Phenotypic and genotypic variant of MDR-Mycobacterium tuberculosis multiple isolates in the same tuberculosis episode, Rio de Janeiro, Brazil

Assuming that the IS6110-restriction fragment length polymorphism (RFLP) changes at a constant rate of 3.2 years, this methodology was applied to demonstrate, for the first time, variant patterns of Mycobacterium tuberculosis (MTB) in multiple isolates obtained at short time intervals from sputum and blood of an HIV+ patient with multiple admissions to the Emergency Room and to the multidrug-resistant tuberculosis (MDR-TB) Reference Center of a secondary-care hospital in Rio de Janeiro, Brazil. In sputum, the IS6110-RFLP appeared in isolates with two variant patterns with 10 and 13 IS6110 copies. However, blood presented only the pattern corresponding to 10 copies, suggesting compartmentalization. With regard to the exact match of 10 of 13 bands, this may be a subpopulation with the same clonal origin and this may be related to the IS6110 transposition. A susceptibility test demonstrated an MDR profile (INH R, RIF R, SM R, and EMB R), with the sputum isolate also exhibiting EMB S (R = resistant; S = sensitive). A gene mutation confirmed resistance only to streptomycin. There was agreement between the results of the phenotypic test and the clinical response to MDR-TB treatment, suggesting serious implications with regard to treatment administration based exclusively on molecular methods, and calling attention to the fact that more effective control strategies against the emergence of MDR strains must be implemented by the TB control program to prevent transmission of MDR-MTB strains at health facilities in areas highly endemic for TB.

Phenotypic and genotypic variant of MDR-Mycobacterium tuberculosis multiple isolates in the same tuberculosis episode, Rio de Janeiro, Brazil.

Assuming that the IS6110-restriction fragment length polymorphism (RFLP) changes at a constant rate of 3.2 years, this methodology was applied to demonstrate, for the first time, variant patterns of Mycobacterium tuberculosis (MTB) in multiple isolates obtained at short time intervals from sputum and blood of an HIV+ patient with multiple admissions to the Emergency Room and to the multidrug-resistant tuberculosis (MDR-TB) Reference Center of a secondary-care hospital in Rio de Janeiro, Brazil. In sputum, the IS6110-RFLP appeared in isolates with two variant patterns with 10 and 13 IS6110 copies. However, blood presented only the pattern corresponding to 10 copies, suggesting compartmentalization. With regard to the exact match of 10 of 13 bands, this may be a subpopulation with the same clonal origin and this may be related to the IS6110 transposition. A susceptibility test demonstrated an MDR profile (INH(R), RIF(R), SM(R), and EMB(R), with the sputum isolate also exhibiting EMB(S) (R = resistant; S = sensitive). A gene mutation confirmed resistance only to streptomycin. There was agreement between the results of the phenotypic test and the clinical response to MDR-TB treatment, suggesting serious implications with regard to treatment administration based exclusively on molecular methods, and calling attention to the fact that more effective control strategies against the emergence of MDR strains must be implemented by the TB control program to prevent transmission of MDR-MTB strains at health facilities in areas highly endemic for TB.

IgA antibody responses to Mycobacterium tuberculosis recombinant MPT-64 and MT-10.3 (Rv3019c) antigens in pleural fluid of patients with tuberculous pleurisy.

SETTING: Microbiological tests lack sensitivity for pleural tuberculosis (TB) and histopathology is expensive, time consuming and needs specialised personnel. Immunoassay (ELISA) may be a promising approach in this respect. OBJECTIVE: To evaluate the reactivity of IgA antibody to MPT-64 and MT-10.3 recombinant mycobacterial protein antigens in pleural fluid as a marker of pleural TB, based on the fact that IgA is the main antibody in the mucosa/serosa of the gastrointestinal and respiratory tract. METHOD: Anti-MPT-64 and MT-10.3 IgA response was determined by ELISA in 72 patients with pleural TB and 27 with other pleural conditions. RESULTS: High sensitivities for IgA were measured against MPT-64 (52/72, 72%) and MT-10.3 (52/72,72%) antigens. Combining the sensitivities of both antigens, further increase in sensitivity (55/72, 76%) was obtained with no loss of specificity (96%). Similar IgA reactivity was obtained from culture-negative and culture-positive specimens. In eight pleural TB patients with human immunodeficiency virus (HIV) co-infection, the sensitivity was 88% (7/8). CONCLUSION: To our knowledge, this is the first description of the presence of IgA antibody pleural TB effusion reactive to MPT-64 and MT-10.3, with sensitivity similar to histopathological examination, which is presently considered the gold standard for pleural TB.

The usefulness of serum adenosine deaminase 2 (ADA2) activity in adults for the diagnosis of pulmonary tuberculosis.

Rapid diagnosis of Mycobacterium tuberculosis remains an obstacle for therapy of tuberculosis (TB). Adenosine deaminase isoform 2 (ADA2) is produced by activated macrophages and has been used for diagnosis of TB from extra-pulmonary sites. However, few studies adequately address whether serum ADA2 activity is useful for diagnosis of active pulmonary tuberculosis (PTB). We prospectively measured serum ADA2 activity in 110 patients with pulmonary disease (65 cases with active PTB and 45 cases with other respiratory diseases) and 78 healthy volunteers (eight with tuberculin skin test positive). The serum ADA2 for the diagnosis of PTB had the sensitivity of 36.9%, the specificity of 84.5%, the positive predictive value of 10.9% and the negative predictive value of 96.2%. We concluded that serum ADA2 activity is neither useful to diagnosis of active PTB nor to differentiate from other respiratory diseases.

Evaluation of Etest strips for rapid susceptibility testing of Mycobacterium tuberculosis.

In this study, our objective was to evaluate Etest strips containing exponential gradients of isoniazid (INH), rifampin (RIF), and streptomycin (STR) for susceptibility testing of Mycobacterium tuberculosis. M. tuberculosis isolates were tested for antimicrobial susceptibilities by the standard proportion method using Löwenstein-Jensen (LJ) medium and by the Etest. The MICs determined by the Etest were obtained at 5, 7, or 10 days. In some strains with Etest-discrepant results, radiometric susceptibility testing (BACTEC) was performed to determine a consensus result. M. tuberculosis concordance between the two methods was 97% (86 of 89 isolates) for RIF, 96% for INH (84 of 87 isolates), and 80% (61 of 76 isolates) for STR. Most of the MICs determined by the Etest were easy to interpret and readable within 5 days. Results correlated well with those obtained by the LJ proportion and BACTEC methods for INH and RIF. However, a high proportion of false-sensitive and false-resistant results were observed, most often for STR. We also observed that variations in the inoculum size of M. tuberculosis isolates affected the MICs to a substantial degree. These discrepancies, along with the expense of the media, the Etest strips, and the specialized equipment required (CO2 incubator), make this method less useful in developing countries.

Mycobacterium tuberculosis typing: usefulness of DRE-PCR to confirm cross-contamination in the mycobacteriology laboratory of a general reference hospital for AIDS.

In this study two molecular typing methods, a simple double repetitive element PCR-based assay and the standardized restriction fragment length polymorphism (RFLP), were used to confirm cross-contamination in the mycobacteriology laboratory. Clinical specimens from 12 patients, submitted for acid-fast bacilli stain smear and processed for culture in Lowenstein-Jensen on the same day, resulted in positive bacterioscopy (+++) and confluent growth only for one of the patients. The specimens from all the other patients but two were smear-negative and culture-positive, with one or two colonies. None of them had clinical symptoms and radiological findings for active tuberculosis (TB). The suspicion of false-positive cultures arose when a health care worker who had had a PPD skin test conversion, claimed to be healthy and had no TB symptoms, was found to have a positive sputum culture. DRE-PCR demonstrated that all nine cultures typed belonged to one cluster, further confirmed by RFLP. Although DRE-PCR has been found to be poorly reproducible, it has enough discriminatory power to be useful for rapid epidemiological investigation in selected settings.

Estudo da transmissão horizontal de Mycobacterium avium-intracellulare em suínos

Experimental infections were performed to study the horizontal transmission of Mycobacterium avium-intracellulare (MAI) in swine. Twelve 35 to 42 day-old pigs were divided into three groups of four pigs each. Each group was housed in separate rooms, and inoculated with three MAI strains: VPS1, SC1 and ATCC-13950. After inoculation, four non-inoculated swines were added to each group and maintained in the same pen up to 150 days of age, when were slaughtered. Other four non-inoculated swines were maintained as a control group. The infection was diagnosed by the tuberculin test at 140 days of age, by detection of macroscopic lesions at slaughter and histopathologic and bacteriologic examination of tissues collected at necropsy. Culture of fecal samples were made during the experiment indicating active excretion of MAI between 14 and 42 days post-inoculation. Transmission of MAI among pigs kept in the same pen was observed with all the three strains used. The most evident transmission was with strain SC1, in which four "in contact" pigs were infected, followed by ATCC-13950 (two pigs) and VPS1 (one pig). These results indicate that infected swine can be an important source of infection, favoring the dissemination of this disease.

O trabalho teve como objetivo estudar a transmissão horizontal de Mycobacterium avium-intracellulare (MAI) entre suínos. Doze suínos com 35 a 42 dias de idade, divididos em três grupos de quatro e alojados em salas independentes, foram inoculados por via oral com as cepas VPS1, SC1 e ATCC-13950 de Mycobacterium avium-intracellulare. A seguir, quatro suínos não inoculados foram colocados em cada grupo e mantidos na mesma baia até o abate aos 150 dias da idade. Outro grupo de quatro suínos não inoculados foi utilizado como controle. Pelo exame de fezes verificou-se que houve excreção ativa de MAI entre 14 e 42 dias pós-inoculação. A transmissão da infecção foi avaliada pelo teste de tuberculina aos 140 dias de idade e pelos exames de patologia macro e microscópica e de isolamento do MAI de órgãos. Os quatro suínos em contato com aqueles inoculados com a cepa SC1 foram contaminados, enquanto que com a cepa VPS1 apenas um e com a ATCC-13950 somente dois. Concluiu-se que houve transmissão de MAI entre os suínos mantidos na mesma baia, com as três cepas utilizadas, mas foi mais evidente com a cepa SC1, indicando que o suíno pode ser importante fonte de infecção na disseminação da doença.

Imunidade celular e humoral na hanseníase bordeline tuberculoid (BT) / Humoral and cellular immunity in bordeline tuberculoid (BT) leprosy

Foram estudados 20 pacientes com diagnóstico de hanseníase bordeline tuberculoid (BT), classificados segundo os critérios de Ridley & Jopling, bem como dois pacientes com forma Tuberculoid tuberculoid (TT), todos com baciloscopia negativa, exceto um, que apresentou índice baciloscópico 1+. Todos os pacientes foram avaliados quanto a sua capacidade de resposta imune humoral ao DBSA (antígeno sintético semelhante ao glicolipídeo fenólico I, específico do M, leprae) e 18 pacientes foram submetidos a testes de avaliaçåo da resposta imunocelular in vivo (teste Mitsuda) e in vitro (linfoproliferaçåo e produçåo de interferon-gama) frente ao Mycobacterium leprae. Observamos que 90 por cento dos pacientess apresentaram resultados negativos quanto à pesquisa de IgM anti-DBSA pelo método imunoenzimático ELISA (densidade óptica , 0,27), o que demonstra ser este teste inadequado para a detecçåo de pacientes paucibacilares. Quanto aos testes de imunidade celular, oito pacientes (44,4 por cento) apresentaram teste de Mitsuda positivo (>= 5mm), sendo os demais considerados negativos. Cerca de 89 por cento dos pacientes tiveram teste de Mitsuda maior ou igual a 3mm. Doze pacientes (66,7 por cento) tiveram resposta linfoproliferativa positiva (índice estimulatório >= 3,0) para o M. leprae. Vinte e dois por cento dos pacientes apresentaram níveis de interferon-gama acima do limite de positividade (40 U/ml). Houve 66,7 por cento de correlaçåo entre os testes de Mitsuda e interferon-gama; 55,6 por cento de correlaçåo entre os testes in vitro (linfoproliferaçåo e interferon-gama). Quando estes três testes foram considerados em conjunto, uma correlaçåo de 38,9 por cento foi observada. Este estudo demonstra a heterogeneidade do comportamento imunológico mediado por células e anticorpos em pacientes com hanseníase BT, apesar de todos histologicamente serem capazes de conter a multiplicaçåo bacilar e de formar granulomas epitelióides

Relationship mbetween immune response and clinical manifestations in patients with tuberculosis

Cutaneous reactivityy to purified protein derivative (PPD) and antibody levels were investigated in 122 adults and 28 children with tuberculosis. IgG anti-PPD levelks (measured by ELISA and reported as absorbance at 405 nm) were higher in adult patients with the disease for more than one year (0.533 ñ 0.391, N=38 vs 0.224 ñ 0.256, n+50 in patients with the disease for less than one year) as well as inn children with disseminated tuberculosis (0.138 ñ 0.137, N=11, vs 0.072 ñ 0.55, N=15 in children with localized disease). The cut-off values (mean + 2SD) for healthy children and adults were 0.09 and 0.22 absorbance at 405 nm. In both adult and pediatric patients, cutaneous reactivity to PPD was inversely correlated with antibody levels. The present data provide additional evidence for the existence of an unstable spectrum of immune response in tuberculosis patients in whom changes in clinical situation are dynamic

Use of the anti-PGL-I antibody ELISA and the Mitsuda reaction in early diagnosis of leprosy

Cutaneous reaction to the Mitsuda antigen and anti-PGL-I IgM antibodies (ELISA) were determined in 134 leprosy patients, 290 household contacts, 52 healthy controls and 43 tuberculous individuals. The multibacillary patients did nort developed cell-mediated immunity (CMI), although they presented high levels of IgM (absorbance at 492 m > 0.5). The paucibacillatry patients presented CMI, although in varying degrees, and IgM levels did not exceed 0.5 absorbance units. Most of the contacts (107) showed a Mitsuda-postive test, and 25 of then were anti-PGL-I IgM seropositive (absorbance < 0.5 but > 0.22), although none became ill during the twoyear follow-up. Of the 17 Mitsuda-negative contacts, two wxhibited an immunological status of lepromatous leprosy (negative Mitsuda test and positive serology; absorvance > 0.05) and became ill (one borderline lepromatous and one indeterminate leprosy). These results show that the immunological status of lepromatous leprosy can appear prior to clinical symptoms, and thus serology for anti-PGL-I IgM together with the Mitsuda test can be useful in an activbe search for new preclinical cases among high risk populations

The dot-ELISA test for the detection of anti-PGL-1 IgM in leprosy patients and their contacts

Since dot-ELISA has recently been reported to be a sensitive, simple and method, we have compared it with the conventional microplate ELISA method. Sera of 124 leprosy patients, 136 household and professional contacts, and 92 controls were tested for a antibodies against a Mycobacterium leprae antigen using dot-ELISA on nitrocellulose membrane filters and microplate ELISA. The sensitive of the techniques was similar for multibacillary patients, but dot-ELISA was less sensitive for paucibacillary patients although it was more specific (100%) than ELISA (93,4%). Of 21 household contacts that gave a response by ELISA, 3 were also positive by dot-ELISA; one of these 3 developed indeterminate leprosy 12 months later and the other was diagnosed as borderline lepromatous after 28 months. These data indicate that dot-ELISA has a high spedificity and can be a useful tool in field evaluation

IgM immunoglobulins reacting with the phenolic glycolipid-1 antigen from Mycobacterium leprae in sera of leprosy patients and their contacts

For the first time in Brazil it was investigated the occurrence of IgM anti-PGL-1 in the sera of household contacts of leprozy patients using the ELISA methodology. The sera of the multipatients. It was observed a high subclinical infection incidence among household contacts (19.4%). The percentage of leprosy development was 5% (1/21) among the seropositive contact group. This finding suggests that serology could be useful as prognostic test, but for better definition is necessary to tet a population from endemic area for long period time