Jyotish Chandra Samantaray

Successful treatment of Acanthamoeba meningitis with combination oral antimicrobials

Acanthamoeba was implicated as the causative agent of chronic meningitis in three apparently immunocompetent children. Diagnosis was established by cerebrospinal fluid wet mount examination and culture. Two children improved rapidly with combination oral therapy composed of trimethoprim-sulfamethoxazole, rifampin and ketoconazole.

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.

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.

Two-dimensional difference gel electrophoresis (DIGE) analysis of sera from visceral leishmaniasis patients

IntroductionVisceral leishmaniasis is a parasitic infection caused by Lesihmania donovani complex and transmitted by the bite of the phlebotomine sand fly. It is an endemic disease in many developing countries with more than 90% of the cases occurring in Bangladesh, India, Nepal, Sudan, Ethiopia and Brazil. The disease is fatal if untreated. The disease is conventionally diagnosed by demonstrating the intracellular parasite in bone marrow or splenic aspirates. This study was carried out to discover differentially expressed proteins which could be potential biomarkers.MethodsSera from six visceral leishmaniasis patients and six healthy controls were depleted of high abundant proteins by immunodepletion. The depleted sera were compared by 2-D Difference in gel electrophoresis (DIGE). Differentially expressed proteins were identified the by tandem mass spectrometry. Three of the identified proteins were further validated by western blotting.ResultsThis is the first report of serum proteomics study using quantitative Difference in gel electrophoresis (DIGE) in visceral leishmaniasis. We identified alpha-1-acidglycoprotein and C1 inhibitor as up regulated and transthyretin, retinol binding protein and apolipoprotein A-I as down regulated proteins in visceral leishmaniasis sera in comparison with healthy controls. Western blot validation of C1 inhibitor, transthyretin and apolipoprotein A-I in a larger cohort (n = 29) confirmed significant difference in the expression levels (p < 0.05).ConclusionsIn conclusion, DIGE based proteomic analysis showed that several proteins are differentially expressed in the sera of visceral leishmaniasis. The five proteins identified here have potential, either independently or in combination, as prognostic biomarkers.

Clinical and molecular characteristics of nosocomial meticillin-resistant Staphylococcus aureus skin and soft tissue isolates from three Indian hospitals

We analysed risk factors for nosocomial meticillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections (SSTIs) in three Indian hospitals. We also determined antimicrobial resistance patterns and genotypic characteristics of MRSA isolates using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphylococcal cassette chromosome (SCCmec) typing. Medical records of 709 patients admitted to three tertiary hospitals with nosocomial S. aureus SSTIs were clinically evaluated. Antimicrobial susceptibility testing of patient isolates was performed in accordance with Clinical and Laboratory Standards Institute guidelines, with meticillin and mupirocin resistance confirmed by multiplex polymerase chain reaction. PFGE analysis of 220 MRSA isolates was performed, followed by MLST and SCCmec typing of a selected number of isolates. MRSA was associated with 41%, 31% and 7.5% of infections at the three hospitals, respectively. Multiple logistic regression analysis identified longer duration of hospitalisation [odds ratio (OR): 1.78; OR: 2.83 for >or=20 days], intra-hospital transfer (OR: 1.91), non-infectious skin conditions (3.64), osteomyelitis (2.9), neurological disorders (2.22), aminoglycoside therapy (1.74) and clindamycin therapy (4.73) as independent predictors for MRSA SSTIs. MRSA isolates from all three hospitals were multidrug resistant, with fifteen clones (I-XV) recognised. A majority of the strains possessed type III cassette. The common sequence type (ST) 239 was considered the signature MLST sequence for PFGE clone III. This major MRSA clone III was closely related to the UK EMRSA-1 and was significantly more resistant to antibiotics. Dissemination of multidrug-resistant MRSA clones warrants continuous tracking of resistant genotypes in the Indian subcontinent.

Early versus delayed initiation of antiretroviral therapy for Indian HIV-Infected individuals with tuberculosis on antituberculosis treatment.

BackgroundFor antiretroviral therapy (ART) naive human immunodeficiency virus (HIV) infected adults suffering from tuberculosis (TB), there is uncertainty about the optimal time to initiate highly active antiretroviral therapy (HAART) after starting antituberculosis treatment (ATT), in order to minimize mortality, HIV disease progression, and adverse events.MethodsIn a randomized, open label trial at All India Institute of Medical Sciences, New Delhi, India, eligible HIV positive individuals with a diagnosis of TB were randomly assigned to receive HAART after 2-4 or 8-12 weeks of starting ATT, and were followed for 12 months after HAART initiation. Participants received directly observed therapy short course (DOTS) for TB, and an antiretroviral regimen comprising stavudine or zidovudine, lamivudine, and efavirenz. Primary end points were death from any cause, and progression of HIV disease marked by failure of ART.FindingsA total of 150 patients with HIV and TB were initiated on HAART: 88 received it after 2-4 weeks (early ART) and 62 after 8-12 weeks (delayed ART) of starting ATT. There was no significant difference in mortality between the groups after the introduction of HAART. However, incidence of ART failure was 31% in delayed versus 16% in early ART arm (p = 0.045). Kaplan Meier disease progression free survival at 12 months was 79% for early versus 64% for the delayed ART arm (p = 0.05). Rates of adverse events were similar.InterpretationEarly initiation of HAART for patients with HIV and TB significantly decreases incidence of HIV disease progression and has good tolerability.Trial registrationCTRI/2011/12/002260

Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry

PURPOSE The purpose of this study was to evaluate the identification of Mycobacterium tuberculosis which is often plagued with ambiguity. It is a time consuming process requiring 4-8 weeks after culture positivity, thereby delaying therapeutic intervention. For a successful treatment and disease management, timely diagnosis is imperative. We evaluated a rapid, proteomic based technique for identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). MATERIALS AND METHODS Freshly grown mycobacterial isolates were used. Acetonitrile/trifluoroacetic acid extraction procedure was carried out, following which cinnamic acid charged plates were subjected to identification by MALDI-TOF MS. RESULTS A comparative analysis of 42 clinical mycobacterial isolates using the MALDI-TOF MS and conventional techniques was carried out. Among these, 97.61% were found to corroborate with the standard methods at genus level and 85.36% were accurate till the species level. One out of 42 was not in accord with the conventional assays because MALDI-TOF MS established it as Mycobacterium tuberculosis (log (score)>2.0) and conventional methods established it to be non-tuberculous Mycobacterium. CONCLUSIONS MALDI-TOF MS was found to be an accurate, rapid, cost effective and robust system for identification of mycobacterial species. This innovative approach holds promise for early therapeutic intervention leading to better patient care.

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.

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.

Fatal haemophagocytic syndrome and hepatitis associated with visceral leishmaniasis.

Haemophagocytic syndrome (HPS) secondary to infections occurs due to excessive, non-malignant proliferation of histiocytes, with resultant haemophagocytosis. The syndrome is essentially treatable, provided timely etiological diagnosis is achieved. In this report, we present a rare case of a child who hailed from Uttaranchal and presented with severe hepatitis. Bone marrow examination revealed an unexpected diagnosis of HPS secondary to visceral leishmaniasis. Despite initiating appropriate antileishmanial treatment, the child had a fatal outcome.