QuantiFERON-TB Gold plus testing for the detection of LTBI among health care workers in major TB hospitals of the Northern Kyrgyz Republic.

BACKGROUND: Health care workers (HCW) are at increased risk of TB infection due to their close contact with infected patients with active TB. The objectives of the study were (1) to assess the prevalence of LTBI among HCW in the Northern Kyrgyz Republic, and (2) to determine the association of LTBI with job positions or departments. METHODS: HCWs from four TB hospitals in the Northern Kyrgyz Republic were tested with the interferon-gamma release assay (IGRA) Quantiferon-TB Gold plus (QFT) for the detection of an immune response to TB as marker of TB infection. Age was controlled for as a confounder. Univariate and multivariable analysis were performed using logistic regression to assess the association of the risk factors (job position, and department) with having a QTF positive result. Firth's penalized-likelihood estimates were used to account for the small-sample size. Pairwise comparisons using the Bonferroni correction (conservative) and comparisons without adjusting for multiple comparisons (unadjusted) were used to identify the categories where differences occurred. RESULTS: QFT yielded valid results for 404 HCW, with 189 (46.7%) having a positive test. In the National Tuberculosis Center there was an increased odds to have a positive QFT test for the position of physician (OR = 8.7, 95%, CI = 1.2-60.5, p = 0.03) and laboratory staff (OR = 19.8, 95% CI = 2.9-135.4, p < 0.01) when administration staff was used as the baseline. When comparing departments for all hospitals combined, laboratories (OR 7.65; 95%CI 2.3-24.9; p < 0.001), smear negative TB (OR 5.90; 95%CI 1.6-21.8; p = 0.008), surgery (OR 3.79; 95%CI 1.3-11.4; p = 0.018), and outpatient clinics (OR 3.80; 95%CI 1.1-13.0; p = 0.03) had higher odds of a positive QFT result than the admin department. Fifteen of the 49 HCW with follow-up tests converted from negative to positive at follow-up testing. CONCLUSIONS: This is the first report on prevalence and risk factors of LTBI for HCW in the Kyrgyz republic, and results indicate there may be an increased risk for LTBI among physicians and laboratory personnel. Further research should investigate gaps of infection control measures particularly for physicians and laboratory staff and lead to further improvement of policies.

Implementation of whole genome sequencing for tuberculosis diagnostics in a low-middle income, high MDR-TB burden country.

Whole genome sequencing (WGS) is revolutionary for diagnostics of TB and its mutations associated with drug-resistances, but its uptake in low- and middle-income countries is hindered by concerns of implementation feasibility. Here, we provide a proof of concept for its successful implementation in such a setting. WGS was implemented in the Kyrgyz Republic. We estimated needs of up to 55 TB-WGS per week and chose the MiSeq platform (Illumina, USA) because of its capacity of up to 60 TB-WGS per week. The project's timeline was completed in 93-weeks. Costs of large equipment and accompanying costs were 222,065 USD and 8462 USD, respectively. The first 174 WGS costed 277 USD per sequence, but this was skewed by training inefficiencies. Based on real prices and presuming optimal utilization of WGS capacities, WGS costs could drop to 167 and 141 USD per WGS using MiSeq Reagent Kits v2 (500-cycles) and v3 (600-cycles), respectively. Five trainings were required to prepare the staff for autonomous WGS which cost 48,250 USD. External assessment confirmed excellent performance of WGS by the Kyrgyz laboratory in an interlaboratory comparison of 30 M. tuberculosis genomes showing complete agreeance of results.

Implementing contact tracing for tuberculosis in Kyrgyz Republic and risk factors for positivity using QuantiFERON-TB Gold plus.

BACKGROUND: Effective active case finding (ACF) activities are essential for early identification of new cases of active tuberculosis (TB) and latent TB infection (LTBI). Accurate diagnostics as well as the ability to identify contacts at high risk of infection are essential for ACF, and have not been systematically reported from Central Asia. The objective was to implement a pilot ACF program to determine the prevalence and risk factors for LTBI and active TB among contacts of individuals with TB in Kyrgyz Republic using Quantiferon-TB Gold plus (QuantiFERON). METHODS: An enhanced ACF project in the Kyrgyz Republic was implemented in which close and household (home) contacts of TB patients from the Issyk-Kul Oblast TB Center were visited at home. QuantiFERON and the tuberculin skin test (TST) alongside clinical and bacteriological examination were used to identify LTBI and active TB cases among contacts. The association for QuantiFERON positivity and risk factors were analysed and compared to TST results. RESULTS: Implementation of ACF with QuantiFERON involved close collaboration with the national sanitary and epidemiological services (SES) and laboratories in the Kyrgyz Republic. From 67 index cases, 296 contacts were enrolled of whom 253 had QuantiFERON or TST results; of those 103 contacts had LTBI (positive TST or IGRA), and four (1.4%) active TB cases were detected. Index case smear microscopy (OR 1.76) and high household density (OR 1.97) were significant risk factors for QuantiFERON positivity for all contacts. When stratified by age, association with smear positivity disappeared for children below 15 years. TST was not associated with any risk factor. CONCLUSIONS: This is the first time that ACF activities have been reported for Central Asia, and provide insight for implementation of effective ACF in the region. These ACF activities using QuantiFERON led to increase in the detection of LTBI and active cases, prior to patients seeking treatment. Household density should be taken into consideration as an important risk factor for the stratification of future ACF activities.

Population structure of drug-resistant Mycobacterium tuberculosis in Central Asia.

BACKGROUND: Drug-resistant tuberculosis (TB) is a major public health concern threathing the success of TB control efforts, and this is particularily problematic in Central Asia. Here, we present the first analysis of the population structure of Mycobacterium tuberculosis complex isolates in the Central Asian republics Uzbekistan, Tajikistan, and Kyrgyzstan. METHODS: The study set consisted of 607 isolates with 235 from Uzbekistan, 206 from Tajikistan, and 166 from Kyrgyzstan. 24-loci MIRU-VNTR (Mycobacterial Interspersed Repetitive Units - Variable Number of Tandem Repeats) typing and spoligotyping were combined for genotyping. In addition, phenotypic drug suceptibility was performed. RESULTS: The population structure mainly comprises strains of the Beijing lineage (411/607). 349 of the 411 Beijing isolates formed clusters, compared to only 33 of the 196 isolates from other clades. Beijing 94-32 (n = 145) and 100-32 (n = 70) formed the largest clusters. Beijing isolates were more frequently multidrug-resistant, pre-extensively resistant (pre-XDR)- or XDR-TB than other genotypes. CONCLUSIONS: Beijing clusters 94-32 and 100-32 are the dominant MTB genotypes in Central Asia. The relative size of 100-32 compared to previous studies in Kazakhstan and its unequal geographic distribution support the hypothesis of its more recent emergence in Central Asia. The data also demonstrate that clonal spread of resistant TB strains, particularly of the Beijing lineage, is a root of the so far uncontroled MDR-TB epidemic in Central Asia.

Evaluation of the Abbott RealTime MTB and RealTime MTB INH/RIF Assays for Direct Detection of Mycobacterium tuberculosis Complex and Resistance Markers in Respiratory and Extrapulmonary Specimens.

The Abbott RealTime MTB (RT MTB) assay is a new automated nucleic acid amplification test for the detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. In combination with the RealTime MTB INH/RIF (RT MTB INH/RIF) resistance assay, which can be applied to RT MTB-positive specimens as an add-on assay, the tests also indicate the genetic markers of resistance to isoniazid (INH) and rifampin (RIF). We aimed to evaluate the diagnostic sensitivity and specificity of RT MTB using different types of respiratory and extrapulmonary specimens and to compare performance characteristics directly with those of the FluoroType MTB assay. The resistance results obtained by RT MTB INH/RIF were compared to those from the GenoType MTBDRplus and from phenotypic drug susceptibility testing. A total of 715 clinical specimens were analyzed. Compared to culture, the overall sensitivity of RT MTB was 92.1%; the sensitivity rates for smear-positive and smear-negative samples were 100% and 76.2%, respectively. The sensitivities of smear-negative specimens were almost identical for respiratory (76.3%) and extrapulmonary (76%) specimens. Specificity rates were 100% and 95.8% for culture-negative specimens and those that grew nontuberculous mycobacteria, respectively. RT MTB INH/RIF was applied to 233 RT MTB-positive samples and identified resistance markers in 7.7% of samples. Agreement with phenotypic and genotypic drug susceptibility testing was 99.5%. In conclusion, RT MTB and RT MTB INH/RIF allow for the rapid and accurate diagnosis of tuberculosis (TB) in different types of specimens and reliably indicate resistance markers. The strengths of this system are the comparably high sensitivity with paucibacillary specimens, its ability to detect INH and RIF resistance, and its high-throughput capacities.

Comparison of Xpert MTB/RIF with ProbeTec ET DTB and COBAS TaqMan MTB for direct detection of M. tuberculosis complex in respiratory specimens.

BACKGROUND: Nucleic acid amplification assays allow for the rapid and accurate detection of Mycobacterium tuberculosis (MTB) directly in clinical specimens thereby facilitating diagnosis of tuberculosis (TB). With the fully automated Xpert MTB/RIF system (Cepheid) an innovative solution of TB diagnostics has been launched. We performed a direct head-to-head comparison of Xpert MTB/RIF with two widely used commercial assays, ProbeTec ET DTB (DTB) (Becton-Dickinson) and COBAS TaqMan MTB (CTM-MTB) (Roche). METHODS: 121 pre-characterized respiratory specimens (68 culture-positive for MTB complex, 24 culture-positive for non-tuberculous mycobacteria and 29 culture-negative) taken from our frozen specimen bank were tested for the presence of MTB complex by the three assays. RESULTS: Among culture-positive samples (n = 68), overall sensitivity for detection of MTB complex was 74.6%, 73.8%, and 79.1% for Xpert MTB/RIF, CTM-MTB, and DTB, respectively. Within the subgroup of smear-negative TB samples (n = 51) sensitivity was 68% for Xpert MTB/RIF and CTM-MTB and 72% for DTB. Among smear-positive TB samples (n = 17), all (100%) were detected by DTB and 94.1% and 93.3% by Xpert MTB/RIF and CTM-MTB, respectively. Specificity was best for CTM-MTB (100%) and lowest for Xpert MTB/RIF (96.2%) due to misidentification of two NTM samples as MTB complex. CTM-MTB yielded the highest rate of invalid results (4.1%) (0.8% by Xpert MTB/RIF and DTB, respectively). CONCLUSIONS: The direct comparison of Xpert MTB/RIF with CTM-MTB and DTB yielded similar overall performance data. Whereas DTB was slightly superior to Xpert MTB/RIF in terms of sensitivity, at least in the sample collection tested here, CTM-MTB performed best in terms of specificity.

Yersinia enterocolitica palearctica serobiotype O:3/4--a successful group of emerging zoonotic pathogens.

BACKGROUND: High-pathogenic Y. enterocolitica ssp. enterocolitica caused several human outbreaks in Northern America. In contrast, low pathogenic Y. enterocolitica ssp. palearctica serobiotype O:3/4 is responsible for sporadic cases worldwide with asymptomatic pigs being the main source of infection. Genomes of three Y. enterocolitica ssp. palearctica serobiotype O:3/4 human isolates (including the completely sequenced Y11 German DSMZ type strain) were compared to the high-pathogenic Y. enterocolitica ssp. enterocolitica 8081 O:8/1B to address the peculiarities of the O:3/4 group. RESULTS: Most high-pathogenicity-associated determinants of Y. enterocolitica ssp. enterocolitica (like the High-Pathogenicity Island, yts1 type 2 and ysa type 3 secretion systems) are absent in Y. enterocolitica ssp. palearctica serobiotype O:3/4 genomes. On the other hand they possess alternative putative virulence and fitness factors, such as a different ysp type 3 secretion system, an RtxA-like and insecticidal toxins, and a N-acetyl-galactosamine (GalNAc) PTS system (aga-operon). Horizontal acquisition of two prophages and a tRNA-Asn-associated GIYep-01 genomic island might also influence the Y. enterocolitica ssp. palearctica serobiotype O:3/4 pathoadaptation. We demonstrated recombination activity of the PhiYep-3 prophage and the GIYep-01 island and the ability of the aga-operon to support the growth of the Y. enterocolitica ssp. enterocolitica O:8/1B on GalNAc. CONCLUSIONS: Y. enterocolitica ssp. palearctica serobiotype O:3/4 experienced a shift to an alternative patchwork of virulence and fitness determinants that might play a significant role in its host pathoadaptation and successful worldwide dissemination.

Complete genome sequence of Yersinia enterocolitica subsp. palearctica serogroup O:3.

We report here the first finished and annotated genome sequence of a representative of the most epidemiologically successful Yersinia group, Y. enterocolitica subsp. palearctica strain Y11, serotype O:3, biotype 4. This strain is a certified type strain of the German DSMZ collection (DSM no. 13030; Yersinia enterocolitica subsp. palearctica) that was isolated from the stool of a human patient (H. Neubauer, S. Aleksic, A. Hensel, E. J. Finke, and H. Meyer. Int. J. Med. Microbiol. 290:61-64, 2000).

Structures of the arm-type binding domains of HPI and HAI7 integrases.

The structures of the N-terminal domains of two integrases of closely related but not identical asn tDNA-associated genomic islands, Yersinia HPI (high pathogenicity island; encoding siderophore yersiniabactin biosynthesis and transport) and an Erwinia carotovora genomic island with yet unknown function, HAI7, have been resolved. Both integrases utilize a novel four-stranded beta-sheet DNA-binding motif, in contrast to the known proteins that bind their DNA targets by means of three-stranded beta-sheets. Moreover, the beta-sheets in Int(HPI) and Int(HAI7) are longer than those in other integrases, and the structured helical N terminus is positioned perpendicularly to the large C-terminal helix. These differences strongly support the proposal that the integrases of the genomic islands make up a distinct evolutionary branch of the site-specific recombinases that utilize a unique DNA-binding mechanism.

Staphylococcus pettenkoferi sp. nov., a novel coagulase-negative staphylococcal species isolated from human clinical specimens.

Five coagulase-negative, novobiocin-susceptible staphylococcal strains were isolated from human blood cultures in different German and Belgian medical facilities. A novel species, 'Staphylococcus pettenkoferi' was proposed recently to accommodate two of these strains (B3117(T) and A6664), although the name was not validly published. All five strains belonged to the genus Staphylococcus because they were non-motile, Gram-positive, catalase-positive cocci with peptidoglycan type (A3 alpha type L-lys-gly(2-4)-L-Ser-Gly), menaquinone pattern (MK-7, MK-6 and MK-8) and major cellular fatty acids (ai-C(15 : 0), ai-C(17 : 0) and i-C(15 : 0)) that corresponded to those of staphylococci. Phenotypically, the isolates most closely resembled Staphylococcus capitis subsp. capitis and Staphylococcus auricularis, but they could be distinguished from these species by physiological tests and chemotaxonomic investigations. The results of DNA-DNA hybridization, chemotaxonomic investigations and 16S rRNA gene and RNA polymerase B gene (rpoB) sequence analysis enabled strains B3117(T), K6999, 229 and 230 to be differentiated genotypically and phenotypically from known Staphylococcus species, indicating that these isolates are representatives of a novel species. The name Staphylococcus pettenkoferi sp. nov. is proposed for this novel species, with strain B3117(T) (=CIP 107711(T)=CCUG 51270(T)) as the type strain. Due to differences in the results of physiological and chemotaxonomic investigations and DNA-DNA hybridization data, strain A6664 was not included in the description of the novel species.

Independent acquisition of site-specific recombination factors by asn tRNA gene-targeting genomic islands.

Two genomic islands, namely the high-pathogenicity island (HPI) and Ecoc54N target the same asn tRNA genes to integrate into the bacterial chromosome. The HPI encodes the siderophore yersiniabactin in the highly pathogenic Yersinia group (Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica 1B) whilst the Ecoc54N island possibly encodes a polyketide synthase with an unknown function in the uropathogenic Escherichia coli CFT073 strain. HPI encodes the recombinase that promotes site-specific recombination (both integrative and excisive) with its corresponding attachment targets. A recombinase orthologue is also present in Ecoc54N. In addition, the HPI(Yps) of the Y. pestis/Y. pseudotuberculosis evolutionary lineage encodes the excisionase (recombination directionality factor, Xis(HPI)) that facilitates excision of the island. However, no sequence resembling the excisionase gene could be found in Ecoc54N. The rate of the HPI(Yps) excision estimated by real-time PCR was 10(-6) in Y. pseudotuberculosis. The presence of the excisionase increased the efficiency of the excisive recombination only eight fold. However, the introduction of the xis(HPI) in E. coli CFT073 did not influence the excision of Ecoc54N. The Xis(HPI) is encoded by the variable AT-rich part of the HPI(Yps) and substantially differs from its cognate recombinase in A+T content and codon usage. Also the Xis(HPI)-protected region, defined in the HPI attachment site, has suffered several nucleotide substitutions in Ecoc54N that could influence interaction with the excisionase. We propose that the pathogenicity islands (PAIs) targeting asn tRNA genes (PAIs(asn tRNA)) might have acquired recombinase and excisionase (HPI) genes independently and sequentially.

Horizontal transfer of Yersinia high-pathogenicity island by the conjugative RP4 attB target-presenting shuttle plasmid.

The high-pathogenicity island (HPI) encodes a highly efficient yersiniabactin system of iron acquisition responsible for mouse lethality in Yersinia. Although the HPI is widely disseminated among Enterobacteriaceae it lacks functions necessary for its replication and transmission. Therefore, the mechanism of its horizontal transfer and circulation is completely obscure. On the other hand, the HPI is a genetically active island in the bacterial cell. It encodes a functional recombinase and is able to transpose to new targets on the chromosome. Here we report on a possible mechanism of the HPI dissemination based on site-specific recombination of the excised HPI with the attB-presenting (asn tRNA gene) RP4 promiscuous conjugative shuttle plasmid. The resulting cointegrate can be transferred by conjugation to a new host, where it dissociates, and the released HPI integrates into any unoccupied asn tRNA gene target in the genome. This mechanism has been proven both with the 'mini' island carrying only the attP recognition site and genes coding for recombination enzymes and with the complete HPI labelled with an antibiotic resistance marker. After acquisition of the mobilized complete form of the HPI, the ability of the HPI-cured Yersinia enterocolitica WA-TH(-) strain to produce yersiniabactin has been restored. Such 'trapping' of pathogenicity islands and subsequent shuffling to new hosts by a conjugative replicon carrying a suitable attB site could be applied to other functional integrative elements and explain wide dissemination of PAIs.