Clinical characteristics, rapid identification, molecular epidemiology and antifungal susceptibilities of Talaromyces marneffei infections in Shenzhen, China.

Although case series of talaromycosis have been reported in China, their detailed clinical and microbiological characteristics have never been systematically profiled. In this study, we report the clinical characteristics, molecular epidemiology, rapid identification and antifungal susceptibilities of talaromycosis in The University of Hong Kong-Shenzhen Hospital in Shenzhen. Seven cases of talaromycosis were observed since commencement of hospital service in 2012. Three patients were local Shenzhen residents, whereas the other four were immigrants from other parts of China. Two patients were HIV-negative, but with underlying diseases requiring immunosuppressive therapy. Two of the seven patients succumbed. All the seven isolates were successfully identified as T. marneffei by MALDI-TOF MS using Bruker database expanded with in-house generated T. marneffei mass spectra. MLST showed that the seven strains belonged to six different, novel sequences types. Phylogenetic analyses of the concatenated five-locus sequence revealed that the seven strains were scattered amongst other T. marneffei strains. The MICs of itraconazole, isavuconazole, posaconazole and voriconazole against the seven clinical isolates were low but MICs of anidulafungin were high. Underlying diseases other than HIV infection are increasingly important risk factors of talaromycosis. MALDI-TOF MS is useful for rapid identification. Highly diverse T. marneffei sequence types were observed.

Molecular characterization of fluoroquinolone resistance in Mycobacterium tuberculosis: functional analysis of gyrA mutation at position 74.

A PCR-sequencing assay was evaluated for direct detection of mutations in the quinolone resistance-determining region (QRDR) of gyrase A (gyrA) gene in fluoroquinolone-resistant Mycobacterium tuberculosis in respiratory specimens. As determined by gyrA QRDR analysis, complete concordance of genotypic and phenotypic fluoroquinolone resistance was demonstrated. Our results indicate that the assay is a rapid and reliable method for the diagnosis of fluoroquinolone-resistant tuberculosis, facilitating timely clinical management and public health control. Using the assay, we detected a novel gyrA Ala74Ser mutation in M. tuberculosis directly from sputum specimens. The functional effect of the Ala74Ser mutant was verified through the study of the DNA supercoiling inhibitory activity of fluoroquinolones against the recombinant gyrase. The drug-mediated gyrase-DNA cleavage complex model suggests perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation probably disturbs the putative quinolone binding pocket and leads to the reduction of the drug binding affinity. A number of gyrA mutations (Glu21Gln, Ser95Thr, and Gly668Asp) were also characterized to be natural polymorphisms not associated with fluoroquinolone resistance.

Mutations outside the rifampicin resistance-determining region associated with rifampicin resistance in Mycobacterium tuberculosis.

OBJECTIVES: Ninety-six percent of rifampicin resistance in Mycobacterium tuberculosis was shown to be associated with mutations inside the 81 bp rifampicin resistance-determining region (RRDR) located in the centre of the rpoB gene. The detection of rifampicin resistance by targeting the RRDR failed to match with a resistant phenotype in 4% of all cases. Our study aims to identify the mutations outside the RRDR that are associated with rifampicin resistance in M. tuberculosis. METHODS AND RESULTS: Among 50 rifampicin-resistant and 20 rifampicin-susceptible clinical isolates of M. tuberculosis, 2 of the rifampicin-resistant isolates did not harbour any known mutations in the RRDR. Sequencing analysis of the whole rpoB gene identified two rare mutations, V146F and I572F. A molecular structure model based on Thermus thermophilus RpoB revealed that both these substituted amino acids are located in close proximity to the rifampicin-binding pocket of the ß-subunit. Substitutions of simple amino acids for bulky ones are likely to affect the protein-drug interaction. Cloning and transformation of the mutated rpoB gene into wild-type Mycobacterium smegmatis and M. tuberculosis successfully elevated the MIC of rifampicin and conferred the rifampicin resistance phenotype. CONCLUSIONS: Our study showed that amino acid positions 146 and 572 are associated with rifampicin resistance in M. tuberculosis in addition to the RRDR. Molecular assays for identifying rifampicin-resistant M. tuberculosis might be improved in terms of accuracy by including these two positions.