Universal extraction method for gastrointestinal pathogens.

A universal stool extraction method for recovery of nucleic acids (NAs) from gastrointestinal pathogens was developed to support rapid diagnostics for the London 2012 Olympics. The method involved mechanical disruption (bead beating) of the stools, followed by automated extraction and detection using real-time PCR. This method had been used extensively in the Second Infectious Intestinal Disease Study (IID2) for the isolation of NA from bacteria and parasites (and was effective for the robust recovery of Cryptosporidium spp.) but had not been used for enteric viruses. To ensure this method was universally suitable, panels of samples known to contain target bacteria, viruses or parasites were processed in triplicate using the pre-treatment method routinely used for each target and the new extraction method (bead beating). The extracts were tested using real-time PCR and the cycle threshold values were compared. The results from this study showed that bead beating improved yields for the bacterial and parasitic targets and was suitable for the viral targets. The implementation of this universal method should confer cost- and time-saving benefits and streamline the processes required for the characterization of an array of pathogens from faecal samples.

First molecular epidemiology study of Mycobacterium tuberculosis in Kiribati.

Tuberculosis incidence rates in Kiribati are among the highest in the Western Pacific Region, however the genetic diversity of circulating Mycobacterium tuberculosis complex strains (MTBC) and transmission dynamics are unknown. Here, we analysed MTBC strains isolated from culture positive pulmonary tuberculosis (TB) cases from the main TB referral centre between November 2007 and October 2009. Strain genotyping (IS6110 typing, spoligotyping, 24-loci MIRU-VNTR and SNP typing) was performed and demographic information collected. Among 73 MTBC strains analysed, we identified seven phylogenetic lineages, dominated by Beijing strains (49%). Beijing strains were further differentiated in two main branches, Beijing-A (n = 8) and -B (n = 28), that show distinct genotyping patterns and are characterized by specific deletion profiles (Beijing A: only RD105, RD207 deleted; Beijing B: RD150 and RD181 additionally deleted). Many Kiribati strains (59% based on IS6110 typing of all strains) occurred in clusters, suggesting ongoing local transmission. Beijing-B strains and over-crowded living conditions were associated with strain clustering (likely recent transmission), however little evidence of anti-tuberculous drug resistance was observed. We suggest enhanced case finding amongst close contacts and continued supervised treatment of all identified cases using standard first-line drugs to reduce TB burden in Kiribati. Beijing strains can be subdivided in different principle branches that might be associated with differential spreading patterns in the population.

Comparative evaluation of the QIAGEN QIAsymphony® SP system and bioMérieux NucliSens easyMAG automated extraction platforms in a clinical virology laboratory.

BACKGROUND: The QIAGEN QIAsymphony(®) SP is an automated system that can process a variety of different sample types for nucleic acid extraction. OBJECTIVES: To compare this system against the bioMérieux NucliSens easyMAG using a range of common clinical sample types. STUDY DESIGN: Nucleic acid extracts were tested on 6 in-house real time viral PCR assays: quantitative cytomegalovirus (CMV); respiratory and enteric multiplex (10 viruses); influenza A H1N1 2009 specific H1 and N1 assays (sH1/N1); herpes simplex virus (HSV) 1, HSV 2 and varicella zoster virus (VZV) multiplex; norovirus genogroups I and II (Noro GI/II) multiplex. RESULTS: Extraction of the clinical sample by either QIAsymphony(®) SP or NucliSens easyMAG gave similar results for each PCR; CMV viral loads, 52 plasma samples had a mean difference (easyMAG-QIAsymphony(®)) of 0.002 log(10)copies/ml (s.d. 0.536), 52 whole blood samples had a mean difference of -0.232 log(10)copies/ml (s.d. 0.490). Concordance for the qualitative assays were; 64/67 (95.5%) for the respiratory and enteric multiplex, all 28 (100%) for the sH1, sN1 and influenza A matrix multiplex, 33/34 (97%) for the HSV1/HSV2/VZV multiplex and all 15 (100%) for the Noro GI/II. Inter- and intra-run variation, determined for a 10-fold dilution series of CMV (5.20-3.20 log(10)copies/ml), was less than 0.63 log(10)copies/ml. CONCLUSIONS: Our evaluation found the performance of the QIAsymphony(®) SP comparable to the NucliSens easyMAG for a range of sample types commonly extracted in a clinical virology laboratory. In total, 331/343 (96.5%) PCR results were concordant on samples extracted by both platforms.