Ashutosh Panda

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.

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.

A novel Pfs38 protein complex on the surface of Plasmodium falciparum blood-stage merozoites

BackgroundThe Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38 and Pfs12 and three other merozoite surface proteins: Glutamate-rich protein (GLURP), SERA5 and MSP-1 were identified on the Plasmodium falciparum merozoite surface.MethodsRecombinant 6-cys proteins i.e. Pfs38, Pfs12, Pfs41 as well as PfMSP-165 were expressed and purified using Escherichia coli expression system and antibodies were raised against each of these proteins. These antibodies were used to immunoprecipitate the native proteins and their associated partners from parasite lysate. ELISA, Far western, surface plasmon resonance and glycerol density gradient fractionation were carried out to confirm the respective interactions. Furthermore, erythrocyte binding assay with 6-cys proteins were undertaken to find out their possible role in host-parasite infection and seropositivity was assessed using Indian and Liberian sera.ResultsImmunoprecipitation of parasite-derived polypeptides, followed by LC–MS/MS analysis, identified a large Pfs38 complex comprising of 6-cys proteins: Pfs41, Pfs38, Pfs12 and other merozoite surface proteins: GLURP, SERA5 and MSP-1. The existence of such a complex was further corroborated by several protein–protein interaction tools, co-localization and co-sedimentation analysis. Pfs38 protein of Pfs38 complex binds to host red blood cells (RBCs) directly via glycophorin A as a receptor. Seroprevalence analysis showed that of the six antigens, prevalence varied from 40 to 99%, being generally highest for MSP-165 and GLURP proteins.ConclusionsTogether the data show the presence of a large Pfs38 protein-associated complex on the parasite surface which is involved in RBC binding. These results highlight the complex molecular interactions among the P. falciparum merozoite surface proteins and advocate the development of a multi-sub-unit malaria vaccine based on some of these protein complexes on merozoite surface.

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.

Exploring Heme and Hemoglobin Binding Regions of Plasmodium Heme Detoxification Protein for New Antimalarial Discovery

Hemoglobin degradation/hemozoin formation, essential steps in the Plasmodium life cycle, are targets of existing antimalarials. The pathway still offers vast possibilities to be explored for new antimalarial discoveries. Here, we characterize heme detoxification protein, PfHDP, a major protein involved in hemozoin formation, as a novel drug target. Using in silico and biochemical approaches, we identified two heme binding sites and a hemoglobin binding site in PfHDP. Treatment of Plasmodium falciparum 3D7 parasites with peptide corresponding to the hemoglobin binding domain in PfHDP resulted in food vacuole abnormalities similar to that seen with a cysteine protease inhibitor, E-64 (I-1). Screening of compounds that bound the modeled PfHDP structure in the heme/hemoglobin-binding pockets from Maybridge Screening Collection identified a compound, ML-2, that inhibited parasite growth in a dose-dependent manner, thus paving the way for testing its potential as a new drug candidate. These results provide functional insights into the role of PfHDP in Hz formation and further suggest that PfHDP could be an important drug target to combat malaria.

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.

Molecular Detection and Identification of Cryptosporidium viatorum in a Human Immunodeficiency Virus-seropositive Patient

28 Sir, Cryptosporidium was reported by Tyzzer in 1907 and well known to veterinarians since 1953. Since the human immunodeficiency virus (HIV) and acquired immune deficiency syndrome became pandemic, it has brought Cryptosporidium to the forefront as an important pathogen in immunocompromised individuals. Cryptosporidium hominis and Cryptosporidium parvum are the two major species causing human cryptosporidiosis in India, followed by Cryptosporidium felis and Cryptosporidium meleagridis. Here, we describe the first report of Cryptosporidium viatorum in a patient attending our tertiary care center. C. viatorum has been reported in the UK in travelers returning from the Indian subcontinent.[1]

Epidemiological Investigation and Successful Management of Burkholderia cepacia Outbreak in Neuro-Trauma Intensive Care Unit

OBJECTIVE The detailed epidemiological and molecular characterization of an outbreak of Burkholderia cepacia at a neurotrauma intensive care unit of a level 1 trauma centre is described. The stringent infection control interventions taken to successfully curb this outbreak are emphasized. METHODS The clinical and microbiological data for those patients who had more than one blood culture that grew B. cepacia were reviewed. Bacterial identification and antimicrobial susceptibility testing was done using automated Vitek 2 systems. Prospective surveillance, environmental sampling, and multilocus sequence typing (MLST) were performed for extensive source tracking. Intensive infection control measures were taken to further control the hospital spread. RESULTS Out of a total 48 patients with B. cepacia bacteraemia, 15 (31%) had central line-associated blood stream infections. Two hundred and thirty-one environmental samples were collected and screened, and only two water samples grew B. cepacia with similar phenotypic characteristics. The clinical strains characterized by MLST typing were clonal. However, isolates from the water represented a novel strain type (ST-1289). Intensive terminal cleaning, disinfection of the water supply, and the augmentation of infection control activities were done to curb the outbreak. A subsequent reduction in bacteraemia cases was observed. CONCLUSION Early diagnosis and appropriate therapy, along with the rigorous implementation of essential hospital infection control practices is required for successful containment of this pathogen and to curb such an outbreak.

Protein–protein interaction studies reveal the Plasmodium falciparum merozoite surface protein-1 region involved in a complex formation that binds to human erythrocytes

Plasmodium falciparum merozoite surface protein (PfMSP) 1 has been studied extensively as a vaccine candidate antigen. PfMSP-1 undergoes proteolytic processing into four major products, such as p83, p30, p38, and p42, that are associated in the form of non-covalent complex(s) with other MSPs. To delineate MSP1 regions involved in the interaction with other MSPs, here we expressed recombinant proteins (PfMSP-165) encompassing part of p38 and p42 regions and PfMSP-119 PfMSP-165 interacted strongly with PfMSP-3, PfMSP-6, PfMSP-7, and PfMSP-9, whereas PfMSP-119 did not interact with any of these proteins. Since MSP-1 complex binds human erythrocytes, we examined the ability of these proteins to bind human erythrocyte. Among the proteins of MSP-1 complex, PfMSP-6 and PfMSP-9 bound to human erythrocytes. Serological studies showed that PfMSP-165 was frequently recognized by sera from malaria endemic regions, whereas this was not the case for PfMSP-119 In contrast, antibodies against PfMSP-119 showed much higher inhibition of merozoite invasion compared with antibodies against the larger PfMSP-165 fragment. Importantly, anti-PfMSP-119 antibodies recognized both recombinant proteins, PfMSP-119 and PfMSP-165; however, anti-PfMSP-165 antibody failed to recognize the PfMSP-119 protein. Taken together, these results demonstrate that PfMSP-1 sequences upstream of the 19 kDa C-terminal region are involved in molecular interactions with other MSPs, and these sequences may probably serve as a smoke screen to evade antibody response to the membrane-bound C-terminal 19 kDa region.

Biochemical characterization of Plasmodium complement factors binding protein for its role in immune modulation

Complement system is the first line of human defence against intruding pathogens and is recognized as a potentially useful therapeutic target. Human malaria parasite Plasmodium employs a series of intricate mechanisms that enables it to evade different arms of immune system, including the complement system. Here, we show the expression of a multi-domain Plasmodium Complement Control Protein 1, PfCCp1 at asexual blood stages and its binding affinity with C3b as well as C4b proteins of human complement cascade. Using a biochemical assay, we demonstrate that PfCCp1 binds with complement factors and inhibits complement activation. Active immunization of mice with PfCCp1 followed by challenge with Plasmodium berghei resulted in the loss of biphasic growth of parasites and early death in comparison to the control group. The study also showed a role of PfCCp1 in modulating Toll-like receptor (TLR)-mediated signalling and effector responses on antigen-presenting cells. PfCCp1 binds with dendritic cells that down-regulates the expression of signalling molecules and pro-inflammatory cytokines, thereby dampening the TLR2-mediated signalling; hence acting as a potent immuno-modulator. In summary, PfCCp1 appears to be an important component of malaria parasite directed immuno-modulating strategies that promote the adaptive fitness of pathogens in the host.