Entamoeba moshkovskii infections in children, Bangladesh.
ABSTRACT: Entamoeba moshkovskii cysts are morphologically indistinguishable from those of the disease-causing species E. histolytica and the nonpathogenic E. dispar. Although sporadic cases of human infection with E. moshkovskii have been reported, the organism is considered primarily a free-living amoeba. No simple molecular detection tool is available for diagnosing E. moshkovskii infections. We used polymerase chain reaction (PCR) to detect E. moshkovskii directly in stool. We tested 109 stool specimens from preschool children in Bangladesh by PCR; 17 were positive for E. histolytica (15.6%) and 39 were positive for E. dispar (35.8%). In addition, we found that 23 (21.1%) were positive for E. moshkovskii infection, and 17 (73.9%) of these also carried E. histolytica or E. dispar. The high association of E. moshkovskii with E. histolytica and E. dispar may have obscured its identification in previous studies. The high prevalence found in this study suggests that humans may be a true host for this amoeba.
Project description:BACKGROUND: E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples. RESULTS: The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR. CONCLUSION: The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens.
Project description:BACKGROUND: The level of intra-species genetic variation in Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii populations in a localized geographic area, like Puducherry, India, remains unknown. METHODS: In the present study the existence of genetic variation in the nested multiplex polymerase chain reaction (NM-PCR) amplified region of the 16S-like ribosomal RNA genes of E. histolytica, E. dispar and E. moshkovskii was investigated by riboprinting and single strand conformation polymorphism (SSCP) analysis. RESULTS: We found that 70 stool specimens were positive for E. histolytica, 171 stool specimens were positive for E. dispar, and 37 stool specimens were positive for E. moshkovskii by NM-PCR. Ninety liver abscess pus specimens, 21 urine specimens, and 8 saliva specimens were positive for E. histolytica by NM-PCR. Riboprinting analysis detected a mutation in the PCR product of only one E. histolytica isolate from a stool specimen. However, SSCP analysis detected mutations in the PCR products of five E. histolytica isolates and three E. moshkovskii isolates from stool specimens, and one E. histolytica isolate from a saliva specimen. The mutations detected by riboprinting and SSCP analysis were confirmed by sequencing. All the nucleotide sequences showing mutations in this study have already been deposited into the NCBI GenBank database under accession numbers [GenBank: EF682200 to GenBank: EF682208]. CONCLUSION: The present study has revealed the subsistence of mutations in the ribosomal RNA genes of E. histolytica and E. moshkovskii, which points towards the existence of intra-species genetic variation in E. histolytica and E. moshkovskii isolates infecting humans.
Project description:This study investigated the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from a patient population in Sydney, Australia. Stool samples were tested by microscopy and PCR. Five patients were found with E. histolytica infections, while E. dispar and E. moshkovskii were observed in 63 (70.8%) and 55 (61.8%) patients, respectively, by PCR. This is the first study in Australia using molecular techniques to determine the presence of E. histolytica, E. dispar, and E. moshkovskii.
Project description:This study aimed to determine the prevalence of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii (collectively referred to as Entamoeba complex), using microscopic and molecular methods in Kurdistan Province, northwest of Iran. The relationship between positive Entamoeba species and clinical symptoms was also investigated. Eight positive Entamoeba complex, as well as four Entamoeba complex-like isolates, were detected by microscopic stool examination. DNA was extracted from all positive and from 55 randomly selected negative stool samples. PCR was performed using species-specific 18S rRNA primers for the Entamoeba complex. All positive PCR samples were sequenced. In total, 14 (1.01%) out of 1383 isolates, i.e. 12 microscopy-positive and Entamoeba complex-like isolates and two out of 55 microscopy-negative isolates, were identified via PCR and sequencing. Overall, 0.58% (8/1383) of the isolates were E. dispar, 0.14% (2/1383) E. histolytica, 0.07% (1/1383) E. moshkovskii and 0.22% (3/1383) were mixed of E. histolytica and E. dispar. Based on our findings, the prevalence of E. dispar is greater than that of E. histoltyica. On the other hand, a case of E. moshkovskii was reported for the first time in this region. It seems that some gastrointestinal symptoms may be attributed to Entamoeba species.
Project description:Multiplex real-time polymerase chain reaction (PCR) was developed for differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii. Specific primers were designed for all three species, and then differentiation of E. histolytica and E. dispar was achieved simultaneously using a hybridization probe and melting curve analysis, whereas E. moshkovskii was detected with a separate probe under the same condition. This assay detected as little as 0.2 pg of E. histolytica DNA and 2 pg each for E. dispar and E. moshkovskii DNA. Thirty-five clinical samples suspected to be E. histolytica infection by microscopy were tested. The results showed 32 positive samples; four samples were E. histolytica and 28 samples were E. dispar. Interestingly, one E. dispar positive sample showed a mixed infection with E. moshkovskii. This is the first report of E. moshkovskii infection from Thailand and this assay is currently the most rapid and sensitive method to differentiate these human amoebas.
Project description:<h4>Background</h4>Entamoeba moshkovskii is prevalent in developing countries and morphologically indistinguishable from pathogenic Entamoeba histolytica and nonpathogenic Entamoeba dispar. It is not known if E. moshkovskii is pathogenic.<h4>Methods</h4>Mice were intracecally challenged with the trophozoites of each Entamoeba spp. to test the ability to cause diarrhea, and infants in Bangladesh were prospectively observed to see if newly acquired E. moshkovskii infection was associated with diarrhea.<h4>Results</h4>E. moshkovskii and E. histolytica caused diarrhea and weight loss in susceptible mice. E. dispar infected none of the mouse strains tested. In Mirpur, Dhaka, Bangladesh, E. moshkovskii, E. histolytica, and E. dispar were identified in 42 (2.95%), 66 (4.63%), and 5 (0.35%), respectively, of 1426 diarrheal episodes in 385 children followed prospectively from birth to one year of age. Diarrhea occurred temporally with acquisition of a new E. moshkovskii infection: in the 2 months preceding E. moshkvskii-associated diarrhea, 86% (36 of 42) of monthly surveillance stool samples were negative for E. moshkovskii.<h4>Conclusions</h4>E. moshkovskii was found to be pathogenic in mice. In children, the acquisition of E. moshkovskii infection was associated with diarrhea. These data are consistent with E. moshkovskii causing disease, indicating that it is important to reexamine its pathogenicity.
Project description:Entamoeba histolytica, E. dispar and E. moshkovskii are the most frequent species described in human infection where E. histolytica is the only true pathogen. The epidemiology of this infection is complex due to the absence of a routine exam that allows a correct discrimination of the Entamoeba species complex. Therefore, molecular methods appear as the unique epidemiological tool to accomplish the species discrimination. Herein, we conducted a cross-sectional study to determine the frequency of Entamoeba species infections in a group of asymptomatic individuals from a rural area in central Colombia.A total of 181 fecal samples from asymptomatic children under 16 years old from the hamlet La Vírgen, Cundinamarca (Colombia) that voluntarily accepted to participate in the study were collected. The fecal samples were examined by light microscopy and DNA-extracted, subsequently submitted to molecular discrimination of E. dispar/E. histolytica/E. moshkovskii infection based on a multiplex PCR assay targeting the 18S rRNA fragment. To confirm the species description, twenty samples were randomly submitted to DNA sequencing of the aforementioned fragment. By direct microscopic examination, frequency of the complex E. histolytica/E. dispar/E. moshkovskii was 18.8% (34/181). PCR showed a frequency of 49.1% (89/181), discriminated as 23.2% (42/181) that were positive for E. dispar, 25.4% (46/181) for E. moshkovskii and 0.55% (1/ 181) for E. histolytica. Also, mixed infections were detected between E. dispar and E. moshkovskii at 4.42% (8/181) of the samples. Molecular barcoding confirmed the diagnosis depicted by the multiplex PCR assay.This is the first description of E. moshkovskii in Colombia and the second report in South-America to our knowledge. Our results suggest the need to unravel the true epidemiology of Entamoeba infections around the world, including the real pathogenic role that E. moshkovskii may have.
Project description:Entamoeba moshkovskii is a member of the Entamoeba complex and a colonizer of the human gut. We used nested polymerase chain reaction (PCR) to differentiate Entamoeba species in stool samples that had previously been screened by microscopy. Forty-six samples were tested, 23 of which had previously been identified as Entamoeba complex positive by microscopy. Of the 46 specimens tested, we identified nine (19.5%) as E. moshkovskii-positive. In seven of these nine E. moshkovskii-positive samples, either E. dispar or E. histolytica (or both) were also identified, suggesting that co-infections may be common. E. moshkovskii was also detected in both symptomatic and asymptomatic participants. To the best of our knowledge, this is the first report of E. moshkovskii in Kenya.
Project description:The present study was conducted to investigate the clinical outcomes of Entamoeba histolytica infection in symptomatic and asymptomatic Orang Asli (aborigine) communities in Malaysia. Examination was performed on 500 stool samples obtained from Orang Asli communities in 3 different states using formalin-ether concentration, trichrome staining, and single-round PCR techniques. Out of 500 stool samples, single infection of E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii was identified in 3.2%, 13.4%, and 1%, respectively. In addition, 10 samples had mixed infections with E. histolytica and E. dispar. Six samples containing E. dispar were also positive for E. moshkovskii, and only 2 samples had E. histolytica in association with E. dispar and E. moshkovskii. Seventeen E. histolytica-positive samples were from symptomatic subjects, whereas the remaining 11 samples came from asymptomatic subjects. These findings suggest a predominant distribution of pathogenic potential of E. histolytica strains in this community. Therefore, further studies on genotyping of E. histolytica is required, to find out association between E. histolytica genotype and the outcome of the infection.
Project description:A single-round PCR assay was developed for detection and differential diagnosis of the three Entamoeba species found in humans, Entamoeba moshkovskii, Entamoeba histolytica, and Entamoeba dispar, that are morphologically identical as both cysts and trophozoites. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species. PCR generates a 166-bp product with E. histolytica DNA, a 752-bp product with E. dispar DNA, and a 580-bp product with E. moshkovskii DNA. Thirty clinical specimens were examined, and the species present were successfully detected and differentiated using this assay. It was possible to detect as little as 10 pg of E. moshkovskii and E. histolytica DNA, while for E. dispar the sensitivity was about 20 pg of DNA. Testing with DNA from different pathogens, including bacteria and other protozoa, confirmed the high specificity of the assay. We propose the use of this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three morphologically indistinguishable Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys.