Effectiveness of GenoType MTBDRsl in excluding TB drug resistance in a clinical trial.

OBJECTIVES: To assess the performance of the GenoType MTBDRsl v1, a line-probe assay (LPA), to exclude baseline resistance to fluoroquinolones (FQs) and second-line injectables (SLIs) in the Standard Treatment Regimen of Anti-tuberculosis Drugs for Patients With MDR-TB 1 (STREAM 1) trial.METHODS: Direct sputum MTBDRsl results in the site laboratories were compared to indirect phenotypic drug susceptibility testing (pDST) results in the central laboratory, with DNA sequencing as a reference standard.RESULTS: Of 413 multidrug-resistant TB (MDR-TB) patients tested using MTBDRsl and pDST, 389 (94.2%) were FQ-susceptible and 7 (1.7%) FQ-resistant, while 17 (4.1%) had an inconclusive MTBDRsl result. For SLI, 372 (90.1%) were susceptible, 5 (1.2%) resistant and 36 (8.7%) inconclusive. There were 9 (2.3%) FQ discordant pDST/MTBDRsl results, of which 3 revealed a mutation and 5 (1.3%) SLI discordant pDST/MTBDRsl results, none of which were mutants on sequencing. Among the 17 FQ- and SLI MTBDRsl-inconclusive samples, sequencing showed 1 FQ- and zero SLI-resistant results, similar to frequencies among the conclusive MTBDRsl. The majority of inconclusive MTBDRsl results were associated with low bacillary load samples (acid-fast bacilli smear-negative or scantily positive) compared to conclusive results (P < 0.001).CONCLUSION: MTBDRsl can facilitate the rapid exclusion of FQ and SLI resistances for enrolment in clinical trials.

Fluoroquinolone heteroresistance in Mycobacterium tuberculosis: detection by genotypic and phenotypic assays in experimentally mixed populations.

Heteroresistance - the simultaneous presence of drug-susceptible and -resistant organisms - is common in Mycobacterium tuberculosis. In this study, we aimed to determine the limit of detection (LOD) of genotypic assays to detect gatifloxacin-resistant mutants in experimentally mixed populations. A fluoroquinolone-susceptible M. tuberculosis mother strain (S) and its in vitro selected resistant daughter strain harbouring the D94G mutation in gyrA (R) were mixed at different ratio's. Minimum inhibitory concentrations (MICs) against gatifloxacin were determined, while PCR-based techniques included: line probe assays (Genotype MTBDRsl and GenoScholar-FQ + KM TB II), Sanger sequencing and targeted deep sequencing. Droplet digital PCR was used as molecular reference method. A breakpoint concentration of 0.25 mg/L allows the phenotypic detection of ≥1% resistant bacilli, whereas at 0.5 mg/L ≥ 5% resistant bacilli are detected. Line probe assays detected ≥5% mutants. Sanger sequencing required the presence of around 15% mutant bacilli to be detected as (hetero) resistant, while targeted deep sequencing detected ≤1% mutants. Deep sequencing and phenotypic testing are the most sensitive methods for detection of fluoroquinolone-resistant minority populations, followed by line probe assays (provided that the mutation is confirmed by a mutation band), while Sanger sequencing proved to be the least sensitive method.

Disputed rpoB mutations can frequently cause important rifampicin resistance among new tuberculosis patients.

SETTING: Greater Mymensingh area, Bangladesh. OBJECTIVES: To document among new tuberculosis (TB) patients the proportions and treatment outcomes of silent, non-disputed and disputed (generally missed by rapid drug susceptibility testing [DST]) rpoB mutations, and their detection by commercial molecular assays. DESIGN: Retrospective analysis of rpoB sequences from randomly selected ethanol-preserved diagnostic sputum samples; comparison of sequencing with conventional DST results and standard first-line treatment outcome; retesting of samples with mutations using the Xpert MTB/RIF and GenoType MTBDRplus assays. RESULTS: Of 1091 samples, 5.8% failed amplification, and six contained other mycobacteria. In 2005 and 2010, respectively 2/500 (0.4%) and 11/522 (2.1%) amplicons showed non-silent mutations. At least 7/13 of these belonged to the disputed group, with 5/7 patients suffering adverse treatment outcome. One silent mutation went undetected by commercial assays. Following routine DST indications, only three cases with a non-silent mutation were eventually detected. CONCLUSIONS: Disputed rpoB mutations may be responsible for the majority of rifampicin (RMP) resistance among new cases, and lead to adverse outcomes of first-line treatment. Silent mutations do not necessarily cause Xpert or line-probe assay false RMP-resistant results. Molecular RMP DST could greatly simplify resistance surveillance, in addition to offering the best prospects for early and accurate individual diagnosis.

MIR137 variants identified in psychiatric patients affect synaptogenesis and neuronal transmission gene sets.

Sequence analysis of 13 microRNA (miRNA) genes expressed in the human brain and located in genomic regions associated with schizophrenia and/or bipolar disorder, in a northern Swedish patient/control population, resulted in the discovery of two functional variants in the MIR137 gene. On the basis of their location and the allele frequency differences between patients and controls, we explored the hypothesis that the discovered variants impact the expression of the mature miRNA and consequently influence global mRNA expression affecting normal brain functioning. Using neuronal-like SH-SY5Y cells, we demonstrated significantly reduced mature miR-137 levels in the cells expressing the variant miRNA gene. Subsequent transcriptome analysis showed that the reduction in miR-137 expression led to the deregulation of gene sets involved in synaptogenesis and neuronal transmission, all implicated in psychiatric disorders. Our functional findings add to the growing data, which implicate that miR-137 has an important role in the etiology of psychiatric disorders and emphasizes its involvement in nervous system development and proper synaptic function.

Integrated detection of multi- and extensively drug-resistant tuberculosis using the nitrate reductase assay.

It currently takes 2-3 months to obtain a diagnosis for multidrug-resistant (MDR-) and extensively drug-resistant tuberculosis (XDR-TB). We evaluated the rapid non-commercial nitrate reductase assay (NRA), which is capable of the simultaneous detection of MDR- and XDR-TB, and compared the results with the proportion method (PM). The sensitivity was respectively 97%, 99%, 100% and 94.6% for rifampicin (RMP), isoniazid (INH), ofloxacin (OFX) and kanamycin (KM). The specificity was respectively 100%, 95%, 95.7% and 99% for RMP, INH, OFX and KM. The turnaround time for NRA was 10-14 days, compared to 4-6 weeks for the PM. Our study showed that NRA provided sensitive and specific detection of resistance to first- and second-line drugs.

Evaluation of a multiplex-PCR detection in combination with an isolation method for STEC O26, O103, O111, O145 and sorbitol fermenting O157 in food.

The aim of the current study was to evaluate a multiplex PCR (mPCR) detection test combined with the evaluation of a previously described isolation method. Minced beef, raw-milk cheese and sprouted seed samples were inoculated with low amounts (7-58 cfu 25 g(-1)) of non-stressed, cold-stressed or freeze-stressed clinical STEC strains, including serogroups O26, O103, O111, O145, sorbitol fermenting (SF) O157 and non-sorbitol fermenting (NSF) O157. The inoculated pathogen was detected using a 24 h-enrichment followed by an mPCR protocol, and in parallel isolated using an enrichment step of 6 and 24 h, followed by selective plating of the enriched broth and selective plating of the immunomagnetic separation (IMS) product. Recovery results were evaluated and compared. Successful mPCR detection and isolation was obtained for non-stressed and cold-stressed STEC cells in minced beef and raw-milk cheese samples, except for serogroups O111 and SF O157. For freeze-stressed cells and sprouted seed samples, false negatives were often found. Isolation was better after 24 h-enrichment compared to 6 h-enrichment. IMS improved in some cases the isolation of non-stressed and cold-stressed cells belonging to serogroups O111 and O157 from minced beef and raw-milk cheese and freeze-stressed cells of all tested serogroups from minced beef.

Evaluation of the TB Ag MPT64 Rapid test for the identification of Mycobacterium tuberculosis complex.

Rapid identification of Mycobacterium tuberculosis complex in cultured samples is important for starting appropriate treatment. We evaluated the performance of the TB Ag MPT64 Rapid test directly from 131 BACTEC MGIT 960 culture-positive samples: 113 were identified as M. tuberculosis complex and 18 as non-tuberculous mycobacteria. The sensitivity and specificity of the TB Ag MPT64 Rapid test were respectively 96.5% and 100% compared to the polymerase chain reaction. The overall concordance of the TB Ag MPT64 Rapid test was 969%. The TB Ag MPT64 Rapid test is easy, sensitive, and does not require a high level of skill or specific equipment.

Evaluation of the Genotype® MTBDRplus assay as a tool for drug resistance surveys.

SETTING: A national tuberculosis (TB) drug resistance survey in Tanzania. OBJECTIVE: To compare the performance of the Genotype® MTBDRplus line-probe assay (LPA) on smear-positive sputum specimens with conventional culture and isoniazid (INH) plus rifampicin (RMP) drug susceptibility testing (DST). DESIGN: Mycobacterium tuberculosis isolates tested at the Tanzanian Central TB Reference Laboratory (CTRL) were submitted for quality assurance of phenotypic DST to its supranational reference laboratory (SRL), together with ethanol-preserved sputum specimens for LPA DST. RESULTS: Only 321 samples could be tested using LPA; of these, three were identified as being non-tuberculous mycobacteria using CTRL DST. Both tests had 269 sets with interpretable results. CTRL DST yielded almost the same number of interpretable results as LPA, with 90% concordance (κ = 0.612, P < 0.001). Five (1.9%) multidrug-resistant (MDR) strains, 46 (17.1%) resistant to INH only and 0 RMP only, were found by CTRL DST. For the LPA, these results were respectively 5 (1.9%), 26 (9.7%) and 2 (0.7%). With SRL DST as the gold standard, LPA was more accurate than CTRL DST for RMP, but missed almost half the INH-resistant samples. CONCLUSION: LPA applied directly on ethanol-preserved sputum specimens was similar to phenotypic DST in terms of yield of interpretable results. Although probably more accurate for RMP and MDR-TB, it appears to seriously underestimate INH resistance. Considering speed, easy and safe specimen transportation and low infrastructure requirements, LPA DST from sputum can be recommended for surveys in resource-poor settings.

Limited fluoroquinolone resistance among Mycobacterium tuberculosis isolates from Rwanda: results of a national survey.

OBJECTIVES: There is an increasing interest in the possible role of fluoroquinolone antibiotics for the treatment of tuberculosis (TB), but widespread use of these antibiotics for the treatment of other bacterial infections may select for fluoroquinolone-resistant Mycobacterium tuberculosis strains. METHODS: We evaluated fluoroquinolone susceptibility using the proportion method (ofloxacin, critical concentration 2.0 mg/L) in isolates from patients enrolled in a national drug resistance survey in Rwanda from November 2004 to February 2005. RESULTS: Of the 701 M. tuberculosis isolates studied, 617 (88%) were susceptible to all first-line drugs, 32 (4.6%) were multidrug-resistant (MDR) and 52 (7.4%) were resistant to one or more first-line drugs but not MDR. Ofloxacin resistance was found in four (0.6%) of the isolates; three of them being MDR and one susceptible to all first-line drugs. Mutations in the gyrA gene were found in all ofloxacin-resistant strains at codons 80 and 94. CONCLUSIONS: Our finding is not alarming for Rwanda, but highlights the general risk of producing resistance to fluoroquinolones, jeopardizing the potential for these drugs to be used as second-line anti-TB agents in the programmatic management of drug-resistant TB and creating incurable TB strains.

A novel CpG-associated brain-expressed candidate gene for chromosome 18q-linked bipolar disorder.

We previously identified 18q21-q22 as a candidate region for bipolar (BP) disorder and constructed a yeast artificial chromosome (YAC) contig map. Here we identified three potential CpG islands using CCG/CGG YAC fragmentation. Analysis of available genomic sequences using bioinformatic tools identified an exon of 3639 bp downstream of a CpG island of 1.2 kb containing a putative transcription initiation site. The exon contained an open reading frame coding for 1212 amino acids with significant homology to the SART-2 protein; weaker homology was found with a series of sulphotransferases. Alignment of cDNA sequences of corresponding ESTs and RT-PCR sequencing predicted a transcript of 9.5 kb which was confirmed by Northern blot analysis. The transcript was expressed in different brain areas as well as in multiple other peripheral tissues. We performed an extensive mutation analysis in 113 BP patients. A total of nine single nucleotide polymorphisms (SNPs) were identified. Five SNPs predicted an amino acid change, of which two were present in BP patients but not in 163 control individuals.

The European Large Subunit Ribosomal RNA Database.

The European Large Subunit Ribosomal RNA Database compiles all complete or nearly complete large subunit ribosomal RNA sequences available from public sequence databases. These are provided in aligned format and the secondary structure, as derived by comparative sequence analysis, is included. Additional information about the sequences such as literature references and taxonomic information is also included. The database is available from our WWW server at http://rrna.uia.ac.be/lsu/.

Comparative analysis of more than 3000 sequences reveals the existence of two pseudoknots in area V4 of eukaryotic small subunit ribosomal RNA.

The secondary structure of V4, the largest variable area of eukaryotic small subunit ribosomal RNA, was re-examined by comparative analysis of 3253 nucleotide sequences distributed over the animal, plant and fungal kingdoms and a diverse set of protist taxa. An extensive search for compensating base pair substitutions and for base covariation revealed that in most eukaryotes the secondary structure of the area consists of 11 helices and includes two pseudoknots. In one of the pseudoknots, exchange of base pairs between the two stems seems to occur, and covariation analysis points to the presence of a base triple. The area also contains three potential insertion points where additional hairpins or branched structures are present in a number of taxa scattered throughout the eukaryotic domain.

The European small subunit ribosomal RNA database.

The European database of the Small Subunit (SSU) Ribosomal RNA is a curated database that strives to collect all information about the primary and secondary structure of completely or nearly-completely sequenced rRNAs. Furthermore, the database compiles additional information such as literature references and taxonomic status of the organism the sequence was derived from. The database can be consulted via the WWW at URL http://rrna.uia.ac.be/ssu/. Through the WWW, sequences can be easily selected either one by one, by taxonomic group, or by a combination of both, and can be retrieved in different sequence and alignment formats.

The European large subunit ribosomal RNA database.

The European Large Subunit (LSU) Ribosomal RNA (rRNA) database is accessible via the rRNA WWW Server at URL http://rrna.uia.ac.be/lsu/. It is a curated database that compiles complete or nearly complete LSU rRNA sequences in aligned form, and also incorporates secondary structure information for each sequence. Taxonomic information, literature references and other information about the sequences are also available, and can be searched via the WWW interface.

Database on the structure of large subunit ribosomal RNA.

The Antwerp database on large subunit ribosomal RNA now contains 607 complete or nearly complete aligned sequences. The alignment incorporates secondary structure information for each sequence. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available. Information from the database can be downloaded or searched on the rRNA WWW Server at URL http://rrna.uia.ac.be/

Database on the structure of small subunit ribosomal RNA.

Over 11 500 complete or nearly complete sequences are now available from the Antwerp database on small subunit ribosomal RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/

Comparison of two PCRs for detection of Mycobacterium ulcerans.

Two nested PCRs for the detection of Mycobacterium ulcerans were compared by using a collection of 65 clinical specimens. The first method amplifies the gene coding for 16S rRNA, and the second method amplifies a repetitive DNA sequence. The sensitivities of bacterioscopy, culture, 16S rRNA gene PCR, and repetitive-sequence PCR were 29, 34, 80, and 85%, respectively. Compared to the 16S rRNA gene PCR, the repetitive-sequence PCR was faster, easier to perform, and less expensive.

The secondary structure of Nosema apis large subunit ribosomal RNA.

The microsporidia are a group of obligate intracellular eukaryotic parasites, that lack mitochondria. Their ribosomes show several prokaryote-like features. This paper presents the secondary structure of the large subunit ribosomal RNA (LSU rRNA) of the microsporidium Nosema apis. With its 2481 bases, it is the shortest known non-mitochondrial LSU rRNA. The seemingly prokaryote-like features of the molecule cannot be used as evidence for the ancient origin of the microsporidia. The reduction in size can be attributed to changes in the regions of the LSU rRNA that are known to show great variability in length and sequence within the eukaryotes. The lack of fragmentation commonly seen in other eukaryotes may also be a derived feature.

Mycobacterium ulcerans infection in a child from Angola: diagnosis by direct detection and culture.

Buruli ulcer, caused by Mycobacterium ulcerans, is a chronic ulcerative skin disease, found predominantly in central and west Africa and Australia. A boy of 2.5 years of age from Angola was admitted to our hospital with severe kwashiokor and a large ulcer with undermined edges on the left side of the thorax. Further examination revealed anaemia, hypoproteinaemia, bacterial superinfection of the ulcer and intestinal parasites. Histological analysis showed acid-fast bacilli and histopathological changes typical of Buruli ulcer. M. ulcerans was detected by PCR and culture. The patient was treated by surgical excision of diseased skin, followed by split-skin grafting. He also received antibiotic therapy (ciprofloxacin, clarithromycin, rifabutin, and dapsone). After six months, the child was discharged from hospital in good condition. This is the first published case of Buruli ulcer from Angola.

Database on the structure of small ribosomal subunit RNA.

About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/