[Consensus-Based Guidelines for Diagnosis, Prevention and Treatment of Tuberculosis in Children and Adolescents - A Guideline on Behalf of the German Society for Pediatric Infectious Diseases (DGPI)]. / S2k-Leitlinie zur Diagnostik, Prävention und Therapie der Tuberkulose im Kindes- und Jugendalter.

Recently, epidemiological data shows an increase of childhood tuberculosis in Germany. In addition to this, drug resistant tuberculosis becomes more frequent. Therefore, diagnosis, prevention and therapy in childhood and adolescence remain a challenge. Adult guidelines do not work for children, as there are age specific differences in manifestation, risk of progression and diagnostic as well as therapeutic pathways.The German Society for Pediatric Infectious Diseases (DGPI) has initiated a consensus-based (S2k) process and completed a paediatric guideline in order to improve and standardize care for children and adolescents with tuberculosis exposure, infection or disease.Updated dosage recommendations take age dependant pharmacokinetics in the treatment of drug sensitive but also drug resistant tuberculosis in account. In addition to this, there is a detailed chapter on perinatal exposure and disease as well as extrapulmonary manifestations.

[Current microbiological methods in the investigation of mycobacteria]. / Aktuelle mikrobiologische Untersuchungsmethoden bei Mykobakterien.

The rapid and reliable detection of tuberculosis is the main goal of microbiological analyses. This is not only of great value for an early diagnosis and early start of an adequate therapy, but also helps to stop transmission and spread of the disease. Prerequisites for successful detection of mycobacteria are careful selection of patient specimens, proper sampling and appropriate shipping. In addition to the classical microbiological methods such as staining for acid-fast bacteria and culture procedures, newer molecular methods are gaining greater importance (PCR; NAT). TB bacteria and resistance-associated mutations can be detected from the specimens directly, providing an early hint about resistant strains. In positive cultures, M. tuberculosis complex and nontuberculous mycobacteria must be discriminated from each other. Drug susceptibility testing (DST) of all first-line drugs has to be performed from one isolate of each patient and repeated if TB bacteria are still isolated after 2 months of therapy. DST of second-line drugs should follow in case of drug resistance or drug intolerance.

Cost of multidrug resistant tuberculosis in Germany-An update.

BACKGROUND: In 2019, new therapeutic recommendations for multidrug-resistant (MDR-) and extensively drug-resistant (XDR) tuberculosis (TB) were published by the WHO, advocating the use of oral drugs and stepwise composition of antibiotic regimens. To date, the economic consequences of those recommendations in low incidence settings have not been evaluated. OBJECTIVE: To assess the costs of applying the new recommendations against a set of 86 MDR-TB/XDR-TB strains, each with individual phenotypic drug resistance patterns, identified in 2018/2019 by the German National Reference Center for Mycobacteria. METHODS: Hospitalization costs as covered by German statutory health insurance and the loss of productivity due to illness were calculated using the most recent 2018 statistical data. Costs due to combining five agents in the intensive phase and costs of outpatient monitoring were determined by Monte-Carlo simulation covering all treatment options over an 18-month period. Drug costs were compared to those arising under the approach recommended by the WHO in 2016. RESULTS: Hospitalization costs per MDR-TB patient were €30,152 and the mean costs of antimicrobials over a period of 18 months were €66,854 (range €20,671 to €187,444). Total treatment costs, including outpatient monitoring, were €73,551.56 per patient (range €30,114 to €145.878). In addition, we determined an average cost of €11,410.20 due to productivity loss over a period of 6 months sick leave. Despite a shortened minimum recommended treatment duration (18 versus 20 months), the estimated costs were 24.5% higher based on the 2019 recommendations as compared to the 2016 guideline version. CONCLUSION: Higher costs for treating MDR-TB/XDR-TB in Germany are to be expected under the new WHO regimens. However, it must be determined whether treatment duration and costs associated with sick leave may be further reduced in the future through shorter hospital stays and earlier culture conversion.

Differential drug susceptibility patterns of Mycobacterium chimaera and other members of the Mycobacterium avium-intracellulare complex.

OBJECTIVES: To determine MIC distributions for Mycobacterium chimaera, Mycobacterium intracellulare, Mycobacterium colombiense and Mycobacterium avium, and to derive tentative epidemiological cut-off (ECOFF) values. METHODS: A total of 683 bacterial isolates (M. chimaera, n = 203; M. intracellulare, n = 77; M. colombiense, n = 68; M. avium, n = 335) from 627 patients were tested by broth microdilution according to CLSI protocol M24-A2 on Sensititre RAPMYCOI plates. MICs were interpreted based on CLSI breakpoints for clarithromycin, and tentative breakpoints for amikacin, moxifloxacin and linezolid. Tentative ECOFFs were determined by visual approximation and the ECOFFinder algorithm. RESULTS: Modal MIC, MIC50 and MIC90 values were within ± one dilution step from the respective aggregated data set for 47/48 (97.9%), 48/48 (100%) and 48/48 (100%) species-drug combinations. Clarithromycin wild-type populations were mostly classified as susceptible (MIC90 4-8 mg/L; S ≤8 mg/L). Rifabutin MICs were lower than those of rifampicin. Tentative moxifloxacin, linezolid and amikacin breakpoints split wild-type populations. No ECOFFs could be set for rifampicin, ethambutol, ciprofloxacin, isoniazid, trimethoprim/sulfamethoxazole and doxycycline because of truncation of MIC distributions. Agreement between the visually determined and the modelled 97.5% ECOFFs was 90.9%. All 99.0% ECOFFs were one titre step higher than by visual approximation. CONCLUSIONS: Drug susceptibility patterns of M. chimaera are comparable to those of closely related species. Except for clarithromycin, breakpoints for Mycobacterium avium-intracellulare complex should be re-evaluated. Statistical determination of the 99.0% ECOFF may be superior to visual approximation.

[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.

Evaluation of OMNIgene®â€¢SPUTUM reagent for mycobacterial culture.

SETTING: National Mycobacterium Reference Laboratory, Borstel, Germany. OBJECTIVE: To evaluate the effectiveness of OMNIgene®â€¢SPUTUM (OM-S) reagent in comparison with a method using N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) with regard to mycobacterial recovery and contamination of broth and solid cultures. DESIGN: Sputum samples from patients with tuberculosis and other respiratory diseases underwent decontamination with NALC-NaOH-based (MycoDDR™) or OM-S reagent. The decontamination procedure was assigned by block randomisation. Samples were inoculated on Löwenstein-Jensen, Stonebrink and MGIT™ (Mycobacterial Growth Indicator Tubes). Mycobacterial recovery from samples spiked with Mycobacterium tuberculosis following decontamination was determined. RESULTS: Eighty-five samples were randomised to NALC-NaOH and 84 to OM-S reagent. Mycobacterial recovery was significantly lower for samples processed with OM-S reagent compared with the NALC-NaOH method across all media types. Culture contamination was lower with NALC-NaOH reagent on solid media (9.4-12.9% vs. 28.6-29.8%). Growth was not observed in MGIT among samples spiked with 10 600-16 800 colony-forming units of M. tuberculosis following decontamination with OM-S reagent. CONCLUSION: Low mycobacterial recovery, especially in MGIT, observed in the present study suggests that OM-S reagent might not be compatible with the MGIT system. More extensive field evaluations of the OM-S reagent are warranted to demonstrate a significant benefit over currently used methods.

How should discordance between molecular and growth-based assays for rifampicin resistance be investigated?

Molecular tests to detect the presence of Mycobacterium tuberculosis and genetic polymorphisms in the rpoB gene conferring resistance to rifampicin (RMP) have become integral parts of tuberculosis diagnostics worldwide. These assays are often performed sequentially or in parallel to phenotypic drug susceptibility testing. Discordances between molecular and phenotypic tests invariably occur. Root causes range from pre-, post- and analytic errors to co-existence of non-tuberculous mycobacteria, silent mutations, mutations outside the 81 base-pair RMP resistance-determining region, non-canonical mutations conferring increased minimal inhibitory concentrations below the critical concentration in some phenotypic drug susceptibility tests, and heteroresistance. Resolving discordant results is challenging. This guide aims to assist both clinicians and microbiologists in interpreting discordances by providing a structured approach to manage further investigations. Case scenarios are discussed, including the likelihood of occurrence.

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%.

Real-time PCR assay for improved detection of Mycobacterium tuberculosis complex in paraffin-embedded tissues.

OBJECTIVE: To test the usefulness of a commercially available real-time polymerase chain reaction (PCR) kit for the detection of Mycobacterium tuberculosis complex (MTBC) in formalin-fixed, paraffin-embedded tissues. RESULTS: The examination of 24 specimens of patients with a final diagnosis of TB shows that the real-time PCR assay exhibits a higher sensitivity (66.7%) for the detection of MTBC DNA than an alternative in-house IS6110 PCR (33.3%), whereas staining detected acid-fast bacilli in only two cases (8.3%). CONCLUSION: The real-time PCR assay provides a highly sensitive and specific means for the detection of MTBC DNA in histopathological specimens.

Clinical implications of molecular drug resistance testing for Mycobacterium tuberculosis: a TBNET/RESIST-TB consensus statement.

The emergence of drug-resistant strains of Mycobacterium tuberculosis is a challenge to global tuberculosis (TB) control. Although culture-based methods have been regarded as the gold standard for drug susceptibility testing (DST), molecular methods provide rapid information on mutations in the M. tuberculosis genome associated with resistance to anti-tuberculosis drugs. We ascertained consensus on the use of the results of molecular DST for clinical treatment decisions in TB patients. This document has been developed by TBNET and RESIST-TB groups to reach a consensus about reporting standards in the clinical use of molecular DST results. Review of the available literature and the search for evidence included hand-searching journals and searching electronic databases. The panel identified single nucleotide mutations in genomic regions of M. tuberculosis coding for katG, inhA, rpoB, embB, rrs, rpsL and gyrA that are likely related to drug resistance in vivo. Identification of any of these mutations in clinical isolates of M. tuberculosis has implications for the management of TB patients, pending the results of in vitro DST. However, false-positive and false-negative results in detecting resistance-associated mutations in drugs for which there is poor or unproven correlation between phenotypic and clinical drug resistance complicate the interpretation. Reports of molecular DST results should therefore include specific information on the mutations identified and provide guidance for clinicians on interpretation and on the choice of the appropriate initial drug regimen.

Application of the Capilia TB assay for culture confirmation of Mycobacterium tuberculosis complex isolates.

The usefulness of a low-tech rapid test for culture confirmation of Mycobacterium tuberculosis complex, Capilia TB, was tested on 172 mycobacteria-positive clinical samples. The overall sensitivity and specificity were 92.4% and 100%, respectively. In three of nine false-negative isolates a mutation in the mpb64 gene could be detected.

Rifampicin and isoniazid resistance mutations in Mycobacterium tuberculosis strains isolated from patients in Kazakhstan.

OBJECTIVE: To analyse possible associations of specific mutations conferring rifampicin (RMP) and isoniazid (INH) resistance with Beijing and non Beijing genotype strains of Mycobacterium tuberculosis from Kazakhstan. METHOD: Genotypic analysis of 92 multidrug-resistant (MDR), 50 INH but not RMP-resistant (INHr/RMPs) and 10 fully susceptible strains of M. tuberculosis from Kazakhstan was performed. In the MDR group, 59 strains (64.1%), and within the INHr/RMPs group, 32 strains (64.0%) were classified as Beijing genotype. RESULTS: Analysis of the rpoB gene of the MDR strains revealed 10 different mutations in five codons, with rpoB codons 531 (65.2%), 526 (23.9%) and 516 (7.6%) most frequently affected. A significantly higher proportion of the rpoB S531L mutation was found among Beijing genotype strains compared with non Beijing strains (71.2% vs. 46.2%, P = 0.027). All 92 MDR isolates (100%), irrespective of their genotype, carried a mutation in codon 315 of the katG gene (S315T). However, in the INHr/RMPS control group, the S315T mutation was significantly more prevalent in the Beijing than in the non Beijing group (96.9% vs. 71.4%, P = 0.012). CONCLUSION: The high similarity of mutations supports the assumption that transmission of resistant strains is a major reason for the emergence of drug resistance in this region.

Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum.

The phosphinothricin (Pt) N-acetyltransferase gene (pat) of Streptomyces viridochromogenes Tü494 is located on a 0.8-kb BglII fragment [Strauch et al., Gene 63 (1988) 65-74]. By sequencing a 1.3-kb BglII-SstII fragment, an open reading frame representing the pat gene was found. It encodes a polypeptide of 183 amino acids with an Mr of 20,621. The base composition of the pat gene is typical for Streptomyces [70.1 mol% (G + C) in total and 93.5 mol% (G + C) in the third position]. Translation of pat is initiated by a GTG codon which was identified by frameshift mutations in Escherichia coli as well as in Streptomyces lividans. Significant homology of the pat gene was found to the bialaphos-resistance gene (bar) of Streptomyces hygroscopicus [Thompson et al., EMBO J. 9 (1987) 2519-2523]. However, variations were detected in the 5'-noncoding region of the two resistance genes which may reflect differences in regulation. Since Pt is a potent herbicide, the pat gene was modified and introduced into Nicotiana tabacum by Agrobacterium-mediated leaf-disc transformation. The GTG start codon of pat was replaced by ATG. Subsequently the modified pat-coding region was fused to the 35S promoter of the cauliflower mosaic virus. Transgenic plants could directly be selected on Pt-containing medium.

Identification and characterization of phosphinothricin-tripeptide biosynthetic genes in Streptomyces viridochromogenes.

A 4-kb BamHI fragment of Streptomyces viridochromogenes Tü494 carrying phosphinothricin-tripeptide (PTT) biosynthetic genes has been identified by complementation of a nonproducing mutant which is defective in the tripeptide formation step. Nucleotide sequence analysis revealed one incomplete and three complete genes on the cloned fragment. The incomplete gene ('pms) codes for the C terminus of the phosphinomethylmalic acid synthase as determined by comparison with a region from the bialaphos biosynthetic cluster [Shimotohno et al., Agric. Biol. Chem. 54 (1990) 463-470] and with databases. Subcloning experiments showed that the juxtaposing phsA gene is sufficient to restore productivity of the blocked mutant. Analysis of gene disruption and gene replacement mutants confirmed that phsA specifies an enzyme involved in tripeptide formation. Similarities to peptide synthetases indicate that the condensation step follows a thio-template mechanism. A conserved region located in the C terminus of the PhsA protein showed identity to 4'-phosphopantetheine-binding sites of fatty acid and polyketide synthases. In the N terminus, a typical acyl transfer motif has been identified and this may be involved in transthiolation. A similar motif also appears in the deduced product of the third gene (dea), which probably catalyses the deacetylation of N-acetyl-PTT to PTT. The previously described PTT resistance-encoding gene (pat) was located between the phsA and the dea genes.

HOPE-fixation enables improved PCR-based detection and differentiation of Mycobacterium tuberculosis complex in paraffin-embedded tissues.

Standard PCR-based detection of mycobacterial DNA in paraffin-embedded specimens may lack sufficient sensitivity because of the degradation of nucleic acids caused by routinely used formalin fixation. Therefore, we set up an approach that aimed at improving the results by applying the novel HOPE-fixative in PCR-detection of mycobacteria in paraffin-embedded tissues. Comparison of PCR-results using DNA extracted from either HOPE- or formalin-fixed specimens in BCG-infected SCID-mice revealed a more than 100fold enhanced sensitivity for the HOPE-fixed material. Owing to the preservation of DNA from degradation in HOPE-fixed tissues, even differentiation within the M. tuberculosis complex was possible by spoligotyping. We therefore conclude that the HOPE-fixative is a useful tool for molecular pathology that enhances the sensitivity of PCR-based methods for the detection of pathogens in paraffin-embedded tissues compared to formalin-fixation. Owing to the better preserved DNA, improved differentiation of mycobacteria from archived materials is possible. These results promise new and a substantially wider range of possibilities in the field of molecular pathology.

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.

Gene disruption and gene replacement in Streptomyces via single stranded DNA transformation of integration vectors.

For the isolation of single stranded plasmid DNA, various E. coli and E. coli-Streptomyces shuttle plasmids were equipped with the phage f1 replication origin. The transformation of some representative Streptomyces species with plasmid vectors occurred irrespective of whether single or double stranded DNA was used. In contrast, the transformation of Streptomyces was 10 to 100 times more efficient when an integration vector was in the single stranded form as opposed to the double stranded form. Streptomyces viridochromogenes was transformed by single stranded DNA integration vectors in order to replace the pat by the tsr gene and generate mutants unable to synthesize phosphinothricin-tripeptide (PTT).

Genetic and biochemical characterization of the two glutamine synthetases GSI and GSII of the phosphinothricyl-alanyl-alanine producer, streptomyces viridochromogenes Tü494.

The 1410 bp DNA region (glnA) encoding glutamine synthetase I (GSI) from Streptomyces viridochromogenes was amplified by PCR, cloned and sequenced. The molecular mass of the deduced GSI protein (469 residues) was determined to be 50 kDa. The DNA region showed 90% nucleotide identity with the Streptomyces coelicolor A3(2) glnA gene, but no significant nucleotide sequence similarity with the glnII (GSII) gene of S. viridochromogenes. The chromosomal glnA and glnII genes of S. viridochromogenes were disrupted by site-specific mutagenesis. Neither glnA nor glnII single mutants required glutamine for growth and both were normal in their sporulation. Measurement of the GS activity in cultures grown with different nitrogen sources revealed that GSI (heat-stable) and GSII (heat-labile) were always expressed together, with GSI as the predominant activity. It could be proposed that GSI, but not GSII is inactivated by adenylylation under conditions of nitrogen excess. GSI and GSII activities are inhibited by amino acids and by nucleotides.