Current tools available for the diagnosis of drug-resistant tuberculosis.

Drug-resistant tuberculosis (DR-TB) poses a major threat to control of tuberculosis worldwide. Diagnosis and treatment of DR-TB are considerably more difficult than for drug-susceptible tuberculosis (TB) and require higher level infrastructure and proficiency from laboratory specialists and clinicians. The World Health Organization (WHO) End TB Strategy calls for early diagnosis and initiation of appropriate treatment of all persons of all ages with any form of drug-susceptible TB or DR-TB. This requires ensuring access to WHO-recommended rapid diagnostics and universal drug susceptibility testing (DST) for all persons with signs and symptoms of TB. There are a number of laboratory tools available for diagnosis of DR-TB, including phenotypic culture-based DST as well as molecular methods. Optimal and complementary use of the available diagnostic tools at the different levels of the tiered network of TB laboratories, as well as correct interpretation of the diagnostic results provided by them is critical for accurate and timely diagnosis of DR-TB thus enabling effective treatment and care of patients.

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.

Drug susceptibility testing proficiency in the network of supranational tuberculosis reference laboratories.

SETTING: The network of supranational tuberculosis reference laboratories (SRLs). OBJECTIVE: To evaluate the annual SRL Rounds 6-14 of proficiency testing for first-line drug susceptibility testing (DST). DESIGN: Panels consisted of 20-30 cultures (including 10 pairs of duplicate strains), aiming at 50% resistance prevalence with a variety of profiles. The 27 SRLs participating in at least one of these rounds were free to use their preferred DST method. A judicial gold standard of at least 80% concordant 'susceptible' or 'resistant' was used to determine sensitivity, specificity and efficiency; otherwise the strain was excluded. RESULTS: Of 600 strains, 10% were excluded from evaluation. The average SRL sensitivity and specificity varied between rounds, without attaining significance or trends. Both sensitivity and specificity remained at >95% for isoniazid (INH), rifampicin (RMP) and streptomycin and at >80% for ethambutol. The 16 SRLs participating in all rounds performed consistently better. CONCLUSION: The rounds succeeded in comparing the proficiency of laboratories, and should be further promoted for DST quality assessment. However, to function with greater precision and to ultimately improve the clinical relevance of DST, the INH and RMP judicial result gold standard also needs to take into account genotypic and treatment outcome information.

Treatment of varicose tributaries with sclerotherapy with polidocanol 0.5 % foam.

BACKGROUND: Although foam sclerotherapy of varicose tributaries is common in daily practice, scientific evidence for the optimal sclerosant-concentration and session-frequency is still low. This study aimed to increase the knowledge on foam sclerotherapy of varicose tributaries and to evaluate the efficacy and safety of foam sclerotherapy with 0.5 % polidocanol in tributaries with 3-6 mm in diameter. PATIENTS AND METHODS: Analysis of 110 legs in 76 patients. Injections were given every second or third day. A maximum of 1 injection / leg and a volume of 2 ml / injection were administered per session. Controls were performed approximately 6 months and 12 months after the start of therapy. RESULTS: 110 legs (CEAP C2-C4) were followed up for a period of 14.2 +/- 4.2 months. Reflux was eliminated after 3.4 +/- 2.7 injections per leg. Insufficient tributaries were detected in 23.2 % after 6.2 +/- 0.9 months and in 48.2 % after 14.2 +/- 4.2 months, respectively. Only 30.9 % (34 / 110) of the legs required additional therapy. In 6.4 % vein surgery was performed, in 24.5 % similar sclerotherapy was repeated. Significantly fewer sclerotherapy-sessions were required compared to the initial treatment (mean: 2.3 +/- 1.4, p = 0.0054). During the whole study period thrombophlebitis (8.2 %), hyperpigmentation (14.5 %), induration in the treated region (9.1 %), pain in the treated leg (7.3 %) and migraine (0.9 %) occurred. One patient with a history of thrombosis developed thrombosis of a muscle vein (0.9 %). After one year there were just hyperpigmentation (8.2 %) and induration (1.8 %) left. No severe adverse effect occurred. CONCLUSIONS: Foam sclerotherapy with injections of 0.5 % polidocanol every 2nd or 3rd day, is a safe procedure for varicose tributaries. The evaluation of efficacy is difficult, as it can hardly be said whether the detected tributaries in the controls are recurrent veins or have recently developed in the follow-up period. The low number of retreated legs indicates a high efficacy and satisfaction of the patients.

Results of a tuberculosis-specific IFN-gamma assay in children at high risk for tuberculosis infection.

The specificity of the tuberculin skin test (TST) for the diagnosis of latent tuberculosis infection (LTBI) is adversely affected by bacille Calmette-Guérin (BCG) vaccination and infection with non-tuberculous mycobacteria. Interferon-gamma release assays (IGRAs) using TB-specific antigens promise higher specificity. We compared a new IGRA and TST in 184 schoolchildren at high risk for LTBI. The IGRA and TST were positive in 33.2% and 43.5% of the children, respectively (P < 0.001). If studies confirm that this difference is due to higher specificity of this IGRA, it may have an important role to play in the diagnosis of LTBI and identification of children at true risk for TB.

Risk factors associated with default from multidrug-resistant tuberculosis treatment, South Africa, 1999-2001.

SETTING: Multidrug-resistant tuberculosis (MDR-TB) treatment centers in five provinces, South Africa. OBJECTIVES: To estimate the mortality and evaluate risk factors associated with default from MDR-TB treatment. DESIGN: Using registries and a standardized questionnaire, we conducted a case-control study among patients diagnosed and treated for MDR-TB. Cases were defined as patients who began MDR-TB treatment between 1 October 1999 and 30 September 2001 and defaulted from treatment for more than 2 months; controls were defined as patients who began MDR-TB treatment during the same time and were cured, completed or failed. RESULTS: After initial identification and reclassification, 269 cases and 401 controls were confirmed eligible for interview. Further investigation revealed that 74 (27%) cases and 44 (10%) controls had died. Among 96 cases located who consented and were interviewed, 70% had defaulted after receiving at least 6 months of treatment. In a multivariate model, the strongest individual risk factors for default included reporting smoking marijuana or mandrax during treatment, and having an unsatisfactory opinion about the attitude of health care workers. CONCLUSION: Mortality among MDR-TB defaulters was high. Interventions to reduce default from MDR-TB treatment should center on substance abuse treatment, patient education and support and improving provider-patient relationships.

Speaking the same language: treatment outcome definitions for multidrug-resistant tuberculosis.

SETTING: Globally it is estimated that 273000 new cases of multidrug-resistant tuberculosis (MDR-TB, resistance to isoniazid and rifampicin) occurred in 2000. To address MDR-TB management in the context of the DOTS strategy, the World Health Organization and partners have been promoting an expanded treatment strategy called DOTS-Plus. However, standard definitions for MDR-TB patient registration and treatment outcomes do not exist. OBJECTIVE: To propose a standardized set of case registration groups and treatment outcome definitions for MDR-TB and procedures for conducting cohort analyses under the DOTS-Plus strategy. DESIGN: Using published definitions for drug-susceptible TB as a guide, a 2-year-long series of meetings, conferences, and correspondence was undertaken to review published literature and country-specific program experience, and to develop international agreement. RESULTS: Definitions were designed for MDR-TB patient categorization, smear and culture conversion, and treatment outcomes (cure, treatment completion, death, default, failure, transfer out). Standards for conducting outcome analyses were developed to ensure comparability between programs. CONCLUSION: Optimal management strategies for MDR-TB have not been evaluated in controlled clinical trials. Standardized definitions and cohort analyses will facilitate assessment and comparison of program performance. These data will contribute to the evidence base to inform decision makers on approaches to MDR-TB control.

Dihydropteroate synthase and novel dihydrofolate reductase gene mutations in strains of Pneumocystis jirovecii from South Africa.

Dihydropteroate synthase (DHPS) gene mutations have raised concerns about emerging sulfonamide resistance in Pneumocystis jirovecii. DHPS and dihydrofolate reductase (DHFR) gene products were amplified in clinical specimens from South African patients. One of 53 DHPS genes sequenced contained the double mutation Thr55Ala Pro57Ser. DHFR gene mutations detected were Ala67Val and the new mutations Arg59Gly and C278T.

Capacity building for international tuberculosis control through operations research training.

SETTING: In resource-poor countries, few tuberculosis (TB) program staff at the national, provincial, and even district levels have the basic analytical and epidemiological skills necessary for collecting and analyzing quality data pertaining to national TB control program (NTP) improvements. This includes setting program priorities, operations planning, and implementing and evaluating program activities. OBJECTIVES: To present a model course for building capacity in basic epidemiology and operations research (OR). DESIGN: A combination of didactic lectures and applied field exercises were used to achieve the main objectives of the 6-day OR course. These were to increase the understanding of quantitative and qualitative research concepts, study design, and analytic methods, and to increase awareness of how these methods apply to the epidemiology and control of TB; and to demonstrate the potential uses of OR in answering practical questions on NTP effectiveness. As a final outcome, course participants develop OR proposals that are funded and later implemented. RESULTS: Since 1997, this OR course has been conducted nine times in five countries; 149 key NTP and laboratory staff have been trained in OR methods, and 44 OR protocols have been completed or are underway. CONCLUSION: This low-cost model course can be adapted to a wide range of public health issues.

Improved biological and transcriptional activity of monopegylated interferon-alpha-2a isomers.

The addition of polyethyleneglycol (PEG) side chains to interferon alpha-2a improves the serum stability and clinical efficacy. Current commercial PEG-INF formulations such as PEGASYS are heterogeneous and contain multiple monopegylated isomers. We have analyzed the activity of nine, purified monopegylated variants in antiproliferative, antiviral and binding assays, together with a global transcriptional analysis using DNA oligonucleotide microarrays. We show a direct correlation between biological and transcriptional activity for all isomers and an inversed correlation between IFN-receptor 2a affinity and signal transduction. Two out of nine positional isomers have a higher specific biological and transcriptional activity than the mixture, which can be explained by unique structural features of interferon signaling, which involves two distinct receptors. The possible clinical implications are discussed, which might guide the development of pegylated interferons with improved pharmacological properties.

Determinants of drug-resistant tuberculosis: analysis of 11 countries.

SETTING: Eleven countries/territories. OBJECTIVES: Global information on the determinants of drug-resistant tuberculosis (TB) based on representative data is not available. We therefore studied the relationship between demographic characteristics, prior TB treatment, and human immunodeficiency virus (HIV) infection with anti-tuberculosis drug resistance. METHODS: Population-based representative data on new and previously treated patients with TB collected within an international drug resistance surveillance network. RESULTS: Of 9,615 patients, 8,222 (85.5%) were new cases of TB and 1,393 (14.5%) were previously treated cases. Compared with new cases, previously treated cases were significantly more likely to have resistance to one (OR = 2.5,95% CI 2.1-3.0; P < 0.001), two (OR = 4.6, 95%CI 3.7-5.6; P < 0.001), three (OR = 11.5, 95%CI 8.6-15.3; P < 0.001), and four (OR = 18.5, 95% CI 12.0-28.5; P < 0.001) drugs. An approximately linear increase in the likelihood of having multidrug-resistant tuberculosis (MDR-TB) was observed as the total time (measured in months) of prior anti-tuberculosis treatment increased (P < 0.001, chi2 for trend). In multivariate analysis, prior TB treatment for 6-11 months (OR = 7.6, 95% CI 2.6, 22.4; P < 0.001) and > or = 12 months (OR 13.7, 95% CI 4.5-41.6; P < 0.001), but not HIV positivity, was associated with MDR-TB. CONCLUSION: This study shows that prior but ineffective treatment is a strong predictor of drug resistance, and that HIV is not an independent risk factor for MDR-TB. The association between length of treatment and drug resistance may reflect longer treatment as a result of treatment failure in patients with drug resistance; it may also reflect irregular prior treatment for TB, leading to drug resistance.

Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin.

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1-6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482-His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.

Mycobacterium bovis as a zoonosis in the Kruger National Park, South Africa.

SETTING: The Kruger National Park (KNP), Mpumalanga Province, South Africa. OBJECTIVE: The prevalence of tuberculosis caused by Mycobacterium bovis exceeds 70% in African buffalo in the southern region of the KNP. Inter-species transmission (lion, cheetah, baboon, antelope) has also been confirmed. Regular culling of emaciated buffalo and processing of meat and hides constitute routine control policy. Following extensive media coverage of the problem, public health concerns about the transmission of M. bovis to humans, including visitors to the KNP, prompted this investigation. DESIGN: The study was designed to determine the prevalence of infection and/or active disease due to M. bovis among KNP employees selected from three defined risk groups based on occupation category. RESULTS: Of 206 persons screened for active disease by sputum bacteriology, two persons with disease due to M. tuberculosis were identified. No isolate of M. bovis was found. Differential skin testing using three antigens failed to show any degree of M. bovis infection risk, even among high risk occupations. Reasons for these results are discussed. CONCLUSIONS: Bovine tuberculosis was not indicated as an occupational zoonosis in the KNP, nor was aerosol transmission implicated as a mechanism for human infection. Concerns about the public health implications of tuberculosis in buffalo in the KNP have therefore not been validated.

Universal pattern of RpoB gene mutations among multidrug-resistant isolates of Mycobacterium tuberculosis complex from Africa.

SETTING: Multidrug-resistant tuberculosis (MDR-TB) presents an increasing burden in Southern Africa. Rapid diagnostic tests for drug resistance to rifampicin have been developed based on mutation analysis of the rpoB gene. However, geographic differences of underlying mutations have recently been suggested. OBJECTIVE: Drug-resistant strains of Mycobacterium tuberculosis complex from Africa were analysed for geographic differences in frequency and location of rpoB mutations. DESIGN: A random sample of rifampicin-resistant strains was collected from 87 patients with pulmonary MDR-TB treated in 12 hospitals from six different regions of South Africa. In addition, 18 isolates of M. tuberculosis complex from Namibia, Sierra Leone, and Uganda, including 13 isolates of M. africanum, were analyzed. Point mutations were detected by direct sequence analysis of the rpoB gene. RESULTS: Missense mutations were identified for 91 isolates (87%). Double mutations were present in eight (8%) MDR-TB isolates, two of which carried one mutation outside a previously described diagnostic region. We found no geographic differences regarding the frequency and pattern of single rpoB gene mutations. CONCLUSION: Our results confirm that molecular genetic analysis of rifampicin resistance based on a core region within the rpoB gene is universally applicable to strains of M. tuberculosis complex from different geographic regions.

Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis complex from Africa.

A sample of 124 isoniazid (INH)-resistant and 88 susceptible strains of Mycobacterium tuberculosis complex from south, central, and west Africa was analyzed by direct sequence analysis and PCR-restriction fragment length polymorphism analysis of their catalase-peroxidase (katG) genes. Point mutations at codon 315 were found in the genomes of 64% of INH-resistant strains, but no complete deletions were identified. Mutations at codon 463 were independent of INH resistance and were linked to the geographic origins of the strains.