Molecular identification of Mycobacterium bovis and the importance of zoonotic tuberculosis in Mexican patients.

SETTING: The epidemiology of zoonotic tuberculosis (ZTB) in humans in Mexico is poorly known. OBJECTIVE: To identify isolates of Mycobacterium bovis in humans and cattle by polymerase chain reaction (PCR), and establish the clinical and epidemiological importance of ZTB in humans. DESIGN: From 1995 to 2009, 124 isolates from patients with TB and 60 isolates from cattle were analysed. PCR identification was performed using the oxy R gene, and the clinical and epidemiological aspects of ZTB in humans were investigated. RESULTS: PCR identified 93 M. bovis isolates: 35 (28%) from the 124 human isolates and 58 (97%) from the 60 cattle isolates. The sensitivity and specificity of the method were 100%. ZTB in the 35 patients presented as extra-pulmonary TB (EPTB) in 74%: 51% were children, 69% had malnutrition, 51% had consumed unpasteurised milk and 6% had contact with animals; 11% were relapses and 31% died. CONCLUSIONS: PCR using the oxy R gene is highly sensitive, specific and rapid for the identification of M. bois. ZTB is a serious public health problem, and presented as EPTB in children with malnutrition and those who had consumed unpasteurised milk. ZTB provokes relapses and a high mortality rate.

Nested polymerase chain reaction in the diagnosis of cervical tuberculous lymphadenitis in Mexican children.

SETTING: Cervical tuberculous lymphadenitis (CTBL) diagnosis is a critical problem due to the difficulty in culturing Mycobacterium tuberculosis. OBJECTIVE: To evaluate a nested polymerase chain reaction (nPCR) for the diagnosis of CTBL. DESIGN: Thirty-eight children initially diagnosed on the basis of clinical and laboratory criteria as suffering from chronic cervical lymphadenitis were included in the study. Forty-one cervical lymph node specimens were analysed by bacterial staining, culture, cytology or histopathology. The IS6110 DNA sequence of M. tuberculosis complex was amplified by nPCR. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and efficiency were determined for the assay. RESULTS: The sensitivity of nPCR was 96%, the specificity 93%, PPV 96%, NPV 93% and efficiency 95%. Among 25 patients with CTBL, six presented a 'definite' diagnosis (24%) according to established criteria; 10 were classified as 'highly probable' cases (40%) and nine presented a 'possible' diagnosis (36%). The sensitivity of nPCR was higher than the sensitivity of staining (15%), culture (26%) and cytology or histopathology (62.5%) (95%CI P < 0.05, chi(2) P < 0.001). CONCLUSION: The nPCR used is a highly sensitive, specific and efficient method for the diagnosis of CTBL among children.

Epidemiologic Study of Pseudomonas aeruginosa in critical patients and reservoirs.

BACKGROUND: Pseudomonas aeruginosa is a common cause of nosocomial infections, particularly in intensive care units (ICUs). The aim of this study was to characterize P. aeruginosa clinical isolates by comparing antimicrobial susceptibility patterns with the presence of plasmids and to establish the clonal relatedness by pulsed-field gel electrophoresis (PFGE) typing. METHODS: The patients included those with isolation of P. aeruginosa hospitalized for more than 48 h in the ICU from April to May 1998. Environmental and staff cultures were obtained simultaneously. Minimal inhibitory concentrations, plasmid DNA profiles, and PFGE genomic patterns of enzyme restriction chromosomal DNA were compared. RESULTS: Sixty P. aeruginosa isolates were obtained from 197 clinical specimens, 178 environmental samples, and 47 hand cultures of personnel. Antimicrobial resistance was as follows: tobramycin 100%; ticarcillin, cefotaxime, ceftriaxone, ceftazidime, and gentamicin 80%; cefepime 60%; amikacin, ticarcillin/clavulanate, imipenem, and meropenem 40%; piperacillin and norfloxacin 20%; carbenicillin 12%, and ciprofloxacin 0%. Plasmids were detected in 11 isolates (18%). PFGE typing showed that 23 isolates belonged to a common clone (pattern A), identified from five patients, two nurses, and 10 environmental samples. Ten isolates were grouped in four clusters and 27 isolates had unrelated genomic patterns. There was no relationship among DNA genomic patterns, plasmid profiles, and susceptibility patterns. CONCLUSIONS: PFGE demonstrated the existence of a common clone in a critical care area. Reinforcement of infection control measures is needed to avoid horizontal transmission and severe infections.

Primary and acquired resistance of Mycobacterium tuberculosis in Western Mexico.

Resistance of Mycobacterium tuberculosis to antimycobacterial agents is a worldwide problem. The proposite of this study was to analyze the current resistance patterns of patients with initial episodes, as well as relapses, due to M. tuberculosis in western Mexico. From January 1993 to February 1999 a total of 237 strains of M. tuberculosis (120 from initial cases and 117 from relapse cases) were analyzed. Two hundred and four (86%) strains were isolated from the lower respiratory tract, and 33 strains (14%) from extrapulmonary sites. Twenty-three percent of M. tuberculosis isolated from patients with initial episodes were resistant to both isoniazid and rifampin, and 52% of M. tuberculosis isolated from relapse cases were also resistant to both isoniazid and rifampin.

Rapid and efficient detection of extra-pulmonary Mycobacterium tuberculosis by PCR analysis.

SETTING: The diagnosis of extra-pulmonary tuberculosis (EPTB) remains an important clinical problem, primarily because of the inadequate sensitivity of conventional bacteriologic methods for detecting Mycobacterium tuberculosis in extra-pulmonary specimens. OBJECTIVE: To evaluate whether a IS6110-based polymerase chain reaction (PCR) method can be utilized to detect M. tuberculosis in non-pulmonary specimens. DESIGN: Specimens from 286 Mexican patients with a presumptive clinical diagnosis of EPTB were prospectively examined by Ziehl-Neelsen staining, mycobacterial culture on Löwenstein-Jensen slants, and by PCR. The DNA for PCR was extracted by the buffer lysis method and phenol-guanidine thiocyanate-chloroform. Primers that amplify a 200 bp fragment from the insertion-like M. tuberculosis sequence element IS6110 were utilized. RESULTS: Our results demonstrate that this PCR method is highly specific (100%) for identifying M. tuberculosis from a variety of specimens including cerebrospinal fluid (CSF), pleural fluid, ascitic fluid, pericardial fluid, urine, and lymph node exudate. Moreover, the sensitivity of PCR for detecting M. tuberculosis in CSF (94%), pleural fluid (94%), ascitic fluid and other extrapulmonary specimens (93%) greatly exceeds the sensitivity of conventional smear and culture methods. CONCLUSION: These results demonstrate that PCR can be a highly specific and sensitive aid in the detection of M. tuberculosis from extra-pulmonary specimens.

Biotypes and serotypes of Haemophilus influenzae of clinical isolates from Mexican children.

BACKGROUND: Infectious diseases caused by H. influenzae type b are considered preventable through vaccination with Hib conjugate vaccines. Some countries which follow Hib vaccination programs are close to eradication of the disease. In Mexico in particular, little epidemiological information is available. METHODS: In this study, 90 clinical strains of H. influenzae were obtained from Mexican children who were treated in four pediatric hospitals in Puebla City, and were diagnosed with invasive or localized infectious diseases. The strains were identified by standard bacteriological methods. Biotyping was done by Kilian criteria and serotyping by coagglutination. RESULTS: H. influenzae infections were found in children younger than 5 years of age, and 68.8% of the children were younger than 24 months. Sixty percent of the isolates belonged to serotype b, 31.1% were nontypeable, and 7.7% were considered non-type b. Serotype b was the most frequent isolate associated with invasive infectious diseases; however, nontypeable strains were isolated more frequently from children with otitis, sinusitis, conjunctivitis, and bronchial secretion. Non-type b serotypes were isolated from invasive and non-invasive infections in few cases. Biotypes I and IV were the most frequent isolates of H. influenzae. CONCLUSIONS: This study emphasizes the urgent need for an Hib-conjugated vaccine to achieve immunization in a pediatric population.

The absence of genetic markers for streptomycin and rifampicin resistance in Mycobacterium avium complex strains.

Mycobacterium avium, Mycobacterium intracellulare complex (MAC) bacilli are an important cause of bacteraemia in AIDS patients but treatment is complicated by their resistance to the usual antimycobacterial agents. In this study of 20 strains of MAC none was found to have the mutations associated with resistance to rifampicin and streptomycin in M. tuberculosis suggesting that MAC have unique mechanisms for resistance to these agents.

Molecular mechanisms of multiple drug resistance in clinical isolates of Mycobacterium tuberculosis.

The molecular mechanisms of resistance to streptomycin, rifampin, and isoniazid in 53 Mycobacterium tuberculosis clinical isolates were examined. Twenty-five of 44 streptomycin-resistant strains had mutations in the rpsL gene and 5 of these had rrs gene perturbations. The region of the rpoB gene that is associated with resistance to rifampin was altered in 28 of 29 rifampin-resistant strains. Mutations in known genetic markers of isoniazid resistance were detected in 25 of 42 isoniazid-resistant isolates: 20 strains had katG gene alterations and 5 had perturbations in the inhA operon. Of the 20 multiply resistant strains with reduced sensitivity to streptomycin, rifampin, and isoniazid, 11 had mutations in genetic markers associated with resistance to each of these three drugs. These studies suggest that the primary mechanism of multiple drug resistance in tuberculosis is the accumulation of mutations in individual drug target genes.