Molecular appraisal of intestinal parasitic infection in transplant recipients.

BACKGROUND & OBJECTIVES: Diarrhoea is the main clinical manifestation caused by intestinal parasitic infections in patients, with special reference to transplant recipients who require careful consideration to reduce morbidity and mortality. Further, molecular characterization of some important parasites is necessary to delineate the different modes of transmission to consider appropriate management strategies. We undertook this study to investigate the intestinal parasitic infections in transplant recipients with or without diarrhoea, and the genotypes of the isolated parasites were also determined. METHODS: Stool samples from 38 transplant recipients comprising 29 post-renal, two liver and seven bone marrow transplant (BMT) recipients presenting with diarrhoea and 50 transplant recipients (42 post-renal transplant, eight BMT) without diarrhoea were examined for the presence of intestinal parasites by light microscopy using wet mount, modified Ziehl-Neelsen staining for intestinal coccidia and modified trichrome staining for microsporidia. Genotypes of Cryptosporidium species were determined by multilocus genotyping using small subunit ribosomal (SSUrRNA), Cryptosporidium oocyst wall protein (COWP) and dihydrofolate reductase (DHFR) as the target genes. Assemblage study for Giardia lamblia was performed using triose phosphate isomerase (TPI) as the target gene. Samples were also screened for bacterial, fungal and viral pathogens. RESULTS: The parasites that were detected included Cryptosporidium species (21%, 8/38), Cystoisospora (Isospora) belli (8%, 3), Cyclospora cayetanensis (5%, 2), G. lamblia (11%, 4), Hymenolepis nana (11%, 4), Strongyloides stercoralis (3%, 1) and Blastocystis hominis (3%, 1). Multilocus genotyping of Cryptosporidium species at SSUrRNA, COWP and DHFR loci could detect four isolates of C. hominis; two of C. parvum, one of mixed genotype and one could not be genotyped. All the C. hominis isolates were detected in adult post-renal transplant (PRT) recipients, whereas the C. parvum isolates included a child with BMT and an adult with PRT. Clostridium difficle, cytomegalovirus and Candida albicans were found in 2, 3 and 2 patients, respectively. INTERPRETATION & CONCLUSIONS: In the present study, C. hominis was observed as an important parasite causing intestinal infections in transplant recipients. Multilocus genotyping of Cryptosporidium species could detect four isolates of C. hominis; two of C. parvum, one of mixed genotype and one could not be genotyped. Genotyping of G. lamblia revealed that assemblage B was most common.

Development and evaluation of molecular methods for detection of Cryptosporidium spp. in human clinical samples.

Cryptosporidiosis is predominantly a gastrointestinal disease of humans and other animals, caused by various species of protozoan parasites representing the genus Cryptosporidium. Detection of Cryptosporidium spp. in human clinical samples is central to the prevention, surveillance and control of cryptosporidiosis, particularly given that there is presently no broadly applicable treatment regimen for this disease. A non-radioactive, genus specific DNA dot blot hybridization assay was developed using Digoxigenin (DIG) labelled probes to detect Cryptosporidium DNA in human clinical samples. Four hundred fifty (n = 450) clinical samples were subjected to microscopic examination, Polymerase Chain Reaction assay (PCR), Dot blot hybridization assay and Real Time PCR assay. A total of forty-one (n = 41) samples were positive by microscopy, forty-two (n = 42) by both PCR assay and dot blot hybridization assay and forty-three (n = 43) by Real Time PCR assay. Dot blot hybridization assay with a sensitivity of 95.5% and specificity of 99.75% could be an ideal choice for routine investigation of a large number of samples in a clinical setting as well as field.

Intestinal Parasitosis in Relation to Anti-Retroviral Therapy, CD4(+) T-cell Count and Diarrhea in HIV Patients.

Intestinal parasitic infections are one of the major causes of diarrhea in human immunodeficiency virus (HIV) seropositive individuals. Antiretroviral therapy has markedly reduced the incidence of many opportunistic infections, but parasite-related diarrhea still remains frequent and often underestimated especially in developing countries. The present hospital-based study was conducted to determine the spectrum of intestinal parasitosis in adult HIV/AIDS (acquired immunodeficiency syndrome) patients with or without diarrhea with the levels of CD4(+) T-cell counts. A total of 400 individuals were enrolled and were screened for intestinal parasitosis. Of these study population, 200 were HIV seropositives, and the remaining 200 were HIV uninfected individuals with or without diarrhea. Intestinal parasites were identified by using microscopy as well as PCR assay. A total of 130 (32.5%) out of 400 patients were positive for any kinds of intestinal parasites. The cumulative number of parasite positive patients was 152 due to multiple infections. A significant association of Cryptosporidium (P<0.001) was detected among individuals with CD4(+) T-cell counts less than 200 cells/µl.

MALDI-TOF mass spectrometry proteomic based identification of clinical bacterial isolates.

BACKGROUND & OBJECTIVES: Pathogenic bacteria often cause life threatening infections especially in immunocompromised individuals. Therefore, rapid and reliable species identification is essential for a successful treatment and disease management. We evaluated a rapid, proteomic based technique for identification of clinical bacterial isolates by protein profiling using matrix-assisted laser desorption-ionization time - of - flight mass spectrometry (MALDI-TOF MS). METHODS: Freshly grown bacterial isolates were selected from culture plates. Ethanol/formic acid extraction procedure was carried out, followed by charging of MALDI target plate with the extract and overlaying with α-cyano-4 hydroxy-cinnamic acid matrix solution. Identification was performed using the MALDI BioTyper 1.1, software for microbial identification (Bruker Daltonik GmbH, Bremen, Germany). RESULTS: A comparative analysis of 82 clinical bacterial isolates using MALDI -TOF MS and conventional techniques was carried out. Amongst the clinical isolates, the accuracy at the species level for clinical isolates was 98.78%. One out of 82 isolates was not in accordance with the conventional assays because MALDI-TOF MS established it as Streptococcus pneumoniae and conventional methods as Streptococcus viridans. INTERPRETATION & CONCLUSIONS: MALDI - TOF MS was found to be an accurate, rapid, cost-effective and robust system for identification of clinical bacterial isolates. This innovative approach holds promise for earlier therapeutic intervention leading to better patient care.

Cryptosporidium species subtypes and associated clinical manifestations in Indian patients.

AIM: Present hospital based study was carried out at our tertiary care centre with an aim to study the distribution of Cryptosporidium species subtypes in patients with complaints of diarrhea. BACKGROUND: Cryptosporidium species are one of the important causative agents of parasitic diarrhea, amongst which Cryptosporidium hominis (C.hominis) and Cryptosporidium parvum (C.parvum) are the two major species that are associated with human cryptosporidiosis. METHODS: Four hundred and fifty (n=450) diarrheic patients complaining of different types of diarrhea were enrolled in the present study. Both microscopic and molecular diagnostic methods were used for the detection as well as for identification of Cryptosporidium species and its speciation and subtyping. RESULTS: Forty one (n=41) and forty three (n=43) patients were positive for Cryptosporidium species by microscopy and Polymerase chain reaction (PCR) assay respectively. Of these 43 cases, 70% (30/43) were identified as C. hominis and 21% (9/43) was as C. parvum, 7% (3/43) was as Cryptosporidium felis (C.felis) and 2% (1/43) as Cryptopsoridium viatorum (C. viatorum) respectively . Upon subtyping of C. hominis and C. parvum, 16 subtypes belonging to 8 different subtype families could be identified. The frequency of different families were Ia (13%, 5/39), Ib (15%, 6/39), Id (18%, 7/39), Ie (30%, 12/39) and IIa (5%, 2/39), IIc (8%, 3/39), IId (8%, 3/39) and IIe (3%, 1/39). CONCLUSION: Our study results strongly suggest and reinforces the fact that most of the human cryptosporidiosis is anthroponotic and we expect that present molecular epidemiological data will provide more insight to unravel the changing clinical paradigm of human cryptosporidiosis at large.

Molecular Characterization and Subtyping of Blastocystis Species in Irritable Bowel Syndrome Patients from North India.

Blastocystis species has been extensively studied in recent few years to establish its pathogenecity. Present study was designed to identify and examine the association of Blastocystis sp. and its subtypes with Irritable Bowel Syndrome (IBS).Blastocystis sp. detected using wet-mount microscopy, trichrome staining, in-vitro culture and Polymerase Chain Reaction (PCR) assay in a cohort of IBS patients (n = 150) and healthy controls (n = 100). Isolates of Blastocystis sp.were subtyped using Sequence Tagged Site and representative samples were sequenced at SSUrRNA locus.A total of sixty five isolates of Blastocystis sp. were identified [IBS (n = 50); Controls (n = 15)] of which 91% belonged to ST3 and 9% belonged to ST1. No other subtypes could be identified. Statistically significant association was observed between Blastocystis sp. and IBS patients; however no particular subtype could be ascertained to any particular clinical type of IBS.The frequency of occurrence of Blastocystis sp. was more in IBS patients as compared to the controls and ST3 being the most prevalent subtype. The genetic polymorphism of SSU-rRNA gene amongst the different Blastocystis sp.isolates found in this study reinforces the fact that these organisms are genetically highly divergent.

Prevalence of Naegleria fowleri in Environmental Samples from Northern Part of India.

Naegleria fowleri the causative agent of Primary Amoebic Meningoencephalitis, is ubiquitously distributed worldwide in various warm aquatic environments and soil habitats. The present study reports on the presence of Naegleria spp. in various water bodies present in Rohtak and Jhajjar district, of state Haryana, India. A total of 107 water reservoirs were screened from summer till autumn (2012 and 2013). In order to isolate Naegleria spp. from the collected water samples, the water samples were filtered and the trapped debris after processing were transferred to non-nutrient agar plates already seeded with lawn culture of Escherichia coli. Out of total 107 water samples, 43 (40%) samples were positive by culture for free living amoeba after incubation for 14 days at 37°C. To identify the isolates, the ITS1, 5.8SrDNA and ITS2 regions were targeted for PCR assay. Out of total 43 positive samples, 37 isolates were positive for Naegleria spp. using genus specific primers and the most frequently isolated species was Naegleria australiensis. Out of 37 Naegleria spp. positive isolates, 1 isolate was positive for Naegleria fowleri. The sequence analysis revealed that the Naegleria fowleri strain belonged to Type 2.

Molecular detection of DHFR gene polymorphisms in Pneumocystis jirovecii isolates from Indian patients.

INTRODUCTION: Pneumocystis pneumonia (PCP) is an opportunistic life-threatening infection, especially for immunocompromised individuals. A trimethoprim-sulfamethoxazole (TMP-SMX) combination is commonly used for the treatment of PCP, targeting both dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes. Several studies have already shown that polymorphisms in the DHPS gene are associated with drug resistance. The present study analyzed DHFR gene polymorphisms in Pneumocystis jirovecii recovered from clinical samples from patients admitted to a tertiary care health center in New Delhi, India. METHODOLOGY: Detection of P. jirovecii was performed using Gomori methenamine silver staining (GMS) and nested polymerase chain reaction (PCR) assay targeting the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) gene. The DHFR gene was amplified using nested PCR protocol and was sequenced for detection of polymorphisms. RESULTS: Of 180 clinical samples, only 4% (7/180) were positive by GMS staining, and 10% (18/180) were positive by mt LSU rRNA PCR assay. Of these 18 positive samples, only 77% (14/18) were amplified by the DHFR gene PCR assay. A total of 16 nucleotide substitutions were observed in 42% (6/14) samples targeted for the DHFR gene, of which 8 nucleotide substitutions were synonymous and the rest were non-synonymous. CONCLUSIONS: The DHFR gene mutations found in this study may possibly indicate an association of process likely to contribute to therapeutic failure or an evolutionary process, and warrant continuous monitoring.

MALDI-TOF mass spectrometry for rapid identification of clinical fungal isolates based on ribosomal protein biomarkers.

This study aimed to evaluate the identification of clinical fungal isolates (yeast and molds) by protein profiling using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS). A total of 125 clinical fungal culture isolates (yeast and filamentous fungi) were collected. The test set included 88 yeast isolates (Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida parapsilosis, Candida rugosa, Candida tropicalis and Cryptococcus neoformans) and 37 isolates of molds (Alternaria spp., Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Cunninghamella spp., Histoplasma capsulatum, Microsporum gypseum, Microsporum nanum, Rhizomucor spp. and Trichophyton spp.). The correlation between MALDI TOF MS and conventional identification for all these 125 fungal isolates included in the study was 87.2% at the species level and 90.4% at the genus level. MALDI TOF MS results revealed that the correlation in yeast (n=88) identification was 100% both at the genus and species levels whereas, the correlation in mold (n=37) identification was more heterogeneous i.e. 10.81% isolates had correct identification up to the genus level, 56.7% isolates had correct identification both at the genus and species levels, whereas 32.42% isolates were deemed Not Reliable Identification (NRI). But, with the modification in sample preparation protocol for molds, there was a significant improvement in identification. 86.4% isolates had correct identification till the genus and species levels whereas, only 2.7% isolates had Not Reliable Identification. In conclusion, this study demonstrates that MALDI-TOF MS could be a possible alternative to conventional techniques both for the identification and differentiation of clinical fungal isolates. However, the main limitation of this technique is that MS identification could be more precise only if the reference spectrum of the fungal species is available in the database.