High-throughput multiplex quantitative polymerase chain reaction method for Giardia lamblia and Cryptosporidium species detection in stool samples.
ABSTRACT: Giardia lamblia and Cryptosporidium species belong to a complex group of pathogens that cause diseases hampering development and socioeconomic improvements in the developing countries. Both pathogens are recognized as significant causes of diarrhea and nutritional disorders. However, further studies are needed to clarify the role of parasitic infections, especially asymptomatic infections in malnutrition and stunting. We developed a high-throughput multiplex quantitative polymerase chain reaction (qPCR) method for G. lamblia and Cryptosporidium spp. detection in stool samples. The sensitivity and specificity of the method were ensured by analyzing confirmed positive samples acquired from diagnostics laboratories and participating in an external quality control round. Its capability to detect asymptomatic G. lamblia and Cryptosporidium spp. infections was confirmed by analyzing stool samples collected from 44 asymptomatic 6-month-old infants living in an endemic region in Malawi. Of these, five samples were found to be positive for G. lamblia and two for Cryptosporidium spp. In conclusion, the developed method is suitable for large-scale studies evaluating the occurrence of G. lamblia and Cryptosporidium spp. in endemic regions and for clinical diagnostics of these infections.
Project description:Enteric pathogens have been related to child undernutrition. Whereas there are lots of data on enteric bacterial microbiota and infections, much less is known about the incidence of prevalence of intestinal colonisation with viruses or important parasitic species. This study assessed the presence of selected viruses and parasites in stools of 469, 354, 468 Malawian children at 6, 12 and 18 months. We also assessed environmental predictors of the presence of viruses and parasites among 6-month infants. Microbial presence was documented using real-time polymerase chain reaction (PCR). Enteroviruses were identified in 68%, 80% and 81% of the stool samples at 6, 12 and 18 months children, rhinovirus in 28%, 18% and 31%, norovirus in 24%, 22% and 16%, parechovirus in 23%, 17% and 17%, rotavirus in 3%, 1% and 0.6%, Giardia lamblia in 9.6%, 23.5% and 26%, and Cryptosporidium (spp.) in 6%, 8% and 2% of the 6, 12 and 18 months stool samples. Dry season (May-October) was associated with a low infection rate of enterovirus, norovirus and Cryptosporidium (spp.). Higher father's education level, less number of person in the household and higher sanitation were associated with a low infection rate of enterovirus, norovirus and rotavirus, respectively. The results suggest that the prevalence of asymptomatic viral and parasitic infections is high among Malawian children and that the family's living conditions and seasonality influence the rate of infections.
Project description:The associations between enteric pathogenic parasites and growth in infants in São Tomé were explored using a refined anthropometric approach to recognize early growth faltering. A birth cohort study was conducted with follow-up to 24 months of age. Microscopic examination for protozoa and soil-transmitted helminths was performed. Anthropometric assessments included: z-scores for weight-for-length (WLZ), length-for-age (LAZ), weight (WAVZ) and length velocities (LAVZ), length-for-age difference (LAD), and wasting and stunting risk (?-1 SD). Generalized additive mixed effects regression models were used to explore the associations between anthropometric parameters and enteric parasitic infections and cofactors. A total of 475 infants were enrolled, and 282 completed the study. The great majority of infants were asymptomatic. Giardia lamblia was detected in 35.1% of infants in at least one stool sample, helminths in 30.4%, and Cryptosporidium spp. in 14.7%. Giardia lamblia and helminth infections were significantly associated with mean decreases of 0.10 in LAZ and 0.32 in LAD, and of 0.16 in LAZ and 0.48 in LAD, respectively. Cryptosporidium spp. infection was significantly associated with a mean decrease of 0.43 in WAVZ and 0.55 in LAVZ. The underestimated association between subclinical parasitic enteric infections and mild growth faltering in infants should be addressed in public health policies.
Project description:BACKGROUND: The relevance of Cryptosporidium infections for the burden of childhood diarrhoea in endemic settings has been shown in recent years. This study describes Cryptosporidium subtypes among symptomatic and asymptomatic children in rural Ghana to analyse subtype-specific demographic, geographical, seasonal and clinical differences in order to inform appropriate control measures in endemic areas. METHODOLOGY/PRINCIPAL FINDINGS: Stool samples were collected from 2232 children below 14 years of age presenting with and without gastrointestinal symptoms at the Agogo Presbyterian Hospital in the rural Ashanti region of Ghana between May 2007 and September 2008. Samples were screened for Cryptosporidium spp. by PCR and isolates were classified into subtypes based on sequence differences in the gp60 gene. Subtype specific frequencies for age, sex, location and season have been determined and associations with disease symptoms have been analysed within a case-control study. Cryptosporidium infections were diagnosed in 116 of 2232 (5.2%) stool samples. Subtyping of 88 isolates revealed IIcA5G3 (n = 26, 29.6%), IbA13G3 (n = 17, 19.3%) and IaA21R3 (n = 12, 13.6%) as the three most frequent subtypes of the two species C. hominis and C. parvum, known to be transmitted anthroponotically. Infections peak at early rainy season with 67.9% and 50.0% of infections during the months April, May and June for 2007 and 2008 respectively. C. hominis infection was mainly associated with diarrhoea (odds ratio [OR] = 2.4; 95% confidence interval [CI]: 1.2-4.9) whereas C. parvum infection was associated with both diarrhoea (OR = 2.6; CI: 1.2-5.8) and vomiting (OR = 3.1; 95% CI: 1.5-6.1). CONCLUSIONS/SIGNIFICANCE: Cryptosporidiosis is characterized by seasonal anthroponotic transmission of strains typically found in Sub-Saharan Africa. The infection mainly affects young infants, with vomiting and diarrhoea being one of the leading symptoms in C. parvum infection. Combining molecular typing and clinical data provides valuable information for physicians and is able to track sources of infections.
Project description:Infections by the protozoan enteroparasites Giardia duodenalis and Cryptosporidium spp are a major cause of morbidity in children attending day care facilities in developed countries. In this cross-sectional study, we aimed to estimate the occurrence and genotype frequencies of these pathogens in children attending day care centers in Majadahonda, Central Spain. To do so, single stool samples were obtained from 90 children and tested for the presence of G duodenalis and Cryptosporidium spp by conventional microscopy and immunochromatography. Positive results by these techniques were subsequently confirmed by immunofluorescence microscopy. G duodenalis-positive samples were subjected to molecular characterization studies by multilocus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of the parasite. G duodenalis assemblages were confirmed by restriction fragment length polymorphism analyses and sequencing. A socioepidemiological questionnaire was used to identify variables potentially associated with giardiasis/cryptosporidiosis in the population of children under investigation. Overall, G duodenalis and Cryptosporidium spp were detected in 15.5% and 3.3% of stool samples, respectively. Giardiasis and cryptosporidiosis were found in 3/3 and 2/3 day care centers, respectively, affecting mainly infants aged 13 to 24 months. A total of 8 G duodenalis isolates were confirmed as subassemblage BIV, all of them belonging to asymptomatic children. Attempts to genotype Cryptosporidium isolates failed. None of the variables considered could be associated with higher risk of infection with giardiasis or cryptosporidiosis. These results clearly indicate that asymptomatic infections with G duodenalis and Cryptosporidium spp are frequent in <3-year-old children in Central Spain.
Project description:Cryptosporidium is a leading cause of moderate to severe childhood diarrhea in resource-poor settings. Understanding the natural history of cryptosporidiosis and the correlates of protection are essential to develop effective and sustainable approaches to disease control and prevention.Children (N = 497) were recruited at birth in semiurban slums in Vellore, India, and followed for 3 years with twice-weekly home visits. Stool samples were collected every 2 weeks and during diarrheal episodes were tested for Cryptosporidium species by polymerase chain reaction (PCR). Serum samples obtained every 6 months were evaluated for seroconversion, defined as a 4-fold increase in immunoglobulin G directed against Cryptosporidium gp15 and/or Cp23 antigens between consecutive sera.Of 410 children completing follow-up, 397 (97%) acquired cryptosporidiosis by 3 years of age. PCR identified 1053 episodes of cryptosporidiosis, with an overall incidence of 0.86 infections per child-year by stool and serology. The median age for the first infection was 9 (interquartile range, 4-17) months, indicating early exposure. Although infections were mainly asymptomatic (693 [66%]), Cryptosporidium was identified in 9.4% of diarrheal episodes. The proportion of reinfected children was high (81%) and there was clustering of asymptomatic and symptomatic infections (P < .0001 for both). Protection against infection increased with the order of infection but was only 69% after 4 infections. Cryptosporidium hominis (73.3%) was the predominant Cryptosporidium species, and there was no species-specific protection.There is a high burden of endemic cryptosporidiosis in southern India. Clustering of infection is suggestive of host susceptibility. Multiple reinfections conferred some protection against subsequent infection.
Project description:BACKGROUND:Intestinal parasites such as Cryptosporidium spp., Giardia lamblia and Entamoeba histolytica can cause severe diarrhea, especially among children in developing countries. This study aims to determine the frequency of Cryptosporidium spp., Giardia lamblia and Entamoeba histolytica in children with diarrhea and identify risk factors for infection. METHODOLOGY:We conducted a cross-sectional study in children aged 0-168 months hospitalized with diarrhea in three regions of Mozambique, from June 2014 to January 2018. Following consent, caretakers were interviewed and a single stool specimen was collected from each child to diagnose Cryptosporidium spp., G. lamblia and E. histolytica using commercial immune-enzymatic assay (TechLab, Inc, Blacksburg, VA, USA). Anthropometric data were collected from the clinical reports. Multivariable logistic regression models were built to identify risk factors for Cryptosporidium spp. and G. lamblia infection. RESULTS:Twenty-one percent of all specimens (212/1008) presented at least one parasitic infection. Cryptosporidium spp. infection was the most common 12.0% (118/985), followed by G. lamblia 9.7% (95/983) and E. histolytica 2.0% (20/1004). Risk factors for infection by Cryptosporidium spp. were: provenience (children from Nampula province showed the highest risk, OR: 8.176; CI: 1.916-34.894; p-value < 0.01); animal contact (children with animal contact had a protective effect OR: 0.627; CI: 0.398-0.986; p-value < 0.05); underweight (children severely underweight showed a risk of 2.309; CI: 1.310-4.069; p-value < 0.05). Risk factors for infection by G. lamblia were: age (group with highest risk, 60-168 months (OR: 2.322; CI: 1.000-5.393, p-value > 0.05)); and living in a household with five or more members (OR: 2.141; CI: 1.286-3.565, p-value < 0.01). CONCLUSIONS:Parasitic infection is common among children with diarrhea. Routine testing, standard treatment, and assessment for risk exposure of children with diarrhea should be implemented at health facilities in Mozambique.
Project description:Entamoeba histolytica, Giardia lamblia, and Cryptosporidium are three of the most important diarrhea-causing parasitic protozoa. For many years, microscopic examination of stool samples has been considered to be the "gold standard" for diagnosis of E. histolytica, G. lamblia, and C. parvum infections. Recently, more specific and sensitive alternative methods (PCR, enzyme-linked immunosorbent assay, and direct fluorescent-antibody assay) have been introduced for all three of these parasitic infections. However, the incorporation in a routine diagnostic laboratory of these parasite-specific methods for diagnosis of each of the respective infections is time-consuming and increases the costs of a stool examination. Therefore, a multiplex real-time PCR assay was developed for the simultaneous detection of E. histolytica, G. lamblia, and C. parvum in stool samples. The multiplex PCR also included an internal control to determine efficiency of the PCR and detect inhibition in the sample. The assay was performed on species-specific DNA controls and a range of well-defined stool samples, and it achieved 100 percent specificity and sensitivity. The use of this assay in a diagnostic laboratory would provide sensitive and specific diagnosis of the main parasitic diarrheal infections and could improve patient management and infection control.
Project description:The parasitic causes of diarrhea have historically been identified by use of microscopy; however, the use of this technique does not allow one to distinguish between subspecies or genotypes of parasites. Our objective was to determine, by use of modern diagnostic methods, the proportion of diarrhea cases in Bangladesh attributable to Cryptosporidium hominis, Cryptosporidium parvum, Entamoeba histolytica, and Giardia lamblia assemblages A and B.A prospective case-control study was performed involving 3646 case patients (both children and adults) who presented with diarrhea to the Dhaka hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh, and 2575 control subjects with asymptomatic infection. Parasitic infection was detected by use of a stool parasite antigen test, and the parasite load and the species and/or genotypes were determined by use of polymerase chain reaction (PCR).Cryptosporidium species and E. histolytica were more prevalent in patients with acute diarrhea than in healthy control subjects, for all ages (2.1% vs. 1.4%; P = .039) and, specifically, for those 0-12 months of age (2.2% vs. 0.4%; P = .009). G. lamblia assemblage A was also more prevalent in case patients with diarrhea than in healthy control subjects (20% vs. 5%; P = .001). For case patients with diarrhea, the parasite load in feces, as measured by quantitative real-time PCR cycle threshold, was not higher that that for control subjects with asymptomatic infection. Case patients with diarrhea and cryptosporidiosis were less likely to have abdominal pain, compared with control subjects (15% vs. 37%; P = .001); case patients with amebiasis more likely to have visible blood in stool, compared with control subjects (8% vs. 1.6%; P = .001); and case patients with giardiasis more likely to be dehydrated, compared with control subjects (81% vs. 71%; P = .001).E. histolytica, C. hominis, C. parvum, and G. lamblia assemblage A infections are important causes of diarrheal illness in Bangladesh.
Project description:We collected clinical and morphologic data from children with diarrhea attending 3 diverse hospitals/clinics in Accra. Stool samples were tested for rotavirus and Cryptosporidium spp. In all, 58% of the children with diarrhea had rotavirus infections, 25% of which were of the G3 sero/genotype. The most common strains were G3P  (18.8%) and G2P  (12.5%). Cryptosporidium spp. infections were uncommon (3/143, 2.0%).
Project description:BackgroundProtozoan enteroparasites Cryptosporidium species and Giardia duodenalis are major contributors to the burden of gastrointestinal illness in children globally, whereas the stramenopile Blastocystis species has been associated with irritable bowel syndrome and skin disorders.AimTo investigate the carriage of these parasites in voluntary asymptomatic schoolchildren (4‒14 years) in 12 different primary and secondary schools in Leganés (Madrid, Spain).MethodsIn a prospective cross-sectional study, stool samples and epidemiological questionnaires on demographics and potential risk factors were collected from participating schoolchildren. Detection of enteric parasites was conducted by PCR-based methods and confirmed by sequence analysis. We calculated prevalence and odds ratios (OR) with logistic regression.ResultsStool samples and questionnaires were provided by 1,359 schoolchildren from 12 schools. The individual prevalence for any parasite was 28%; Blastocystis sp.: 13%; G. duodenalis: 18%; Cryptosporidium spp.: 1%. Two schoolchildren were infected with all three species and 53 with two species. Multivariable risk factor analysis using logistic regression models indicated that an existing infection with one parasite increased the odds for an additional infection with another parasite. The odds of Blastocystis sp. carriage increased up to the age of 10 years and being female increased the odds of Cryptosporidium spp. infection. Washing vegetables before preparing a meal was protective for Blastocystis sp. infection.ConclusionWe detected a larger than expected proportion of asymptomatic cases in the participanting schoolchildren. Further investigation of asymptomatic children should be considered. Good hygiene measures should be encouraged for individuals of all ages to protect from protozoal infections.