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Background and Aims Nontuberculous mycobacteria (NTM) are mainly distributed as important emerging pathogens in patients with chronic or immunosuppressive diseases. Accurate identification of causative species is crucial for proper treatment and patient follow-up. However, several difficulties are associated with phenotypic and molecular diagnostic methods for precise identification at the species level due to shared metabolic and genetic characteristics. We undertook this study to evaluate the application of the phylogenetic method based on hsp65 gene into Telenti's PCR-restriction enzyme analysis (PRA) for molecular identification of NTM. Methods The study population was comprised of 1646 Mycobacterium clinical isolates (AFB positive) collected from 2008–2011, of which 537 (32.6%) were MNT identified by PRA analysis. DNA sequencing of hsp65 in 53 isolates (10%) was performed. Sequence identification through NCBI-Basic Local Alignment Search Tool (BLAST) achieved correct identification in 23 isolates. Phylogenetic trees including hsp65 available GenBank sequences for all described genres of NTM and hsp65 obtained sequences were constructed using Mega 5.05 software. We compared sequence identification based on phylogenetic clustering and BLAST similarity search. Results Phylogenetic clustering allowed more specific differentiation of closely related species and clearer identification in comparison with BLAST; 30 Mycobacterium species (this is the first report of isolation of some of these from clinical samples in Mexico) were identified in this way. Conclusions The proposed 440 bp hsp65 phylogenetic method allows a better identification tool to differentiate Mycobacterium species and is useful to complement diagnosis and epidemiological surveillance of NTM.

Background Nocardia identification has been based on biochemical and morphological characteristics. However, molecular biology techniques allow a better characterization of species and biotypes that are related to invasive diseases. Methods Twelve isolates of Nocardia spp. were obtained from sputum of patients with tuberculosis under retreatment. Identification was done based on morphological characteristics, biochemical tests (casein, tyrosine, xanthine, gelatin, and urea) and molecular biology techniques (PCR-RFLP) using restriction enzymes Msp I, Hin fI, Bsa HI, Hae III and Bst EII. Results Biochemical tests identified the 12 isolates as Nocardia asteroides . PCR-RFLP technique identified nine isolates to species and biotype level: five as N. asteroides type II, two as N. asteroides type VI, and two as N. asteroides type I. The remaining three isolates were identified as follows: one to species level as N. farcinica and two at genus level as Nocardia sp. Conclusions Significant statistical differences between the use of traditional techniques and PCR-RFLP were not found at genus level, but there were important differences at species and biotype level. Biochemical tests identified correctly the actinomycete isolates as belonging to Nocardia genus, but at N. asteroides complex level were not able to discern among their different species. PCR-RFLP is a rapid, non-expensive, and reliable method that allows to discriminate the N. asteroides complex species, identifying biotypes related to invasive disease. Our results suggest that the hospital environment was not a contamination source.

Objective. To compare three methods: Biochemical tests, high-performance liquid chromatography (HPLC) and polymerase chain reaction-restriction fragments length polymorphism (PCR-RFLP), for the identification of mycobacteria, and to perform a cost-benefit analysis to define an optimum identification algorithm. Material and methods. One-hundred-and-seven mycobacteria isolates were identified by the three methods at Instituto de Diagnostico y Referencia Epidemiologicos, between February of 1999 and January of 2000 and the results were compared with those of a reference laboratory using the Q-Cochran statistical test. Results. PCR-RFLP was the most rapid and specific procedure but also the most expensive; biochemical tests excelled for identification of Mycobacterium tuberculosis, but were lengthy and expensive for other mycobacteria; HPLC ranked in the middle for price, speed and specificity. Conclusions. Considering the expected proportion of M. tuberculosis, the following algorithm was proposed: Initially, biochemical tests should be performed; if the results indicate a non-tuberculous mycobacteria, the isolate should be analyzed with HPLC; if results are unclear, the isolate should be analyzed using PCR-RFLP. Isolates showing a previously undescribed PCR-RFLP pattern should be characterized by DNA sequencing.

Dual typing (VP4 and VP7) of rotavirus obtained from 257 Mexican children during three epidemiological seasons was performed by reverse transcription-PCR. The P1G1 genotype was the most prevalent (40%), followed by P1G3 (19%) and P2G2 (16%). Thirty-one specimens (12%) presented mixed infections, while some genotypes were not found. This is the first dual typing of isolates from diarrhea cases in Mexico.

Background Consequences of drug-resistant tuberculosis (TB) in developing countries using directly observed treatment, short-course (DOTS), are not well defined. Objective To determine the impact of drug resistance on clinical outcome and transmission of TB under programmatic conditions. Patients and Methods A prospective cohort and molecular epidemiologic study was conducted in southern Mexico. Between March 1995 and February 1998 all patients with persistent cough whose sputa had acid-fast bacilli (AFB) underwent clinical and mycobacteriologic evaluation (species identification, drug susceptibility testing, and IS6110-based genotyping). Treatment was provided in accordance with Mexico's National Tuberculosis Program. Clinical and microbiologic outcomes and molecular epidemiologically defined transmission were measured. Results Mycobacterium tuberculosis was isolated from 238 of the 284 AFB smear–positive persons. The overall rate of resistance was 28.4% (new, 20.7%; retreated, 54.7%), and 10.8% (new, 3.3%; retreated, 35.8%) had multi–drug-resistant TB (ie, resistance to isoniazid and rifampin). After treatment, 75% (new, 81.0%; retreated, 52.8%) were cured, 8% (new, 7.8%; retreated, 7.5%) abandoned therapy, 9% (new, 3.9%; retreated, 28.3%) had treatment failure, and 4% (new, 3.3%; retreated, 7.5%) died. Another 2% of patients relapsed, and 9% died during a median of 24.4 months of follow-up. Drug-resistance was a strong independent risk factor for treatment failure. Being infected with multi–drug-resistant TB was the only factor associated with a decreased likelihood of being in a restriction fragment length polymorphism cluster. Conclusions Despite the use of DOTS, patients with drug-resistant TB had a dramatically increased probability of treatment failure and death. Although multi–drug-resistant TB may have a decreased propensity to spread and cause disease, it has a profoundly negative impact on TB control.