ß-d-Glucan and Aspergillus Galactomannan assays in the diagnosis of invasive fungal infections.

BACKGROUND: With an increase in the number of patients who are immunosuppressed or immunocompromised there has been an increase in invasive fungal infection (IFI) in the past few decades with its associated high morbidity and mortality, ranging from 60% to 90%. The critical problem is the identification of the causative fungus and initiation of appropriate therapy. Hence, there is a requirement for better diagnostic methods for IFI. Detection of markers for the presence of fungi during early stage of the infection, such as constituents of the cell wall or fungal DNA, is essential for timely diagnosis of IFI. Galactomannan (GM) which is a cell wall surface antigen is the most studied diagnostic marker, followed by 1,3 ß-d-Glucan (BG) which is seen in deep layers of cell wall. METHODS: We have assessed the effectiveness of Galactomannan/ß-d-Glucan for the early diagnosis of IFI in immunosuppressed patients in our tertiary care setting. RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value of GM assay were 45%, 93%, 86% and 63% respectively, while the BG assay showed a sensitivity of 78%, specificity of 85%, Positive predictive value (PPV) 84% and Negative predictive value (NPV) 79%. CONCLUSION: BG assay is better for detection of IFI in patients with immunosuppression. However, a combination of both BG and GM assays would be the best approach as BG assay is highly sensitive, while the GM assay is highly specific for diagnosing IFI.

Speciation of fungi using real time PCR with molecular beacons: Can we solve the enigma of diagnosis of invasive fungal disease?

BACKGROUND: Invasive fungal diseases (IFDs) are difficult to diagnose and associated with high mortality rates, especially in the immunosuppressed. Species of Aspergillus and Candida are the cause of majority of invasive fungal disease however IFDs are also caused by Fusarium, Zygomycetes, Trichosporon, etc. Early detection is crucial for appropriate antifungal therapy. Blood cultures usually fail to isolate filamentous fungi, while detection of circulating beta-d-glucan or galactomannan antigens show variable sensitivity and specificity. There is a need of reliable, sensitive and specific diagnostic tests for IFDs. METHODS: A real-time Polymerase Chain Reaction (PCR) assay with a universal primer/molecular beacon system was developed for detecting and speciating most of the pathogenic fungi implicated in IFD. A single-reaction assay was designed targeting a carefully selected region of the ITS2 and ITS5 subunits of the fungal rDNA gene along with four molecular beacons capable of differential hybridization to the amplicons of different species. This generated a signature set of melting temperatures using the standard strains. The assay was tested on clinical specimens from patients with suspected invasive fungal disease. RESULTS: The assay was tested on 72 clinical samples and 72 healthy controls. Of these, 22 clinical samples (6/8 proven; 13/29 probable; 3/35 possible IFD, classified by the EORTC/MSG criteria) were positive by PCR and generated a set of melting temperatures enabling identification of the causative fungus. The assay was negative in all healthy controls. CONCLUSION: The molecular beacon assay is a promising tool providing a rapid method for detection and monitoring of invasive fungal disease in immunosuppressed patients.

Evaluation of three DNA extraction methods from fungal cultures.

Polymerase chain reaction (PCR) based assays have been developed to amplify DNA of fungal pathogens as culture-based detection methods show low sensitivity. In order to perform a sensitive, specific, and reliable PCR based assay, the availability of pure DNA as well as an easy-to-perform DNA extraction protocol is essential. The existing protocols for DNA extraction used for bacteria or viruses show poor release of fungal DNA. In this study, we evaluated three different methods of DNA extraction and compared their efficacy in the extraction of DNA from filamentous fungi, yeasts, and dermatophytes commonly isolated in our laboratory. It was found that the Fungi/Yeast Genomic DNA Isolation Kit (Norgen Biotek Corp, Ontario, Canada) demonstrated satisfactory extraction of DNA from all the fungi analyzed as compared to that of the Qiamp DNA extraction kit (Qiagen GmbH, Dusseldorf, Germany) or the Phenol Chloroform Isoamyl alcohol extraction method which failed to extract amplifiable DNA from many of the fungal species. Thus, we recommend the use of Fungi/Yeast Genomic DNA Isolation Kit (Norgen) with modifications for the extraction of DNA from fungal cultures.

Species distribution and antimicrobial resistance pattern of Coagulase-negative Staphylococci at a tertiary care centre.

BACKGROUND: Coagulase-negative Staphylococci (CoNS), previously dismissed at contaminants, have now emerged as an important cause of nosocomial infections especially in patients with implants and prosthetic devices. They are a well-known cause of bloodstream infections, urinary tract infections, wound infections, prosthetic valve endocarditis and eye infections. This study was conducted with an aim to identify CoNS at the species level from various clinical samples and determine the antimicrobial resistance pattern of these isolates. METHODS: This cross sectional study was carried out from September 2011 to February 2014 in which 150 non-repetitive clinical isolates of CoNS were identified at the species level by conventional phenotypic methods. Complete antimicrobial susceptibility profile was also determined by Kirby Bauer disc diffusion method. Susceptibility testing to vancomycin was done by E-test method. RESULTS: Only three species of CoNS were isolated, the most common being Staphylococcusepidermidis (60%) followed by Staphylococcussaprophyticus (27.3%) and Staphylococcushemolyticus (12.7%). Most S. epidermidis were isolated from blood and intravascular catheter tip samples, whereas all S. saprophyticus were isolated from urine samples of female patients. All isolates were found to be resistant to penicillin, but were susceptible to glycopeptides and linezolid and showed variable resistance to fluoroquinolones, aminoglycosides and macrolides. CONCLUSION: CoNS are emerging nosocomial pathogens and should not always be overlooked as contaminants. However, growth of CoNS from blood cultures and intravascular catheter tips should be clinically correlated and carefully interpreted. As many CoNS strains exhibit drug resistance, antimicrobial susceptibility profile should be determined prior to treatment of these infections.