In vitro adhesion and invasion by Cladosporium sphaerospermum in human bronchial epithelial cells (BEAS-2B) and human pulmonary alveolar epithelial cells (HPAEpiC)

@#Cladosporium spores are ubiquitous in indoor and outdoor environment and may potentially trigger allergic responses upon inhalation. To date, there is limited investigation on the fate of Cladosporium spores after being inhaled into the respiratory tract. This study was conducted to investigate the interaction of Cladosporium sphaerospermum with Human Bronchial Epithelial Cells (BEAS-2B) and Human Pulmonary Alveolar Epithelial Cells (HPAEpiC). C. sphaerospermum conidia were harvested and co-cultured with BEAS-2B or HPAEpiC cells for 72 hours. At each time point (30 minutes, 2, 4, 24, 48 and 72 hours), adherence and invasion of the cells by C. sphaerospermum conidia (and hyphae) were investigated by immunofluorescence staining. This study demonstrated the adherence and internalization of C. sphaerospermum conidia within these epithelial cells. In addition, the conidia were able to germinate and invade the epithelial cells. The ability of the fungal conidia to adhere, internalize, germinate and invade both the bronchial and alveolar epithelial cells of the respiratory tract in vitro might contribute to the understanding of the pathogenesis of Cladosporium in respiratory infection and allergy in vivo. INTRODUCTION Cladosporium species is a member of the phylum Ascomycota. The common species include C. herbarum, C. cladosporioides and C. sphaerospermum. This genus has worldwide distribution. Aerobiological studies reported that majority of fungal spores in outdoor air is from the phyla Ascomycota and Basidiomycota, while Cladosporium is one of the most studied allergenic Ascomycetes fungi (Knutsen et al., 2012). Cladosporium spores are found abundantly in indoors and outdoors at approximately 18/m3 and 141/m3 respectively (Codina et al., 2008). As an imperfect dematiaceous fungus, Cladosporium species causes opportunistic infections such

Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Candida glabrata, Candida parapsilosis and Candida rugosa from two major hospitals in Kuala Lumpur, Malaysia

@#The purpose of this study is to characterize 3 non-albicans Candida spp. that were collected from two major hospitals in a densely populated area of Kuala Lumpur for their susceptibilities to azole and genetic background. Fifteen non-albicans Candida clinical isolates in two major hospitals in Kuala Lumpur area of Malaysia were collected by convenience sampling during 2007 and 2010. The genetic diversity of 15 non-albicans Candida species comprising C. glabrata (n = 5), C. parapsilosis (n = 5) and C. rugosa (n = 5) were assessed by RAPD-PCR typing. Strains were initially identified using biochemical tests and CHROMagar Candida medium. Fluconazole and voriconazole susceptibilities were determined by E-test method. Commercial kits were used for DNA extraction and amplification with RAPD primers (OPA02, OPA03 and OPA08). PCR conditions were optimized and simultaneous identification was possible by agarose gel electrophoresis of PCR products and the bands obtained were analyzed using BioNumerics Applied Maths v.6.6 software. The RAPD primers used in this study generated 100% polymorphic profile. Cluster analysis using the RAPD-PCR profile showed 12.5-25% similarity among the strains. The genetic diversity was based on the strain susceptibility towards both the azoles, site of isolation and place according to their unique banding patterns. In contrast, strains susceptible to azoles were found to be genetically similar with clonal dissimilarity. The use of OPA02, OPA03 and OPA08 primers in differentiating non-albicans Candida spp. underscores the higher resolution of RAPD-PCR as a reliable tool for strain/species level differentiation.

Genetic diversity, antifungal susceptibility and enzymatic characterisation of Malaysian clinical isolates of Candida glabrata

@#Candida glabrata has been reported as the second or third most common yeast species isolated from patients with vaginitis and invasive candidiasis. This study was aimed to determine the genetic diversity, antifungal susceptibility and enzymatic profiles of C. glabrata isolated from vaginal and blood samples in the Medical Microbiology Diagnostic Laboratory, University Malaya Medical Centre. A random amplified polymorphic DNA (RAPD) analysis method, using M13 and (GTG)5 primers, was used for strain differentiation of C. glabrata isolates. Antifungal susceptibility testing of C. glabrata isolates was determined using E-test against amphotericin B, caspofungin, fluconazole and voriconazole and microbroth dilution method against clotrimazole. The enzymic profiles of C. glabrata were determined using APIZYM semi-quantitation kit and egg-yolk agar method. A total of 14 RAPD patterns were identified amongst C. glabrata isolates investigated this study. Susceptibility to amphotericin B, caspofungin, fluconazole and voriconazole was noted. Approximately one third of the isolates demonstrated resistance to clotrimazole (MIC>1 μg/ml). A single isolate of C. glabrata was resistant to caspofungin (MIC:1.5 μg/ml). Enzymatic activities of acid and alkaline phosphatase, aminopeptidases, esterase and lipase and phospholipase were detected in the C. glabrata isolates. The genetic diversity and antifungal susceptibility profiles of C. glabrata isolates were presented in this study. Continued surveillance and monitoring of the incidence and antifungal resistance in C. glabrata isolates is necessary.

Comparison of Anyplex II RV16 assay with conventional methods for detection of respiratory viruses

Early detection of viral etiologies of acute respiratory tract infections of patients affects management and disease control in pediatric patients. In this study, the performance of Anyplex II RV16 assay (Seegene, Seoul, Korea) was evaluated by comparing with viral culture and direct immunofluorescence staining of clinical specimens for detection of respiratory viruses in patients. A total of 168 respiratory specimens were collected from 122 patients from November 2012 to May 2013 at the time of admission to the University of Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. The Anyplex II RV16 assay, viral culture, and direct immunofluorescence staining were positive in 74.4%, 18.5% and 14.9% of the specimens, respectively. HRV was the predominant virus detected by the Anyplex II RV16 assay. In 47 cases, two or more respiratory viruses were detected by the Anyplex II RV16 assay, which were missed by conventional methods. The performance of the Anyplex II RV16 assay was better than viral culture and direct immunofluorescence staining of clinical specimens for the detection of respiratory viruses. The implementation of the Anyplex II RV16 assay in hospital laboratories will provide rapid diagnosis of major viral infections of the respiratory tract.

Identification of local clinical Candida isolates using CHROMagar CandidaTM as a primary identification method for various Candida species

The objective of our study was to study the effectiveness of CHROMagar CandidaTM as the primary identification method for various clinical Candida isolates, other than the three suggested species by the manufacturer. We studied 34 clinical isolates which were isolated from patients in a local teaching hospital and 7 ATCC strains. These strains were first cultured in Sabouraud dextrose broth (SDB) for 36 hours at 35ºC, then on CHROMagar plates at 30ºC, 35ºC and 37ºC. The sensitivity of this agar to identify Candida albicans, Candida dubliniensis, Candida tropicalis, Candida glabrata, Candida rugosa, Candida krusei and Candida parapsilosis ranged between 25 and 100% at 30ºC, 14% and 100% at 35ºC, 56% and 100% at 37ºC. The specificity of this agar was 100% at 30ºC, between 97% and 100% at 35ºC, 92% and 100% at 37ºC. The efficiency of this agar ranged between 88 and 100% at 30ºC, 83% and 100% at 35ºC, 88% and 100% at 37ºC. Each species also gave rise to a variety of colony colours ranging from pink to green to blue of different colony characteristics. Therefore, the chromogenic agar was found to be useful in our study for identifying clinical Candida isolates

Salmonellosis in persons infected with human immunodeficiency virus: a report of seven cases from Malaysia.

Persons infected with human immunodeficiency virus (HIV) have an increased risk of salmonellosis when compared to the general population. We describe seven such patients with Salmonella bacteremia, of whom two had recurrent salmonellosis. In the latter two cases the infection was unusually severe, characterized by widespread infection, bacteremia and relapse, despite standard antimicrobial therapy. HIV-infected individuals will benefit from education on the source of Salmonella, mode of acquisition and prevention through safe food handling and food preparation practices. Because of the difficulty of eradicating Salmonella infection in patients with acquired immunodeficiency syndrome, long-term suppressive treatment with antimicrobials is warranted.

Human immunodeficiency virus type 1 subtypes among Malaysian intravenous drug users.

The HIV-1 genetic variation in 60 infected Malaysian intravenous drug users (IDU) was studied by comparison of the nucleotide sequences and their predicted amino acid sequences in the V3 loop of the external glycoprotein gp120. In this study, HIV-1 B, C and E subtypes were identified among Malaysian IDU, with HIV-1 B being the predominant subtype (91.7%). HIV-1 C and HIV-1 E were minority subtypes among Malaysian IDU. Analysis of the amino acid alignment of the C2-V3 region of the env gene suggests a genetic relationship between Thai and Malaysian B and E subtype strains. This study serves as a baseline for monitoring HIV-1 genetic diversity and spread in Malaysia.