Diagnosis of extraintestinal microsporidiosis is always hampered due to non-specific symptoms and difficulty in diagnosis. This study aimed to compare the diagnostic utility of blood and faecal-based polymerase chain reaction (PCR) to detect microsporidiosis in immunocompromised patients. A total of 42 immunocompromised patients consisting of HIV-infected and chemotherapy-treated patients were enrolled. Paired faecal and blood samples were collected and subjected to PCR to detect Enterocytozoon bieneusi and Encephalitozoon spp. Faecal samples were microscopically screened for microsporidia spores. Overall, 42.9% (18/42) of patients were positive for microsporidiosis. Of this, 19.0% (8/42) and 4.8% (2/42) were positive by blood and stool PCR respectively. Meanwhile, 33.3% (14/42) of the faecal specimens were microscopically positive. Among the positive patients, 22.2% (4/18) had microsporidia confirmed by blood PCR and stool microscopy, suggestive of dissemination. Interestingly, the stool specimen in which microsporidia spores were detected via microscopy is not positive via PCR method. This highlights the limitation of the faecal-based detection method and the important use of blood samples for diagnosing extraintestinal microsporidiosis. Only E. bieneusi species were detected in all PCR-positive samples. This study highlights the diagnostic value of blood PCR in diagnosing extraintestinal microsporidiosis infections.
Abstract. The species identification of Enterocytozoon bieneusi and Encephalitozoon intestinalis is only possible using transmission electron microscopy (TEM), mo lecular techniques and immunofluorescence antibody assays (IFA). In this study, 50 positive and 50 negative fecal specimens for microsporidial spores using the Weber modified trichrome (WMT) staining technique were examined using IFA-MAbs. Of the 100 specimens examined, the microsporidial spores identified by IFA-MAbs were Enterocytozoon Bieneusi 42 (75%) Encephalitozoon intestinalis 7 (12.5%) and mixed infections 7 (12.5%). The sensitivity and specificity of IFA-MAbs in detecting microsporidial spores were 98% and 86%, respectively. The agreement between the WMT staining technique and IFA-MAbs was statistically significant by Kappa statistics (K = 0.840; p < 0.001). E. bieneusi was the commonest Microsporidia species isolated from the studied population; the presence of microsporidial spores detected by IFA-MAbs should be confirmed by other methods.
Enterocytozoon bieneusi is an emerging opportunistic pathogen infecting humans, and both domestic and wild pigs are known to harbour zoonotic genotypes. There remains a paucity of information on the prevalence and epidemiology of this enteropathogen in Southeast Asia. The present study was undertaken to determine the molecular prevalence and risk factors associated with E. bieneusi infection among commercially farmed pigs in Malaysia. Faecal samples were collected from 450 pigs from 15 different farms and subjected to nested PCR amplification of the ribosomal internal transcribed spacer (ITS) gene of E. bieneusi. Phylogenetic analysis involved 28 nucleotide sequences of the ITS region of E. bieneusi. An interviewer-administered questionnaire provided information on the animal hosts, farm management systems and environmental factors and was statistically analysed to determine the risk factors for infection. The prevalence of E. bieneusi infection was relatively high (40.7%). The highest prevalence (51.3%) was recorded among the piglets, while the adults showed the lowest level of infection (31.3%). Multivariate analysis indicated that age of the pigs, distance of the farm from human settlement and farm management system were significant risk factors of infection. Three genotypes (EbpA, EbpC and Henan-III) detected among the pigs are potentially zoonotic. The high prevalence of E. bieneusi among locally reared pigs, the presence of zoonotic genotypes and the spatial distribution of pig farms and human settlements warrant further investigation on the possibility of zoonotic transmission.
Most studies of opportunistic infections focus on immunocompromised patients. However, there is a lack of information on microsporidiosis in healthy people (immunocompetent) worldwide. This study aimed to detect and identify microsporidia species in immunocompetent Orang Asli living in Pahang, Malaysia. Orang Asli is a collective term for a group of indigenous people that usually reside in the interior regions of Peninsular Malaysia. They comprise about 0.7% of the total population in Malaysia and 76% of them lived below the poverty line i.e., poor housing conditions with the lack of access to safe drinking water and adequate sanitation, contaminated environment, high illiteracy rate and unhygienic practices by these people. Stool samples were collected from 209 Orang Asli and analyzed for detecting the presence of Enterocytozoon bieneusi and Encephalitozoon intestinalis by polymerase chain reaction assay targeting small subunit ribosomal RNA gene. E. bieneusi was detected in 8 individuals (3.83%). This infection was commonly found in males than females (5.2% vs. 2.7%). All infected Orang Asli were adults, with a mean age of 44years. Diarrhea and other gastrointestinal symptoms were reported in one case (12.5%) among individuals infected with this species. These findings clearly show that exposure to E. bieneusi may actually be common than reported. The accurate detection and identification of microsporidian species by molecular technique will improve therapy, clinical manifestations and prognosis of this infection, as no antiparasitic therapy has been approved for E. bieneusi. It is hoped that these findings will allow the formulation of better health management and disease prevention advisories, and improvement in the standards of health in similar communities.
Microsporidia are obligate intracellular parasitic fungi causing chronic diarrhea, particularly among immunocompromised patients. The current method used for diagnosis is based on conventional microscopy, which does not differentiate parasites at the species level. The present study was carried out to identify microsporidian species in immunocompromised patients. From March 2016 to March 2017, a total of 289 archived stool samples were examined microscopically for microsporidian spores using Gram-chromotrope Kinyoun (GCK) stain. Positive stool samples by microscopy were subjected to polymerase chain reaction and sequencing for species identification. Based on microscopy examination, the overall prevalence of microsporidian infections was 32.2% (93/289; 95% CI = 27.1-37.8). Of these stool samples, 45 samples were successfully amplified and confirmed as Enterocytozoon bieneusi. No Encephalitozoon intestinalis was detected. Accurate identification of species might help clinicians to decide appropriate management strategies as dissemination risks and treatment response vary for different species, hence improving the management of microsporidian infections.