Bijay Ranjan Mirdha

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.

Double-blinded randomized controlled trial for immunomodulatory effects of Tulsi (Ocimum sanctum Linn.) leaf extract on healthy volunteers.

ETHNOPHARMACOLOGICAL RELEVANCE Tulsi (Ocimum sanctum Linn.) is considered as a sacred herb and traditionally it is believed that consumption of Tulsi leaf on empty stomach increases immunity. Experimental studies have shown that alcoholic extract of Tulsi modulates immunity. MATERIALS AND METHODS The present study was designed to evaluate the immunomodulatory effects of ethanolic extract of Tulsi leaves through a double-blinded randomized controlled cross-over trial on healthy volunteers. Three hundred milligrams capsules of ethanolic extracts of leaves of Tulsi or placebo were administered to 24 healthy volunteers on empty stomach and the results of 22 subjects who completed the study were analyzed. The primary objective was to study the levels of Th1 and Th2 cytokines (interferon-γ and interleukin-4) during both pre and post intervention period in blood culture supernatants following stimulation with lipopolysaccharide and phytohaemagglutinin. Other immunological parameters such as T-helper and T-cytotoxic cells, B-cells and NK-cells also were analyzed using Flowcytometry. RESULTS Statistically significant increase in the levels of IFN-γ (p=0.039), IL-4 (p=0.001) and percentages of T-helper cells (p=0.001) and NK-cells (p=0.017) were observed after 4 weeks in the Tulsi extract intervention group in contrast to the placebo group. CONCLUSIONS These observations clearly ascertain the immunomodulatory role of Tulsi leaves extract on healthy volunteers.

Hymenolepis nana: A Common Cause of Paediatric Diarrhoea in Urban Slum Dwellers in India

The prevalence of intestinal parasitic infections was studied for a period of 5 years (April 1996-April 2001) among urban slum dwellers. All age groups were represented in the study. Parasitological examinations were performed on 939 faecal specimens collected on a household basis. The total prevalence of pathogenic parasites was 33.6 per cent. No significant age and sex differences in pathogenic parasites were observed. The prevalence of intestinal helminths and pathogenic protozoa was as follows: Hymenolepis nana (9.9 per cent), Ascaris lumbricoides (8.5 per cent), Giardia lamblia (8.4 per cent) and Entamoeba histolyticaldispar (3.7 per cent). Thirty-four E. histolytica/dispar positive samples were cultured and speciation was done using polymerase chain reaction (PCR). The predominant isolate was E. dispar compared to E. histolytica. The notable finding of the present study was high prevalence of Hymenolepis nana compared with other parasitic infections in slum dwellers.

Diagnostic significance of nested polymerase chain reaction for sensitive detection of Pneumocystis jirovecii in respiratory clinical specimens

A total of 327 clinical specimens, including both invasive and noninvasive samples, obtained from 275 patients with various types of underlying immunocompromised conditions and a clinical suspicion of Pneumocystis pneumonia (PCP) were subjected to 2 different nested polymerase chain reaction (PCR) assays. The target genes used for nested PCR were mitochondrial large subunit ribosomal RNA (mtLSU rRNA) and internal transcribed spacer (ITS) region. The results were compared with a single-round PCR targeting major surface glycoprotein (MSG) gene. Amplification was successful in 16% of cases by mtLSU rRNA nested PCR, in 14.5% by ITS nested PCR, and in 10.9% by MSG PCR. The nested mtLSU rRNA PCR was found to be more sensitive (100% sensitive and 98.7% specific) and useful in detecting PCP for its use in routine diagnosis in our settings. Thus, this assay may be quite useful in the identification of patients who are in the early stage of Pneumocystis jirovecii infection with an organism load that could not be easily detected by the single-step PCR.

High frequency of parasitic and viral stool pathogens in patients with active ulcerative colitis: report from a tropical country.

Objective. Diarrhoeal relapses in patients with ulcerative colitis (UC) may be associated with enteric infections and its diagnosis may lessen avoidable exposure to corticosteroids and/or immunosuppressants. The purpose of this study was to assess the frequency of stool pathogens (parasitic and viral) in patients with active UC. Material and methods. This prospective cross-sectional study included 49 consecutive patients (32 M, 17 F, mean age 35.8±12 years) with active UC. Three stool samples were collected from each patient and examined for parasitic infection. Rectal biopsies were obtained during sigmoidoscopy to demonstrate cytomegalovirus (CMV) inclusion bodies and to conduct qualitative polymerase chain reaction (PCR) for CMV and herpes simplex virus (HSV) DNA detection. Results. Median duration of illness was 3.9±3.7 years and 83.7% of the patients had moderate to severe disease. The prevalence of parasitic infections in UC was 12%. The organisms isolated were Strongyloides stercoralis in 4%, Ankylostoma duodenale in 4%, Cryptosporidium in 2% and Entamoeba histolytica in 2% of the patients. The prevalence of CMV and HSV in rectal biopsies using qualitative PCR was 8% and 10%, respectively. No predictive factor was identified with CMV superinfection in patients with active UC. Conclusions. In India there is a high prevalence of parasitic and viral infections in patients with active UC. The results of the study suggest that, in tropical countries with a known high prevalence of parasitic diseases, aggressive evaluation for parasitic and viral infections should be carried out, as early identification and prompt treatment of such infections can improve the clinical course of patients with active UC.

Ocular toxocariasis in a North Indian population.

Sera form 68 patients aged 1-30 years, suffering from posterior pole granuloma, peripheral granuloma, uveitis, endophthalmitis, optic neuritis and with a clinical diagnosis of suspected ocular toxocariasis, were tested for the presence of anti-Toxocara antibodies. Antibodies to Toxocara were detected in 11 (17 per cent) subjects less than 15 years old and three (4 per cent) subjects more than 15 years of age using enzyme-linked immunosorbent assay. None of the controls sera (other helminthic diseases) were positive for anti-Toxocara antibody.

Improved detection of Pneumocystis jirovecii infection in a tertiary care reference hospital in India

We prospectively examined 143 clinical samples from 115 patients including both HIV infected (n=53) and HIV uninfected immunocompromized (n=62) patients, with lung infiltrates and with clinical features suggestive of Pneumocystis carinii pneumonia/ PneumoCystis Pneumonia (PcP), using both microscopic techniques as well as PCR assay. Clinical samples in the present study consisted of bronchoalveolar lavage (BAL), tracheal aspirate (TA), nasopharyngeal aspirate (NPA), sputum and gastric aspirate (GA). Another group of 21 individuals with other respiratory diseases not compatible with PcP served as control during the study period of 15 months. Overall, P. jirovecii positivity rate by PCR was 12.17% (14/115 patients) compared to 3.4% (4/115) by microscopy. None of the specimens in the control group was positive by any of the techniques used. All PCR negative patients including cases and controls showed no evidence of PcP. After resolution of the discrepant results upon review of the clinical data, the sensitivity and specificity were 100% and 99%, respectively, for PCR and 30.7% and 100%, respectively, for microscopy by GMS staining. Thus, our data support the significance of PCR assay for confirming and improving the diagnosis of PcP in high-risk patients.

Refractory giardiasis: A molecular appraisal from a tertiary care centre in India

PURPOSE The intestinal flagellate Giardia lamblia includes many genetically distinct assemblages, of which assemblage A and B, predominantly infect humans. Nitroimidazoles derivatives (metronidazole and tinidazole) and nitazoxanide are some of the therapeutic agents for treatment of giardiasis. Nevertheless, some individuals with giardiasis are non-responsive to standard therapy. The present study highlights cases of refractory giardiasis and attempts to elucidate if genetic heterogeneity in the parasite is associated with treatment failure. MATERIALS AND METHODS Three stool samples were obtained on three consecutive days from 4000 patients with diarrhoea and were microscopically examined for the detection of trophozoites, and/or cysts, using both normal saline and Lugols iodine. A hemi-nested polymerase chain reaction (PCR) assay using triose phosphate isomerase (tpi) as the target gene was performed to determine the assemblages. Sequencing of the PCR products of the patients showing failure to treatment of giardiasis was also performed. RESULTS Two per cent (82/4000) of the total patients were microscopically positive for Giardia lamblia in the stool samples. All these patients were treated with metronidazole/tinidazole as per the standard regimens. However, eight patients showed treatment failure to giardiasis as stool examinations were repeatedly positive even after treatment with multiple courses of anti-giardial therapy. Genetic characterisation of all eight Giardia isolates showed that they belonged to Assemblage B and had homogeneous sequences. These patients were either treated with extended regimens or with combination therapy of anti-giardials. CONCLUSION In our experience, combination of two or more drugs for a longer duration is the treatment modality to treat refractory giardiasis.

Genotypic variation of Pneumocystis jirovecii isolates in India based on sequence diversity at mitochondrial large subunit rRNA

Pneumocystis pneumonia (PCP), a common and serious opportunistic infection in immunocompromised patients, is caused by Pneumocystis jirovecii (formerly known as Pneumocystis carinii f. sp. hominis). The aim of the present study was to describe the prevalence and distribution of genotypes of P. jirovecii based on sequence polymorphisms at mitochondrial large subunit ribosomal RNA (mt LSU rRNA) region in both HIV and non-HIV immunocompromised individuals with a positive PCR result for PCP in a tertiary health care centre in northern India. From January 2005 to October 2008, 50 patients [22 HIV-seropositive individuals, 10 post-renal transplant (PRT) recipients, 3 cancer patients, and 15 patients with various other kinds of immunosuppression] were found to be positive for P. jirovecii using PCR at the mt LSU rRNA gene. Genotyping of the positive samples was performed at the mt LSU rRNA locus. Genotype 2 was the most common accounting for 42% of total types. This was followed by the genotypes 3 (24%), 1 (20%), and 4 (8%). Mixed infection was observed in 3 cases (6%). The rates of genotype distribution were similar in HIV-seropositive individuals, cancer patients, and in patients with other kinds of immunosuppression. In the PRT recipients, genotype 1 was the most prevalent type (80%). This is the first study describing the prevalence of genotypes in HIV-infected and HIV-uninfected, immunocompromised patients based on the mt LSU rRNA gene from the Indian subcontinent. The most prevalent genotype observed was type 2 in contrast to many studies from other parts of the world where genotype 1 was the most prevalent type, suggesting geographical variation.

Latex agglutination test : an adjunct to the laboratory diagnosis of pyogenic bacterial meningitis

Gram stain, culture and latex agglutination test (LAT) of cerebrospinal flud were performed in 50 patients clinically diagnosed as suffering from pyogenic bacterial meningitus. Using all the three techniques, an aetiological diagnosis was made in 27 (54%).Neisseria meningitidis, Streptococcus pneumoniae and H. influenzae were the infecting organisms in 21 cases (44%). There were 12 additional cases in which LAT was the only clue to the diagnosis as compared to conventional techniques.Propionibacterium acnes was isolated from one case of anaerobic meningitis. It is concluded that LAT is an adjunct to conventional techniques in the diagnosis of pyogenic bacterial meningitis, where the latter tests fail.