Multicentre study to establish interpretive criteria for clofazimine drug susceptibility testing.

To conduct a multicentre study to establish the critical concentration (CC) for clofazimine (CFZ) for drug susceptibility testing (DST) of Mycobacterium tuberculosis on the MGIT™960™ system using the distribution of minimum inhibitory concentrations (MIC) and genotypic analyses of Rv0678 mutations. In phase I of the study, the MIC distribution of laboratory strains (H37Rv and in vitro-selected Rv0678 mutants) and clinical pan-susceptible isolates were determined (n = 70). In phase II, a tentative CC for CFZ (n = 55) was proposed. In phase III, the proposed CC was validated using clinical drug-resistant tuberculosis (DR-TB) isolates stratified by Rv0678 mutation (n = 85). The MIC distribution of CFZ for laboratory and clinical pan-susceptible strains ranged between 0.125 µg/ml and 0.5 µg/ml. As the MIC values of DR-TB isolates used for phase II ranged between 0.25 µg/ml and 1 µg/ml, a CC of 1 µg/ml was proposed. Validation of the CC in phase III showed that probably susceptible and probably resistant Rv0678 mutants overlapped at 1 µg/ml. We therefore recommend a CC of 1 µg/ml, with additional testing at 0.5 µg/ml to define an intermediate category. This was the first comprehensive study to establish a CC for routine phenotypic DST of CFZ using the MGIT960 system to guide therapeutic decisions. .

Ethambutol-resistance testing by mutation detection using MTBDRsl.

AIMS AND OBJECTIVES: Despite the successes in managing drug-susceptible TB, drug-resistant (DR) tuberculosis is a major challenge to the effectiveness of National Tuberculosis Program in Armenia, placing the country in the list of 18 high-burden countries for MDR-TB in the WHO European Region. Estimated burden of MDR-TB in 2012 was 9.4 (7-12) and 43 (38-49) among retreatment TB cases. A total of 92 laboratory confirmed cases had been reported to the WHO (57 new and 35 previously treated) out of 511 cases tested for MDR-TB. GenoType MTBDRsl is a new molecular kit designed for rapid identification of the resistance to the second-line antituberculosis drugs with a single strip. The aim of this study was to identify the mutation that confers resistance to ethambutol (EMB) in Mycobacterium tuberculosis in comparison with the phenotypic drug susceptibility test (DST). Ethambutol is used in M/XDR-TB regimens only if it is susceptible by DST results. METHODS: A set of 173 drug resistances TB isolates during 2011 and 2012 period, being either acid fast bacterium positive or negative but culture positive resistant to isoniazid, rifampin, or both according to the GenoType MTBDR plus assay were consecutively tested, using the GenoType MTBDRsl (MTBDR plus version 1.0 and MTBDRsl version 2.0 Hain Lifescience, Nehren). embB gene analysis and the results were compared to phenotypic EMB testing (DST). The DNA preparation method used as described recommended by the manufacturer. RESULTS: Genotypic analysis identified mutations at codon 306 of the embB gene in 20.8% (36/173) and miss band of wild type in 12.71% (22/173) of the resistant isolates in comparison to only 14.45% (25/173) of those that were phenotypically resistant to EMB by DST MGIT liquid media. Mutation locus were identified in embB MUT1B and embB MUT1A 8.67% (15/173) and 12.13% (21/173), simultaneously. Phenotypic retesting in MGIT demonstrated that 45 (306 of the embB gene and miss band of wild type together) genotypically resistant isolates were phenotypically resistant to EMB. This implies that 58.62% (33/58) of EMB resistance had been phenotypically missed by routine laboratory procedures. EMB resistance was closely linked to multidrug resistance (MDR); 70.69% (41/58) of the EMB-resistant isolates were resistant to both isoniazid and rifampicin. CONCLUSION: Implementation of more accurate diagnosis of EMB resistance may enhance patient management in Armenia, as standardized treatment of MDR-TB with second-line drugs is currently dependent on the outcome of the EMB resistance test.

Inactivation of Mycobacterium bovis ssp. caprae in high-temperature, short-term pasteurized pilot-plant milk.

Experiments to determine the efficacy of high temperature, short time (HTST) pasteurization of milk in terms of inactivation of pathogenic microorganisms were mainly performed between 1930 and 1960. Among the target organisms were Mycobacterium bovis and Mycobacterium tuberculosis. As a result, the Codex Alimentarius prescribes that HTST treatment of milk should lead to a significant reduction of pathogenic microorganisms during milk pasteurization. Due to the development of improved methods for the detection of survivors and of more advanced heating technology, verification of this requirement seemed to be necessary. To address recent outbreaks of tuberculosis in cattle caused by M. bovis ssp. caprae (M. caprae) in the southern regions of Germany, this organism was tested and compared with M. bovis ssp. bovis (M. bovis). Experiments were performed in a pilot plant for HTST pasteurization of milk with 3 strains of M. caprae and 1 strain of M. bovis. In preliminary trials at a fixed holding time of 25 s, the temperature at which significant inactivation occurred was 62.5°C for all strains. To determine D-values (decimal reduction times) for the inactivation kinetics, the strains were tested at 65, 62.5, and 60°C at holding times of 16.5, 25, and 35 s. At 65°C, the D-values of all strains ranged from 6.8 to 7.8 s, and at 62.5°C, D-values ranged from 14.5 to 18.1 s. Low inactivation was observed at 60°C. When the low slope of the inactivation curve allowed calculation of a D-value, these ranged from 40.8 to 129.9 s. In terms of log10 reductions, the highest values for all strains were 4.1 to 4.9 log at 65°C, with a holding time of 35 s. The tested strains of M. caprae and M. bovis showed similar low resistance to heat. Standard HTST treatment should result in a high reduction of these organisms and thus the requirements of the Codex Alimentarius for inactivation of pathogens by this process are far exceeded.

Cost of multi drug resistance tuberculosis in Germany.

OBJECTIVES: 4220 new cases of tuberculosis (TB) were reported in Germany in 2012; of those, 65 cases were multidrug-resistant TB (MDR-TB) or extensively multidrug-resistant TB (XDR-TB) cases. However, there is only limited information on the economic consequences of drug resistance patterns on the treatment costs of MDR-and XDR-TB patients. METHODS: On the basis of drug susceptibility of the single MDR-TB/XDR-TB strains the direct medical costs of suitable therapies were calculated according to the current guidelines of the World Health Organization (WHO) and those of the German Central Committee against Tuberculosis. These costs were combined with hospital and outpatients monitoring costs and followed the most recent German invoicing system and health statistics. Total drug and monitoring costs and were determined by Monte-Carlo simulation comprising all different options. RESULTS: According to this, the mean drug costs were €51,113.22 (range €19,586.14 to €94,767.90). The weighted costs for hospitalization were €26,000.76 per patient compared to only €2,192.13 for primary outpatients; the total treatment costs of MDR-TB amounted to €64,429.23. These are joined by the costs due to loss of productivity, varying between €17,721.60 and €44,304. From a societal perspective, the total cost per MDR-TB/XDR-TB case reach an amount between €82,150 and €108,733 per case, respectively. CONCLUSION: Cost analyses based on strain resistance patterns allow more reliable estimates of the real costs of treating MDR-TB/XDR-TB than do methods that ignore this factor. Advantageously, they demonstrate the economic impact of drug-resistant TB in low-incidence countries. Costs of productivity loss is of new importance because of the length of MDR-XDR therapy, but its true share of total costs has still to be determined.

Characterisation of pyrazinamide-resistant Mycobacterium tuberculosis strains isolated in Poland and Germany.

BACKGROUND: Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug that is generally administered with isoniazid, rifampicin, ethambutol and streptomycin. OBJECTIVE: To analyse the correlation between phenotypic resistance to PZA and genotype to find out whether the great diversity in pncA mutations is epidemiologically useful in tracing the transmission of PZA-resistant Mycobacterium tuberculosis strains among patients. MATERIALS AND METHODS: The study included 71 PZA-resistant M. tuberculosis strains isolated from 62 Polish and 9 German patients. All strains were analysed using minimal inhibitory concentration value determination, pncA mutation analysis, spoligotyping, 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and insertion sequence (IS) 6110 restriction fragment length polymorphism (RFLP) fingerprinting. RESULTS: In 63 isolates, 37 (88.7%) different mutations in the pncA gene were observed, 13 of which had not been previously reported; 11 molecular families with the same MIRU-VNTR and IS6110-RFLP pattern were found. The same mutation was identified in three families, while different ones were identified in the remaining families. CONCLUSION: Mutations in the pncA gene are a major cause of PZA resistance in M. tuberculosis. pncA mutation analysis can be used to obtain valuable additional information, but should be applied with caution for epidemiological analysis.

First national survey of anti-tuberculosis drug resistance in Azerbaijan and risk factors analysis.

SETTING: Civilian population of the Republic of Azerbaijan. OBJECTIVES: To determine patterns of anti-tuberculosis drug resistance among new and previously treated pulmonary tuberculosis (TB) cases, and explore their association with socio-demographic and clinical characteristics. DESIGN: National cross-sectional survey conducted in 2012-2013. RESULTS: Of 789 patients (549 new and 240 previously treated) who met the enrolment criteria, 231 (42%) new and 146 (61%) previously treated patients were resistant to any anti-tuberculosis drug; 72 (13%) new and 66 (28%) previously treated patients had multidrug-resistant TB (MDR-TB). Among MDR-TB cases, 38% of new and 46% of previously treated cases had pre-extensively drug-resistant TB (pre-XDR-TB) or XDR-TB. In previously treated cases, 51% of those who had failed treatment had MDR-TB, which was 15 times higher than in relapse cases (OR 15.2, 95%CI 6-39). The only characteristic significantly associated with MDR-TB was a history of previous treatment (OR 3.1, 95%CI 2.1-4.7); for this group, history of incarceration was an additional risk factor for MDR-TB (OR 2.8, 95%CI 1.1-7.4). CONCLUSION: Azerbaijan remains a high MDR-TB burden country. There is a need to implement countrywide control and innovative measures to accelerate early diagnosis of drug resistance in individual patients, improve treatment adherence and strengthen routine surveillance of drug resistance.

[Recommendations for diagnosis and treatment of nontuberculous mycobacterioses of the German Central Committee against tuberculosis and the German Respiratory Society]. / Empfehlungen zur Diagnostik und Therapie nichttuberkulöser Mykobakteriosen des Deutschen Zentralkomitees zur Bekämpfung der Tuberkulose (DZK) und der Deutschen Gesellschaft für Pneumologie und Beatmungsmedizin (DGP).

Nontuberculous mycobacterioses comprise a group of diseases caused by mycobacteria which do not belong to the Mycobacterium (M.) tuberculosis complex and are not ascribed to M. leprae. These mycobacteria are characterized by a broad variety as to environmental distribution and adaptation. Some of the species may cause specific diseases, especially in patients with underlying immunosuppressive diseases, chronic pulmonary diseases or genetic predisposition, respectively. Worldwide a rising prevalence and significance of nontuberculous mycobacterioses can be recognized. The present recommendations summarise actual aspects of epidemiology, pathogenesis, clinical aspects, diagnostics - especially microbiological methods including susceptibility testing -, and specific treatment for the most relevant species. Diagnosis and treatment of nontuberculous mycobacterioses during childhood and in HIV-infected individuals are described in separate chapters.

[Tuberculosis infection control - recommendations of the DZK]. / Infektionsprävention bei Tuberkulose - Empfehlungen des DZK.

The epidemiological situation of tuberculosis (TB) in Germany has improved considerably during the past few years. However, those in unprotected contact with infectious tuberculosis patients frequently and/or over longer periods of time and/or intensively continue to have a higher risk for TB infection. Rapid diagnosis, prompt initiation of effective treatment, and adequate infection control measures are of particular importance to prevent infection. The present recommendations depict the essentials of infection control as well as specific measures in the hospital (isolation, criteria for its duration and technical requirements, types of respiratory protection, disinfection measures, waste disposal). The specific requirements for outpatients (medical practice), at home, for ambulance services, and in congregate settings, including prisons, are also addressed. Compared with the previous recommendations the pattern of respiratory protection measures has been simplified. As a rule, hospital staff and those visiting infectious tuberculosis patients are advised to wear respiratory protection that satisfies the criteria of FFP2-masks (DIN EN 149), while patients should wear mouth-nose protectors (surgical masks) in the presence of others and outside the isolation room. A detailed depiction of criteria for isolation and its duration in smear positive and only culturally confirmed pulmonary tuberculosis has been added.

[International Standards of Tuberculosis Care (ISTC)--comments from the German point of view]. / International Standards of Tuberculosis Care (ISTC)--Kommentierung aus deutscher Sicht.

The "International Standards for Tuberculosis Care" (ISTC) were developed by the World Health Organisation (WHO) and others to provide internationally agreed and, if possible, evidence-based standards for tuberculosis care including the care by private providers who are not part of national tuberculosis programmes or health-care systems. Hence, the ISTC primarily address resource-restrained countries with high tuberculosis prevalence. In this article, the German translation of the 21 standards from 2009 is presented - addressing diagnostic and therapeutic standards, co-infection (especially with HIV) and public-health issues. The accompanying comments show how these standards have to be modified for Germany due to the medical resources available here and country-specific characteristics respectively.

[Tuberculosis infection control--recommendations of the DZK]. / Infektionsprävention bei Tuberkulose--Empfehlungen des DZK.

The epidemiological situation of tuberculosis (TB) in Germany has improved considerably during the past few years. However, those in unprotected contact with infectious tuberculosis patients frequently and/or over longer periods of time and/or intensively continue to have a higher risk for TB infection. Rapid diagnosis, prompt initiation of effective treatment, and adequate infection control measures are of particular importance to prevent infection. The present recommendations depict the essentials of infection control as well as specific measures in the hospital (isolation, criteria for its duration and technical requirements, types of respiratory protection, disinfection measures, waste disposal). The specific requirements for outpatients (medical practice), at home, for ambulance services, and in congregate settings, including prisons, are also addressed. Compared with the previous recommendations the pattern of respiratory protection measures has been simplified. As a rule, hospital staff and those visiting infectious tuberculosis patients are advised to wear respiratory protection that satisfies the criteria of FFP2-masks (DIN EN 149), while patients should wear mouth-nose protectors (surgical masks) in the presence of others and outside the isolation room. A detailed depiction of criteria for isolation and its duration in smear positive and only culturally confirmed pulmonary tuberculosis has been added.

WHO guidelines for the programmatic management of drug-resistant tuberculosis: 2011 update.

The production of guidelines for the management of drug-resistant tuberculosis (TB) fits the mandate of the World Health Organization (WHO) to support countries in the reinforcement of patient care. WHO commissioned external reviews to summarise evidence on priority questions regarding case-finding, treatment regimens for multidrug-resistant TB (MDR-TB), monitoring the response to MDR-TB treatment, and models of care. A multidisciplinary expert panel used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach to develop recommendations. The recommendations support the wider use of rapid drug susceptibility testing for isoniazid and rifampicin or rifampicin alone using molecular techniques. Monitoring by sputum culture is important for early detection of failure during treatment. Regimens lasting ≥ 20 months and containing pyrazinamide, a fluoroquinolone, a second-line injectable drug, ethionamide (or prothionamide), and either cycloserine or p-aminosalicylic acid are recommended. The guidelines promote the early use of antiretroviral agents for TB patients with HIV on second-line drug regimens. Systems that primarily employ ambulatory models of care are recommended over others based mainly on hospitalisation. Scientific and medical associations should promote the recommendations among practitioners and public health decision makers involved in MDR-TB care. Controlled trials are needed to improve the quality of existing evidence, particularly on the optimal composition and duration of MDR-TB treatment regimens.

[Significance of local and general risk factors for the pathogenesis of pulmonary, non-tuberculous mycobacteriosis in non-AIDS patients]. / Bedeutung der lokalen und allgemeinen Risikofaktoren für die Pathogenese der pulmonalen nicht-tuberkulösen Mykobakteriosen bei Nicht-AIDS-Patienten.

BACKGROUND: Pre-existing underlying bronchopulmonary diseases and relative impairments of the immune system are risk factors that predispose to the development of pulmonary infections with non-tuberculous mycobacteria (NTM), even if the impairment is not severe. METHODS: In a prospective study n = 111 patient diagnoses between 1992 and 2004 were included. The criterion for inclusion was laboratory evidence of non-tuberculous mycobacteria. The local risk factors and general risks were recorded for each case and the total number of risks for each patient was counted. Risk profiles were drawn up and risk scores calculated for different groups. RESULTS: N = 66 patients met the ATS criteria for NTM disease. The disease rates for the most frequent species varied widely ( M. AVIUM complex 57 %, M. KANSASII 100 %, M. XENOPI 73 %). Older women (> 65 years) with M. AVIUM complex were rarely ill. The risk factors were almost equally frequent for patients meeting criteria for disease status and those who did not and patients under 65 years of age had fewer local risk factors than older patients. Patients with M. GORDONAE showed fewer local risk factors than patients with M. AVIUM complex or M. XENOPI. CONCLUSIONS: Even local risk factors predispose towards infections with mycobacteria and do not only lead to disease after infection. Bullous changes of the lungs, cavities and bronchiectasis are local risk factors, but can also develop as sequelae of mycobacteriosis. There is sufficient evidence to support the continued use of the concept of colonisation alongside those of infection with and infection without disease status. In our region, a thorough evaluation is needed to establish whether older women with M. AVIUM complex actually have mycobacteriosis.

Mycobacterium tuberculosis strains with highly discordant rifampin susceptibility test results.

The objectives of this study were to investigate the origin of highly discordant rifampin (rifampicin) (RMP) drug susceptibility test results obtained for Mycobacterium tuberculosis strains during proficiency testing. Nine Supra-National Tuberculosis Reference Laboratories tested the RMP susceptibilities of 19 selected M. tuberculosis strains, using standard culture-based methods. The strains were classified as definitely resistant (R) (n = 6) or susceptible (S) (n = 2) or probably resistant (PR) (n = 8) or susceptible (PS) (n = 3) based on rpoB mutations and treatment outcome. All methods yielded a susceptible result for the two S and three PS strains lacking an rpoB mutation and a resistant result for one R strain with a Ser531Leu mutation and one PR strain with a double mutation. Although the remaining 12 R and PR strains had rpoB mutations (four Asp516Tyr, three Leu511Pro, two Leu533Pro, one each His526Leu/Ser, and one Ile572Phe), they were all susceptible by the radiometric Bactec 460TB or Bactec 960 MGIT methods. In contrast, only one was susceptible by the proportion method on Löwenstein-Jensen medium and two on Middlebrook 7H10 agar. Low-level but probably clinically relevant RMP resistance linked to specific rpoB mutations is easily missed by standard growth-based methods, particularly the automated broth-based systems. Further studies are required to confirm these findings, to determine the frequency of these low-level-resistant isolates, and to identify technical improvements that may identify such strains.

Liquid culture for Mycobacterium tuberculosis: proceed, but with caution.

Attempts to improve the diagnosis of tuberculosis (TB) in high-burden countries has resulted in significant funding and initiatives to change the method of diagnosis of TB from light microscopy supplemented with X-ray to a sophisticated diagnostic algorithm based on the latest technological innovations. Such activities are overdue and should be welcomed, but the lack of skills and support available to interpret and use the results represents a danger. The introduction of new diagnostic methods, particularly liquid culture, should be carefully structured according to the local situation, failing which frustration and the disruption of previously underdeveloped but adequately functioning laboratories may result.

[Modern laboratory diagnostics for mycobacteria]. / Moderne mykobakteriologische Labordiagnostik.

Early diagnosis of tuberculosis is one of the most important tools for the interruption of transmission chains and the elimination of the disease. Even today the final diagnosis of tuberculosis has to be confirmed finally by the bacteriological detection of mycobacteria in sputa or other specimens (1 - 4). The suspicion of tuberculosis can be corroborated by chest X-ray and clinical symptoms. Immunologically based assays like the tuberculin skin-test and the new interferon-gamma-release-assays enable the diagnosis of tuberculosis infection, but cannot distinguish between active and latent tuberculosis. [nl]Therefore, the main bacteriological tools in tuberculosis diagnostics remain the following: the microscopic detection of acid-fast bacteria in the specimens, the cultural isolation of the bacteria in liquid media and subsequent identification with molecular methods and the rapid direct identification with nucleic acid amplification techniques (NAT). Drug susceptibility testing has been enhanced by application of the more rapid liquid media and, at least for some drugs, the time to detection can be further shortened by the usage of molecular-based techniques.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis (EUCAST document E.DEF 8.1)--report of the Subcommittee on Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis of the European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID).

This review describes the methods available for drug susceptibility testing of Mycobacterium tuberculosis. The methods have been developed over several decades and are restricted to specialised centres in most European countries, as they are technically demanding, require appropriate isolation facilities and can be difficult to interpret. The absolute concentration, resistance ratio and proportion methods can all give accurate results, provided that they are carefully quality-controlled and standardised. Automated rapid culture and molecular methods have been evaluated at large reference centres and in multicentre collaborations, and perform well for testing susceptibility to most first- and second-line anti-tuberculosis drugs. Accuracy is more important than rapid testing, and this is most reliably achieved if drug susceptibility tests are done in a small number of well-equipped, experienced laboratories that participate and perform well in an international drug susceptibility testing quality assessment scheme. The WHO Supranational Laboratory Quality Control Network offers a global scheme that assesses the ability of participating laboratories to identify isoniazid, rifampicin, ethambutol and streptomycin resistance. Second-line drug resistance testing is currently being standardised, and such testing should only be performed at the national reference laboratories in western and central European countries because of the relatively small number of cases and the concomitant difficulty of maintaining testing proficiency in multiple centres performing small numbers of tests. There is a need to expand international external quality assessment to include second-line drug susceptibility testing.

Application of the Genotype MTBDR assay directly on sputum specimens.

The Genotype MTBDR assay was tested for its capability to detect rifampicin (RMP) and isoniazid (INH) resistance (r) and susceptibility (s) directly from 42 smear-positive sputum specimens (15 RMPr/INHr, 2 RMPs/INHr and 25 RMPs/INHs Mycobacterium tuberculosis complex strains). The concordance between the MTBDR assay and conventional drug susceptibility testing was 100%.

Recommended standards for modern tuberculosis laboratory services in Europe.

The principles underpinning these standards are that any tuberculosis laboratory-based diagnostic procedure should be performed by appropriately trained staff, working to standardised operating procedures in appropriately equipped and safe laboratories, against clear national and international proficiency and quality standards. Quality should be the pre-eminent criteria, not cost. The standards are technologically feasible, but initially may not be within the financial capacity of all laboratories. There is a requirement for government and international donors to adequately fund an appropriate safe infrastructure to enable staff to deliver accurate and timely results at whatever level of activity they are performing. There is a need for national reference laboratories to train a new cadre of mycobacterial laboratory experts. This will require the funding of appropriate individuals at these centres to train and assist in the implementation of good laboratory practice and evaluation to build sustainable capacity. Further operational research is needed to establish the optimal configuration of new technologies to determine isoniazid, rifampicin and second-line drug susceptibility in mycobacterial cultures and also, increasingly, directly on specimens. Improved integration of laboratory medicine as a core part of all tuberculosis programmes is needed to achieve and maximise the potential of new developments.

No associations of human pulmonary tuberculosis with Sp110 variants.

BACKGROUND: After a recent report on the role of the Ipr1 gene in mediating innate immunity in a mouse model of Mycobacterium tuberculosis infection, the human Ipr1 homologue, Sp110, was considered a promising candidate for an association study in human tuberculosis. METHODS: In a sample of >1000 sputum positive, HIV negative West African patients with pulmonary tuberculosis and >1000 exposed, apparently healthy controls, we have genotyped 21 Sp110 gene variants that were either available from public databases, including HapMap data, or identified by DNA re-sequencing. RESULTS: No significant differences in the frequencies of any of the 21 variants were observed between patients and controls. This applied also for HapMap tagging variants and the corresponding haplotypes, when including sliding window analyses with three adjacent variants, and when stratifying controls for positivity and negativity according to the results of intradermal tuberculin (purified protein derivative, PPD) skin tests. DNA re-sequencing revealed 13 novel Sp110 variants in the 5'-UTR, exons, and adjacent intronic regions. CONCLUSIONS: Based on the results obtained in this case-control study, the hypothesis that Sp110 variants and haplotypes might be associated with distinct phenotypes of human M tuberculosis infection is doubtful.