Evaluation of the clinical sensitivity and specificity of the BD Max™ Enteric Bacterial Panel for molecular detection of pathogens for acute gastroenteritis in the Singaporean population.

PURPOSE: Acute gastroenteritis (AGE) is caused by a wide range of pathogens. Culture methods for the detection of bacterial pathogens is time consuming and labour intensive. This study compared a same-day-to-result commercial molecular method using BD Max™ Enteric Bacterial Panel against conventional culture and laboratory-developed PCR assays (LDTs), and characterised the epidemiology of bacterial AGE in Singapore. METHODOLOGY: PCRs for Campylobacter spp., Salmonella spp., Shigella spp./Enteroinvasive Escherichia coli (EIEC) and Shiga toxin-producing E. coli (STEC)/Shigella dysenteriae were performed on the BD Max™ platform. Concurrent routine bacterial culture ("reference standard") was performed for Campylobacter, Salmonella, Shigella, Vibrio and Aeromonas spp. In the event of a discrepancy, an "expanded reference standard" (bacterial culture with LDT) was used. RESULTS: There were 299 stool specimens in the study, with no bacterial pathogens detected in 190 samples (63.5%). The positive samples (n = 109,36.5%) were detected with Salmonella (n = 57,19.1%), Campylobacter (n = 28,9.4%), Vibrio parahaemolyticus (n = 6,2.0%), Shigella/EIEC (n = 6,2.0%), ETEC (n = 4,1.3%), STEC (n = 2,0.7%), Aeromonas (n = 2,0.7%), Plesiomonas shigelloides (n = 1,0.3%) and 3(1.0%) co-infections. Compared to the "expanded reference standard", conventional culture missed 38/112 (33.9%) pathogens. Conversely, testing by BD Max™ alone failed to detect 17 pathogens. BD Max™ reported seven (2.3%) false-positive results. CONCLUSIONS: BD Max™ increased the detection rate of bacterial AGE pathogens in the panel, but was limited by the absence of detection capability for Vibrio and Aeromonas spp.

Development and validation of a real-time multiplex PCR assay for the detection of dermatophytes and Fusarium spp.

Introduction: Onychomycosis is a debilitating, difficult-to-treat nail fungal infection with increasing prevalence worldwide. The main etiological agents are dermatophytes, which are common causative pathogens in superficial fungal mycoses. Conventional detection methods such as fungal culture have low sensitivity and specificity and are time-consuming.Aim: The main objective of this study was to design, develop and validate a real-time probe-based multiplex qPCR assay for the detection of dermatophytes and Fusarium species.Methodology: The performance characteristics of the qPCR assays were evaluated. The multiplex qPCR assays targeted four genes (assay 1: pan-dermatophytes/Fusarium spp.; assay 2: Trichophyton rubrum/Microsporum spp.). Analytical validation was accomplished using 150 fungal isolates and clinical validation was done on 204 nail specimens. The performance parameters were compared against the gold standard (fungal culture) and expanded gold standard (culture in conjunction with sequencing).Results: Both the single-plex and multiplex qPCR assays performed well especially when compared against the expanded gold standard. Among the 204 tested nail specimens, the culture method showed that 125 (61.3 %) were infected with at least one organism, of which 40 yielded positive results for dermatophytes and Fusarium spp. These target organisms detected include 20 dermatophytes and 22 Fusarium spp. The developed qPCR assays demonstrated excellent limit of detection, efficiency, coefficient of determination, analytical and clinical sensitivity and specificity.Conclusion: The multiplex qPCR assays were reliable for the diagnosis of onychomycosis, with shorter turn-around time as compared to culture method. This aids in the planning of treatment strategies to achieve optimal therapeutic outcome.

Evaluation of bacterial recovery and viability from three different swab transport systems.

This study evaluated three types of swab transport systems for organism recovery. Swabs with transport media were further assessed for organism viability over 24  hours over a range of different storage temperatures. Test organisms consisted of aerobes, fastidious aerobes and anaerobes. Swabs were tested according to the standardised quantitative elution method published by the Clinical Laboratory Standards Institute (CLSI; M-40A). There were substantial differences in primary organism recovery, with recovery rates from different swabs ranging from <0.1% to 78% for Streptococcus pyogenes. Similar differences were noted for other test organisms. In general, the flocked swab (ESwab) demonstrated highest rates of recovery for aerobic organisms, while higher rates of recovery of Fusobacterium nucleatum were demonstrated from a standard swab (Transwab). When considering organism viability, no single swab fulfilled all the criteria stipulated by the M-40A standard for all organism/temperature combinations. Organism viability was marginally better for the gel-based swab transport systems as compared to the liquid-media based ESwab. Significant differences between swab transport systems were demonstrated, including differences for primary organism recovery and viability. The ESwab showed the best recovery of organisms, while gel-based media demonstrated marginally better bacterial viability for most tested retention times and temperatures.