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Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for the detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive by specific real-time PCR, of which 21 were positive by the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). In conclusion, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotypes was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting nonviral causes of IK, including Acanthamoeba.

Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses., Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for the detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive by specific real-time PCR, of which 21 were positive by the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). In conclusion, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotypes was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting nonviral causes of IK, including Acanthamoeba.

Blastocystis is a genus of common single-celled intestinal parasitic protists with an unsettled role in human health and disease. Being a stable component of intestinal microbiota, once established, the Blastocystis parasite appears more common in healthy individuals than in patients with infectious, functional, or inflammatory bowel disease. Recent data suggest that the parasite is associated with certain gut microbiota profiles and health indices. Convincing data and tools differentiating asymptomatic colonization from infection are yet to be demonstrated. Although the parasite may elicit disease under certain circumstances, the focus on Blastocystis may be shifting from a clinical to a public health perspective.

Entamoeba polecki is a parasite of human and nonhuman primates, other mammals, and birds. Due to overlapping morphological features, cysts of E. polecki may be confused with those of other Entamoeba species commonly found in human fecal samples, including immature cysts of Entamoeba histolytica . Although the presence of E. polecki in human Entamoeba -positive stool samples may be rare, its prevalence is likely underestimated due to such confusion. Here, we give examples of diagnostic approaches applied so far and summarize data on the molecular epidemiology of E. polecki , including host specificity and phylogeography. Moreover, we evaluate a novel diagnostic conventional PCR developed for the screening of fecal samples for E. polecki . The assay was highly sensitive and specific when used on genomic DNA extracted directly from stool and Swedish wastewater samples. The PCR enabled the identification of all four subtypes (ST1 to ST4) of E. polecki by PCR product sequencing. Most (23/28) subtyped E. polecki- positive samples detected in patients in Sweden between 2002 and 2015 reflected colonization by ST4 and were seen in travelers/foreigners. Two and three human cases of ST2 and ST3, respectively, were also detected. Subtypes 1, 2, and 3 were detected in 3/21 wastewater samples, suggesting local endemicity of these E. polecki subtypes; interestingly, ST4 was not detected in wastewater. In conclusion, the current PCR assay enables simple and cost-effective screening of fecal and wastewater samples for E. polecki . Human cases of E. polecki appear to involve primarily ST4, while E. polecki detected in wastewater may be primarily of animal origin.

Species of Sarcocystis are Apicomplexan parasites requiring intermediate and definitive hosts to complete their life cycle. Humans are one of many natural host species and may serve as both intermediate and definitive hosts. However, the extent and public health significance of human Sarcocystis infection are incompletely known. In this minireview, we provide an update on the epidemiology and diagnosis of human sarcocystosis and propose some tools that could contribute to a better understanding of the clinical significance and epidemiology of Sarcocystis infections.

This study describes the prevalence and genotype distribution of Pneumocystis jirovecii obtained from nasopharyngeal (NP) swabs from immunocompetent Cuban infants and toddlers with whooping cough (WC). A total of 163 NP swabs from 163 young Cuban children with WC who were admitted to the respiratory care units at two pediatric centers were studied. The prevalence of the organism was determined by a quantitative PCR (qPCR) assay targeting the P. jirovecii mitochondrial large subunit (mtLSU) rRNA gene. Genotypes were identified by direct sequencing of mtLSU ribosomal DNA (rDNA) and restriction fragment length polymorphism (RFLP) analysis of the dihydropteroate synthase (DHPS) gene amplicons. qPCR detected P. jirovecii DNA in 48/163 (29.4%) samples. mtLSU rDNA sequence analysis revealed the presence of three different genotypes in the population. Genotype 2 was most common (48%), followed in prevalence by genotypes 1 (23%) and 3 (19%); mixed-genotype infections were seen in 10% of the cases. RFLP analysis of DHPS PCR products revealed four genotypes, 18% of which were associated with resistance to sulfa drugs. Only contact with coughers (prevalence ratio [PR], 3.51 [95% confidence interval {CI}, 1.79 to 6.87]; P = 0.000) and exposure to tobacco smoke (PR, 1.82 [95% CI, 1.14 to 2.92]; P = 0.009) were statistically associated with being colonized by P. jirovecii . The prevalence of P. jirovecii in infants and toddlers with WC and the genotyping results provide evidence that this population represents a potential reservoir and transmission source of P. jirovecii .

Dientamoeba fragilis is an intestinal protozoan of debated clinical significance. Here, we present cross-sectional and longitudinal observations on D. fragilis in children aged 0 to 6 years from a 1-year multi-day-care-center cohort study set in Copenhagen, Denmark. The inclusion period for the cohort was 2009 through 2012. Stool samples collected from the children were accompanied by questionnaires completed by the parents or guardians of the children. Using real-time PCR, D. fragilis was detected in the first stool sample from 97 of 142 (68.3%) children. We evaluated the associations between seven plausible risk factors (age, sex, having siblings, having domestic animals at home, having had infant colic, recent history of intake of antibiotics, and recent history of travel abroad) as well as six reported symptoms (lack of appetite, nausea, vomiting, abdominal pain, weight loss, and diarrhea) and testing positive for D. fragilis . The final multivariable model identified being >3 years old and having a history of recent travel abroad as risk factors for testing positive for D. fragilis . Moreover, univariable analyses indicated that having siblings was a risk factor. There was no statistical association between a recent history of gastrointestinal symptoms and testing positive for D. fragilis . Among the 108 children who were represented by ≥2 samples and thus included in the longitudinal analysis, 32 tested negative on the first sample and positive later, and the last sample from each of the 108 children was positive. The results are in support of D. fragilis being a common enteric commensal in this population.

Recent studies have demonstrated the effectiveness and applicability of PCR-based methods for the detection of intestinal helminths in stool samples ([1][1][–][2][5][3]); however, successful DNA extraction is essential. We compared DNA extractions using NucliSENS easyMag, with and without a

Blastocystis is a common intestinal parasite of unsettled clinical significance, which is not easily detected by standard parasitological methods. The genus comprises at least 13 subtypes (STs) (which likely represent separate species), 9 of which have been found in humans. Recent data indicate that at least one of the subtypes is associated with intestinal disease. A quantitative TaqMan 5′ nuclease real-time PCR (TaqMan PCR) including an internal process control (IPC) was developed for the detection of Blastocystis and shown to be applicable to genomic DNAs extracted directly from feces. The assay enabled successful amplification of DNAs from all relevant subtypes within the genus (ST1 to ST9). For assay evaluation, 153 samples previously tested by xenic in vitro culture (XIVC) were screened by the TaqMan assay. A total of 49/51 samples positive by XIVC and 13/102 samples negative by XIVC were positive by the TaqMan assay; samples positive by the TaqMan assay and negative by XIVC were subsequently tested by conventional PCR, and amplicons could be identified to the subtype level by sequencing in 69% of the cases. Compared to the TaqMan assay, XIVC had a sensitivity of 79%. This is the first time that a genus-specific, probe-based, internal-process-controlled real-time PCR assay for the detection Blastocystis has been introduced.

Opisthorchis viverrini is an important food-borne trematode in Southeast Asia. The infection causes significant morbidity in terms of hepatobiliary diseases and cholangiocarcinoma. The aim of this study was to improve the sensitivity of the PCR-based diagnosis of O. viverrini infection. A new fecal DNA extraction protocol for the detection of O. viverrini DNA using cetyltrimethyl-ammoniumbromide to remove PCR inhibitor was used and compared with the commercial stool kit method. The sensitivity of the new test was 79.3%, compared with the 44.8% of the previous method ( P < 0.01). PCR-positive tests identified several cases judged parasite negative by the parasitological method (28.6%), indicating the new test's advantage in the diagnosis of individuals with light infections.