Miriam Bobadilla-del-Valle

Association of diabetes and tuberculosis: impact on treatment and post-treatment outcomes

Objective To determine the clinical consequences of pulmonary tuberculosis (TB) among patients with diabetes mellitus (DM). Methods We conducted a prospective study of patients with TB in Southern Mexico. From 1995 to 2010, patients with acid-fast bacilli or Mycobacterium tuberculosis in sputum samples underwent epidemiological, clinical and microbiological evaluation. Annual follow-ups were performed to ascertain treatment outcome, recurrence, relapse and reinfection. Results The prevalence of DM among 1262 patients with pulmonary TB was 29.63% (n=374). Patients with DM and pulmonary TB had more severe clinical manifestations (cavities of any size on the chest x-ray, adjusted OR (aOR) 1.80, 95% CI 1.35 to 2.41), delayed sputum conversion (aOR 1.51, 95% CI 1.09 to 2.10), a higher probability of treatment failure (aOR 2.93, 95% CI 1.18 to 7.23), recurrence (adjusted HR (aHR) 1.76, 95% CI 1.11 to 2.79) and relapse (aHR 1.83, 95% CI 1.04 to 3.23). Most of the second episodes among patients with DM were caused by bacteria with the same genotype but, in 5/26 instances (19.23%), reinfection with a different strain occurred. Conclusions Given the growing epidemic of DM worldwide, it is necessary to add DM prevention and control strategies to TB control programmes and vice versa and to evaluate their effectiveness. The concurrence of both diseases potentially carries a risk of global spreading, with serious implications for TB control and the achievement of the United Nations Millennium Development Goals.

Does DOTS work in populations with drug-resistant tuberculosis?

BACKGROUND Directly observed therapy (DOTS) is the main strategy for prevention and control of tuberculosis worldwide. However, its effect on tuberculosis transmission in populations with moderate rates of drug-resistant disease is not known. METHODS This population-based prospective study in southern Mexico between March, 1995, and February, 2000, was based on passive case finding and detection of acid-fast bacilli in sputum samples to diagnose pulmonary tuberculosis. We also used cultures, drug-susceptibility testing, bacterial genotyping, and monitoring of treatment outcomes. FINDINGS We enrolled 436 patients; the HIV seroprevalence rate was 2%. We used three indicators to monitor continuing tuberculosis transmission: the incidence rate of pulmonary tuberculosis, which decreased by 54.4% between 1995 and 2000, from 42.1 to 19.2 per 10(5) population (p=0.00048); the percentage of clustered pulmonary tuberculosis cases, which decreased by 62.6% from 22% to 8% (p=0.02); and the rate of primary drug resistance, which decreased by 84.0% from 9.4 to 1.5 per 10(5) population (p=0.004). Rates of multidrug-resistant (MDR) tuberculosis also decreased (p<0.0001). The case-fatality ratio was 12% for MDR tuberculosis (five of 41), 7% for strains resistant to at least one drug after exclusion of MDR (four of 55), and 3% for pansusceptible strains (nine of 272). There were 13 treatment failures (11%) in 1995 and one (2%) in 2000 (p=0.012). INTERPRETATION Even in settings with moderate rates of MDR tuberculosis, DOTS can rapidly reduce the transmission and incidence of both drug-susceptible and drug-resistant tuberculosis. However, further interventions, such as drug-susceptibility testing and standardised or individualised treatment regimens, are needed to reduce mortality rates for MDR tuberculosis.

Gender differentials of pulmonary tuberculosis transmission and reactivation in an endemic area

Background: In most low income countries there are twice as many cases of tuberculosis (TB) reported among men than among women, a difference commonly attributed to biological and epidemiological characteristics as well as socioeconomic and cultural barriers in access to health care. The World Health Organization has encouraged gender specific comparisons in TB rates to determine whether women with TB are less likely than men with TB to be diagnosed, reported, and treated. A study was undertaken to identify gender based differences in patients with pulmonary TB and to use this information to improve TB control efforts. Methods: Individuals with a cough for more than 2 weeks in southern Mexico were screened from March 1995 to April 2003. Clinical and mycobacteriological information (isolation, identification, drug susceptibility testing and IS6110 based genotyping, and spoligotyping) was collected from those with bacteriologically confirmed pulmonary TB. Patients were treated in accordance with official norms and followed to ascertain treatment outcome, retreatment, and vital status. Results: 623 patients with pulmonary TB were enrolled. The male:female incidence rate ratio for overall, reactivated, and recently transmitted disease was 1.58 (95% CI 1.34 to 1.86), 1.64 (95% CI 1.36 to 1.98), and 1.41 (95% CI 1.01 to 1.96), respectively. Men were more likely than women to default from treatment (adjusted OR 3.30, 95% CI 1.46 to 7.43), to be retreated (hazard ratio (HR) 3.15, 95% CI 1.38 to 7.22), and to die from TB (HR 2.23, 95% CI 1.25 to 3.99). Conclusions: Higher rates of transmitted and reactivated disease and poorer treatment outcomes among men are indicators of gender differentials in the diagnosis and treatment of pulmonary TB, and suggest specific strategies in endemic settings.

Nested Polymerase Chain Reaction for Mycobacterium tuberculosis DNA Detection in Aqueous and Vitreous of Patients with Uveitis

BACKGROUND Tuberculosis may be a lethal disease. Its ocular manifestations are commonly associated with severe difficulties in diagnosis and therapy; furthermore, it may cause blindness. DNA amplification methods may allow early detection of small amounts of Mycobacterium tuberculosis DNA to afford the possibility of prompt diagnosis. We evaluated a nested polymerase chain reaction (nPCR) assay for detection of Mycobacterium tuberculosis DNA in aqueous and vitreous. METHODS In a case-control study, 22 cases of diagnosed TB uveitis (three HIV-infected patients) and 38 controls (18 HIV-infected patients) with other types of uveitis (syphilis, nine; cytomegalovirus, seven; toxoplasmosis, five; herpes simplex, one; autoimmune vasculitis, eight; Vogt-Koyanagi-Harada, four; pars planitis, one; serpinginous choroiditis, one; Wegener granulomatosis, one; and Fuchs iridocyclitis, one studied). Samples from aqueous or vitreous were cultured and analyzed by nPCR for presence of M. tuberculosis nucleic acids. We used two sets of primers corresponding to IS6110 region coding for 219 bp and 123 bp DNA sequences. RESULTS Results were confirmed by Southern blot. All samples were tested by PCR simultaneously for Herpes simplex I, Herpes zoster, cytomegalovirus (CMV) and Toxoplasma gondii. nPCR was positive in 17 cases (77.2%) and only in three controls (8.8%) p = 0.022. All cultures were negative. Southern blot confirmed all positive nPCR tests. According to our definition of cases, there were five false negative results: two in patients with pulmonary tuberculosis; two in patients with tuberculous lymphadenitis, and one with positive skin test and hematuria. There were three cases considered false positives for nPCR: one with autoimmune vasculitis, and two with toxoplasmic uveitis. CONCLUSIONS nPCR for TB in ocular fluids was positive in the majority of cases of ocular TB. This method is useful in early confirmation of ocular tuberculosis.

Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis Isolates from India and Mexico by a Molecular Beacon Assay

ABSTRACT We assessed the performance of a rapid, single-well, real-time PCR assay for the detection of rifampin-resistant Mycobacterium tuberculosis by using clinical isolates from north India and Mexico, regions with a high incidence of tuberculosis. The assay uses five differently colored molecular beacons to determine if a short region of the M. tuberculosis rpoB gene contains mutations that predict rifampin resistance in most isolates. Until now, the assay had not been sufficiently tested on samples from countries with a high incidence of tuberculosis. In the present study, the assay detected mutations in 16 out of 16 rifampin-resistant isolates from north India (100%) and in 55 of 64 rifampin-resistant isolates from Mexico (86%) compared to results with standard susceptibility testing. The assay did not detect mutations (a finding predictive of rifampin susceptibility) in 37 out of 37 rifampin-susceptible isolates from India (100%) and 125 out of 126 rifampin-susceptible isolates from Mexico (99%). DNA sequencing revealed that none of the nine rifampin-resistant isolates from Mexico, which were misidentified as rifampin susceptible by the molecular beacon assay, contained a mutation in the region targeted by the molecular beacons. The one rifampin-susceptible isolate from Mexico that appeared to be rifampin resistant by the molecular beacon assay contained an S531W mutation, which is usually associated with rifampin resistance. Of the rifampin-resistant isolates that were correctly identified in the molecular beacon assay, one contained a novel L530A mutation and another contained a novel deletion between codons 511 and 514. Overall, the molecular beacon assay appears to have sufficient sensitivity (89%) and specificity (99%) for use in countries with a high prevalence of tuberculosis.

Unique Gene Expression Profiles in Infants Vaccinated with Different Strains of Mycobacterium bovis Bacille Calmette-Guérin

ABSTRACT Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) has variable efficacy in preventing tuberculosis. We hypothesized that some of this variation might be due to differences among BCG strains. To test this, neonates in Orizaba, Mexico, were vaccinated with one of three different BCG strains (BCG-Brazil [BBCG], BCG-Denmark [DBCG], or BCG-Japan [JBCG]). One year after vaccination, peripheral blood mononuclear cells (PBMC) were obtained and recall immune responses to culture filtrate proteins (CFP) of Mycobacterium tuberculosis were evaluated using quantitative real-time PCR. CFP-activated PBMC from BBCG- and DBCG-immunized children expressed high levels of cytokines characteristic of an adaptive immune response (gamma interferon, interleukin-2β [IL-12β], and IL-27), while those from children immunized with JBCG did not. In contrast, vaccination with JBCG resulted in significantly greater expression of cytokines characteristic of a proinflammatory immune response (IL-1α, IL-1β, IL-6, and IL-24) in PBMC activated with CFP compared to PBMC from children vaccinated with BBCG or DBCG. Thus, different strains of BCG can activate different immune pathways, which may affect long-term vaccine efficacy.

Tuberculosis in ageing: high rates, complex diagnosis and poor clinical outcomes

BACKGROUND worldwide, the frequency of tuberculosis among older people almost triples that observed among young adults. OBJECTIVE to describe clinical and epidemiological consequences of pulmonary tuberculosis among older people. METHODS we screened persons with a cough lasting more than 2 weeks in Southern Mexico from March 1995 to February 2007. We collected clinical and mycobacteriological information (isolation, identification, drug-susceptibility testing and IS6110-based genotyping and spoligotyping) from individuals with bacteriologically confirmed pulmonary tuberculosis. Patients were treated in accordance with official norms and followed to ascertain treatment outcomes, retreatment, and vital status. RESULTS eight hundred ninety-three tuberculosis patients were older than 15 years of age; of these, 147 (16.5%) were 65 years of age or older. Individuals ≥ 65 years had significantly higher rates of recently transmitted and reactivated tuberculosis. Older age was associated with treatment failure (OR=5.37; 95% CI: 1.06-27.23; P=0.042), and death due to tuberculosis (HR=3.52; 95% CI: 1.78-6.96; P<0.001) adjusting for sociodemographic and clinical variables. CONCLUSIONS community-dwelling older individuals participate in chains of transmission indicating that tuberculosis is not solely due to the reactivation of latent disease. Untimely and difficult diagnosis and a higher risk of poor outcomes even after treatment completion emphasise the need for specific strategies for this vulnerable group.

Prevalence of Latent and Active Tuberculosis among Dairy Farm Workers Exposed to Cattle Infected by Mycobacterium bovis

Background Human tuberculosis caused by M. bovis is a zoonosis presently considered sporadic in developed countries, but remains a poorly studied problem in low and middle resource countries. The disease in humans is mainly attributed to unpasteurized dairy products consumption. However, transmission due to exposure of humans to infected animals has been also recognized. The prevalence of tuberculosis infection and associated risk factors have been insufficiently characterized among dairy farm workers (DFW) exposed in settings with poor control of bovine tuberculosis. Methodology/Principal Findings Tuberculin skin test (TST) and Interferon-gamma release assay (IGRA) were administered to 311 dairy farm and abattoir workers and their household contacts linked to a dairy production and livestock facility in Mexico. Sputa of individuals with respiratory symptoms and samples from routine cattle necropsies were cultured for M. bovis and resulting spoligotypes were compared. The overall prevalence of latent tuberculosis infection (LTBI) was 76.2% (95% CI, 71.4–80.9%) by TST and 58.5% (95% CI, 53.0–64.0%) by IGRA. Occupational exposure was associated to TST (OR 2.72; 95% CI, 1.31–5.64) and IGRA (OR 2.38; 95% CI, 1.31–4.30) adjusting for relevant variables. Two subjects were diagnosed with pulmonary tuberculosis, both caused by M. bovis. In one case, the spoligotype was identical to a strain isolated from bovines. Conclusions We documented a high prevalence of latent and pulmonary TB among workers exposed to cattle infected with M. bovis, and increased risk among those occupationally exposed in non-ventilated spaces. Interspecies transmission is frequent and represents an occupational hazard in this setting.

Comparison of Sodium Carbonate, Cetyl-Pyridinium Chloride, and Sodium Borate for Preservation of Sputa for Culture of Mycobacterium tuberculosis

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is essential for a successful treatment of patients, mainly those with a previous history of antituberculosis therapy. Susceptibility testing of strains requires their isolation from sputum samples within 24 to 48 h of collection and storage at 2° to 4°C. If the sample is left at room temperature or in refrigeration for longer periods of time, the recovery of M. tuberculosis decreases to 63% and contamination rises to 18% (3). Both problems occur when the laboratory is located far from the patients home, when refrigeration is not available, or when transportation to the laboratory is inadequate, all common situations in developing countries. Despite these problems, no preservatives are presently used to improve the rate of isolation of M. tuberculosis from sputum. In previous studies, sodium carbonate (SC), cetyl-pyridinium chloride (CP), and sodium borate (SB) proved to be good preservatives of this bacterium (1, 5, 6-8). We compared the efficacy of these three compounds in preserving the viability of M. tuberculosis in 58 sputum samples positive for acid-fast bacilli (AFB) from 23 patients with pulmonary tuberculosis who lived in a rural area of Mexico. The study was approved by the Institutional Review Board of our institution. All samples were initially processed in the local laboratory, in Huauchinango Puebla. Each sample was divided into four equal aliquots and placed in a 50-ml conical test tube with one of the following: (i) SC, 75 mg (J. T. Baker, Xalostoc, Mexico); (ii) 5% SB, 800 μl (J. T. Baker); (iii) 1% CP, equal volume (Sigma Aldrich Chemical Co., St. Louis, Mo.); and (iv) no chemical (control). Aliquots were left at room temperature (25 to 35°C) for 5 to 18 days; 8 samples were stored for 5 days, 17 for 6 days, 13 for 7 days, 6 for 8 days, 8 for 9 days, 1 for 15 days, 3 for 16 days, and 2 for 18 days. Aliquots were then sent to our laboratory where they were digested and were decontaminated with 0.5% N-acetyl-cisteine and 2% NaOH (4). Part of the sediment was smeared and was stained first with auramine-rhodamine (AR) (Sigma-Aldrich) and was then stained with Ziehl-Neelsen (ZN) (Sigma-Aldrich) to confirm results. The remaining sediment was resuspended in Na2HPO4-KH2PO4 (0.067 M); 0.5 ml was inoculated in Lowenstein-Jensen (LJ) medium (Becton Dickinson, Mexico City, Mexico) and another 0.5 ml in mycobacterial growth indicator tube (MGIT) medium (Becton Dickinson, Sparks, Md.). The remaining sediment was kept at 4°C for 15 days and was redigested if the first culture became contaminated. LJ was incubated at 37°C in 7.5% CO2 and was examined weekly for 8 weeks. MGIT was incubated in the MGIT 960 instrument (Becton Dickinson). M. tuberculosis was identified in positive cultures by DNA probe (Gene Probe, San Diego, Calif.) (2). AR and ZN stains were AFB positive in 91.4% of smears from samples preserved with SC, 96.6% with SB, and 98.3% of the controls. In contrast, of these samples preserved with CP only, 31% were AR positive and 37.9% were ZN positive. M. tuberculosis was isolated from at least one of the four aliquots in all 58 sputum samples. AFB were cultured in LJ and MGIT from all samples preserved with SC; however, due to contamination with bacteria and fungi, M. tuberculosis was identified in only 86 and 98% of samples, respectively (Table ​(Table1).1). From samples preserved in CP, 98% grew M. tuberculosis when cultured in LJ, and in contrast, only 71% grew M. tuberculosis when cultured in MGIT. The recovery of M. tuberculosis was significantly lower in samples preserved with SB and in controls, mainly due to contamination (Table ​(Table1).1). We observed viability of M. tuberculosis in samples preserved with all compounds for 5 to 18 days. TABLE 1. Comparison of three compounds for recovery of M. tuberculosis from 58 sputum samples Although this study did not include a systematic analysis of the maximum effective storage time of sputum with the different compounds, we can conclude that the recovery and staining of M. tuberculosis were best when sputum was preserved in SC and was cultured in liquid media. Similar yields were obtained when sputum was preserved in CP and cultured in LJ.

Vancomycin-resistant enterococci, Mexico City.

To the Editor: Vancomycin-resistant Enterococcus (VRE) has become an important nosocomial pathogen because of its rapid spread, limited therapy options, mortality, and the possibility of transfer of vancomycin resistance to other pathogens such as Staphylococcus aureus. Vancomycin-resistant E. faecium (VREF) and E. faecalis were first described in 1988 (1,2).They have become major nosocomial pathogens, but their prevalence in Latin America has remained <2% (3). In Mexico, VRE has rarely been reported (4,5). In a recent study in Mexico City, 100% (n = 60) of the isolates of E. faecium and E. faecalis were susceptible to vancomycin (6). From May 2004 to April 2005, the rate of vancomycin resistance among all Enterococcus isolates was 0.27%. However, in May 2005 the first fully VREF was isolated at our hospital, and the rate of vancomycin resistance was 6.23% (a 23-fold increase) during the following 12-month period. We performed a retrospective study to describe the isolates and the characteristics of patients with VREF. All VREF isolates from May 2005 through April 2006 were included. We collected demographic and clinical data. For the final identification of the isolates, the VITEK system (bioMerieux, Lyon, France) with VITEK GPI cards (bioMerieux, Inc., Durham NC, USA) were used. Antimicrobial drug susceptibility was tested by using the VITEK GPS-111 card and confirmed by MIC determination that used broth microdilution. Resistance to vancomycin and teicoplanin was confirmed by E-test (AB Biodisk, Solna, Sweden). An isolate was considered vancomycin resistant when the MIC was ≥32 μg/mL and was considered to have high-level resistance when the MIC was ≥256 μg/mL. A PCR for detection of the vanA or vanB genotype was used (7). Isolates were characterized by pulsed-field gel electrophoresis (PFGE) (8,9); a dendrogram was constructed with the GelCompare II 4.0 software (Applied Maths, Kortrijk, Belgium), and the similarity was compared with the Dice coefficient. In the study period, VREF was isolated from 27 patients. The median age was 40 years (range 22–84 years). VREF was isolated from the abdomen in 14 patients (51.9%); 11 isolates were from an abscess, 2 from infected surgical sites, and 1 from ascites. An additional 8 isolates were from the urinary tract (29.6%), 2 from the bloodstream (7.4%), 2 from soft-tissue (7.4%), and 1 (3.7%) from bone. Residence in the general medical wards during the isolation of VREF was most common, 17 (63%) cases, followed by 6 (22.2%) in the intensive care unit. The remaining 4 (14.8%) were distributed in other areas. Median time of hospitalization before the isolation was 21 days (range 1–84 days). Twenty-five patients (92.6%) had a central line, 12 (44.4%) had mechanical ventilation, and 20 (74.1%) previous surgery. Of the last group, 17 (85%) of 20 had abdominal surgery. Twenty-four patients (88.8%) received an antimicrobial drug before the isolation of VREF: third- or fourth-generation cephalosporins (89%), metronidazole (70.4%), aminoglycosides (70.4%), vancomycin (66.7%), carbapenems (66.7%), amoxicillin or ampicillin (48.1%), antifungal agents (48.1%); and <20% received quinolones, trimethoprim-sulfamethoxazole, colistin, macrolides, and antimycobacterial or antiviral agents. The median time of antimicrobial drug use was 11 days (range 1–84 days). During hospitalization, 7 patients died (crude death rate, 25.9%), 5 of them from sepsis with at least another microorganism isolated; the remaining 2 died of gastrointestinal hemorrhage. All isolates of E. faecium had a vancomycin MIC ≥256 μg/mL and a vanA phenotype (teicoplanin resistance); 26 (96.3%) had vanA genotype. Only 1 isolate of E. faecium was classified as non–vanA, non vanB, even though it demonstrated high-level resistance to vancomycin and teicoplanin. Resistance to other antimicrobial agents was as follows: ampicillin and ciprofloxacin, 100%; high-level gentamicin, 48.2%; quinupristin/dalfopristin, 7.4%; and linezolid, 0%. PFGE analysis showed several genotypes of E. faecium; however, 18 of 26 of the isolates had <3 band differences from the predominant strain classified as type A. One isolate of E. faecium could not be typed (Figure). Figure Pulsed-field gel electrophoresis (PFGE) banding patterns of chromosomal DNA of 26 isolates of vancomycin-resistant enterococci. There is a clear predominant type, classified as type A (≥80% similarity), composed of 18 isolates of Enterococcus ... As in most tertiary-care centers, our PFGE data suggest that a heterogenous population of VREF exists, but a particular clone established itself as the dominant strain. Although infection control measures are well established in our hospital, in disseminated outbreaks caused by several different clones, infection control measures and control of vancomycin use have shown only limited efficacy. This suggests selection pressure by antimicrobial drugs other than vancomycin (10). Early detection of VREF is of extreme importance because of the possibility that the vanA gene may be transferred to a variety of gram-positive microorganisms, including S. aureus. The rate of isolation of VREF at our hospital increased considerably during the last year. Even though the number of patients is small, we consider this finding to be of utmost importance, since VREF seems to be emerging in Mexico. To our knowledge, this is the first well-documented outbreak of high-level resistance to vancomycin in enterococci in Mexico. Further research is needed to determine if the problem is limited to our hospital or if it is a nationwide trend.