Captive Cheetahs (Acinonyx Jubatus) with Gastrointestinal Symptoms-A Pilot Study.

Simple SummaryIn recent years, many studies have demonstrated the effectiveness of probiotics in acute and chronic gastrointestinal disorders in both humans and animals. The objective of this study is to evaluate the effect of a specific probiotic mixture in cheetahs. In the wild, cheetah populations have drastically reduced due to habitat destruction, human–wildlife conflict and illegal wildlife trade. In captivity, chronic gastrointestinal diseases have a high prevalence. Based on our results, it can be concluded that probiotics may be helpful as a dietary supplement in cheetahs suffering from gastrointestinal disease.Cheetahs (Acinonyx jubatus) are classified as “vulnerable” species due to the low numbers persisting in the wild. Gastrointestinal diseases are very common in this species when they are kept in captivity, in particular gastritis. Clinical signs are predominantly characterized by vomiting, diarrhea, weight loss and anorexia. In this study, we evaluated the efficacy of a multi-strain probiotic in two groups of cheetahs: Group A (n = 4)—rescued cheetahs housed at the Cheetah Conservation Fund (Otjiwarongo, Namibia); Group B (n = 9)—captive cheetahs housed in Italian zoos. Animals showed gastrointestinal signs of different severity, and were positive for Helicobacter spp., detected by PCR in stool samples. Two sachets of probiotic formulation were administered to all cheetahs once a day for 21 consecutive days. Clinical conditions (appetite loss, vomiting, stool consistency and Body Condition Score) before (T0) and after 21 days of probiotic administration (T1) were then compared using a simplified Feline Chronic Enteropathy Activity Index (FCEAI) score. A slight but not significant improvement in the scores was observed in Group A, which had mild intestinal symptoms, while a significant decrease in vomiting and stool consistency (**p < 0.01) scores was observed in Group B, which had more pronounced symptoms. Results suggest that high concentrations of live probiotics can be of help in managing gastrointestinal signs in cheetahs.

Use of the PCR in a Combined Methodological Approach for the Study of Human Fascioliasis in an Endemic Area.

Fascioliasis is a worldwide distributed trematodiasis considered a neglected disease. Diagnosis in humans has been traditionally based on parasitological and immunological techniques. Recently we reported the use of the PCR in stool samples for the individual diagnosis. The purpose of this study was to evaluate human fascioliasis by a combination of diagnostic methods in an area where the disease is highly endemic in animals. We studied all the inhabitants (N = 240) of Tatón village, Argentina, by Fasciola hepatica rproCL1-ELISA. Among them, we continued the study with 13 cases that had at least two positive serological tests, who performed a questionnaire, physical examination, abdominal ultrasonography, and collection of blood and faeces. Blood/serum samples were used for Fh rproCL1-ELISA and liver function tests. Faeces were used for parasitological analysis and PCR of a repetitive fragment of Fasciola sp. Among the 13 patients, 9 presented symptoms of biliary colic. All patients repeated positive serology. F. hepatica eggs were not detected. PCR was positive in 11 cases. This is the first report employing an approach based on the combination of methods for the evaluation of human fascioliasis in an endemic area, which includes molecular tools with a high value in detecting low infections.

Comparison of commercial and in-house real-time PCR platforms for 15 parasites and microsporidia in human stool samples without a gold standard

IntroductionA test comparison of in-house and commercial real-time PCR (qPCR) kits for the detection of human parasites and microsporidia in stool samples was conducted without a gold standard. Three different commercial kits were included in the comparison, with a range of 3–15 different PCR targets, while 14 targets were covered by in-house testing, so not all 16 target pathogens were covered by all assays. MethodsResidual materials from nucleic acid extractions of stool samples with very high likelihood of being colonized or infected by at least one enteric parasite species or microsporidia were tested. In all, 500 DNA samples were analyzed, but due to limited sample volume, only 250 of the 500 samples were tested per assay. Each sample was assessed with the qPCR platforms being compared and cycle threshold (Ct) values were included in a descriptive comparison. ResultsDepending on the assay applied, qPCR detected per 250 tested samples Giardia duodenalis (184–205), Blastocystis spp. (174–183), Trichuris trichiura (118–120), Ascaris lumbricoides (79–96), Necator americanus (78–106), Hymenolepis nana (40–42), Cryptosporidium spp. (27–36), Dientamoeba fragilis (26–28), Schistosoma spp. (13–23), Enterobius vermicularis (8–14), Entamoeba histolytica (7–16), Strongyloides stercoralis (6–38), Cyclospora spp. (6–13), Taenia spp. (1–4), microsporidia (1–5), and Ancylostoma spp. (1–2). Inter-assay agreement kappa was almost perfect (0.81–1) for Dientamoeba fragilis, Hymenolepis nana, Cryptosporidium spp., and Ascaris lumbricoides, substantial (0.61–0.8) for Necator americanus, Blastocystis spp., Ancylostoma spp., Giardia duodenalis, Schistosoma spp., Trichuris trichiura, and Enterobius vermicularis, moderate (0.41–0.6) for Entamoeba histolytica, fair (0.21–0.4) for microsporidia, slight (0–0.2) for Cyclospora spp. and Strongyloides stercoralis, and poor (<0) for Taenia spp. ConclusionsVarying inter-assay agreement makes interpretation of microsporidia and parasite PCR in stool samples challenging. Intra-assay agreement had been controlled during the developing of the assays. Future studies, e.g., with optimized nucleic acid procedures and including microscopically characterized samples, are advisable.

Molecular Study of Norovirus in Pediatric Patients with Gastroenteritis

Aim:The aim of the present study was to detect the prevalence of norovirus and genotypes determination by real-time PCR among children below 18 years as an etiology of acute gastroenteritis and to compare rapid detection of norovirus by Enzyme-Linked Immunoassay (ELISA) to virus detection by real-time PCR.Methods:The research was a cross-sectional study conducted on children below 18 years complaining of community-acquired acute gastroenteritis. A stool sample was subjected to direct-antigen detection by ELISA for norovirus and molecular study by real-time polymerase chain reaction.Results:The study included 200 children with acute gastroenteritis with a mean age of 6.7±3.8 years. Norovirus antigen was detected by EIA in 34.5% and by real-time PCR in 30.5% of studied children with genotype GII, the predominant detected genotype (80.97%). Both real-time PCR and antigen detection of norovirus were positive in 43 (70.5%) of the children and negative in 113(81.3%) of the studied children. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy for antigen detection by ELISA were 70.5%, 81.3%, 62.3%, 86.3% and 78%, respectively. Comparison between patients positive for norovirus and those negative for norovirus by real-time PCR revealed non-significant difference as regards age, sex, the season of occurrence and residence.Conclusion:The present study highlights that norovirus prevalence is common among pediatric patients with gastroenteritis above 5 years with GII genotype as the prevalent genotype. There was a significant correlation between positive and negative results of antigen detection of norovirus by ELISA and detection of RNA of norovirus by real-time PCR in stool samples. However, the screening for norovirus by ELISA has limited sensitivity and needs to be associated with a molecular method for accurate diagnosis of sporadic cases of gastroenteritis.

Determinants of Campylobacter infection and association with growth and enteric inflammation in children under 2 years of age in low-resource settings

Campylobacter species infections have been associated with malnutrition and intestinal inflammation among children in low-resource settings. However, it remains unclear whether that association is specific to Campylobacter jejuni/coli. The aim of this study was to assess the association between both all Campylobacter species infections and Campylobacter jejuni/coli infections on growth and enteric inflammation in children aged 1–24 months. We analyzed data from 1715 children followed from birth until 24 months of age in the MAL-ED birth cohort study, including detection of Campylobacter species by enzyme immunoassay and Campylobacter jejuni/coli by quantitative PCR in stool samples. Myeloperoxidase (MPO) concentration in stool, used as a quantitative index of enteric inflammation, was measured. The incidence rate per 100 child-months of infections with Campylobacter jejuni/coli and Campylobacter species during 1–24 month follow up were 17.7 and 29.6 respectively. Female sex of child, shorter duration of exclusive breastfeeding, lower maternal age, mother having less than 3 living children, maternal educational level of <6 years, lack of routine treatment of drinking water, and unimproved sanitation were associated with Campylobacter jejuni/coli infection. The cumulative burden of both Campylobacter jejuni/coli infections and Campylobacter species were associated with poor growth and increased intestinal inflammation.

Initial Reverse Transcription is Essential for Reliable Helicobacter pylori 23S rDNA Real-Time PCR Diagnostics from Fecal Samples.

A sensitive, non-invasive molecular test for identifying Helicobacter pylori (H. pylori) in stool samples is important in the context of a "test-and-treat strategy". The goal of this study was to elucidate the benefit of an initial reverse transcription (RT) step for the sensitivity of H. pylori 23S rDNA Real-time PCR in stool samples. We compared a LightMix® Modular Helicobacter 23S rDNA PCR assay without (LightMix® PCR) and with an initial reverse transcription step (LightMix® RT-PCR) assay for rapid detection of Helicobacter pylori in fecal samples. For maximum sensitivity, all Lightmix® RT-PCR assays were performed in triplicate for each sample. Additionally, the LightMix® RT-PCR was compared to H. pylori AG ELISA. Direct detection of H. pylori in feces by Lightmix® PCR was less sensitive than LightMix® RT-PCR. Only 2 out of 44 stool samples (4.5%) from patients with diarrhea were positive for H. pylori in the LightMix® PCR assay. In contrast, a reverse transcription step prior Lightmix® PCR increased the assay sensitivity markedly (19 out of 44 positives, 43.2%). When re-testing 65 samples initially analyzed with H. pylori AG ELISA with LightMix® RT-PCR, the detection rate for H. pylori was similarly increased from 23.1% (15/65) with H. pylori AG ELISA to 43.1% (28/65) with H. pylori LightMix® RT-PCR. 21.5% of the 28 H. pylori positive samples were positive in 1/3, 32% in 2/3, and 46.5% in 3/3 triple RT-PCR approaches. When re-testing the 28 LightMix® RT-PCR positive samples by LightMix® PCR, only 17.9% (5/28) RT-PCR positive samples were positive in PCR. LightMix® RT-PCR is much more sensitive than Lightmix® PCR. In comparison to H. pylori AG ELISA, the LightMix® RT-PCR shows a markedly higher sensitivity. An initial reverse transcription step is crucial for reliable Helicobacter pylori 23S rDNA Real-time PCR diagnostics. The triple RT-PCR approach can additionally improve the detection of H. pylori in fecal samples. Thus, the method presented provides a highly sensitive, noninvasive assay to detect H. pylori in fecal samples with potential advantages compared to other H. pylori detection methods currently used in routine diagnostics.

Clinical and biological features of enteroviral meningitis among adults and children and factors associated with severity and length of stay

BackgroundEnterovirus (EV) meningitis is the most common form of meningitis. Clinical and biological manifestations may be non-specific, leading to prolonged and costly investigations. ObjectivesTo determine the different aspects of EV meningitis and the variables associated with length of stay (LOS) in hospital independently of patients’ age. Study designSingle center retrospective study of all EV PCR positive CSF samples during 3.5 years in Bordeaux University Hospital, France. Results172 patients were included. 65 were under 3 years old and 49 over 18 years old. 10% of patients had severe forms of the disease. 47 patients (27.3%) had normal CSF count and in 63 patients (36.6%) polynuclear cells predominated in CSF. Procalcitonin, Hoens’ score or PCR in stool samples appeared as good markers for enteroviral diagnosis. Time elapsed before PCR results was associated with LOS (p = .002) and should help in limiting investigations in case of aseptic meningitis. ConclusionRapid availability of EV PCR reduces LOS for patients and contributes to diminish unnecessary procedures and further tests.

Tu1239 - The Application Value of CMV DNA PCR in Stool Samples from Patients with Ulcerative Colitis

Tu1239 - The Application Value of CMV DNA PCR in Stool Samples from Patients with Ulcerative Colitis

P162 The value of CMV DNA PCR in stool samples from IBD patients

P162 The value of CMV DNA PCR in stool samples from IBD patients

Simultaneous development of Kawasaki disease following acute human adenovirus infection in monozygotic twins: A case report

BackgroundThe etiology of Kawasaki disease (KD) remains unknown. However, many studies have suggested that specific genetic factors and/or some infectious agents underlie the onset of KD. Previous studies have suggested that human adenovirus (HAdV) is one of the triggering pathogens of KD. Here, we report monozygotic twin boys who sequentially developed KD in conjunction with acute HAdV type 3 (HAdV-3) infection.Case presentationThe patients were four-year-old monozygotic twin boys. The elder brother developed a high fever and was diagnosed with HAdV infection with an immunochromatographic kit for HAdV (IC-kit). He was transferred to our institute after persistent fever for 7 days. On admission, he already fulfilled all the diagnostic criteria for KD. His laboratory data were as follows: WBC, 9700/μl; CRP, 2.42 mg/dl; IFN-γ, 99.8 pg/ml; and TNF-α, 10.9 pg/ml. He received intravenous immunoglobulin (IVIG) and aspirin and responded well, with no coronary artery abnormalities. The younger brother, who was also IC-kit-positive, was hospitalized on the same day as his elder brother after persistent fever for 3 days. His data on admission were as follows: WBC, 12,600/μl; CRP, 5.54 mg/dl; IFN-γ, 105.0 pg/ml; and TNF-α, 33.6 pg/ml. Although he developed all of the typical KD symptoms by day 4, his fever subsided spontaneously on day 6 without IVIG or aspirin. However, he developed a dilation of the coronary artery in the region of the left circumflex artery bifurcation on day 10. His coronary artery dilation had resolved 3 months after onset. HAdV-3 DNA was detected with PCR in stool samples from both patients, and HAdV3 was isolated from the younger brother’s stool sample. Serum neutralizing antibodies to AdV3 were also significantly elevated in both patients, suggesting seroconversion.ConclusionsThere have been few reports of the simultaneous development of KD in monozygotic twins. Notably, both twins had an acute HAdV-3 infection immediately before they developed KD. These cases strongly suggest that KD was triggered by HAdV-3 infection, and they indicate that specific immune responses to some pathogens (such as HAdV-3), arising from genetic susceptibility, play a critical role in the pathogenesis of KD.

A molecular Approach for Evaluation of Experimental Trials of Anti Schistosomal Vaccination in Murine Models

Background and study aim: Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believed that the best long term strategy to control schistosomiasis is through immunization with anti-schistosomiasis vaccines. This study aims at assessment of the efficacy of different potential anti-schistosomal vaccines (as crude soluble egg antigens (SEA), soluble worm antigen preparation (SWAP) and combined SEA & SWAP) by parasitological and molecular studies in experimental murine models. Materials and Methods: Sixty male laboratory bred Swiss Albino mice were used and divided into six groups; control normal (G1), control infected by ± 80 cercariae by S.C. route (G2), Freund’s adjuvant (adj.) received then infected (G3), SEA+adj. received then infected (G4), SWAP+ adj. received then infected (G5) and combined (SEA+SWAP) + adj. received then infected (G6). A schedule of sensitization, immunization and schistosomiasis challenge were followed and performed on different mice groups. Mice were euthanized 10 weeks post-infection. Potential vaccine efficacy was investigated by parasitological and molecular studies including egg count/gram stool using modified Kato thick smear,liver egg load, oogram pattern in the liver and stool PCR to detect S. mansoni egg DNA in stools of studied mice. Results: The combined (SEA+SWAP) vaccine caused the highest significant reduction in the fecal egg count followed by SWAP then SEA antigens. On the other hand, the highest percentage reduction in eggs/gram liver tissue was attributed to the combined (SEA+SWAP) followed by SEA then SWAP antigens. Regarding oogram results, the combined (SEA+ SWAP) antigens were more efficient in increasing the number of dead ova with highly significant reduction in the number of mature & immature ova, followed by SEA then SWAP antigens. The lowest percentage of S. mansoni egg DNA detected by PCR in stool samples was encountered with the combined (SEA+ SWAP), followed by SEA then SWAP antigens. Conclusion:The parasitological and PCR-based assessment studies denoted that the combined (SEA+SWAP) vaccine candidate was the most effective in protection against schistosomiasis challenge. The results of parasitological and molecular studies were nearly similar but the molecular study was more sensitive, definite and accurate.

FOOD SENSITIZATION IN CHILDREN WITH OPISTHORCHIS FELINEUS INVASION

Aim: to determine the mechanism of food sensitization in children with chronic Opisthorchis felineus invasion. Patients and methods: during the epidemiological study the groups of patients (7–10 years) with chronic opisthorchiasis (n =237) and children without chronic opisthorchiasis (n =496) were formed. The investigation included interviewing of parents / guardians, measurement of total IgE, specific IgE to food allergens and component-resolved diagnostic, real-time PCR in stool samples. Results: the chronic opisthorchiasis invasion in children is associated with reduced risk of food sensitization, compared with non-infected group (9,7 vs 16,94%, OR =0,53; 95% CI 0,31–0,88; p =0,01). Opisthorchiasis of high intensity negatively correlated with increased level of specific IgE to food allergens in serum (OR =0,46; 95% CI 0,24–0,91; p =0,023). The association between level of total IgE and intensity of opisthorchiasis combinated with food sensitization was found. The sensitization to allergens class I (cyp c1, cor a11, gal d2, pru p3) is more prevalent in patients with opisthorchiasis, in uninfected children the sensitization to bet v1-homologues (mal d1, pru p1, cor a1, ara h8) is most common. Conclusions: the epidemiological data on the effect of the Opisthorchis felineus invasion on mechanisms of food sensitization in children were obtained.

Outbreak of hepatitis A in an orphanage in Pune city, India

Aims: An outbreak of hepatitis was reported in an orphanage, located in the Pune city, India. Investigations were carried out to identify the etiological agent, source of infection and subclinical infections Study design: Descriptive study. Place and Duration of Study: An orphanage, located in the Pune city, India. Study was carried out in August - September 2014. Methodology: The children in the orphanage were investigated for hepatitis. Clinical, epidemiological findings were recorded and sanitary survey was done. Sera collected from children were tested for anti HAV IgM antibodies, anti HEV IgM antibodies, HBsAg, and serum alanine amino transferase levels. Stools were processed for HAV RNA detection, sequencing and phylogenetic analysis. Results: Fourteen suspected jaundice cases were line listed (boys:7, girls:7). Overall clinical attack rate was 15.1% (14/93). Among all 93 resident children, blood samples 83.9% (78/93) and stool samples 67.7% (78/93) could be investigated. Presence of anti HAV IgM antibodies were detected in 20 children (14 symptomatic and 6 asymptomatic). Presence of HAV RNA detected in 16 stool samples and all HAV RNA sequences clustered with genotype IIIA. Potential source of the infection was probably food served to children. Conclusion: An outbreak of HAV in children was confirmed by serology and virus detection by PCR in stool samples. It would be advisable to vaccinate all children above one year of age in an orphanage set ups.

Clinical relevance of positive human parechovirus type 1 and 3 PCR in stool samples

Human parechoviruses (HPeV) cause symptoms ranging from severe neonatal infections to mild gastrointestinal and respiratory disease. Use of PCR and genotyping has markedly improved the detection rate of HPeV but has simultaneously raised questions about the clinical relevance of positive tests. This retrospective study correlates positive HPeV1 or HPeV3 PCR tests in stools from children with their symptoms to determine clinical relevance. Children with HPeV1- or HPeV3-positive stool samples, as detected by real time RT-PCR and direct genotyping, between 2004 and 2008 were selected. Clinical data were retrospectively collected from the patient’s files and results were compared. One hundred and thirty-eight children with positive HPeV1 (n = 112) or HPeV3 (n = 26) stool samples were identified. Significantly more HPeV3-infected children were neonates or infants younger than 6 months of age. Meningitis or sepsis-like illnesses were diagnosed most frequently and were found in significantly younger children. Almost half of HPeV1-infected children had an underlying disease. Mild gastrointestinal disease was seen most frequently in these children. There was no clear correlation between viral load (Ct value) and severity of symptoms. In conclusion, HPeV3 detected by PCR in stool samples is associated with clinically relevant disease. For HPeV1, a positive stool sample is mainly associated with symptoms in children with underlying disease.

Epidemiological investigation of two parallel gastroenteritis outbreaks in school settings

BackgroundTwo parallel gastroenteritis outbreaks occurred in an elementary school and a neighboring kindergarten in Kilkis, Northern Greece in 2012. The aim of the study was the investigation of these two parallel outbreaks as well as their possible source.MethodsTwo retrospective cohort studies were performed to identify the mode and the vehicle of transmission as well as the possible connection between them.ResultsElementary school and kindergarten populations of 79.9% (119/149) and 51.1% (23/45) respectively, participated in the study. Case definition was satisfied by 65 pupils from the elementary school and 14 from the kindergarten. For elementary school, 53 cases were considered primary cases of the outbreak and were included in the analysis. Based on the results of the multivariate analysis, consumption of tap water was the only statistically significant independent risk factor of gastroenteritis (RR = 2.34, 95% C.I.: 1.55-3.53).; a finding supported by the shape of the epidemic curve which referred to a common point source outbreak with secondary cases. For kindergarten, no statistically significant risk factor was identified, and the epidemic curve supported a person-to-person transmission according univariate analysis. Norovirus GI and GII and human Adenovirus were detected by Real Time PCR in stool samples from seven children of elementary school, but stool samples were not collected by children of the kindergarten.ConclusionsEven though the etiological agent of the outbreak was not verified, combined epidemiological and laboratory results were in favor of a waterborne viral gastroenteritis outbreak at the elementary school, followed by a person to person spread at the kindergarten.

Parasitic infections and immune function: Effect of helminth infections in a malaria endemic area

According to the hygiene hypothesis, reduced exposure to infections could explain the rise of atopic diseases in high-income countries. Helminths are hypothesised to alter the host's immune response in order to avoid elimination and, as a consequence, also reduce the host responsiveness to potential allergens. To elucidate the effect of current helminth infections on immune responsiveness in humans, we measured cytokine production in a rural Ghanaian population in an area with multiple endemic parasites including malaria, intestinal helminths and protozoa. Multiplex real-time PCR in stool samples was used for the detection of four gastrointestinal helminths, of which only Necator americanus was commonly present. A similar assay was used to test for Giardia lamblia in stool samples and malaria infection in venous blood samples. Levels of the cytokines interleukin (IL)-10, tumour necrosis factor (TNF)-α, IL-17, IL-6, IL-13, and interferon (IFN)-γ were determined in whole-blood samples ex vivo-stimulated either with lipopolysaccharide (LPS) and zymosan (for innate cytokine production) or the T-cell mitogen phytohaemagglutinin (PHA). There were no significant differences in either innate or PHA-stimulated cytokine production dependent on current N. americanus infection. Plasmodium falciparum malarial infection was associated with a pro-inflammatory response indicated by increased innate production of TNF-α, IL-17 and IL-6. There was no clear pattern in cytokine responses dependent on G. lamblia-infection. In conclusion, in this rural Ghanaian population current N. americanus infections are not associated with altered immune function, while infection with P. falciparum is associated with pro-inflammatory innate immune responses.

High Prevalence of Human Parechovirus (HPeV) Genotypes in the Amsterdam Region and Identification of Specific HPeV Variants by Direct Genotyping of Stool Samples

Human parechoviruses (HPeV) are widespread pathogens belonging to the Picornavirus family. Six genotypes, which have predominantly been isolated from children, are known. Data on prevalence of HPeV genotypes are generally based on cell culture, which may underestimate the prevalence of certain HPeV strains that are difficult to grow. We studied 1,824 stool samples from 1,379 children (<5 years old) sent to the Academic Medical Center, Amsterdam, The Netherlands, between 2004 and 2006. Samples were screened using specific human enterovirus (HEV) and HPeV real-time PCRs based on the 5' untranslated region. A high percentage of HPeV infections (16.3%), comparable to the percentage of HEV infections (18.4%), were found by PCR in stool samples. HPeV-positive stool samples were directly genotyped based on the VP1 region for the first time to avoid a culture bias. HPeV1 was found to be the most prevalent type. The majority of the HPeV1 strains clustered separately from the prototype strain, Harris, which has not been reported to circulate lately. However, we could identify three strains as HPeV1 Harris. HPeV3 was identified as the second most predominant type during 2004 and 2006 but was not found in 2005. HPeV4 to -6 were found in smaller numbers. One strain could not be associated with a known HPeV type (VP1 gene nucleotide similarity: 71%), possibly indicating a new genotype. Also, we report the first identification of three HPeV5 strains and one HPeV1 strain with a different motif at the C-terminal end of VP1, where the arginine-glycine-aspartic acid (RGD) motif is normally located.