Phylogenetic analysis of genetically distinct Enterocytozoon bieneusi infecting renal transplant recipients.

Enterocytozoon bieneusi (E. bieneusi), infecting renal transplant (RT) recipients may be transmitted anthroponotically or zoonotically. Accordingly, we aimed to, a) evaluate genotypes of E. bieneusi ínfecting RT recipients, and b) infer phylogenetic interpretation on transmission of different genotypes among infected hosts. Stool samples of 22 RT recipients infected with microsporidia (identified using modified trichrome staining) were subjected to species identification. All E. bieneusi positive samples were subjected to genotyping. The phylogenetic tree was constructed using Mega 5 software. Of 22 microsporidia infected RT recipients, 21 (95.5%) had E. bieneusi. ITS sequences of 21 E. bieneusi were classified into eight genotypes (Ind1 to Ind8). Among them, 4 (Ind5 to Ind8) were novel. Genotypes Ind2, Ind3, Ind4, Ind7 and Ind8 showed close sequence similarity to genotypes reported exclusively from humans. Phylogenetic analysis further supported their anthroponotic transmission. Genotypes Ind1, Ind5 and Ind6 showed close sequence similarity to genotypes reported from both animals and humans. Phylogenetic analysis further supported their zoonotic transmission. Anthroponotic transmission of E. bieneusi was more common among males (11, 100% vs. 7/10, 70%; P = 0.05), presenting with diarrhea (11, 100% vs. 6/10, 60%; P = 0.02) and watery stool (10/11, 91% vs. 5/10, 50%; P = 0.03).

Identification and genotyping of Enterocytozoon bieneusi among human immunodeficiency virus infected patients.

Microsporidia cause diarrhea among human immunodeficiency virus (HIV) infected patients worldwide. Enterocytozoon bieneusi and Encephalitozoon intestinalis are the most common species infecting HIV patients. Various genotypes of E. bieneusi are transmitted from human to human (anthroponotic route) or from animal to human (zoonotic route). However, there is no study from India on genotypes of E. bieneusi among infected hosts. Therefore, we aimed to (a) study the prevalence, clinical symptoms, and species identification of microsporidia among HIV infected patients and (b) perform a genotypic analysis of E. bieneusi and a phylogenetic interpretation of the transmission of different genotypes among infected hosts. Two hundred and twenty-two HIV-infected patients and 220 healthy controls (HC) were tested for the presence of microsporidia using modified trichrome (MT) staining and PCR. Demographic, clinical and laboratory parameters were studied. Species identification was performed using PCR-RFLP. All E. bieneusi isolates were subjected to genotypic and phylogenetic analysis. Patients with HIV [n=222, age 37.4±10.4y, 169 (76%) male] were more commonly infected with microsporidia than the HC [n=220, age 34.5±6.5y, 156 (71%) male], using MT stain and PCR [4/222, 1.8% vs. 0/220, p=0.04]. Patients infected with microsporidia more commonly presented with diarrhea than those not infected with microsporidia [4, 100% vs. 98/218, 45%; p=0.04]. E. bieneusi was detected in all patients with microsporidia. Four novel genotypes (Ind1 to Ind4) were identified. Ind1 showed 95% similarity with genotype L (AF267142.1) reported in cats (Germany). Genotypes Ind2 to Ind4 showed 94-96% similarity to host-specific genotype A (AF101197.1) reported in humans. Phylogenetic analysis mainly showed an anthroponotic route of transmission (3/4), while the zoonotic route (1/4) was also observed. The prevalence of microsporidia among HIV-infected patients was 1.8%. Patients with microsporidia commonly present with diarrhea. E. bieneusi is the most common species infecting the study population. Four novel genotypes of E. bieneusi were identified, suggesting presumptive transmission mainly through the anthropological route.

Evaluation of Immunoflourescence antibody assay for the detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis.

Detection of microsporidia at the species level is important for therapeutic purpose. The available techniques, modified trichrome (MT) staining cannot differentiate between species, while polymerase chain reaction (PCR) requires a reference laboratory and skilled technical staff. Immunoflourescence antibody (IFA) assay is another technique, which can differentiate among commonest species of microsporidia. However, there are very limited studies on its efficacy worldwide. Therefore, we aimed to evaluate IFA assay for the detection of microsporidia and differentiation among commonest species, Enterocytozoon bieneusi (E. bieneusi) and Encephalitozoon intestinalis infecting immunocompromised patients. Stool samples from 200 immunocompromised patients (19 with microsporidia and 181 without microsporidia using MT staining) were tested for species identification by PCR-RFLP and IFA assay. Sensitivity, specificity, diagnostic accuracy, and positive and negative predictive values were calculated as per standard formulae. Kappa statistics was used to assess the agreement between three tests. Of 200 immunocompromised patients, 21 and 20 patients had microsporidia using PCR and IFA assay, respectively. IFA assay and PCR identified E. bieneusi in all patients infected with microsporidia. Considering MT stain as gold standard, sensitivity and specificity of IFA assay was 100 and 99.4 %, respectively. Upon considering PCR as gold standard, sensitivity and specificity of IFA assay was 95.2 and 100 %, respectively. Diagnostic accuracy of IFA assay was 99.5 % along with its high test agreement with MT staining and PCR (K = 0.915, p = 0.049; K = 0.973, p = 0.027). IFA assay is highly sensitive and specific technique for detecting and identifying species of microsporidia among immunocompromised patients. E. bieneusi was the commonest species identified.

Effect of fixed space maintainers and removable appliances on oral microflora in children: An in vivo study.

BACKGROUND: Oral habits and caries if left untreated may result in crowding and arch length discrepancy in developing dentition. Therefore, appliances are used to maintain the arch length and for proper relationship between dental arches. However, its insertion may cause an increase in bacterial concentration. AIM: This study aimed to evaluate the growth of Streptococcus mutans, Lactobacillus sp., and Candida albicans in saliva during the first 6 months of orthodontic therapy. MATERIALS AND METHODS: Twenty children in the age group of 6-15 years were selected with regard to indication of orthodontic treatment, and subsequently grouped as patients to be treated with fixed space maintainers or removable appliances. Unstimulated saliva was collected in a sterile container at baseline and at 1-month, 3-month, and 6-month recalls for every selected child. Samples collected were processed for bacterial culture in different culture media at different time intervals. STATISTICAL ANALYSIS: SPSS (Statistical Package for Social Sciences) Version 15.0 statistical software was carried for bacterial counts. Chi-square test and t-test were performed to know the effects of each variable and to reveal the statistical significance. RESULT: Bacterial counts of Streptococcus mutans, Lactobacillus sp., and Candida albicans were found to be statistically significant (P 0≤ 0.001), (P < 0.05), and (P < 0.001), respectively in both the groups at all intervals. CONCLUSION: At different time intervals, the total numbers of bacterial count of Streptococcus mutans were comparatively higher, followed by Lactobacillus sp. and Candida albicans.

Comparative evaluation of staining techniques and polymerase chain reaction for diagnosis of intestinal microsporidiosis in immunocompromised patients.

CONTEXT: Microsporidia, which causes chronic diarrhoea in immunocompromised hosts, are often missed. The commonest diagnostic techniques include modified trichrome (MT) stain; however, it requires expertise and does not identify the species, which is important therapeutically. Other diagnostic techniques include Calcoflour white staining and polymerase chain reaction (PCR). Data on comparative utility of different diagnostic techniques are scanty. AIM: Comparison of Calcoflour white, MT staining and PCR for the diagnosis of intestinal microsporidiosis. SUBJECTS AND METHODS: Fecal samples of consecutive immunocompromised patients were evaluated for Microsporidia using Calcoflour white, MT stain and PCR. Species were identified by restriction fragment length polymorphism using HindIII and HinfI. Presence of Microsporidia by two or more techniques was considered true positive. Absence of Microsporidia by all three techniques was taken as true negative. RESULTS: Of 730 patients, Microsporidia was detected in 28 (3.8%), 250 (34.2%) and 30 (4.1%) patients by MT, Calcoflour white stains and PCR, respectively. Enterocytozoon bieneusi was identified in all 30 (4.1%) patients. 30 (4.1%) and 479 (65.6%) patients were true positive and true negative, respectively. Sensitivity and specificity of Calcoflour white, MT stains and PCR were 100%, 93.8%, 96.8% and 68.5%, 100% and 99.8%, respectively. Diagnostic accuracy of MT stain and PCR was superior to Calcoflour white (99.6% vs. 69.8%; P < 0.05). CONCLUSIONS: Though Calcoflour white stain is a highly sensitive, but it is nonspecific technique. MT stain and PCR with high sensitivity, specificity and diagnostic accuracy are useful diagnostic techniques. Furthermore, PCR is useful for species identification, which has therapeutic implications.

Antibiofilm and membrane-damaging potential of cuprous oxide nanoparticles against Staphylococcus aureus with reduced susceptibility to vancomycin.

The antimicrobial effects of copper ions and salts are well known, but the effects of cuprous oxide nanoparticles (Cu2O-NPs) on staphylococcal biofilms have not yet been clearly revealed. The present study evaluated Cu2O-NPs for their antibacterial and antibiofilm activities against heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and vancomycin-intermediate S. aureus (VISA). Nanoscaled Cu2O, generated by solution phase technology, contained Cu2O octahedral nanoparticles. Field emission electron microscopy demonstrated particles with sizes ranging from 100 to 150 nm. Cu2O-NPs inhibited the growth of S. aureus and showed antibiofilm activity. The MICs and minimum biofilm inhibitory concentrations ranged from 625 µg/ml to 5,000 µg/ml and from 2,500 µg/ml to 10,000 µg/ml, respectively. Exposure of S. aureus to Cu2O-NPs caused leakage of the cellular constituents and increased uptake of ethidium bromide and propidium iodide. Exposure also caused a significant reduction in the overall vancomycin-BODIPY (dipyrromethene boron difluoride [4,4-difluoro-4-bora-3a,4a-diaza-s-indacene] fluorescent dye) binding and a decrease in the viable cell count in the presence of 7.5% sodium chloride. Cu2O-NP toxicity assessment by hemolysis assay showed no cytotoxicity at 625 to 10,000 µg/ml concentrations. The results suggest that Cu2O-NPs exert their action by disruption of the bacterial cell membrane and can be used as effective antistaphylococcal and antibiofilm agents in diverse medical devices.

A series on intestinal strongyloidiasis in immunocompetent and immunocompromised hosts.

BACKGROUND: Strongyloidiasis, endemic in tropical areas, may be asymptomatic in immunocompetent subjects or may cause potentially fatal hyper-infection in immunocompromised patients. METHODS: Of the 13,885 patients referred to the parasitology laboratory at our tertiary care referral center for stool microscopy, 15 were diagnosed as strongyloidiasis over a 6 year period. We assessed these patients retrospectively. RESULTS: Most patients were young (median age 32 years, range 3-66) males (12, 80%). Seven patients (46.6%) were immunocompromised. All patients were symptomatic, and symptoms included chronic diarrhea (4, 26.7%), acute diarrhea (1,6.7%), abdominal pain (6, 40%), weight loss (3, 20%), cough (2, 13.33%), vomiting (1, 6.7%), anemia (10, 66.7%) and eosinophilia (3, 20%). Thirteen patients (86.6%) were diagnosed on first stool microscopy. Duodenal biopsy showed normal histology in twelve (80%) and partial villous atrophy in one (6.7%) patient. Stool microscopy also revealed giardiasis and cryptosporidiosis in one patient each. Nine patients responded well to ivermectin and albendazole, one died and five were lost to follow-up. CONCLUSIONS: In endemic areas, even immunocompetent subjects may suffer from symptomatic strongyloidiasis and associated eosinophilia is uncommon.