Haiana Charifker Schindler

Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients

OBJECTIVE To evaluate the performance of nested PCR (nPCR) in detecting the Mycobacterium tuberculosis complex in blood samples of patients suspected of having TB, in order to determine its potential for use as an auxiliary tool in the laboratory diagnosis of TB in children. METHODS Detection of the M. tuberculosis complex in blood samples using as a target the insertion sequence IS6110 of the genomic DNA of the bacillus. Blood samples of 120 patients were evaluated. All of the patients were under 15 years of age at the time of their treatment at public hospitals in the city of Recife, Brazil (between January of 2003 and August of 2005). Attending physicians at the hospitals diagnosed TB based on the criteria recommended by the American Thoracic Society. The nPCR amplified a 123-bp fragment with outer oligonucleotides (IS1/IS2) and, in the subsequent reaction, using inner oligonucleotides (IS3/IS4), generating an 81-bp amplicon. RESULTS Active or latent TB was found in 65 patients, TB was ruled out in 28 suspected cases, and 27 patients were TB-free (controls). The sensitivity of nPCR was 26.15% and was significantly higher for the extrapulmonary form of the disease (55.56%) than for the pulmonary form (18.18%). The specificity was 92.73%. CONCLUSIONS Despite the difficulties in diagnosing TB in children and the low number of cases evaluated in the present study, nPCR in blood samples proved to be a rapid and specific technique, albeit one with low sensitivity. In order to establish its true usefulness in the diagnosis of paucibacillary forms, especially extrapulmonary TB, further studies need to be carried out with a larger sample of children and analyzing biological specimens other than blood.

Susceptibility to mycobacterium tuberculosis infection in HIV-positive patients is associated with CARD8 genetic variant

Abstract:HLA and other genetic variants, playing an important role in innate and adaptive immunity, are known to influence tuberculosis (TB) development in HIV-1–positive (HIV+) patients. Because inflammasome genes contribute to HIV-1 susceptibility, we investigated the possible association between polymorphisms in inflammasome genes with HIV-1 and Mycobacterium tuberculosis coinfection (HIV+TB+) in a case/control cohort of Brazilian individuals. Nineteen single-nucleotide polymoprhims in 8 inflammasome genes (NLRP1, NLRP3, AIM2, CARD8, CASP1, IL1B, IL1R, and HSP90) were analyzed in HIV+TB+ Brazilian patients (from Recife, Pernambuco). CARD8 rs6509365 polymorphism was associated with HIV+TB+ (P = 5 × 10−5), suggesting a predisposing role of this variant in M. tuberculosis susceptibility in HIV+ subjects (odds ratio = 2.45). This effect is even strong when this allele is combined to CARD8 rs2043211 single-nucleotide polymoprhim. The results of this study support the novel association between CARD8 gene and HIV+TB+ coinfection, demonstrating that inflammasome genetics could influence HIV-1 infection and the development of opportunistic infection.

Regulamentações, conflitos e ética da pesquisa médica em países em desenvolvimento

O paciente e o sujeito que pode satisfazer as necessidades e interesses de um investigador medico em sua pesquisa. Esse conflito intrinseco torna-se mais evidente e assume particularidades quando se consideram os projetos de pesquisa envolvendo os ensaios clinicos em paises em desenvolvimento. Nesses paises, as populacoes-alvo tem pouco acesso a servicos de saude, pouca compreensao sobre os riscos do estudo e tambem menor capacidade de pleitear judicialmente no caso de prejuizo. Em geral, os debates sobre etica em pesquisa nos paises industrializados sao caracterizados pela abordagem da doenca na dimensao biomedica e pela visao neoliberal da economia e comercio. De fato, a maior parte das pesquisas biomedicas tem sido dirigida para beneficiar comunidades ja privilegiadas. Portanto, e necessario que se minimize o risco de exploracao dos individuos de paises em desenvolvimento. Assim, o presente artigo apresenta uma visao da protecao etica para pesquisas em seres humanos de paises em desenvolvimento.

MBL2 gene polymorphisms and susceptibility to tuberculosis in a northeastern Brazilian population

The innate immune system represents the first line of host defense against pathogens. Genetics factors regulating the immune responses play a role in the susceptibility to infectious diseases, such as tuberculosis (TB). We analyzed MBL2 promoter and exon 1 functional single nucleotide polymorphisms (SNPs) in a group of 155TB patients and 148 healthy controls in order to evaluate their influence on the onset of infection and TB development. There was no association between MBL2 -550 HL promoter polymorphisms and susceptibility to develop TB, but heterozygous -221 Y/X genotype was significantly more frequent in pulmonary TB patients than controls. Moreover, MBL2 exon 1 O allele, was significantly associated with susceptibility to TB development in general (p=0.023, OR=1.61, 95% CI 1.05-2.49) and pulmonary TB (p=0.0008, OR=2.16, 95% CI 1.35-3.46); C allele at codon 57, as well as A/C genotype, were significantly more frequent in TB patients than in controls. Our results indicate that MBL2 polymorphisms, especially at codon 57, could be considered as risk factors for TB development.

Evaluation of a nested-pcr for Mycobacterium tuberculosis detection in blood and urine samples

The polymerase chain reaction (PCR) and its variations, such as the nested-PCR, have been described as promising techniques for rapid diagnosis of tuberculosis (TB). With the aim of evaluating the usefulness of a nested-PCR method on samples of blood and urine of patients suspected of tuberculosis we analyzed 192 clinical samples, using as a molecular target the insertion element IS6110 specific of M. tuberculosis genome. Nested-PCR method showed higher sensitivity in patients with extrapulmonary tuberculosis (47.8% and 52% in blood and urine) when compared to patients with the pulmonary form of the disease (sensitivity of 29% and 26.9% in blood and urine), regardless of the type of biological sample used. The nested-PCR is a rapid technique that, even if not showing a good sensitivity, should be considered as a helpful tool especially in the extrapulmonary cases or in cases where confirmatory diagnosis is quite difficult to be achieved by routine methods. The performance of PCR-based techniques should be considered and tested in future works on other types of biological specimens besides sputum, like blood and urine, readily obtainable in most cases. The improving of M. tuberculosis nested-PCR detection in TB affected patients will give the possibility of an earlier detection of bacilli thus interrupting the transmission chain of the disease.

Evaluation of a IS6110-Taqman real-time PCR assay to detect Mycobacterium tuberculosis in sputum samples of patients with pulmonary TB.

Evaluate the IS6110‐Taqman system performance in sputum samples from patients with pulmonary tuberculosis from health services in north‐eastern Brazil as a diagnostic laboratory tool for pulmonary tuberculosis.

Immunological diagnosis of tuberculosis based on recombinant antigens ESAT-6 and CFP-10 in children from an endemic area in Northeast Brazil.

Diagnostic tests for tuberculosis (TB) using interferon gamma (IFN‐γ) responses produced by T lymphocytes after stimulation by early secretory antigen target 6 (ESAT‐6), culture filtrate protein 10 (CFP‐10) or purified protein derivate (PPD) were carried out using ELISA (enzyme‐linked immunosorbent assay) in whole blood culture supernatants from children with suspected TB disease (n = 21), latent TB infection (LTBI; n = 17) and negative controls (NC; n = 21) from Recife, Pernambuco, Brazil. The results were analysed using the ROC (receiver operating characteristic) curves and the areas under the curve (AUC) generated varied from 0.5 to 1.0 with higher values indicating increased discriminatory ability. Comparisons of AUCs were made using non‐parametric assumptions, and the differences were considered significant if P < 0.05. The ROC curve showed a statistical difference (P = 0.015) between the LTBI and NC groups with an AUC of 0.731, TB disease and NC (AUC = 0.780; P = 0.002) and a group with TB (latent infection + disease, n = 38) and NC (AUC = 0.758; P = 0.001) when the antigen used was ESAT‐6. No statistical difference was found between the groups when CFP‐10 or PPD was used. In conclusion, the ESAT‐6 test may be the most appropriate for diagnosis of childhood TB, both LTBI and TB disease, when associated with epidemiological and clinical data, especially in endemic areas such as Brazil.

Perfil epidemiológico da filariose linfática em crianças residentes em áreas endêmicas

OBJECTIVE Lymphatic filariasis still represents a major public health problem in the city of Recife. In spite of the fact that previous surveys had already shown high frequency of microfilaraemia in pediatric population, the prevalence of filarial disease and the microfilaraemic pattern of this group were unknown. This paper describes the clinical-epidemiological pattern of filariasis in children and adolescents living in two highly endemic areas of Recife. METHODS The parasitological survey was done through a census carried out between December 1990 and July 1991. Thick drop technique (45 micro l) was performed on a total of 1,464 children and adolescents between the ages of 5 and 14, of whom 967 were submitted to clinical examination. Positive cases had their blood recollected (60 micro l) to measure the microfilaraemic density. RESULTS The microfilaraemia prevalence was 6.4 %. In the age groups of 5 to 9 and 10 to 14 a microfilaraemia prevalence of respectively 4.6% and 8.3% was observed. The microfilaraemic density varied from 3 to 864 microfilariae per 60 microl of blood, there having been no statistically significant difference between the sexes and age groups (p<0.05). 6 cases (0.6 %) of acute filarial disease and 11 of chronic filarial disease (1.1%) were identified, hydrocele being the principal manifestation found. Lymphadenopathy was found in 22% of the children, statistical association with microfilaraemia being observed (p<0.001). CONCLUSIONS The results of the parasitological survey show the strong presence of children in the contingent of microfilaraemic individuals, indicating an early and intense exposure to filariasis in the population studied.

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

INTRODUCTION The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. METHODS The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) RESULTS: Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identification of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the final diagnosis from the attending physician. CONCLUSIONS Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifiable using conventional biochemical and phenotypic techniques.

Preliminary results in the immunodiagnosis of tuberculosis in children based on T cell responses to ESAT-6 and PPD antigens

The aim of this work was to study the difference in interferon gamma (IFN-gamma) production by T lymphocytes after early secretory antigen target 6 (ESAT-6) or purified protein derivate (PPD) stimulation in whole blood culture supernatants from children with suspected tuberculosis (TB) disease (n = 21), latent TB infection (n = 16) and negative controls (NC) (n = 22) from an endemic area in Brazil. The concentration of IFN-gamma (pg/ml) was measured by enzyme linked immunosorbent assay and the differences in the IFN-gamma levels for each group were compared and evaluated using an unpaired Students t-test; p values < 0.05 were considered significant. Measurement of IFN-gamma levels after ESAT-6 stimulation raised the possibility of early diagnosis in the latent TB group (p = 0.0030). Nevertheless, the same group showed similar responses to the NC group (p > 0.05) after PPD stimulation. The IFN-gamma assay using ESAT-6 as an antigenic stimulus has the potential to be used as a tool for the immunodiagnosis of early TB in children.