Specific anti-Yersinia G- and M-antibodies in healthy blood donors and in patients with alimentary intoxication

The work deals with the results of determination of specific antibodies in blood donors of Moscow and Tula and in patients with alimentary toxicoinfection, made with the use of enzyme immunoassay on the basis of Yersinia enterocolitica lipopolysaccharides (LPS), serovars O3 and O9. The sera of patients with alimentary toxicoinfection were found to yield positive reactions with Y. enterocolitica LPS in 35.9% of cases (the number of such reactions obtained with blood donor sera was 3 times less). The presence of cross reactions between Y. enterocolitica LPS and the microsomal antigens of the thyroid gland was established. A high detection rate of antibodies to the microsomal antigens of the thyroid gland among blood donors of Tula was registered.     

A two-step strategy for molecular typing of multidrug-resistant Mycobacterium tuberculosis clinical isolates from Poland

Tuberculosis (TB) continues to be one of the most challenging public health problems in the world. An important contributor to the global burden of the disease is the emergence and spread of drug-resistant and particularly multidrug-resistant Mycobacterium tuberculosis strains (MDR), defined as being resistant to at least isoniazid and rifampicin. In recent years, the introduction of different DNA-based molecular typing methods has substantially improved the knowledge of the epidemiology of TB. The purpose of this study was to employ a combination of two PCR-based genotyping methods, namely spoligotyping and IS6110-Mtb1/Mtb2 PCR to investigate the clonal relatedness of MDR M. tuberculosis clinical isolates recovered from pulmonary TB patients from Poland. Among the 50 isolates examined, 28 (56%) were clustered by spoligotyping, whereas IS6110-Mtb1/Mtb2 PCR resulted in 16 (32%) clustered isolates. The isolates that clustered in both typing methods were assumed to be clonally related. A two-step strategy consisting of spoligotyping as a first-line test, performed on the entire pool of isolates, and IS6110-Mtb1/Mtb2 PCR typing as a confirmatory subtyping method, performed only within spoligotype-defined clusters, is an efficient approach for determining clonal relatedness among M. tuberculosis clinical isolates.     

IS6110 restriction fragment length polymorphism of Mycobacterium tuberculosis isolated from patients with pulmonary tuberculosis in Campinas,Brazil: evidence of intercontinental distribution of strains

Tuberculosis (TB) is a major concern in developing countries. In Brazil, few genotyping studies have been conducted to verify the number of IS6110 copies present in local prevalent strains of Mycobacterium tuberculosis, the distribution and clustering of strains. IS6110 DNA fingerprinting was performed on a sample of M. tuberculosis isolates from patients with AFB smear-positive pulmonary TB, at a hospital in Brazil. The IS6110 profiles were analyzed and compared to a M. tuberculosis database of the Houston Tuberculosis Initiative, Houston, US. Seventy-six fingerprints were obtained from 98 patients. All M. tuberculosis strains had an IS6110 copy number between 5-21 allowing for differentiation of the isolates. Human immunodeficiency virus infection was confirmed in nearly half the patients of whom data was available. Fifty-eight strains had unique patterns, while 17 strains were grouped in 7 clusters (2 to 6 strains). When compared to the HTI database, 6 strains matched isolates from El Paso, Ciudad de Juarez, Houston, and New York. Recently acquired infections were documented in 19% of cases. The community transmission of infection is intense, since some clustered strains were recovered during the four-year study period. The intercontinental dissemination of M. tuberculosis strains is suspected by demonstration of identical fingerprints in a distant country.     

IS6110 restriction fragment length polymorphism typing of Mycobacterium tuberculosis isolates from East Azerbaijan Province of Iran

To investigate the genetic variation among Mycobacterium tuberculosis isolates in the East Azerbaijan Province of Iran and to evaluate the level of and risk factors for recent transmission of tuberculosis (TB), we performed IS6110-based restriction fragment length polymorphism analysis of strains, isolated from 105 patients during the period of September 2002 to March 2003 in TB centers and university hospitals of the province. Among 105 isolates, 81 different IS6110 patterns were found, of which 70 were observed only once and 11 were shared by two to eight isolates. Ninety-six isolates (91.4%) were found to have more than five copies of IS6110 and together with high patterns polymorphism, shows that IS6110-RFLP typing could be useful for studying the epidemiology of TB in Azerbaijan. The minimum estimated rate of recent transmission was 23%, suggesting that the degree of recent transmission in East Azerbaijan Province is relatively low. Clustering was not associated with age, sex or site of infection of TB but drug-resistant isolates were less likely to be clustered than sensitive isolates (p < 0.05).     

Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of mycobacteria

A fast, sensitive and cost-effective multiplex-PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex-PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co-infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex-PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.     

Molecular evidence for independent occurrence of IS6110 insertions at the same sites of the genome of Mycobacterium tuberculosis in different clinical isolates

Several characteristics of Mycobacterium tuberculosis (e.g., conserved genome and low growth rate) have severely restricted the study of the microorganism. The discovery of IS6110 raised hopes of overcoming these obstacles. However, our knowledge of this IS element is relatively limited; even its two basic characteristics (transposition mechanism and target site selection) are far from well understood. In this study, IS6110 insertions in ipl loci (iplA and iplB) in two collections of clinical isolates of M. tuberculosis from different geographic locations, one from Scotland and the other from Thailand, were investigated. Five different IS6110 insertions in the loci were identified: ipl-4::IS6110, ipl-5::IS6110, ipl-11::IS6110, ipl-12::IS6110, and ipl-13::IS6110. An attempt to establish the phylogenetic relationship of the isolates containing these insertions was unsuccessful, suggesting that some of these insertions may have arisen from more than one event. This possibility is further supported by the observation that IS6110 copies existed in the same site but with different orientations in different isolates, and the insertion site of ipl-1::IS6110 harbored IS6110 copies in both iplA and iplB in different strains. All these suggest the independent occurrence of IS6110 insertions at the same sites of the genome of M. tuberculosis in different clinical isolates. The implications of this finding are discussed.     

IS6110 fingerprinting of sensitive and resistant strains (1991-1992) of Mycobacterium tuberculosis in Colombia

The standardized method to study the polymorphism of IS 6110 was used to characterize 53 isolates of Mycobacterium tuberculosis obtained during 1991-1992 from 14 regions in Colombia. In Valle region cluster rate was 25% (4/16). The mean number of IS6110 band was 10 +/- 3. Similarity between strains was of 60% in 81% of strains and this tended to be correlated with geographic origin. For the first time M. tuberculosis without IS6110 bands in restriction fragment length polymorphism analysis was found in Colombia. Additional studies are necessaries in order to best characterize the situation in relation to human immunodeficiency virus epidemic and recent changes in tuberculosis control program.     

Evaluation of reasons of the MDR M. tuberculosis strains dissemination by analysis of the rifampicin and/or isoniazid resistant isolates

During the last years in Novosibirsk region of Russia the rate of TB patients infected by MDR strains of M. tuberculosis has been constantly increasing. This increase may occur as a result of the spontaneously mutated mycobacterium selection during treatment of patients or as a result of primary infection by the resistant M. tuberculosis, or also, as a result of both reasons in combination. If the main reason of MDR strain dissemination is selection of resistant bacterium during patient treatment, the equal apportionment of the dominated mutation into the mycobacterium genotypes would be observed. If the main reason is the primary infection by resistant M. tuberculosis, the unequal apportionment would be revealed. For deeper understanding of the main reasons of the fast MDR strains spreading in the region, the distribution of the main mutations over genotypes of strains in Novosibirsk (170 isolates) and Tomsk prison (51 isolates) was investigated. Mutations in rpoB gene associated with the rifampicin resistance and in katG (isoniazid resistance) were detected by biochips. M. tuberculosis genotypings were carried out by IS6110 PCR typing or MIRU typing, in the last method the twelve loci (MIRU 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40) have been used. The most frequent mutation in the rpoB gene was Ser531-->Leu (60-70% of the rifampicin resistant strains) and Ser315-->Thr in gene katG (80% of the isoniazid resistant M. tuberculosis). Both in Novosibirsk and in Tomsk prison the rates of clustered cases transmissions were high (69 and 63% respectively). Analysis of the distribution of the dominated mutations Ser531-->Leu (rpoB) and Ser315-->Thr (katG) revealed that all of them were detected in each clusters, but in Novosibirsk there were only two clusters, in which the percentage of strains, containing mutation Ser531-->Leu (rpoB) were higher (85.7% and 77.7% respectively, P < 0.05), then in others. Among the Tomsk prison's clusters it was revealed one in which the proportion of the Ser3 15-->Thr mutation in katGwas higher (96.4%, P < 0.05). The nonuniform distribution of the dominated mutations highlighted that the epidemic spread of drug-resistant strains of M. tuberculosis in region resulted from the selection of them during patient treatment and the subsequent transmission by TB patients.     

Differentiation of the Mycobacterium tuberculosis W-Beijing family strains from the Russian Federation by the VNTR-typing

Recent phylogenetic studies allowed the Mycobacterium tuberculosis complex to be divided into a number of the strain families. The W-Beijing family is one of most widespread M. tuberculosis variants frequently causing epidemic outbreaks. This family is genetically homogenous and conserved so that ETR A, B, C, D, E - typing is insufficient for the W-Beijing differentiation. All W-Beijing isolates have common profile (42435). This led to the false clustering in the molecular epidemiology study, especially in the region of predominance of the W-Beijing family. In this investigation we searched for the VNTR loci with high evolution rate, which were polymorphic in the W-Beijing genome. Eleven VNTR-loci were assayed in the DNA panel of 99 M. tuberculosis isolates from the tuberculosis patients in North-West and West-Siberian regions of Russia during the period from 2000 to 2001. Ninety nine strains of M. tuberculosis were divided into 74 VNTR-types, 51 isolates of the W-Beijing family were subdivided into 30 VNTR-types. The Hunter-Gudson index (HGDI) for all studied loci (ETR-A, ETR-C, ETR-E, V, V2, V3, V4, V5, V6, V10, V11) was close to one of the IS6110 RFLP indices being "the gold standard" of the M. tuberculosis complex genotyping. The V2, V3 loci located in the sequences of the PPE gene family, were highly polymorphic and more discriminative then others (HGDI is about 0.8). The congruence between the IS6110 RFLP-typing and 11 loci VNTR-typing was measured during genotyping for 23 isolates of the W-Beijing family. The isolates were divided into 9 genotypes by the IS6110 RFLP and into 13 variants by the VNTR-typing. The profiles correlation coefficient was 0.767689 that reflected the differences in the rate and type of the given genome target evolution.     

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.     

结核分枝杆菌耐链霉素和乙胺丁醇分子机制的研究

目的:研究结核分枝杆菌耐链霉素和乙胺丁醇的rpsL和emb B基因突变情况,探讨耐药基因突变与耐药性的关系。方法:通过传统药敏实验和聚合酶链反应(PCR)--单链构象多态性(SSCP)技术初步鉴定62株临床分离株的药敏和rps L、emb B基因。结果:与结核菌标准株H37Rv对照,分析30例TB菌耐链霉素(SM)的rps L基因,发现其突变率为70.0%(21/30),分析29例耐乙胺丁醇(EMB)的emb B基因,该基因的突变率为65.5%(19/29)。结论:部分结核分枝杆菌耐SM和EMB是由于其rps L、emb B基因突变所致,PCR-SSCP银染技术可能成为测定部分结核分枝杆菌耐药的简便、快速的方法。     

Distribution of spoligotyping defined genotypic lineages among drug-resistant Mycobacterium tuberculosis complex clinical isolates in Ankara, Turkey

Background

Investigation of genetic heterogeneity and spoligotype-defined lineages of drug-resistant Mycobacterium tuberculosis clinical isolates collected during a three-year period in two university hospitals and National Tuberculosis Reference and Research Laboratory in Ankara, Turkey.

Methods and Findings

A total of 95 drug-resistant M. tuberculosis isolates collected from three different centers were included in this study. Susceptibility testing of the isolates to four major antituberculous drugs was performed using proportion method on Löwenstein–Jensen medium and BACTEC 460-TB system. All clinical isolates were typed by using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) methods. Seventy-three of the 95 (76.8%) drug resistant M. tuberculosis isolates were isoniazid-resistant, 45 (47.4%) were rifampicin-resistant, 32 (33.7%) were streptomycin-resistant and 31 (32.6%) were ethambutol-resistant. The proportion of multidrug-resistant isolates (MDR) was 42.1%. By using spoligotyping, 35 distinct patterns were observed; 75 clinical isolates were grouped in 15 clusters (clustering rate of 79%) and 20 isolates displayed unique patterns. Five of these 20 unique patterns corresponded to orphan patterns in the SITVIT2 database, while 4 shared types containing 8 isolates were newly created. The most prevalent M. tuberculosis lineages were: Haarlem (23/95, 24.2%), ill-defined T superfamily (22/95, 23.2%), the Turkey family (19/95, 20%; previously designated as LAM7-TUR), Beijing (6/95, 6.3%), and Latin-America & Mediterranean (LAM, 5/95 or 5.3%), followed by Manu (3/95, 3.2%) and S (1/95, 1%) lineages. Four of the six Beijing family isolates (66.7%) were MDR. A combination of IS6110-RFLP and spoligotyping reduced the clustering rate from 79% to 11.5% among the drug resistant isolates.

Conclusions

The results obtained showed that ill-defined T, Haarlem, the Turkey family (previously designated as LAM7-TUR family with high phylogeographical specifity for Turkey), Beijing and LAM were predominant lineages observed in almost 80% of the drug-Resistant M. tuberculosis complex clinical isolates in Ankara, Turkey.     

Drug resistance and genotypes of strains of Mycobacterium tuberculosis isolated from human immunodeficiency virus-infected and non-infected tuberculosis patients in Bauru,São Paulo,Brazil

Little is known about transmission and drug resistance of tuberculosis (TB) in Bauru, State of S o Paulo. The objective of this study was to evaluate risk factors for transmission of Mycobacterium tuberculosis strains in this area. Strains were collected from patients attended at ambulatory services in the region and susceptibility towards the main first line antibiotics was determined and fingerprinting performed. A total of 57 strains were submitted to susceptibility testing: 23 (42.6%) were resistant to at least one drug while 3 (13%) were resistant against both rifampicin and isoniazide. Resistant strains had been isolated from patients that had not (n = 13) or had (n = 9) previously been submitted to anti-TB treatment, demonstrating a preoccupying high level of primary resistance in the context of the study. All strains were submitted to IS6110 restriction fragment length polymorphism (IS6110-RFLP) and double repetitive element PCR (DRE-PCR). Using IS6110-RFLP, 26.3% of the strains were clustered and one cluster of 3 patients included 2 HIV-infected individuals that had been hospitalized together during 16 days; clustering of strains of patients from the hospital was however not higher than that of patients attended at health posts. According to DRE-PCR, 55.3% belonged to a cluster, confirming the larger discriminatory power of IS6110-RFLP when compared to DRE-PCR, that should therefore be used as a screening procedure only. No clinical, epidemiological or microbiological characteristics were associated with clustering so risk factors for transmission of TB could not be defined in the present study.     

Activation of natural tularemia foci of the field-meadow and steppe types on the territory of Tula Province 1977-1978

Natural tularemia foci of the meadow and steppe type are extremely stable and become active in those years when the most favourable living conditions for rodents appear. For the first time during the last 30 years a great increase in the number of common voles, accompanied by widely spread epizooty covering the whole territory of the Tula region, was observed. House mice, common field mice, harvest mice and black rats were also involved in this epizooty and 235 tularemia patients with all clinical forms of the disease were registered, the pulmonary form of the disease being prevalent. The cases of the disease were observed among both urban and rural population. In spite of a high morbidity rate, no cases of group infection were registered in domestic conditions and among agricultural workers due to the existence of the numerous immune layer among the population. The formation of this layer resulted from planned vaccinal prophylaxis covering, on the average, 86.3% of the rural population of the region.     

Genetic Diversity of Mycobacterium tuberculosis from Guadalajara,Mexico and Identification of a Rare Multidrug Resistant Beijing Genotype

Determining the genetic diversity of M. tuberculosis strains allows identification of the distinct Mycobacterium tuberculosis genotypes responsible for tuberculosis in different regions. Several studies have reported the genetic diversity of M. tuberculosis strains in Mexico, but little information is available from the state of Jalisco. Therefore, the aim of this study was to determine the genetic diversity of Mycobacterium tuberculosis clinical isolates from Western Mexico. Sixty-eight M. tuberculosis isolates were tested for susceptibility to first-line drugs using manual Mycobacteria Growth Indicator Tube method and genotyped using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) pattern analyses. Forty-seven (69.1%) isolates were grouped into 10 clusters and 21 isolates displayed single patterns by spoligotyping. Three of the 21 single patterns corresponded to orphan patterns in the SITVITWEB database, and 1 new type that contained 2 isolates was created. The most prevalent lineages were T (38.2%), Haarlem (17.7%), LAM (17.7%), X (7.4%), S (5.9%), EAI (1.5%) and Beijing (1.5%). Six (12.8%) of the clustered isolates were MDR, and type 406 of the Beijing family was among the MDR isolates. Seventeen (26.2%) isolates were grouped into 8 clusters and 48 isolates displayed single patterns by IS6110-RFLP. Combination of IS6110-RFLP and spoligotyping reduced the clustering rate to 20.0%. The results show that T, Haarlem, and LAM are predominant lineages among clinical isolates of M. tuberculosis in Guadalajara, Mexico. Clustering rates indicated low transmission of MDR strains. We detected a rare Beijing genotype, SIT406, which was a highly resistant strain. This is the first report of this Beijing genotype in Latin America.     

Studies of Polymorphic DNA Fingerprinting and Lipid Pattern of Mycobacterium tuberculosis Patient Isolates in Japan

Strain differentiation by DNA restriction fragment length polymorphism (RFLP) has been used mainly for the epidemiological purpose of Mycobacterium tuberculosis infection. In this study, we tried to connect the molecular and phenotypic characteristics of M. tuberculosis patient isolates by comparing the DNA fingerprints obtained by RFLP using IS6110 and lipid patterns using two-dimensional thin-layer chromatography (2-D TLC) with silica gel, since M. tuberculosis has a lipid-rich cell envelope which contributes to the virulence and immunomodulatory properties. We found that 66 isolates of M. tuberculosis from tuberculosis patients showed that the occurrence of IS6110 varied from 1 to 24 copies. The IS6110 patterns were highly variable among isolates. Fifty different RFLP patterns were observed, and 12 RFLP patterns were shared by two or more strains. By computerized analysis of the RFLP patterns of M. tuberculosis patient isolates, we found that 95% of the isolates fell into seven clusters, from A to G, with at least two isolates in each (> 30% similarity). Among the cellular lipids, the phospholipid composition did not differ by strain, whereas the glycolipid pattern differed markedly. Especially, the relative concentration of cord factor and sulfolipid, both of which were known as virulent factors, varied by strain. The fingerprints of some strains showed an association between the DNA and glycolipid patterns, even though some of the same DNA fingerprint strains showed differences in lipid patterns. Among the patient isolates, M. tuberculosis strain 249 possessed a specific glycolipid with 2-O-methyl-L -rhamnose and L-rhamnose, which is rarely found in other strains. This glycolipid showed serological activity against the sera of tuberculosis patients, even if the reactivity was not as strong as trehalose dimycolate. It also showed the inhibition of phagosome-lysosome fusion in macrophages, suggesting involvement with virulence. These results suggest that RFLP analysis using IS6110 is useful for clustering the human isolates of M. tuberculosis, however, for further strain differentiation on virulence, a lipid analysis provides more information.     

Random Postoperative Day-3 Cortisol Concentration as a Predictor of Hypothalamic-Pituitary-Adrenal Axis Integrity after Transsphenoidal Surgery

ObjectiveTo determine whether a random postoperative day-3 cortisol value of 10 μg/dL or greater is predictive of adrenal sufficiency 3 to 10 weeks after transsphenoidal surgery (TSS) and during long-term clinical follow-up.MethodsWe retrospectively reviewed the case records of patients who underwent TSS at our institution between 1991 and 2008. Inclusion criteria were as follows: random cortisol measured on the morning of postoperative day 3, adrenal dynamic testing performed 3 to 10 weeks after TSS, and clinical assessment of the hypothalamicpituitary-adrenal (HPA) axis at least 6 months after TSS.ResultsA total of 466 patients underwent TSS at our institution during the study period. Eighty-three patients met study inclusion criteria. Sensitivity of a random postoperative day-3 serum cortisol value of 10 μg/dL or greater for the prediction of adrenal sufficiency at a median follow-up of 42 days was 64.81% (95% confidence interval, 50.6%-77.32%), with an odds ratio of 3.1 (95% confidence interval, 1.08-8.58). Specificity was 62.1% (95% confidence interval, 42.3%-79.3%). At a median follow-up of 500 days, only 2 patients with a postoperative day-3 cortisol value of 10 μg/dL or greater required hydrocortisone replacement, both of whom had multiple anterior pituitary hormone deficiencies and evidence of pituitary dysfunction during the perioperative period.ConclusionsIn the appropriate clinical context, a postoperative day-3 cortisol value of 10 μg/dL or greater accurately predicts the integrity of the HPA axis. The final decision regarding corticosteroid replacement should be personalized, considering the postoperative day-3 cortisol level, the clinical context in which the measurement was obtained, and any evidence of concomitant pituitary dysfunction in the perioperative period. (Endocr Pract. 2011;17:717-726)     

Molecular characterization of Mycobacterium tuberculosis isolates from Izmir, Turkey

In recent years, molecular typing methods have been used in epidemiologic studies of Mycobacterium tuberculosis isolates in various areas of the world. However, there have been few data on this issue in Turkey. We describe the molecular characterization of 56 Mycobacterium tuberculosis isolates recovered from individual patients in Izmir and the surrounding area by three different molecular methods. Isolated M. tuberculosis strains were characterized by IS6110 RFLP, spoligotyping and major genetic group designation. In total, 51 RFLP and 35 spoligopatterns were identified. Fourteen (25%) isolates were indicated as low copy number. Based on three genotypic characterization methods together, five clusters with two isolates each were identified. Most of the isolates (98.2%) were assigned as genetic groups 2 or 3. Only one isolate was identified as Beijing family strain (principal genetic group 1). The shared international clades were found to be Beijing-family, var T1 (ST 37), LAM (Latin-American-Mediterranean) 7 (ST 41), LAM 9 (ST 42), Haarlem 1 (ST 47), Haarlem 3 (ST 50) and T1 (ST 53). In this study, IS6110 RFLP, spoligotyping and major genetic group designation were found to be useful methods for molecular epidemiologic studies.     

A Novel Molecular Strategy for Surveillance of Multidrug Resistant Tuberculosis in High Burden Settings

Background

In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR.

Methods

A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discriminatory Indexes (HGI) and Wallace coefficients were compared between the methods.

Results

Overall clustering rates were high by both IS6110-RFLP (52.8%) and MIRU-VNTR (45.8%), indicative of on-going transmission. Both 24-loci MIRU-VNTR and IS6110-RFLP had similar HGI (0.972 and 0.973, respectively), with close numbers of unique profiles (87 vs. 70), clustered isolates (129 vs. 146), and cluster sizes (2 to 26 vs. 2 to 25 isolates). Spoligotyping alone was the least discriminatory (80.1% clustering, HGI 0.903), with 28 unique types. However, the discriminatory power of spoligotyping was improved when combined with pncA sequencing using the SpoNC approach (61.8% clustering, HGI 0.958). A high proportion of MDR-TB isolates had mutations in pncA (68%, n = 145), and pncA mutations were significantly associated with clustering (p = 0.007 and p = 0.0013 by 24-loci MIRU-VNTR and IS6110-RFLP, respectively), suggesting high rates of resistance to pyrazinamide among all MDR-TB cases and particularly among clustered cases.

Conclusion

We conclude that SpoNC provides good discrimination for MDR-TB surveillance and early identification of outbreaks in South Africa, with 24-loci MIRU-VNTR applied for pncA wild-type strains as needed.     

Typing of Mycobacterium tuberculosis strains isolated in Community Health Centers of Rio de Janeiro City, Brazil

Fingerprinting of Mycobacterium tuberculosis strains from tuberculosis (TB) patients attended in Community Health Centers (CHCs) of Rio de Janeiro was performed to verify possible risk factors for TB transmission. A prospective community-based study was performed during the period of July 1996 to December 1996 by collecting sputum samples of 489 patients in 11 different CHCs in four different planning areas (APs) of the city. Bacteriological, clinical, and epidemiological information was collected and M. tuberculosis genotypes defined after restriction fragment length polymorphism (IS6110-RFLP) and double repetitive element (DRE) fingerprinting of RFLP-clustered cases. Risk factors for TB transmission were looked for using three levels of cluster stringency. Among 349 (71%) positive cultures obtained, IS6110-RFLP typing could be performed on strains from 153 different patients. When using identity of RFLP patterns as cluster definition, 49 (32%) of the strains belonged to a cluster and none of the clinical or epidemiologic characteristics was associated with higher clustering levels. However, higher clustering level was observed in the AP including the central region of the city when compared to others. This strongly suggests that more recent transmission occurs in that area and this may be related with higher incidence of TB and HIV in this region.