Mycobacterium tuberculosis multi-drug-resistant strain M induces IL-17+ IFNγ- CD4+ T cell expansion through an IL-23 and TGF-ß-dependent mechanism in patients with MDR-TB tuberculosis.

We have reported previously that T cells from patients with multi-drug-resistant tuberculosis (MDR-TB) express high levels of interleukin (IL)-17 in response to the MDR strain M (Haarlem family) of Mycobacterium tuberculosis (M. tuberculosis). Herein, we explore the pathways involved in the induction of Th17 cells in MDR-TB patients and healthy tuberculin reactors [purified protein derivative healthy donors (PPD+ HD)] by the M strain and the laboratory strain H37Rv. Our results show that IL-1ß and IL-6 are crucial for the H37Rv and M-induced expansion of IL-17+ interferon (IFN)-γ- and IL-17+ IFN-γ+ in CD4+ T cells from MDR-TB and PPD+ HD. IL-23 plays an ambiguous role in T helper type 1 (Th1) and Th17 profiles: alone, IL-23 is responsible for M. tuberculosis-induced IL-17 and IFN-γ expression in CD4+ T cells from PPD+ HD whereas, together with transforming growth factor (TGF-ß), it promotes IL-17+ IFN-γ- expansion in MDR-TB. In fact, spontaneous and M. tuberculosis-induced TGF-ß secretion is increased in cells from MDR-TB, the M strain being the highest inducer. Interestingly, Toll-like receptor (TLR)-2 signalling mediates the expansion of IL-17+ IFN-γ- cells and the enhancement of latency-associated protein (LAP) expression in CD14+ and CD4+ T cells from MDR-TB, which suggests that the M strain promotes IL-17+ IFN-γ- T cells through a strong TLR-2-dependent TGF-ß production by antigen-presenting cells and CD4+ T cells. Finally, CD4+ T cells from MDR-TB patients infected with MDR Haarlem strains show higher IL-17+ IFN-γ- and lower IL-17+ IFN-γ+ levels than LAM-infected patients. The present findings deepen our understanding of the role of IL-17 in MDR-TB and highlight the influence of the genetic background of the infecting M. tuberculosis strain on the ex-vivo Th17 response.

Modelling the feasibility of bovine tuberculosis eradication in Argentina.

The ability of countries to control and eradicate bovine tuberculosis (TB) has been jeopardised by various epidemiological and ecological features of the disease. The authors have used epidemiological modelling to develop an analytical framework to assess the likely success of a national TB eradication programme in Argentina. Study results suggest that the current control programme is financially feasible in the long term. However, considering that the costs of the TB eradication programme in Argentina are entirely borne by the producer, the initial investment required and the long-term horizon needed to gain revenue may prevent producers from endorsing the programme. Regionalised programmes that allow differential control strategies to be implemented in specific regions may increase the likelihood of success. This methodological approach could be extended to design and evaluate control and eradication programmes for TB and other infectious diseases in other regions of the world.

AIDS pacient's long-term battle with multiply recurrent tuberculosis: reinfection or reactivation?

The advent of Mycobacterium tuberculosis strain genotyping has allowed differentiation between disease relapse and exogenous re-infection. We report here a remarkable case of multiply recurrent tuberculosis in a patient living with HIV. Between 1995 and 2009, a young HIV-infected intravenous drug user, who was reluctant to comply with anti-retroviral treatment, underwent at least five tuberculosis episodes caused by three distinct M. tuberculosis strains sharply differentiated by drug susceptibility profile, genotype and infectious source. Eventually, the patient died during a relapse of tuberculosis due to a notorious multidrug-resistant outbreak-strain, which infected him during a prolonged hospitalization in the epicentre of such outbreak. Whether recurrent tuberculosis is due to a new infection or to reactivation of a previous one is a century-long controversial question. In our patient, both conditions alternated throughout his 15 years of living with HIV. Cases such as this might not be exceptional in certain underprivileged suburban areas of Argentina and should raise concern over three pending issues in tuberculosis control policies, namely secondary preventing therapy, institutional infection control and patient follow-up throughout the health network system.

Combined approach to the identification of clinically infrequent non-tuberculous mycobacteria in Argentina.

SETTING: Over 150 potentially pathogenic non-tuberculous mycobacteria (NTM) species have been described, posing an onerous challenge for clinical laboratory diagnosis. OBJECTIVE: To evaluate different approaches for the identification of 40 clinically relevant NTM isolates whose species were not reliably identified using our routine diagnostic workflow comprising phenotypic tests and hsp65 polymerase chain reaction restriction analysis. DESIGN: We used 1) sequencing analysis of four conserved gene targets: 16S rRNA, rpoB, hsp65 and sodA; 2) two commercial reverse hybridisation assays; and 3) protein analysis using matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). RESULTS: Combined, but not individual, sequence analysis allowed reliable species identification for 30/40 (75%) isolates, including species previously unknown to be circulating in Argentina. Commercial kits outperformed our routine identification in only 5/35 isolates, and misclassified many more. MALDI-TOF MS accurately identified species in 22/36 (61%) isolates and did not misidentify any. CONCLUSIONS: Commercial kits did not resolve the problem of species of NTM isolates that elude identification. Combined DNA sequence analysis was the approach of choice. MALDI-TOF MS shows promise as a powerful, rapid and accessible tool for the rapid identification of clinically relevant NTM in the diagnostic laboratory, and its accuracy can be maximised by building up a customised NTM spectrum database.

Multidrug-resistant tuberculosis outbreak among transvestite sex workers, Buenos Aires, Argentina.

We describe the first outbreak of multidrug-resistant tuberculosis (MDR-TB) that occurred in Argentina among transvestite sex workers, and actions undertaken for its control. In Buenos Aires city, transmission was documented between 2001 and 2004 by conventional and molecular methods in a hotel where transvestites used to reside and work. The source case was traced back to 1998. Six secondary cases were diagnosed and treated. Thirty-two contacts were investigated. The outbreak strain had formerly caused nosocomial transmission in Rosario, a city 300 km from Buenos Aires. Our findings highlight the difficulties controlling MDR-TB in Argentina.

Multicenter study of MTT and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs.

OBJECTIVE: A multicentre evaluation was performed to assess two rapid low-cost methods, MTT (3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) and resazurin assays, for testing the susceptibility of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin (RMP), isoniazid (INH), ethambutol (EMB) and streptomycin (SM). METHODS: Thirty coded M. tuberculosis strains were sent to seven laboratories located in Latin America, representing six countries. Each site performed the colorimetric assays, MTT and resazurin, blind for the first-line drugs RMP, INH, EMB and SM. The minimum inhibitory concentration results obtained were compared to the conventional proportion method on Lowenstein-Jensen medium. RESULTS: After establishing the breakpoint concentrations, excellent results were obtained for RMP, INH and EMB, with levels of specificity and sensitivity of between 96% and 99%. CONCLUSION: MTT and resazurin assays are promising, accessible new alternative methods for middle- and low-resource countries that need low-cost methods to perform rapid susceptibility testing of M. tuberculosis to key anti-tuberculosis drugs.

Comparative molecular study of Mycobacterium tuberculosis strains, in times of antimicrobial drug resistance.

Strains of Mycobacterium tuberculosis were compared using two DNA fingerprinting techniques: Restriction Fragment Length Polymorphism (RFLP) and Double-Repetitive-Element-PCR (DRE-PCR). Two of these strains: IH1 (susceptible to isoniazid) and IH2 (resistant to isoniazid) were recovered from cases of pulmonary tuberculosis which occurred in two brothers who lived together. The first one was recognized on July 1999, and the second was diagnosed one year later. IH1 and IH2 showed the same pattern of bands with both molecular tests. These results suggest that single drug chemoprophylaxis may occasionally select resistant strains for that drug, which can eventually cause disease and be recognized through these tests. Strains IH3, IH4 and IH5 were obtained from sputum samples of 3 different patients, and intra-laboratory cross-contamination was suspected when it was realized that the 3 positive materials had been consecutively processed the same day by the same worker in the same biological safety cabinet. Again, the 3 strains revealed identical band patterns with RFLP and DRE-PCR, confirming the posed suspicion. The results with DRE-PCR were obtained after only 8 hours of work, without the need for subcultures. This procedure allows quick correction of treatment conducts, avoiding unnecessary exposure of people and bacteria to antimicrobial drugs.

Detection of circulating antibodies to purified protein derivative by enzyme-linked immunosorbent assay: its potential for the rapid diagnosis of tuberculosis.

An enzyme-linked immunosorbent assay for detecting antibodies to purified protein derivative was evaluated as a rapid method for serodiagnosis of childhood tuberculosis. Its specificity for IgG antibodies was 0.98 as determined in 55 sera from nontuberculous children who showed no significant effect of previous Bacillus Calmette-Guérin vaccination on the production of specific antibodies. Results were negative in 29 of 33 (87.9%) tuberculin-positive children and in 18 of 20 (90.0%) contacts, none of whom had evidence of tuberculosis. The sensitivity of this test was 0.51 as determined in 49 sera from bacteriologically confirmed cases; 17 of 27 smear positive cases and 8 of 22 children with positive cultures were detected. Results were positive in 32 of 114 (28.1%) patients with a diagnosis of tuberculosis not confirmed by microbiology. Consequently whereas a negative result does not rule out tuberculosis, a positive result is a strong indication of the disease. The IgM antibody determination yielded much less discriminative results.

Use of IS901 and IS1245 in RFLP typing of Mycobacterium avium complex: relatedness among serovar reference strains, human and animal isolates.

SETTING: Mycobacterium avium complex (MAC) includes major acquired immune deficiency syndrome (AIDS)-associated pathogens. Formerly, MAC serotyping was used for epidemiological purposes. Recently, restriction fragment length polymorphism (RFLP) typing has become available. OBJECTIVE: Examination of the usefulness of insertion sequence IS1245 in RFLP typing of MAC isolates and the association with IS901 RFLP. DESIGN: Ninety-four serovar reference strains were compared with 144 clinical and animal MAC isolates in RFLP typing. RESULTS: All but four strains containing M. avium-specific-rRNA possessed IS1245. Most human isolates showed polymorphic multiband IS1245 patterns, which were associated with serovars 4, 6 and 8. Sequential clinical isolates obtained at up to five years' distance displayed indistinguishable/closely related patterns. Eleven M. paratuberculosis isolates showed indistinguishable six-band patterns. All 29 MAC isolates from 23 bird species, 7/23 from mammals and 1/81 clinical isolates showed an IS1245 three-band pattern, associated with serovars 1, 2 and 3. All these IS1245 'bird' type strains showed closely related IS901 RFLPs. Only three IS1245 'non-bird' type strains contained IS901, but exhibited completely different RFLP patterns. CONCLUSION: IS1245-RFLP typing is useful for the classification of M. avium and epidemiology of most human isolates. The highly conserved IS901 and IS1245 RFLPs among 'bird' type isolates provide proof that these strains constitute a separate taxon within the MAC.

Bovine tuberculosis in Latin America and the Caribbean: current status, control and eradication programs.

Out of the approximately 300 million head which constitute the bovine population in Latin America and the Caribbean, 80 million are found in countries where rates of Mycobacterium bovis infection are very low or nil. The remaining 220 million are found in countries with either a moderate to high prevalence or where no recent information is available. Argentina and Brazil, both have huge cattle populations with estimated prevalences higher than 1%, and together may harbour 3.5 million infected cattle. Information on the impact of M. bovis on human health in the Region is scarce and does not include data on infection of children. In Argentina, human tuberculosis of bovine origin was found to be mainly an occupational disease, transmitted by aerosol. Control or eradication has been achieved in several countries in the Region by use of the tuberculin test followed by sacrifice of reactors. In countries such as Cuba, where the prevalence is already very low, area tuberculin testing is being replaced by slaughter surveillance and epidemiological trace-back. Other countries, where the prevalence is high (e.g. Chile, Paraguay, Peru and Argentina), promote regional campaigns based on the decision and active participation of cattle farmers. Recent diagnostic developments based on the in vitro measurement of humoral and cellular immune responses could be an aid in control and eradication campaigns, provided their usefulness is demonstrated in field trials. In heavily infected areas complementary or alternative strategies should also be proposed, aiming at lowering the prevalence rates prior to the application of the test and slaughter method.

Reciprocal cellular and humoral immune responses in bovine tuberculosis.

A sandwich ELISA for the detection of gamma interferon showed higher sensitivity and specificity than an indirect ELISA for mycobacterial antibodies in the diagnosis of bovine tuberculosis. Circumstantial evidence of an inverse relationship between cellular and humoral immune responses to Mycobacterium bovis was found in cattle with natural infection.

The administration of foot and mouth disease vaccine with oil adjuvant and its influence on the diagnosis of bovine tuberculosis.

The possible influence of vaccination with oil adjuvanted foot and mouth disease vaccines on the tuberculin response was investigated in 32 normal guinea pigs and 190 non-tuberculous bovines. Circulating anti-Mycobacterium bovis IgG antibodies were analysed by an enzyme-linked immunosorbent assay (ELISA), in order to determine the effect of the vaccination on the humoral response against mycobacteria in cattle. Control animals were either nonvaccinated or injected with aluminium hydroxide adjuvanted vaccine. Administration of foot and mouth disease vaccine had no apparent influence on the tuberculin responses of either guinea pigs or cattle, nor did it influence the level of anti-M. bovis antibodies in cattle.

[Humoral response to mycobacteria in patients with Crohn disease]. / Respuesta humoral a micobacterias en pacientes con enfermedad de Crohn.

The recent recovery of Mycobacterium paratuberculosis from tissues of patients with Crohn's disease has highlighted the possible etiologic role of this microorganism in the disease. However, the immunological evidence generated by various groups supporting this hypothesis is as yet inconclusive. A specific antibody response might be masked in these patients by the wide antigenic homologies prevailing within the genus Mycobacterium. The present study was undertaken with the purpose of exploring the humoral response to M. paratuberculosis in patients with Crohn's disease, by means of a cross-absorption procedure recently proposed for unveiling the presence of specific antibodies in bovine paratuberculosis. Antibodies IgG to M. paratuberculosis were investigated by enzyme-linked immunosorbent assay in 90 serum samples from 17 patients with Crohn's disease, 23 patients with ulcerative colitis an 14 with other bowel diseases. Samples from 86 subjects without bowel disease (healthy individuals and patients with tuberculosis, mycobacterioses and fungal diseases) were also included as controls. The specificity of these antibodies was explored by the absorption of sera with an ubiquitous Mycobacterium (M. phlei). The results were compared to those obtained by similar ELISA tests employing M. avium or M. tuberculosis as antigens. A faint humoral response to M. paratuberculosis and M. tuberculosis was detected in patients with Crohn's disease. Cross-absorption with M. phlei did not disclose a specific response nor was an increase in antibody levels detected in patients studied periodically. Sera from patients with ulcerative colitis and other bowel diseases also showed a slight reaction to mycobacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

[Evaluation of an enzyme immunoassay for the rapid diagnosis of paucibacillary tuberculosis in adults]. / Evaluación del enzimoinmunoensayo para el diagnóstico rápido de la tuberculosis paucibacilar del adulto.

The ELISA has been extensively evaluated as a serodiagnostic method for tuberculosis. However, there is scarce information about its application to cases that cannot be diagnosed by microscopic examination: those with closed lesions or undergoing early stages of the disease. Since a reliable serological test might substantially contribute to their prompt detection, the objective of the present study was to determine the diagnostic value of an ELISA applied to adult smear-negative cases of tuberculosis. Sera from 235 patients with active tuberculosis--176 pulmonary and 59 extrapulmonary cases--and 181 control subjects were tested for IgG antibodies to PPD by ELISA. Eleven cases of non tuberculous mycobacterial (MOTT) disease and 33 cases of mycosis were also included in this group. With the adopted cut-off value, 73.9% (105/142) of smear positive and 52.7% (49/93) of smear negative tuberculosis cases, were correctly classified. Particularly in the latter, the test was positive in 55.2% (32/58) of patients with positive cultures for Mycobacterium tuberculosis and in 48.6% (17/35) of patients diagnosed by clinical, radiological and or histopathological findings. No antibody activity was demonstrated in 92.7% of sera from the control population which included 92 healthy volunteers, 32 non tuberculous diseased subjects and 13 household contacts of smear-positive cases. Among those control subjects who were skin tested, ELISA results were not related to the tuberculin reactivity: 93.7% (30/32) of tuberculin negative and 95.2% (40/42) of tuberculin positive healthy individuals had no detectable antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Hospital transmission of multidrug-resistant Mycobacterium tuberculosis in Rosario, Argentina.

Multidrug-resistant tuberculosis has emerged over the last two years at Carrasco Hospital, located in Rosario city. Nosocomial transmission among 7 AIDS patients admitted into the same ward between June and December/94 was supported by temporal clustering of cases, matching drug susceptibility, and identical IS6110 fingerprints. Among 8 non-HIV chronic cases without evidence of reciprocal contact outside the hospital, two additional clusters of 2 and 4 cases, respectively, were identified. The latter was found to be generated by a strain genetically related to the one that infected AIDS patients. It is hypothesized that an ancestor strain, common to both, might have been brought into the hospital long before the outbreak was first suspected.

[Evaluation of four antigens for the detection of anti-Mycobacterium bovis antibodies by enzyme immunoassay]. / Evaluación de cuatro antígenos para la detección de anticuerpos anti-Mycobacterium bovis por enzimoinmunoensayo.

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of bovine tuberculosis through the detection of specific seric antibodies has recently been developed in our laboratory. In order to assess its reproducibility and select the most adequate antigen, four bovine PPDs from different sources were evaluated in parallel: PPD M. bovis strain AN5, CEPANZO standard (CPZ), PPD M. bovis strain AN5, European Economic Community standard (EEC), PPD M. bovis strain AN5, prepared from non heated bacilli, killed by phenol (P) and PPD. M. bovis BCG strain prepared at the Pasteur Institute, Paris (BCG). Sera from 22 healthy cattle from tuberculosis free area and 20 bacteriologically confirmed tuberculous animals were employed in simultaneous assays. Antibody mean and standard deviations from healthy cattle expressed as optical density (OD) values were 45 +/- 22 when CPZ was used as antigen, 24 +/- 10 with EEC, 103 +/- 56 with P and 56 +/- 20 with BCG. Mean O.D. from tuberculous cattle were 588 +/- 158, 510 +/- 234, 782 +/- 138 and 441 +/- 189 with antigens CPZ, EEC, P and BCG respectively. A close correlation was observed when results obtained with EEC and P were compared with that of CPZ (r: 0.97 and 0.94 respectively). A lower specificity was achieved when BCG was used as antigen being also lower its correlation with the results obtained with CPZ (r: 0.87). It is concluded that our ELISA would achieve similar sensitivity and specificity if CPZ, EEC and P were used as antigens. On the other hand, BCG would not be suitable for this assay.

[Study of Mycobacterium bovis in milk using bacteriological methods and the polymerase chain reaction]. / Estudio de Mycobacterium bovis en leche mediante métodos bacteriológicos y reacción en cadena de la polimerasa.

The frequency of Mycobacterium bovis detection in milk samples obtained from infected animals was explored in an intensive dairy area in Argentina. To this end, an "in house" polymerase chain reaction (PCR) method was developed using Mycobacterium tuberculosis Complex specific INS1-INS2 primers, and its performance was compared with that of bacteriological methods. The decontamination procedures previous to culture reduced M. bovis viability. The pathogen was identified in milk samples from 1 of 143 infected cows and in none of 43 uninfected ones. Even though PCR sensitivity was found to be 2-20 times higher than that of bacteriology in experimentally inoculated milk samples, all 186 field samples resulted negative by PCR, including the bacteriologically-confirmed one. In spite of the high prevalence of bovine tuberculosis in Argentinian dairy herds, the detection of M. bovis in milk is an unusual finding.

[False-positive cultures due to cross contamination in tuberculosis laboratories]. / Falsos cultivos positivos por contaminación cruzada en laboratorios de tuberculosis.

Fifteen episodes of Mycobacterium tuberculosis laboratory cross-contamination suspected between 1996 and 2001 at 6 laboratories in Buenos Aires City and suburbs were investigated by IS6110 RFLP. Thirteen episodes were confirmed. Even though BACTEC 460 produced the highest number of confirmed episodes in a single laboratory, the most extended one occurred while employing conventional culture procedures in solid medium. The double repetitive element-polymerase chain reaction (DRE-PCR) was applied to 8 of these episodes and produced concordant results with those of the RFLP. The DRE-PCR appears to be a valuable tool for the prompt identification of false positive cultures. The timely rectification of defects in laboratory protocols can avert false diagnoses of tuberculosis and unnecessary prolonged treatments.

Differences in the robustness of clusters involving the Mycobacterium tuberculosis strains most frequently isolated from immigrant cases in Madrid.

Tuberculosis cases infected by the same Mycobacterium tuberculosis (MTB) strain are considered to be clustered and involved in a transmission chain. Large clusters are assumed to represent active transmission chains in a population. In the present study, we focused on the analysis of large clusters defined by IS6110-restriction fragment length polymorphism (RFLP) typing in the immigrant population in Madrid. We identified 12 large clusters (involving 43% of the isolates) comprising 4-23 representatives. We proposed a gradient of epidemiological certainty for these large clusters. For a cluster to be considered robust and a good indicator of recent transmission, the MTB strain involved should not have been identified in a geographically and epidemiologically unrelated population and the cluster had to be re-confirmed by another highly discriminative molecular marker (MIRU-VNTR). The clusters that we discovered were classified into three categories: high, intermediate and low expected epidemiological value. In the largest cluster in the study (cluster M6; 23 representatives), failures by both criteria were identified: the representative seven-band RFLP pattern was also the most prevalent in the unrelated population (25 cases) and the cluster was fully split by MIRU-15, suggesting a lack of epidemiological value. The RFLP pattern representative of this cluster was also identified in 64 isolates from five countries in the Latin American genotype database, and again proved to be heterogeneous according to the MIRU-15 analysis. Specific analysis of large clusters, combined with the application of criteria for evaluating their robustness, could help identify uninformative clusters and target epidemiological resources towards those clusters with higher expected epidemiological value.