Hsp65 phylogenetic assay for molecular diagnosis of nontuberculous mycobacteria isolated in Mexico.

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.

Skin biopsy: a pillar in the identification of cutaneous Mycobacterium tuberculosis infection.

INTRODUCTION: The present study aimed to establish the frequency and clinical characteristics of cutaneous tuberculosis among Mexican adult patients. METHODOLOGY: Ninety-five patients with clinically compatible lesions to cutaneous tuberculosis participated in the study. All patients were HIV negative and none of them had previous anti-TB treatment. A skin biopsy was taken from every patient suspected of having tuberculosis, and a histopathologic examination was performed as follows: Ziehl-Neelsen staining; culturing of mycobacteria by Löwenstein-Jensen (L-J) medium; Mycobacteria Growth Indicator Tube detection via BACTEC (MGIT-360); and polymerase chain reaction (PCR) with the sequence of insertion IS6110 for Mycobacterium tuberculosis complex. RESULTS: Tuberculosis was confirmed in 65 out of 95 cases (68.4%). Identified lesions were scrofuloderma (42 cases, 64.6%); lupus vulgaris (12 cases, 18.4%); warty tuberculosis (six cases, 9.2%); and papulonecrotic tuberculoid (five cases; 7.7%). The Ziehl-Neelsen staining was positive for acid fast bacilli in nine cases (13.8%) and 48 patients were positive for the PCR amplification (73.8%). All skin biopsies resulted positive for tuberculosis. A positive clinical response to the specific treatment was considered a confirmation for tuberculosis. The noninfectious etiology corresponded to 30 cases (31.6%). CONCLUSIONS: Tuberculosis in developing countries is still an important cause of skin lesions which must be studied via histopathological examination and culture due to their low bacillary load. A PCR test is necessary to obtain faster confirmation of the disease and to establish an early, specific and effective treatment.

Oseltamivir-resistant pandemic (H1N1) 2009 virus, Mexico.

During May 2009-April 2010, we analyzed 692 samples of pandemic (H1N1) 2009 virus from patients in Mexico. We detected the H275Y substitution of the neuraminidase gene in a specimen from an infant with pandemic (H1N1) 2009 who was treated with oseltamivir. This virus was susceptible to zanamivir and resistant to adamantanes and oseltamivir.

Bell's palsy. A prospective, longitudinal, descriptive, and observational analysis of prognosis factors for recovery in Mexican patients.

OBJECTIVE: To determine the prognosis factors in Mexican patients with Bell's palsy. DESIGN: We designed a prospective, longitudinal, descriptive, and observational analysis. Two hundred and fifty one patients diagnosed with Bell's palsy at the National Institute of Rehabilitation were included. We studied the sociodemographic characteristics, seasonal occurrence, sidedness, symptoms, and therapeutic options to determine the prognostic factors for their recovery. RESULTS: Thirty-nine percent of patients had a complete recovery and 41.5% had an incomplete recovery. Marital status, gender, etiology, symptoms, sidedness, House-Brackmann grade, and treatments did not represent significant prognostic factors for recovery. Age > 40 years (OR = 2.4, IC 95% 1.3-4.3, p = 0.002) and lack of physical therapy (OR = 6.4, IC 95% 1.4-29.6, p = 0.006) were significant prognostic factors for incomplete recovery. Familial palsy resulted to be a protective prognostic factor against an incomplete recovery (OR = 0.54, IC 95% 0.28-1.01, p = 0.039). This protection factor was only significant in female patients (OR = 0.41, p = 0.22) but not in male patients (OR = 1.0, p = 0.61). CONCLUSIONS: The proportion of cases with incomplete recovery was high. The age > 40 years and lack of physical therapy were the only significant prognostic factors for an incomplete recovery.

Biochemical identification and molecular characterization (PCR-RFLP) of Nocardia isolates from sputum.

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 MspI, HinfI, BsaHI, HaeIII and BstEII. 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.

Comparison among three methods for mycobacteria identification

Objetivo. Comparar tres métodos: pruebas bioquímicas, cromatografía líquida de alta resolución (HPLC, por sus siglas en inglés) y reacción en cadena de la polimerasa-polimorfismo del tamaño de fragmentos de restricción (PCR-RFLP) para identificar micobacterias a nivel especie, analizando costo-beneficio y proponiendo un algoritmo de identificación. Material y métodos. Entre febrero de 1999 y enero de 2000, en los laboratorios del Instituto de Diagnóstico y Referencia Epidemiológicos se tipificaron 107 aislados de micobacterias y los resultados se compararon con los obtenidos en un laboratorio de referencia utilizando la prueba estadística Q de Cochran. Resultados. Se encontró que el PCR-RFLP fue el método más específico y rápido pero también el más caro. Las pruebas bioquímicas fueron confiables para la identificación de Mycobacterium tuberculosis, pero lentas e inespecíficas para otras micobacterias. El HPLC estuvo en un nivel medio tomando en cuenta costo, tiempo y especificidad. Conclusiones. Considerando la proporción esperada de M. tuberculosis, se propone el siguiente algoritmo: si las pruebas bioquímicas indican una micobacteria no tuberculosa, el aislado será analizado por HPLC; si la identificación no es clara, el aislado será analizado usando PCR-RFLP. Si el aislado no pertenece a un patrón descrito, se identificará por secuenciación de ADN.