Evaluation of the BD MAX Enteric Parasite Panel for the detection of Cryptosporidium parvum/hominis, Giardia duodenalis and Entamoeba histolytica.

PURPOSE: Conventional laboratory detection methods for gastrointestinal parasites are time consuming, require considerable technical expertise and may suffer from poor analytical sensitivity. This study sought to evaluate the automated BD MAX Enteric Parasite Panel (EPP) for the detection of Cryptosporidium parvum/hominis, Entamoeba histolytica and Giardia duodenalis.Methodolgy. A total of 104 known positive samples (43 Cryptosporidium parvum/hominis and 61 G. duodenalis), 15 simulated samples (E. histolytica and other Entamoeba species) and 745 patient stool samples, submitted for enteric pathogen culture and microscopy, were inoculated into BD MAX EPP sample buffer tubes (SBTs). All specimens were blinded and tested within 7 days of SBT inoculation using the BD MAX EPP assay with results compared to those generated by microscopy.Results/Key findings. Combining the results from the known positive samples and anonymously tested patient samples, the sensitivity of the BD MAX EPP assay was 100 % for both Cryptosporidium spp. and G. duodenalis. Specificities of 99.7 and 98.9 % were calculated for the detection of Cryptosporidium spp. and G. duodenalis respectively. Insufficient clinical specimen data was available to determine the performance of the assay for E. histolytica detection. CONCLUSIONS: The findings of this study indicate that the BD MAX EPP is suitable for the detection of Cryptosporidium parvum/hominis and G. duodenalis from clinical specimens with reduced hands-on time and complexity compared to microscopy. Results for the detection of E. histolytica were promising although further work is required to evaluate the assay for the detection of this pathogen.

Evaluation of the Luminex xTAG Gastrointestinal Pathogen Panel and the Savyon Diagnostics Gastrointestinal Infection Panel for the detection of enteric pathogens in clinical samples.

Infectious gastrointestinal disease is caused by a diverse array of pathogens, and is a challenging syndrome to correctly diagnose and manage. Conventional laboratory diagnostic methods are often time-consuming and frequently suffer from low detection rates. Two commercial multiplex nucleic acid amplification tests [Luminex xTAG Gastrointestinal Pathogen Panel (GPP) and Savyon Diagnostics Gastrointestinal Infection Panel (GIP)] were applied to 1000 stored diarrhoeal clinical stool samples. The Luminex xTAG GPP and Savyon GIP detected Campylobacter in 42/44 and 44/44 culture-positive samples, Salmonella in 4/4 and 3/4 culture-positive samples, Shigella in 1/1 culture-positive sample, Clostridium difficile toxin in 32/35 ELISA-positive samples, and Giardia in 6/6 wet-preparation-microscopy-positive samples, respectively. When the Luminex GPP assay was used concurrently with conventional methods for 472 clinical samples, it detected Campylobacter in 22/22 culture-positive samples, Salmonella in 1/1 culture-positive sample, Clostridium difficile toxin in 14/14 ELISA-positive samples and Giardia in 4/4 wet-preparation-microscopy-positive samples. The pathogen/toxin detection rate for conventional methods in both sample sets was <10%. The Luminex xTAG GPP detection rate was 24.8% in the stored samples and 32.6% in the concurrently tested samples. The Savyon GIP detection rate was 22.5%. From stored samples, 2.4% of Luminex xTAG GPP detections and 3.1% of Savyon GIP detections could not be confirmed using alternative nucleic acid amplification tests. Enhanced detection rates resulted from increased detection of pathogens routinely sought using conventional methods and were also due to ascertainment of micro-organisms that current testing strategies do not diagnose. Use of multiplex nucleic acid amplification tests will allow clinical laboratories to diagnose infectious gastroenteritis in more patients with diarrhoeal disease by increasing the sensitivity of pathogen detection and by reducing the selective bias of current strategies. The clinical and economic impact of these results warrants further investigation.

Is confirmatory testing of Roche cobas 4800 CT/NG test Neisseria gonorrhoeae positive samples required? Comparison of the Roche cobas 4800 CT/NG test with an opa/pap duplex assay for the detection of N gonorrhoeae.

OBJECTIVES: Recently marketed nucleic acid amplification tests (NAAT) for the detection of Neisseria gonorrhoeae (NG) have improved specificity over previous generation assays. A study to assess the necessity for confirmation of Roche cobas 4800 NG positive samples was undertaken by the Public Health Wales Microbiology Molecular Diagnostic Unit in Cardiff. METHODS: Classical NG culture identification was compared to cobas 4800 (DR-9), opacity (opa) gene and porA pseudogene (pap) results. Confirmatory NAATs (opa/pap) were performed prospectively for 120 cobas 4800 NG positive urogenital and extragenital samples. Retrospective supplementary NAAT and sequence analysis of additional cobas 4800 NG positive extragenital samples was also carried out. RESULTS: Of the 188 classically identified clinical NG isolates, 184 were identified as NG in all 3 molecular targets. Two isolates were only detected by 2 molecular targets. A further 2 isolates were culture false-positives. Combining the results from prospective and retrospective testing, the sensitivity and negative predictive value for cobas 4800 NG detection for urogenital, rectal and oropharyngeal samples was 100%. Specificity for all sample types was greater than 99.7%. Positive predictive value was 96.0% and 96.4% for urogenital and rectal specimens, respectively, and 88.6% for oropharyngeal samples. CONCLUSIONS: Molecular tests could be used for culture confirmation where available. Roche cobas 4800 Chlamydia trachomatis/Neisseria gonorrhoeae (CT/NG)CT/NG gonorrhoea diagnosis is superior to culture with urogenital and rectal positives not requiring confirmation. Roche cobas 4800 oropharyngeal NG detection findings warrant further prospective study of routine confirmatory testing accounting for its cost and clinical usefulness.

Development and evaluation of a real-time nucleic acid sequence based amplification assay for rapid detection of influenza A.

The development and introduction of effective treatment for influenza A in the form of neuraminidase inhibitors have made the rapid diagnosis of infection important especially in high-risk populations. The aim of this study was to develop a real-time nucleic acid sequenced based amplification (NASBA) using a molecular beacon that could detect a wide range of influenza A subtypes and strains in a single reaction by targeting a conserved region of the influenza genome, and to evaluate its sensitivity and specificity against traditional laboratory techniques on a range of clinical samples usefulness during the 2003/2004 influenza season. The results demonstrated the assay to be highly sensitive and specific, detecting <0.1 TCID50 of virus stock. Three hundred eighty-nine clinical samples were tested in total from two patient groups. Overall, the real-time NASBA assay detected 64% (66/103) more influenza positive samples than cell culture and direct immunofluorescence (IF) and, therefore, was shown to be more sensitive in detecting influenza A in a wide range of respiratory samples than traditional methods. In conclusion, the real-time influenza A assay demonstrated clinical usefulness in both hospital and community populations.

Evidence for a dynamic host-parasite relationship in e-negative hepatitis B carriers.

Anti-hepatitis Be (HBe) carriers are perceived as having low infectivity, with hepatitis B virus (HBV) DNA levels far below those seen in the HBeAg carrier. However, the temporal stability of HBV DNA in anti-HBe carriers remains poorly characterised. UK Department of Health guidelines use HBV DNA levels to define whether HBV-infected health care workers may perform exposure-prone procedures. Two samples separated by 1-23 years available from 147 carriers were analysed for precore variants and HBV DNA levels. Among 15 HBeAg carriers, HBV DNA was maintained at high levels. There was a 5 log(10) fold reduction in DNA in 11 individuals who developed anti-HBe during follow-up evaluation. Proportional changes in HBV DNA levels in anti-HBe carriers were similar to those in HBeAg carriers, although there was a trend for changes in viral DNA to be more marked in anti-HBe carriers followed up for longer periods. Closer sampling in 20 anti-HBe carriers demonstrated large fluctuations of DNA levels over short periods. Serum transaminases and precore mutant status at the outset failed to predict those in whom HBV DNA levels fluctuated. HBV DNA was below the detection threshold (<400 copies/ml) in 36 anti-HBe carriers at first sampling and remained so in all but 5 of these carriers. Twelve individuals who were previously viraemic lost detectable HBV DNA during follow-up evaluation. While HBV DNA levels are found to fluctuate in carriers, our results indicate that once below the threshold of detectability, levels are unlikely to rise. This is an important factor when assessing health care workers for exposure-prone procedures.