Bacteriological confirmation of pulmonary tuberculosis in children with gastric aspirates in Germany, 2002-2010.

OBJECTIVE: To assess bacteriological confirmation of pulmonary tuberculosis (TB) in children using nucleic acid amplification tests (NAAT) and culture of gastric aspirates in Germany. DESIGN: We analysed 2002-2010 TB notification data to determine the use of gastric aspirates, NAAT and culture performance, including test agreement among pulmonary TB patients aged <15 years (grouped into <1, 1-4, 5-9 and 10-14 years). RESULTS: Gastric aspirates were used among 59% (769/1307) of the patients with available diagnostic information. For 454 patients, gastric aspirates were the only reported specimen, and both NAAT and culture were performed. Among these, culture was positive in 53% (95%CI 48-58), NAAT in 48% (95%CI 44-53) and either test in 63% (95%CI 59-68), with an overall test agreement of 74% (95%CI 70-78). Infants < 1 year had the highest positivity rate (79%, 95%CI 68-88, either test). Test agreement was the highest among 10-14 year olds (79%, 95%CI 67-89). CONCLUSIONS: Routine notification data document a wide use of gastric aspirates and high yield of both NAAT and culture for bacteriological confirmation of TB with gastric aspirates, particularly in infants. Imperfect test agreement supports the combined use of molecular assays and culture-whenever available-in the diagnosis of childhood TB.

Laboratory diagnosis of paediatric tuberculosis in the European Union/European Economic Area: analysis of routine laboratory data, 2007 to 2011.

Laboratory confirmation of paediatric tuberculosis (TB) is frequently lacking. We reviewed the range of routine laboratory tests and their performance in different biological samples used to diagnose active TB in children. A questionnaire-based survey was conducted among the European Reference Laboratory Network for TB followed by collection of routine laboratory data on 10,549 paediatric samples tested in 2007 to 2011 at six reference laboratories (in Croatia, Germany, Italy, Latvia, Lithuania and the United Kingdom (UK)). The questionnaire showed that all laboratories used rapid assays. Non-respiratory samples were collected more often in Germany (135/275, 49.1%) and the UK (490/2,140, 22.9%) compared with Croatia (138/2,792, 4.9%), Latvia (222/2,401, 9.2%) and Lithuania (76/1,549, 4.9%). Overall laboratory positivity rates (isolation of Mycobacterium tuberculosis complex and/or identification of its nucleic acids in a sample) were higher in lymph node and gastric aspirate samples (14/203 (6.9%) and 43/1,231 (3.5%)) than in sputum samples (89/4,684 (1.9%)). Pooled sensitivity, specificity, positive and negative predictive values and accuracy of molecular assays assessed against solid or liquid culture were 79.2%, 93.6%, 67.1%, 96.5% and 91.6%, respectively. A more intensive approach in obtaining gastric aspirate and non-respiratory samples may increase laboratory confirmation of paediatric TB. Major effort is needed in optimisation and validation of molecular tests in these samples.

The Colour Test for drug susceptibility testing of Mycobacterium tuberculosis strains.

SETTING: Tartu, Estonia. OBJECTIVE: To assess the performance and feasibility of the introduction of the thin-layer agar MDR/XDR-TB Colour Test (Colour Test) as a non-commercial method of drug susceptibility testing (DST). DESIGN: The Colour Test combines the thin-layer agar technique with a simple colour-coded quadrant format, selective medium to reduce contamination and colorimetric indication of bacterial growth to simplify interpretation. DST patterns for isoniazid (INH), rifampicin (RMP) and ciprofloxacin (CFX) were determined using the Colour Test for 201 archived Mycobacterium tuberculosis isolates. Susceptibilities were compared to blinded DST results obtained routinely using the BACTEC™ Mycobacteria Growth Indicator Tube™ (MGIT) 960 to assess performance characteristics. RESULTS: In all, 98% of the isolates produced interpretable results. The average time to positivity was 13 days, and all results were interpretable. The Colour Test detected drug resistance with 98% sensitivity for INH, RMP and CFX and 99% for multidrug-resistant tuberculosis. Specificities were respectively 100% (95%CI 82-100), 88% (95%CI 69-97) and 91% (95%CI 83-96) and 90% (95%CI 74-98). Agreement between the Colour Test and BACTEC MGIT 960 were respectively 98%, 96%, 94% and 97%. CONCLUSION: The Colour Test could be an economical, accurate and simple technique for testing tuberculosis strains for drug resistance. As it requires little specialist equipment, it may be particularly useful in resource-constrained settings with growing drug resistance rates.

Diagnosis of tuberculosis and drug resistance: what can new tools bring us?

This is an exciting time for tuberculosis (TB) diagnostics. The technology for rapid diagnosis of TB and rifampicin (RMP) resistance in pulmonary sputum smear-positive specimens is well advanced, and assays have high specificity with good sensitivity. Nevertheless, the current sensitivity of TB detection means that these assays still cannot replace the standard diagnostic methods for TB or conventional drug susceptibility testing (DST). In extra-pulmonary specimens, the performance of molecular tools varies and should be considered separately for each specimen type. Evidence for the use of these assays for TB and drug resistance detection in individuals co-infected with TB and the human immunodeficiency virus (HIV) is limited. As the positive predictive value for RMP resistance reaches ≥ 90% only when the prevalence of RMP resistance in new TB patients is >15%, which is rare globally, many cases with such resistance will be false-resistant, emphasising the need for a secondary confirmative test. Similarly, increased (or incorrect) diagnosis of TB may compromise programme effectiveness by increasing the numbers of individuals requiring anti-tuberculosis treatment, unless it is carefully planned. For the future, 1) assays with greater sensitivity for TB detection are needed; 2) rapid diagnostics for paediatric TB are important, and there is a need for carefully designed studies, including those involving HIV-positive children; 3) more clinical data need to be obtained from longitudinal studies, especially related to the influence of rapid diagnostics on disease outcome; and 4) point-of-care tests using untreated sputum, blood or urine and little or no equipment would be of immeasurable benefit. Although great progress has been made, we are not there yet.

Performance of the GenoType® MTBDRPlus assay in routine settings: a multicenter study.

Former Soviet Union countries including the Baltic States (Latvia, Lithuania, and Estonia) are hot spots for an emerging epidemic of drug resistant tuberculosis (TB). As a part of the development of a co-ordinated network of centers for diagnostic trials across Eastern Europe we conducted a retrospective multicenter analysis of the performance of the GenoType® MTBDRPlus assay for TB identification and susceptibility to isoniazid (INH) and rifampicin (RIF) in routine settings. A total of 1,045 primary samples, 1045 TB cultures derived from these specimens and 306 separate M. tuberculosis isolates tested in 2007-2010 at four participating sites (Tartu, Estonia; Riga, Latvia; Vilnius, Lithuania; and Samara, Russian Federation) were included in the analysis. The pooled sensitivity and specificity values for RIF and INH were 95.3% and 95.5%, 89.9 and 87.1%, respectively; there were no statistically significant variations in performance across sites. The proportion of multidrug resistant (MDR) strains in the collections ranged from 21.8% (in Estonia) to 55.9% (in Russia). In a routine non-trial context, the assay reliably detected both rifampicin and isoniazid resistance. The absence of statistically significant differences between sites suggested that the comparable performance obtained using these assays has helped demonstrate the formation of a successful diagnostic trial network.

Evaluation of two molecular assays for rapid detection of mycobacterium tuberculosis resistance to fluoroquinolones in high-tuberculosis and -multidrug-resistance Settings.

The Russian Federation is a high-tuberculosis (TB)-burden country with high rates of drug resistance, including multidrug and extensive drug resistance to TB (M/XDRTB). Rapid diagnosis of resistance to fluoroquinolones (FQs) using molecular assays is essential for the implementation of appropriate drug regimens and prevention of the transmission of XDR strains. A total of 51 individual MDRTB strains were tested by pyrosequencing of the quinolone resistance determining region of the gyrA gene and the GenoType MTBDRsl assay (Hain Lifescience, GmbH, Nehren, Germany), and the results were evaluated against those obtained by phenotypic drug susceptibility testing (DST). Mutations were detected in 25 (78.1%) FQ-resistant strains, with the majority of mutations (n = 19 [73.0%]) found in codon 94 of the gyrA gene; the novel mutation 1457 Câ†’Τ was found in the gyrB gene. Three mixed allelic variants were detected, which is a well-known phenomenon in areas with high TB and drug-resistant TB rates. The sensitivity and specificity of pyrosequencing (86.2 and 100%, respectively) and MTBDRsl (86.2 and 100%, respectively) were high; however, the results for 5.9% of the analyzed strains were unreadable when MTBDRsl was used. The MTBDRsl and pyrosequencing assays offer a rapid and accurate means for diagnosing resistance to FQs in high-TB-burden areas.

Molecular epidemiology and prevalence of mutations conferring rifampicin and isoniazid resistance in Mycobacterium tuberculosis strains from the southern Ukraine.

Understanding the molecular epidemiology of tuberculosis (TB) and mutations in genes associated with drug resistance may contribute to the development of appropriate interventions to improve tuberculosis control. A structured questionnaire was used to collect basic epidemiological data from 589 patients with radiologically confirmed TB in the Odessa and Nikolaev regions of the Ukraine in 2003-2004. A non-commercial reverse hybridisation assay and DNA sequencing were used to detect mutations associated with rifampicin and isoniazid resistance. Genotyping was performed using multilocus variable number tandem repeat (VNTR) typing and spoligotyping. Mutations conferring rifampicin and isoniazid resistance were detected in 32.9% and 44.0%, respectively, of 225 Mycobacterium tuberculosis isolates from individual consecutive patients. Mutations in codon 531 and codon 315 of the rpoB and katG genes, respectively, were predominant among drug-resistant isolates. Multidrug (MDR) resistance rates were significantly higher among former prison inmates compared with non-prisoners (54.8% vs. 27.3%; RR 2.01; 95% CI 1.35-2.97) and the prevalence of mutations was higher in Beijing strains sharing the VNTR signature 223325173533424 than in other Beijing strains (71.4% vs. 45.7%; RR 1.74; 95% CI 1.17-2.57), suggesting that this group may be responsible for rapid transmission of MDR TB in the southern Ukraine.

Stigma and HIV infection in Russia.

Few studies have examined the personal and social consequences of stigma associated with HIV infection in Russia, a country with one of the most rapidly advancing HIV epidemics globally. By May 2005, Samara Oblast, Russia had 24,022 notified seropositive individuals. Focus-group discussions with randomly sampled seropositive and seronegative individuals, matched by age, gender and education were selected from the general population and used to provide an informal forum for discussion of attitudes to HIV and potentially stigmatizing behavior. The results demonstrated that the perception that HIV was associated with immoral behaviour underpinned stigma. Discriminating attitudes are strongly associated with misperceptions regarding transmission and frequent over-estimation of risks from casual contact. The general population was unforgiving to those who had become infected sexually or through drug use. Infection through a medical procedure or from an assault was perceived as a likely route of infection. Knowledge of population attitudes and perceptions, as well as those who are HIV-positive, is critical for successful interventions and to encourage people to come forward for HIV testing. This research offers insights into the distance that needs to be traveled if stigma is to be addressed in wider efforts to control HIV in Russia.

Costs and outcomes of tuberculosis control in the Russian Federation: retrospective cohort analysis.

We analysed costs and outcomes of tuberculosis care for patients in a traditional Russian tuberculosis control system, using 3-year retrospective cohort data. Of 1749 cases at 3 years of follow-up, 65% were cured, 11.3% (198/1749) still had 'active' or 'chronic' disease, 10.3% had transferred out of the local civilian health care system and 12.7% had died. The mean cost of managing one case over 3 years was 886 US dollars: 1,078 US dollars for bacteriologically confirmed (BK+) cases and 718 US dollars for bacteriologically unconfirmed (BK-) cases. Approximately 60% of treatment costs were incurred in the first 12 months and 40% incurred in the remaining 2 years. Around 60% of the total cost was accounted for by hospital inpatient care. The cost, treatment and outcome of BK+ and BK- cases differed substantially. The cost of treating BK+ cases was 50% higher than treating BK- cases due to higher hospitalization rates and the additional cost of managing BK+ cases that become 'chronic'. While BK+ cases accounted for 55% of total health expenditure on tuberculosis, the share of BK- cases was 45% of the total - due to hospitalization and lengthy periods of follow up. The costs of treating tuberculosis in the Russian tuberculosis control system are very high compared with other high-burden countries due to hospitalization policies and lengthy case management periods. Much of this expenditure can be avoided if the WHO-recommended DOTS strategy is implemented. In particular, the proportion of expenditure for BK- cases is surprisingly high and can be avoided as most of these patients do not need hospitalizing or lengthy periods of follow-up.

The Directly Observed Therapy Short-Course (DOTS) strategy in Samara Oblast, Russian Federation.

BACKGROUND: The World Health Organisation (WHO) defines Russia as one of the 22 highest-burden countries for tuberculosis (TB). The WHO Directly Observed Treatment Short Course (DOTS) strategy employing a standardised treatment for 6 months produces the highest cure rates for drug sensitive TB. The Russian TB service traditionally employed individualised treatment. The purpose of this study was to implement a DOTS programme in the civilian and prison sectors of Samara Region of Russia, describe the clinical features and outcomes of recruited patients, determine the proportion of individuals in the cohorts who were infected with drug resistant TB, the degree to which resistance was attributed to the Beijing TB strain family and establish risk factors for drug resistance. METHODS: Prospective study. RESULTS: 2,099 patients were recruited overall. Treatment outcomes were analysed for patients recruited up to the third quarter of 2003 (n = 920). 75.3% of patients were successfully treated. Unsuccessful outcomes occurred in 7.3% of cases; 3.6% of patients died during treatment, with a significantly higher proportion of smear-positive cases dying compared to smear-negative cases. 14.0% were lost and transferred out. A high proportion of new cases (948 sequential culture-proven TB cases) had tuberculosis that was resistant to first-line drugs; (24.9% isoniazid resistant; 20.3% rifampicin resistant; 17.3% multidrug resistant tuberculosis). Molecular epidemiological analysis demonstrated that half of all isolated strains (50.7%; 375/740) belonged to the Beijing family. Drug resistance including MDR TB was strongly associated with infection with the Beijing strain (for MDR TB, 35.2% in Beijing strains versus 9.5% in non-Beijing strains, OR-5.2. Risk factors for multidrug resistant tuberculosis were: being a prisoner (OR 4.4), having a relapse of tuberculosis (OR 3.5), being infected with a Beijing family TB strain (OR 6.5) and having an unsuccessful outcome from treatment (OR 5.0). CONCLUSION: The implementation of DOTS in Samara, Russia, was feasible and successful. Drug resistant tuberculosis rates in new cases were high and challenge successful outcomes from a conventional DOTS programme alone.

Variability in interpretation of chest radiographs among Russian clinicians and implications for screening programmes: observational study.

OBJECTIVE: To determine variability in interpretation of chest radiographs among tuberculosis specialists, radiologists, and respiratory specialists. DESIGN: Observational study. SETTING: Tuberculosis and respiratory disease services, Samara region, Russian Federation. PARTICIPANTS: 101 clinicians involved in the diagnosis and management of pulmonary tuberculosis and respiratory diseases. MAIN OUTCOME MEASURES: Interobserver and intraobserver agreement on the interpretation of 50 digital chest radiographs, using a scale of poor to very good agreement (kappa coefficient: < or = 0.20 poor, 0.21-0.40 fair, 0.41-0.60 moderate, 0.61-0.80 good, and 0.81-1.00 very good). RESULTS: Agreement on the presence or absence of an abnormality was fair only (kappa = 0.380, 95% confidence interval 0.376 to 0.384), moderate for localisation of the abnormality (0.448, 0.444 to 0.452), and fair for a diagnosis of tuberculosis (0.387, 0.382 to 0.391). The highest levels of agreement were among radiologists. Level of experience (years of work in the specialty) influenced agreement on presence of abnormalities and cavities. Levels of intraobserver agreement were fair. CONCLUSIONS: Population screening for tuberculosis in Russia may be less than optimal owing to limited agreement on interpretation of chest radiographs, and may have implications for radiological screening programmes in other countries.

Tuberculosis, HIV seroprevalence and intravenous drug abuse in prisoners.

High rates of tuberculosis (TB) and HIV are believed to exist in Russian prisons. Prisoners with TB were studied in order to identify the following: 1) prevalence of HIV, and risk factors for HIV and other blood-borne virus infections; and 2) clinical and social factors that might compromise TB treatment effectiveness and/or patient adherence and, hence, encourage treatment failure. A 1-yr cross-sectional prevalence study of 1,345 prisoners with TB was conducted at an in-patient TB facility in Samara, Russian Federation. HIV and hepatitis B and/or C co-infection occurred in 12.2% and 24.1% of prisoners, respectively, and rates were significantly higher than in civilians. Overall, 48.6% of prisoners used drugs, of which 88.3% were intravenous users. Prisoners were more likely to be intravenous drug users and HIV positive compared with civilians with TB, and 40.2% of prisoners shared needles. Two-thirds of prisoners (68.6%) had received previous TB drug therapy (frequently multiple, interrupted courses) and were significantly more likely than civilians to have had previous therapy consistent with the high drug-resistance rates seen. Prisons are major drivers of the tuberculosis and HIV epidemics. Novel strategies are needed to reduce the spread of blood borne diseases, particularly in intravenous drug users.

Rates of drug resistance and risk factor analysis in civilian and prison patients with tuberculosis in Samara Region, Russia.

BACKGROUND: Tuberculosis (TB) and HIV rates continue to escalate in Russia, but true rates for drug resistance, especially multidrug resistant tuberculosis (MDR TB), are unknown. A study was conducted with the aims of identifying first line drug resistance, both in the civilian and prison sectors, for new and previously treated cases; and risk factors for the development of drug resistance. METHODS: A cross sectional survey was undertaken of 600 patients (309 civilians, 291 prisoners) with bacteriologically confirmed pulmonary TB over a 1 year period during 2001-2 in Samara Oblast, Russia. RESULTS: The prevalence of isoniazid, rifampicin, streptomycin, ethambutol and pyrazinamide resistance in new TB cases (civilian and prison patients) was 38.0%, 25.2%, 34.6%, 14.7%, and 7.2%, respectively. The prevalence of MDR TB was 22.7%, 19.8%, and 37.3% in all new cases, new civilian cases, and new prison cases, respectively, with an overall prevalence of 45.5% and 55.3% in previously treated cases. Factors associated with resistance included previous TB treatment for more than 4 weeks, smoking (for isoniazid resistance), the presence of cavitations on the chest radiograph, and imprisonment. HIV was not associated with resistance in all patients. The rates of resistance were significantly higher in prisoners, with rate ratios (RR) of 1.9 (95% CI 1.1 to 3.2) for MDR TB, 1.9 (95% CI 1.1 to 3.2) for rifampicin, and 1.6 (95% CI 1.0 to 2.6) for isoniazid. CONCLUSIONS: Rates of first line drug resistance are high, particularly in prisoners and previously treated cases. TB control programmes should initially focus on standardised treatment to maximise cure, combined with measures to reduce institutional TB spread (particularly in prisons) coupled with early diagnosis of MDR TB to reduce the spread and development of resistance.

Multidrug-resistant tuberculosis in Russia: clinical characteristics, analysis of second-line drug resistance and development of standardized therapy.

The aim of the study presented here was to identify patients with multidrug resistant tuberculosis (MDRTB) in the Samara region of Russia and to analyze the susceptibility of their isolates to second-line drugs in order to develop an empirical, standard, second-line treatment regimen. Treatment of MDRTB can be individualized based on in vitro laboratory analysis or standardized. In the latter case there is still a need to ascertain local second-line drug-resistance patterns. Described here are the clinical characteristics of 251 MDRTB patients identified in the study and the second-line drug susceptibility of 69 MDRTB isolates obtained from them. Antimicrobial resistance to the following agents was detected in the isolates: rifabutin (88.2%), streptomycin (42.8%), amikacin (7.2%), doxycycline (7.4%), ciprofloxacin (4.3%), clofazimine (2.9%), cycloserine (7.4%), and prothionamide (1.5%). The results of the study indicate it is possible to develop a standard, effective, clinical treatment regimen using ethambutol, pyrazinamide, prothionamide, a fluoroquinolone and amikacin.

Detection of mutations associated with isoniazid and rifampin resistance in Mycobacterium tuberculosis isolates from Samara Region, Russian Federation.

High incidence rates of isoniazid-, rifampin-, and multiple-drug-resistant tuberculosis have been reported in countries of the former Soviet Union (FSU). Genotypic (unlike phenotypic) drug resistance assays do not require viable cultures but require accurate knowledge of both the target gene and the mutations associated with resistance. For these assays to be clinically useful, they must be able to detect the range of mutations seen in isolates from the population of tuberculosis patients to which they are applied. Two novel macroarrays were applied to detect mutations associated with rifampin (rpoB) and isoniazid (katG and inhA) resistance. In a sample of 233 isolates from patients in Samara, central Russia, 46.5% of isolates possessed mutations in both the rpoB and the katG (or inhA) genes. Combined results from the macroarrays demonstrated concordance in 95.4 and 90.4% of phenotypically defined rifampin- and isoniazid-resistant isolates, respectively. The contribution of different mutations to resistance was comparable to that reported previously for non-FSU countries, with 90% of rifampin-resistant isolates and 93% of isoniazid resistant isolates due to rpoB531 and katG315 mutations, respectively. The percentage of phenotypically resistant rifampin isolates with no mutations in the rpoB codons 509 to 536 was 4.2%, which was similar to previous reports. Novel macroarrays offer a rapid, accurate, and relatively cheap system for the identification of rifampin-, isoniazid-, and multiple-drug-resistant Mycobacterium tuberculosis isolates.

Tuberculosis control in Samara Oblast, Russia: institutional and regulatory environment.

SETTING: Tuberculosis control programme in Samara Oblast, Russia, funded in part by the government of the United Kingdom. OBJECTIVE: To identify and evaluate institutional and regulatory influences as well as incentives and disincentives that might be amenable to change in the promotion of the DOTS strategy. DESIGN: Multidisciplinary situational analysis through in-depth interviews of stakeholders and review of official federal and oblast documents. RESULTS: Interpretation of traditional notification data is complex because classification and reporting systems differ from World Health Organization principles. Regulations governing financing encourage lengthy hospitalisations and interventions, and provide few incentives to shift policy to ambulatory care. CONCLUSION: Accurate comparability of epidemiological trends and programmatic successes requires equivalent classification and reporting systems. If the DOTS strategy is to be sustainable, changes to financing systems will be needed.