Frequency of Mycobacterium bovis as an etiologic agent in extrapulmonary tuberculosis in HIV-positive and -negative Mexican patients.

Mycobacterium bovis can be an important etiological agent for extrapulmonary (EP) manifestations of tuberculosis, especially in HIV-infected persons. From January 2000 to December 2003, M. bovis as a cause of EP tuberculosis was investigated at the Pneumonology Service, Hospital General de Mexico, Mexico City. Eighty HIV-positive (HIV+) patients and 83 HIV-negative (HIV-) with EP involvement (ganglionar, genitourinary, meningeal, cutaneous, peritoneal, and pericardial) were analyzed using clinical, immunological, bacteriological, histopathological, and molecular biology methods. Mycobacterium species were identified by hsp65-RFLP analysis and species of M. tuberculosis complex isolates by spoligotyping. M. bovis was present in 6 HIV- cases (7.2%; 3 with lymphadenitis and 3 genitourinary) vs 11 in HIV+ cases (13.75%; 7 with lymphadenitis, 3 genitourinary, and 1 meningeal). Favorable response to retroviral and specific M. bovis chemotherapy was observed. Spoligotyping showed a unique profile in each isolate, 16 belonging to BOV1 lineage and 1 to BOV2 lineage. M. bovis is an significant re-emerging cause of EPTB in Mexico. Consumption of unpasteurized dairy products is the most likely source of transmission. Successful treatment depends on the adequate and opportune identification of the agent responsible.

Analysis of antibody response in human dengue patients from the Mexican coast using recombinant antigens.

This study was undertaken to evaluate the feasibility of using recombinant dengue proteins to discriminate between acute dengue infections versus uninfected dengue samples. Dengue virus proteins E, NS1, NS3, and NS4B were cloned as fusion proteins and expressed in Escherichia coli. Recombinant products were tested in 100 serum samples obtained from acute dengue fever cases collected from 3 states of Mexico where dengue is endemic. Sera from 75 healthy individuals living in nonendemic areas for dengue were used as a control group. In sera from the dengue patients group, antibody responses to E protein were demonstrated in 91% of cases and NS1 protein was recognized to various extents (99%) within the first 7 days of infection. The antibody responses to NS3 and NS4B were frequently of low magnitude. Consistent negative antibody responses to all proteins were found in sera from the control group. These data suggest that the glutathione-S-transferase (GST)-dengue fusion proteins may be feasible antigens for a sensitive and specific serological assay.

Peripheral blood CD161+ T cells from asthmatic patients are activated during asthma attack and predominantly produce IFN-gamma.

In humans, T cells expressing the CD161 molecule NKR-P1A constitute around 20% of the circulating CD3(+) cells and are potentially immunoregulatory in several diseases. Their role in asthma is not well known, but they could participate in asthma attacks. To determinate whether activation of CD161(+) T cells and their cytokine production correlate with clinical status of asthma, we analysed blood samples from asthma attack patients (AAP) and stable asthma patients (SAP) in comparison with healthy non-atopic controls (HC). There was a significant higher baseline expression of CD69 on T cells from AAP and the difference was more notorious on CD161(+) T cells; upregulation of CD69 was observed on both CD161(-) and CD161(+) T cells driven by Dermatophagoides pteronyssinus crude extract, whereas polyclonal stimulation with phorbol 12-myristate 13-acetate plus ionomycin predominantly induced IFN-gamma but no IL-4, IL-5 and IL-13 by CD161(+) T cells in all groups; upon polyclonal stimulation, there were more CD161(+) T cells producing IFN-gamma and less CD161(-) T cells producing this cytokine, contrasting with the opposite results observed in SAP and HC groups. Our results indicate that, during asthma attack, CD161(+) T cells are activated and are able to produce predominantly IFN-gamma but no Th2 cytokines. We hypothesize that during an asthma attack, IFN-gamma produced by CD161(+) T cells could help to reestablish the Th1/Th2 equilibrium. These observations may contribute to the understanding of the immune mechanisms involved in asthma attacks.

Prevalence of Escherichia coli and Salmonella spp. in street-vended food of open markets (tianguis) and general hygienic and trading practices in Mexico City.

Street-vendors in Mexico City provide ready-to-eat food to a high proportion of the inhabitants. Nevertheless, their microbiological status, general hygienic and trading practices are not well known. During spring and summer 2000, five tianguis (open markets) were visited and 48 vendors in 48 stalls interviewed. A total of 103 taco dressings were sampled for E. coli and Salmonella spp.: 44 (43%) contained E. coli and 5 (5%) Salmonella (2 S. Enteritidis phage type 8, 1 S. Agona, 2 S. B group). Both E. coli and salmonellas were isolated from three samples. Of Salmonella-positive stalls 80% (4/5) had three or more food-vendors and 80% of vendors were males, compared with 37.3% (16/43) and 46.4% (20/43) in the Salmonella-negative stalls respectively. Food-vendors kept water in buckets (reusing it all day), lacked toilet facilities, and prepared taco dressings the day before which remained at the tianguis without protection for 7.8 h on average. Consumption of street-vended food by local and tourist populations poses a health risk.

Evaluation of the polymerase chain reaction in the diagnosis of miliary tuberculosis in bone marrow smear.

OBJECTIVE: Miliary tuberculosis (MTB) is difficult to diagnose. When prompt diagnosis is necessary, the polymerase chain reaction (PCR) to detect mycobacterial DNA may be valuable. SETTING: Tuberculosis clinic in an academic tertiary-level hospital in Mexico. DESIGN: Bone marrow (BM) aspiration samples from 30 consecutive clinically suspected MTB patients and 58 non-tuberculosis hematologic patients were evaluated by in-house PCR using a fragment of the insertion sequence IS6110; results were compared with those obtained by acid-fast-stained smears, culture in Löwenstein-Jensen medium, histology, and serology. RESULTS: Tuberculosis diagnosis was confirmed in all MTB suspects, 28 by microscopy and culture in pulmonary or extra-pulmonary samples other than BM, and two by clinical and radiologic improvement after antituberculosis treatment. In fresh BM specimens, in-house PCR was positive in 21/30 (70%) suspects, contrasting with only one positive (3.3%) in staining and culture, and four with compatible histologic findings (13.3%). BM samples from the control group showed negative results in bacteriologic and histologic studies, except in nine who had positive PCR results. These nine control cases had malignant processes. CONCLUSION: PCR in aspirates of BM is a useful diagnostic assay in cases of MTB, mainly when bacteriological results are negative.

Tuberculosis is still a major cause of cervical lymphadenopathies in adults from developing countries.

To establish the frequency of infectious aetiology in Mexican adult patients with cervical lymphadenopathies (CLAs), 87 consecutive patients with enlarged cervical lymphatic nodes, HIV negative and without anti-tuberculous treatment, were selected from a tertiary-level speciality concentration hospital. Histopathological studies, investigation of acid-fast bacilli, cultures in Löwenstein Jensen and Mycobacterium growth indicator tube (MGIT) media, and in-house polymerase chain reaction (PCR) with IS6110-based primers for Mycobacterium tuberculosis complex were performed in resected lymphatic nodes. Non-infectious aetiology corresponded to 45 cases (52 %). Tuberculosis was suspected in 42 cases (48%) by histology and confirmed positive results were obtained by staining in 8 (19%), by culture in 23 (55%), and by PCR in 34 (81 %) patients. All were confirmed after therapeutic success. In addition to the epidemiological transition process occurring in Mexico, tuberculosis remains an important cause of CLA. Histopathology with confirmatory studies including PCR can detect tuberculous aetiology.

Underestimation of Mycobacterium tuberculosis infection in HIV-infected subjects using reactivity to tuberculin and anergy panel.

BACKGROUND: This study aimed to evaluate purified protein derivative (PPD) reactivity and its interrelationship with anergy panel and CD4+ lymphocytes in HIV-infected subjects as compared to PPD reactivity in HIV-uninfected individuals in a tuberculosis endemic and high Bacillus Calmette-Guérin (BCG) coverage environment. METHODS: Clients of four Mexico City HIV detection centres were screened for HIV-1 antibodies (ELISA or haemagglutination, Western Blot); reactivity to PPD (Mantoux PPD, 5TU RT-23), Candida (1:1000, 0.1 ml), and tetanus toxoid (10Lf, 0.1 ml); and CD4+ T cells. Active tuberculosis was excluded. Informed consent was obtained. RESULTS: From 5130 clients 1168 subjects were enrolled; of these 801 (68.6%) were HIV positive. Reactivity to PPD among HIV-positive subjects was found in 174 (22%), 261 (32.6%), and 296 (37%), at PPD cutoff levels of > or =10 mm, > or =5 mm, and > or =2 mm as compared to 224 (61%) of 367 HIV-negative individuals' reactors to PPD (> or =10 mm) (P < 0.001). After exclusion of anergic individuals using two cutoff levels for cutaneous allergens (< or =2 mm and < or =5 mm), PPD reactivity between HIV-infected and uninfected individuals continued to be significantly different. Only HIV-infected individuals with CD4+ T cells > or =500 cells/mm3 had similar reactivity to PPD as HIV-uninfected individuals. Variables associated with PPD reactivity were CD4+ T cell counts, BCG scar, HIV infection and age. CONCLUSIONS: PPD reactivity was useful to diagnose tuberculosis infection only among HIV-infected individuals with CD4+ counts > or =500 cells/mm3. Among individuals with lower counts, lowering cutoff levels or using anergy panel did not permit comparable reactivity as that observed among HIV-uninfected individuals.

Gnathostomosis, an emerging foodborne zoonotic disease in Acapulco, Mexico.

Between 1993 and 1997, 98 gnathostomosis cases were clinically identified in Acapulco, Mexico. Intermittent cutaneous migratory swellings were the commonest manifestation. Larvae were identified in 26 cases, while in 72, final diagnosis was made on the basis of epidemiologic data, food habits, and positive enzyme-linked immunosorbent assay and Western blot results.

[Importance of molecular methodology in diagnosis]. / La trascendencia de la metodología molecular en el diagnóstico.

The National Institute for Epidemiological Diagnosis and Reference (INDRE) partially supports epidemiological surveillance programs through the identification of most infectious agents prevalent in the country. The success of a program for the control or eradication of a particular infectious disease mainly depends on the opportune and accurate identification of the corresponding etiologic agent. For laboratory diagnosis at INDRE, both conventional methodology using direct or microscopic examinations of specimens or growth in culture media followed by physiological or immunological characterization of the isolate, as well as new techniques based in biochemical, immunochemical and molecular biology procedures are carried out. Antigens can be detected in clinical samples by ELISAs with polyclonal or monoclonal antibodies. Specific nucleic acids can be extracted, identified and typed with techniques like electrophoresis, hybridization with genomic probes, polymerase chain reaction or fragment restriction length polymorphism. Recombinant molecules or highly purified antigens are being obtained and used for the determination of antibodies, mainly with indirect ELISA, IgM capture-ELISA and Western Blot. The better performance, specificity and sensitivity of these laboratory procedures, provide faster results, with equal or greater accuracy than traditional ones, at lower cost.

Enzyme-linked immunosorbent assay (ELISA) with mycobacterial crude antigens for the sero-epidemiological diagnosis of active tuberculosis.

In search for reliable, nonexpensive procedures for tuberculosis diagnosis suitable for seroepidemiological studies in leprosy-endemic areas, enzyme-linked immunosorbent assays (ELISAs) with whole intact bacilli, whole lipid-free bacilli and protein-enriched soluble extracts from the H37Rv Mycobacterium tuberculosis strain were evaluated. Sera tested came from 47 active, pulmonary tuberculosis adult cases, 60 household contacts of active tuberculosis cases, 20 lepromatous leprosy adult patients, and 67 healthy adult controls obtained from low and high leprosy and tuberculosis endemicity areas. There was no influence of such endemicity levels in the number of positive results in control sera. Antibody levels obtained with each of the antigens in ELISAs were significantly different in tuberculosis patients and the control groups. Ten percent of tuberculosis contacts were positive with some of the antigens and three of them showed suggestive chest radiographs. The best combination for a high number of positive results with tuberculosis sera and low positive results with leprosy sera was the BCG soluble extract (91% and 15%, respectively). This preparation also yielded excellent sensitivity and specificity values for tuberculosis (91.5% and 92.5%, respectively). These data suggest that BCG soluble extract ELISAs could provide helpful information to estimate tuberculosis prevalence only in leprosy-free areas, under a situation of unavailability of purified antigens. In pulmonary cases, sputum microscopic examination and culture have higher sensibility than serodiagnosis; therefore, the utilization of BCG soluble extract ELISAs as a diagnostic aid in individual patients with suspected active tuberculosis only can be useful in extrapulmonary cases.

Antibody response to nitrogenase-positive and -negative Klebsiella pneumoniae strains in juvenile-onset ankylosing spondylitis patients and their first degree relatives: lack of differential recognition of the bacterial nitrogenase.

In the search for the pathogenic consequences of the molecular mimicry between the Klebsiella pneumoniae nitrogenase and the HLA-B27 antigen, sera from individuals belonging to 16 kindreds with juvenile-onset ankylosing spondylitis cases, were analyzed for antibodies against nitrogenase-positive and -negative K. pneumoniae whole bacterial extracts. An initial screening for nitrogenase producing K. pneumoniae strains was performed in 31 clinical isolates. The best nitrogenase producing strain was selected as well as a non producing one for immunoblot analysis using sera from 82 subjects, 55 HLA-B27 positive, of which 26 had some clinical manifestations. Even though electrophoretic patterns were different in both strains, there was no distinctive differential recognition of the 30-40 kDa proteins where the nitrogenase subcomponent which shares the sequence QTDRED with the HLA-B27 molecule is located. On the other hand, strong recognition of a protein of 60 kDa (p60Kp) was detected in 75% of HLA-B27 positive tested subjects independently of their clinical status. Studies on the nature of this protein and its participation in the pathogenesis of ankylosing spondylitis are now in progress.

Prospective immunological follow-up in household contacts of Mexican leprosy patients.

A 6-year prospective study of 79 household contacts of leprosy cases was made in order to correlate the development of the disease with their specific T-cell immunity, measured by the Mitsuda test, and levels of anti-Mycobacterium leprae antibodies determined in three consecutive observations with the FLA-ABS test. Overall in the contacts, 71.7% were Mitsuda positive and 93.6% showed seropositivity, without regard to their age, sex, or leprosy type of their index case. Households were divided into lower-risk and higher-risk groups according to either the paucibacillary or multibacillary character of their index case. The lower-risk group consisted of 19 contacts of 2 tuberculoid (TT) and 5 indeterminate cases. The higher-risk group was made up of 60 household contacts of 18 active lepromatous (LL) cases. All but two contacts in the former group had a positive Mitsuda reaction; the most common antibody titer was 1:160, with a tendency to stabilize or decrease over time. In the two Mitsuda-negative contacts, increased antibody levels were observed. In the higher-risk group, 61.6% were Mitsuda positive and showed a humoral profile similar to those Mitsuda positive in the lower-risk group. In most of the Mitsuda-negative LL contacts, the antibody levels remained constant or progressively increased, suggesting a high probability of active subclinical infection. This assumption was partially supported by the finding of a new borderline lepromatous (BL) leprosy case in the Mitsuda-negative LL contact group. Nevertheless, the contribution of the close and extensive contact with a multibacilliferous case as a risk factor was difficult to evaluate because of the small size of the sample studied.

[Phagocytic capacity of peritoneal exudate cells from rats immunized with a ribosomal preparation of Ty2 Salmonella typhi]. / Capacidad fagocítica de células del exudado peritoneal de ratones inmunizados con una preparación ribosomal de Salmonella typhi Ty2.

It was compared the activity of exudate peritoneal cells (EPC) obtained from CFW mice immunized either with Salmonella typhi Ty2 ribosomal fraction or whole-cell heat inactivated vaccine, both in comparison with EPC from sham-immunized. In the group which received ribosomal preparation, a subcutaneous dose equivalent to 100 micrograms of RNA in incomplete Freund's adjuvant (IFA) was initially used and 14 days after a booster of the same dose in IFA was given. A single dose of whole-cell heat inactivated vaccine, with 10(6) bacteria in IFA was employed subcutaneously in animals of the second group. EPC from controls and immunized mice were withdrawn at periods of 7, 11, 14, 18, 22, 25, 29 and 31 days after immunization and each sample was incubated in vitro in presence of live virulent non-opsonized S. typhi Ty2 in 1:200 cell-bacteria relation. Twenty four hours after cultivation, EPC bacterial capacity was determined after cell disruption and enumeration of survival bacteria were made through viable counts. Results have shown that EPC from mice immunized were more efficient in eliminating intracellular bacteria than those which came from sham-immunized animals. Also, it was found that EPC from mice immunized with ribosomal preparation were more efficient (maximum P = 0.005) than EPC from the mice which received killed whole bacteria.

Demonstration of acid-fast bacilli in skin biopsies from indeterminate leprosy cases.

Because the correct diagnosis of indeterminate leprosy (IL) requires the finding of acid-fast bacilli in skin lesions from clinically and histopathologically suggestive cases, it is necessary to develop a reliable method for this purpose. This paper presents a simple procedure, available to every general laboratory, which consists in obtaining 2 suspensions: SI, by mincing and grinding the tissue in phosphate-buffered saline; and SII, after treating SI with NaOH solution and digesting with trypsin. In 22 IL skin biopsies, bacilli were directly observed in only 3 with the Ziehl-Neelsen (ZN) stain; and with the peroxidase-antiperoxidase method it was impossible to differentiate between nonspecific precipitate and true positive reactions. In contrast, 18 positive results from the same 22 samples were obtained when both SI and SII were evaluated with ZN stain. The logarithmic bacterial index was also increased in at least 7 cases.

Sensitivity and specificity of the FLA-ABS test for leprosy in Mexican populations.

The epidemiological surveillance for leprosy must include several clinical and laboratory procedures. The FLA-ABS test of Abe could be a useful tool for this purpose because it allows the demonstration of an effective contact with Mycobacterium leprae. In order to establish the specificity, sensitivity, and predictability of the FLA-ABS test under Mexican conditions, we studied sera collected from six groups of individuals: 60 healthy donors from a nonendemic area, 57 cases hospitalized for conditions other than infectious diseases from a general hospital in a nonendemic area, 72 patients with active pulmonary tuberculosis, 26 healthy individuals from an endemic area, 100 patients with polar lepromatous leprosy (LLp), and 123 household contacts of patients with LLp. The FLA-ABS test was negative with sera from the first four groups. Strong positive reactions were found in all LLp patients except one; the false-negative results could be attributed to successful treatment and a long-standing cure in this patient. Analysis of these results shows 100% specificity, 99% sensitivity, and predictability values of the test of 100% for positive results and 99% for negative ones. In addition, none of the 20 randomly selected sera from LLp patients were positive with crossreacting mycobacteria. Because 87.8% of the household contacts were positive in the absence of clinical manifestations of leprosy, it is possible to conclude that a positive result by itself is not enough to establish an early diagnosis of the disease, especially among inhabitants of endemic areas.(ABSTRACT TRUNCATED AT 250 WORDS)

The HLA system in the prevalent Mexican Indian group: the Nahuas.

Results of HLA-A, B and C typing as well as haplotype frequencies in the Nahuas, who are the predominant Indian group in Mexico are presented. ABO and Rh blood groups show the genetic homogeneity of this population, since all of them were group O and Rh+. The most frequent antigens and haplotype are in general, the same as in some, but not all Amerindian tribes (Papago, Pimas, Zuñi from North America and Ixils from Guatemala). When compared to other Mongoloids, the HLA pattern is very close to the Japanese population. Antigens Bw39 and Cw4 look like markers of Nahuas and because Bw16 cells from four individuals could not be assigned either as Bw38 or Bw39, and 6 subjects did not type for any of the Bw22 splits, new subtypes of these antigens are probable.