Didi Bang

Rapid genotypic detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis directly in clinical specimens.

ABSTRACT A multiplex PCR DNA strip assay (Genotype MTBDR) designed to detect rifampin (rpoB) and high-level isoniazid (katG) resistance mutations in Mycobacterium tuberculosis isolates was optimized for clinical specimens. Successful genotypic results were achieved with 36 of 38 (95%) smear-positive respiratory specimens, allowing rapid therapeutic adjustments in transmittable drug-resistant tuberculosis.

Mycobacterium arosiense sp. nov., a slowly growing, scotochromogenic species causing osteomyelitis in an immunocompromised child.

A yellow-pigmented, scotochromogenic, slowly growing mycobacterial strain, designated T1921(T), was isolated from the disseminated osteomyelitic lesions of a 7-year-old child with an underlying partial gamma interferon receptor alpha-1 deficiency. Hybridization by the line probe assay indicated the presence of a Mycobacterium species. Sequencing of the 16S rRNA gene, the internally transcribed spacer (ITS) region and the hsp65 and rpoB genes revealed that strain T1921(T) could be differentiated from all recognized species of the genus Mycobacterium. Phylogenetic analysis based on the 16S rRNA gene indicated that strain T1921(T) was related most closely to Mycobacterium intracellulare, whereas analysis based on the ITS and hsp65 and rpoB genes indicated that it was most closely related to Mycobacterium avium. Phenotypic tests were not able to differentiate strain T1921(T) from similar slowly growing mycobacteria. Strain T1921(T) is considered to represent a novel species of the genus Mycobacterium, for which the name Mycobacterium arosiense sp. nov. is proposed. The type strain is T1921(T) (=DSM 45069(T) =ATCC BAA-1401(T)).

Can Molecular Methods Detect 1% Isoniazid Resistance in Mycobacterium tuberculosis?

ABSTRACT Patients may harbor both drug-susceptible and -resistant bacteria, representing heteroresistance. We studied mixtures of isoniazid-resistant and -susceptible Mycobacterium tuberculosis strains. Conventional drug susceptibility testing was the most sensitive method of detection, whereas the line probe assay and sequencing were not able to detect the clinically relevant 1% proportion of resistant bacteria.

Rifampin Heteroresistance in Mycobacterium tuberculosis Cultures as Detected by Phenotypic and Genotypic Drug Susceptibility Test Methods

ABSTRACT Tuberculosis patients may harbor both drug-susceptible and -resistant bacteria, i.e., heteroresistance. We used mixtures of rifampin-resistant and -susceptible Mycobacterium tuberculosis strains to simulate heteroresistance in patient samples. Molecular tests can be used for earlier discovery of multidrug resistance (MDR), but the sensitivity to detect heteroresistance is unknown. Conventional phenotypic drug susceptibility testing was the most sensitive, whereas two line probe assays and sequencing were unable to detect the clinically important 1% resistant bacteria.

Multidrug-resistant tuberculosis: rapid detection of resistance to rifampin and high or low levels of isoniazid in clinical specimens and isolates

The aim of the present study was to evaluate a new improved multiplex polymerase chain reaction (PCR) hybridisation assay to detect multidrug-resistant tuberculosis. The assay, developed to detect rifampin (rpoB) and isoniazid (katG) gene mutations causing Mycobacterium tuberculosis resistance, was recently extended to include inhA gene mutations that code for low-level isoniazid resistance. Interpretable results were obtained in 115 isolates and in all smear-positive clinical specimens. Rifampin resistance was correctly identified in all specimens and in 20 of 21 (95%) multidrug-resistant isolates compared to BACTEC 460TB. Isoniazid resistance correlated in 18 of 22 (82%) specimens, in 31 of 31 (100%) high-level and 24 of 28 (86%) low-level isoniazid-resistant isolates. The assay was rapid, easy to perform and directly applicable in smear-positive specimens. We predict that the assay may be a useful tool to combat and prevent new cases of multi- and extensively drug-resistant tuberculosis.

Isoniazid-resistant tuberculosis in Denmark: Mutations, transmission and treatment outcome

OBJECTIVES A retrospective study on isoniazid-resistant tuberculosis (TB) was conducted in the low-burden country, Denmark (DK). The aim was to describe treatment outcome and transmission and to evaluate a mutation analysis for high- and low-level isoniazid resistance detection. METHODS In the period 2002-2007, all isoniazid-resistant TB-cases were included. Molecular genotyping was performed by standardized IS6110 restriction fragment length polymorphism (RFLP). Identical isoniazid-resistant genotypes, indicating ongoing transmission, were identified from the national RFLP database. An analysis of rifampin (rpoB) and high- or low-level (katG, inhA) isoniazid resistance mutations was performed on subcultured strains. RESULTS There were 1825 culture-confirmed cases, of which 111 (6.1%) had monoresistance or polyresistance to isoniazid. Successful short- and long-term treatment outcome was achieved in 80% and 95%, respectively. Overall, the mutation analysis predicted 94% of isoniazid resistance in 111 strains. The katG S315T1 and inhA C15T1 mutations correctly identified high- and low-level isoniazid resistance in 84% and 84% of the strains, respectively. CONCLUSIONS Isoniazid-resistant TB has a good prognosis in DK. High- and low- level isoniazid resistance does not affect treatment outcome of standard modified treatment. Rapid mutation detection identified the majority of isoniazid-resistant cases however the impact on treatment outcome remains to be determined.

Multidrug-resistant tuberculosis: treatment outcome in Denmark, 1992-2007.

Abstract A retrospective nationwide study including all culture-verified multidrug-resistant (MDR) tuberculosis (TB) cases was performed in Denmark. The aim was to examine the long-term treatment outcome of MDR-TB, to assess if MDR-TB transmission occurs, and to evaluate a rapid mutation analysis detecting rifampin and isoniazid resistance in this cohort. Clinical data were obtained from patient records. A restriction fragment length polymorphism genotype database of all TB cases was compared for identical strains indicating active transmission. Twenty-nine cases of MDR-TB were identified and the incidence was low at 0.5%. Acquired MDR-TB and active transmission was rare. Mutations in rifampin (rpoB) and isoniazid (katG, inhA) genes correctly determined resistance in 100% and 82% of all isolates tested, respectively. Initial treatment success was 89% for 27 MDR-TB patients with available outcome data. Initially 3 patients defaulted; no deaths were reported. Including successfully re-treated default patients and censoring patients who spent <2 y in the cohort, long-term treatment success was achieved for all 26 patients (mean follow-up 8.9 y). MDR-TB has a good prognosis in the high-income, low TB burden country of Denmark. Continued surveillance and rapid detection of resistance mutations directly in smear-positive patients may improve the standard of MDR-TB care.

Performance of the GenoType MTBDRplus assay (v2.0) and a new extended GenoType MTBDRsl assay (v2.0) for the molecular detection of multi- and extensively drug-resistant Mycobacterium tuberculosis on isolates primarily from Lithuania

The emergence of extensively drug-resistant tuberculosis (XDR-TB) hampers infection control. To assess the performance of an extended rapid novel molecular analysis for the detection of resistance conferring mutations to fluoroquinolones (gyrA, gyrB genes) and aminoglycosides/cyclic peptides (16S rRNA rrs gene, eis promotor region) compared to phenotypic susceptibility and sequencing, 43 multidrug-resistant (MDR) and 10 susceptible clinical isolates were analyzed. Results were compared to a previous version. Molecular rifampin (rpoB gene) and isoniazid (katG gene, inhA promotor region) resistance was also analyzed. XDR-TB was confirmed in 13 (30%) MDR isolates. Molecular resistance was detected in 91% ofloxacin-, 83% aminoglycoside/cyclic peptide- and 100% kanamycin-resistant isolates. In conclusion, the novel assay is a useful supplement to phenotypic susceptibility testing in determining the presence of XDR-TB. Molecular kanamycin resistance detection was immensely improved compared to the previous version. Aminoglycoside/cyclic peptide susceptible isolates revealed eis promotor region resistance in 29%, reflecting low-level kanamycin susceptibility challenges.