Complete genome sequences of two strains of Treponema pallidum subsp. pertenue from Indonesia: Modular structure of several treponemal genes.

BACKGROUND:Treponema pallidum subsp. pertenue (TPE) is the causative agent of yaws, a multistage disease endemic in tropical regions in Africa, Asia, Oceania, and South America. To date, seven TPE strains have been completely sequenced and analyzed including five TPE strains of human origin (CDC-2, CDC 2575, Gauthier, Ghana-051, and Samoa D) and two TPE strains isolated from the baboons (Fribourg-Blanc and LMNP-1). This study revealed the complete genome sequences of two TPE strains, Kampung Dalan K363 and Sei Geringging K403, isolated in 1990 from villages in the Pariaman region of Sumatra, Indonesia and compared these genome sequences with other known TPE genomes. METHODOLOGY/PRINCIPAL FINDINGS:The genomes were determined using the pooled segment genome sequencing method combined with the Illumina sequencing platform resulting in an average coverage depth of 1,021x and 644x for the TPE Kampung Dalan K363 and TPE Sei Geringging K403 genomes, respectively. Both Indonesian TPE strains were genetically related to each other and were more distantly related to other, previously characterized TPE strains. The modular character of several genes, including TP0136 and TP0858 gene orthologs, was identified by analysis of the corresponding sequences. To systematically detect genes potentially having a modular genetic structure, we performed a whole genome analysis-of-occurrence of direct or inverted repeats of 17 or more nucleotides in length. Besides in tpr genes, a frequent presence of repeats was found in the genetic regions spanning TP0126-TP0136, TP0856-TP0858, and TP0896 genes. CONCLUSIONS/SIGNIFICANCE:Comparisons of genome sequences of TPE Kampung Dalan K363 and Sei Geringging K403 with other TPE strains revealed a modular structure of several genomic loci including the TP0136, TP0856, and TP0858 genes. Diversification of TPE genomes appears to be facilitated by intra-strain genome recombination events

Reliability of Nucleic Acid Amplification Methods for Detection of Chlamydia trachomatis in Urine: Results of the First International Collaborative Quality Control Study among 96 Laboratories

The first European Quality Control Concerted Action study was organized to assess the ability of laboratories to detect Chlamydia trachomatis in a panel of urine samples by nucleic acid amplification tests (NATs). The panel consisted of lyophilized urine samples, including three negative, two strongly positive, and five weakly positive samples. Ninety-six laboratories in 22 countries participated with a total of 102 data sets. Of 204 strongly positive samples 199 (97.5%) were correctly reported, and of 506 weakly positive samples 466 (92.1%) were correctly reported. In 74 (72.5%) data sets correct results were reported on all samples, and 17 data sets (16.7%) showed either one false-negative or one false-positive result. In another 11 data sets, two or more incorrect results were reported, and two data sets reported a false-positive result on one negative sample. The Roche COBAS Amplicor test was performed in 44 (43%) data sets, the Abbott LCx assay was performed in 31 (30%) data sets, the Roche Amplicor manual assay was performed in 9 (9%) data sets, an in-house PCR was performed in 9 (9%) data sets, the Becton Dickinson ProbeTec ET assay was performed in 5 (4.9%) data sets, and the GenProbe TMA assay was performed in 4 (3.9%) data sets. The results of the Roche Amplicor manual (95.6% correct), COBAS Amplicor (97.0%), and Abbott LCx (94.8%) tests were comparable (P = 0.48). The results with the in-house PCR, BD ProbeTec ET, and GenProbe TMA tests were reported correctly in 88.6, 98, and 92.5% of the tests, respectively. Freeze-drying of clinical urine specimens proved to be a successful method for generating standardized, stable, and easy-to-transport samples for the detection of C. trachomatis by using NATs. Although the results, especially the specificity, for this proficiency panel were better than most quality control studies, sensitivity problems occurred frequently, underlining the need for good laboratory practice and reference reagents to monitor the performance of these assays

Diagnosis of Mycobacterium microti Infections among Humans by Using Novel Genetic Markers

As a result of DNA typing of Mycobacterium microti isolates from animals in the United Kingdom and The Netherlands, we diagnosed four human M. microti infections. These are the first M. microti infections among humans to be reported. Three of the patients were immunocompromised and suffered from generalized forms of tuberculosis. The fourth patient was a 34-year-old immunocompetent male with a persistent cough and undefined X-ray abnormalities. Two of the M. microti infections were recognized by their IS6110 restriction fragment length polymorphism (RFLP) patterns, which showed a high degree of similarity with those of M. microti strains isolated from a pig and a ferret in The Netherlands. The two other human M. microti infections were recognized by using the recently developed DNA fingerprinting method, “spoligotyping,” directly on clinical material. All M. microti isolates from the United Kingdom and The Netherlands were found to contain an exceptionally short genomic direct repeat region, resulting in identical two-spacer sequence reactions in spoligotyping. In contrast, the highly similar IS6110 RFLP patterns of the vole strains from the United Kingdom differed considerably from the RFLPs of all M. microti strains isolated in The Netherlands, suggesting that geographic isolation led to divergent strains in the United Kingdom and on the continent

Multicenter Evaluation of Epidemiological Typing of Methicillin-Resistant Staphylococcus aureus Strains by Repetitive-Element PCR Analysis

Rapid and efficient epidemiologic typing systems would be useful to monitor transmission of methicillin-resistant Staphylococcus aureus (MRSA) at both local and interregional levels. To evaluate the intralaboratory performance and interlaboratory reproducibility of three recently developed repeat-element PCR (rep-PCR) methods for the typing of MRSA, 50 MRSA strains characterized by pulsed-field gel electrophoresis (PFGE) (SmaI) analysis and epidemiological data were blindly typed by inter-IS256, 16S-23S ribosomal DNA (rDNA), and MP3 PCR in 12 laboratories in eight countries using standard reagents and protocols. Performance of typing was defined by reproducibility (R), discriminatory power (D), and agreement with PFGE analysis. Interlaboratory reproducibility of pattern and type classification was assessed visually and using gel analysis software. Each typing method showed a different performance level in each center. In the center performing best with each method, inter-IS256 PCR typing achieved R = 100% and D = 100%; 16S-23S rDNA PCR, R = 100% and D = 82%; and MP3 PCR, R = 80% and D = 83%. Concordance between rep-PCR type and PFGE type ranged by center: 70 to 90% for inter-IS256 PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS256 PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility. None of these assays, however, was sufficiently reproducible for interlaboratory exchange of data