RESUMO
Current international tuberculosis (TB) guidelines recommend the microscopic examination of three sputum specimens for acid-fast bacilli in the evaluation of persons suspected of having pulmonary TB. We conducted a systematic review of studies that quantified the diagnostic yield of each of three sputum specimens. By searching multiple databases and sources, we identified a total of 37 eligible studies. The incremental yield in smear-positive results (in studies using all smear-positive cases as the denominator) and the increase in sensitivity (in studies that used all culture-positive cases as the denominator) of the third specimen were the main outcomes of interest. Although heterogeneity in study methods and results presented challenges for data synthesis, subgroup analyses suggest that the average incremental yield and/or the increase in sensitivity of examining a third specimen ranged between 2% and 5%. Reducing the recommended number of specimens examined from three to two (particularly to two specimens collected on the same day) could benefit TB control programs, and potentially increase case detection for several reasons. A number of operational research issues need to be addressed. Studies examining the most effective and efficient means to utilize current technologies for microscopic examination of sputum would be most useful if they followed an internationally coordinated and standardized approach, both to strengthen the country-specific evidence base and to permit comparison among studies.
Assuntos
Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Humanos , Sensibilidade e Especificidade , Manejo de EspécimesRESUMO
The World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (The Union) recommend direct sputum smear microscopy for tuberculosis (TB) case finding in resource-poor settings. This method is associated with poor sensitivity. Digestion of sputum with bleach prior to smear preparation has been reported to increase sensitivity. Some workers, having reviewed the relevant literature, have called for the WHO and The Union to advocate for a shift to this methodology for TB case finding. This article highlights deficiencies in the scope and detail of available evidence, and cautions against the premature, and possibly counter-productive, adoption of so-called 'bleach microscopy'. Further well-guided research is required to answer policy-relevant gaps in our knowledge about this promising technology.
Assuntos
Ácido Hipocloroso , Oxidantes , Escarro/citologia , Tuberculose/diagnóstico , Tuberculose/prevenção & controle , Humanos , Microscopia/métodos , Organização Mundial da SaúdeRESUMO
OBJECTIVE: To assess the 2012 served available market for tuberculosis (TB) diagnostics in China in the sector served by the China Centre for Disease Control and Prevention (CDC) and the hospital sector in China, including both designated TB hospitals and general hospitals. DESIGN: Test volumes and unit costs were assessed for tuberculin skin tests, interferon-gamma release assays (IGRAs), smear microscopy, serology, cultures, speciation tests, nucleic-acid amplification tests (NAATs), drug susceptibility tests and adenosine-deaminase tests (ADA). Data were obtained from electronic databases (CDC sector) and through surveys (hospital sector), and were estimated for the two sectors and for the country as a whole. Test costs were estimated by staff at China CDC, and using published literature. RESULTS: In 2012, the China CDC and hospital sectors performed a total of 44 million TB diagnostic tests at an overall value of US$294 million. Tests used by the CDC sector were smear microscopy, solid and liquid culture and DST, while the hospital sector also used IGRAs, NAATs, ADA and serology. The hospital sector accounted for 76% of the overall test volume and 94% of the market value. CONCLUSION: China has a very large TB diagnostic market that encompasses a wide range of diagnostic tests, with the majority being performed in Chinese hospitals.
Assuntos
Testes Diagnósticos de Rotina/economia , Testes Diagnósticos de Rotina/métodos , Tuberculose/diagnóstico , Adenosina Desaminase/análise , China , Humanos , Testes de Liberação de Interferon-gama/economia , Testes de Liberação de Interferon-gama/métodos , Microscopia/economia , Microscopia/métodos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/métodos , Teste Tuberculínico/economia , Teste Tuberculínico/métodosRESUMO
BACKGROUND: India represents a significant potential market for new tests. We assessed India's market for tuberculosis (TB) diagnostics in 2013. METHODS: Test volumes and unit costs were assessed for tuberculin tests, interferon-gamma release assays, sputum smear microscopy, serology, culture, speciation testing, nucleic-acid amplification tests (i.e., in-house polymerase chain reaction, Xpert(®) MTB/RIF, line-probe assays) and drug susceptibility testing. Data from the public sector were collected from the Revised National TB Control Programme reports. Private sector data were collected through a survey of private laboratories and practitioners. Data were also collected from manufacturers. RESULTS: In 2013, India's public sector performed 19.2 million tests, with a market value of US$22.9 million. The private sector performed 13.6 million tests, with a market value of US$60.4 million when prices charged to the patient were applied. The overall market was US$70.8 million when unit costs from the ingredient approach were used for the 32.8 million TB tests performed in the entire country. Smear microscopy was the most common test performed, accounting for 25% of the overall market value. CONCLUSION: India's estimated market value for TB diagnostics in 2013 was US$70.8 million. These data should be of relevance to test developers, donors and implementers.
Assuntos
Teste Tuberculínico/economia , Tuberculose/diagnóstico , Tuberculose/economia , Humanos , Índia , Testes de Liberação de Interferon-gama/economia , Microscopia/economia , Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/economia , Reação em Cadeia da Polimerase/economia , Setor Privado/economia , Setor Público/economia , Sensibilidade e Especificidade , Escarro/microbiologiaRESUMO
Rapid and accurate diagnosis of symptomatic patients is a cornerstone of global tuberculosis control strategies. Remarkable progress has recently been made, upgrading the speed and quality of mycobacteriology diagnostic services in industrialized countries, but for most of the world where TB is a large public health burden those gains are still unrealized. Deficiencies in current case-finding tools in disease endemic countries have made it difficult to ensure access to good diagnostics at all health service levels, leaving many patients undiagnosed. Additionally, in well-established TB control programs where diagnostic access has been ensured, efforts to interrupt disease transmission have been hampered by the insensitivity and late detection of smear microscopy. Fortunately, technical progress in diagnostics is resulting in a number of improved tools, including some appropriate for low-income settings. Important work remains, however, before new diagnostic tools can be meaningfully integrated into national TB control programs of high-burden countries and before TB control strategies can take them into account. The design and quality of clinical trials evaluating new diagnostics must be improved, clinical and laboratory services that would allow rapid response to test results need to be enhanced, and basic and operational research to appraise the impact and cost-effectiveness of new diagnostic technologies must be carried out. This paper describes some of the recent advances in TB diagnostic technologies and puts them into perspective for global tuberculosis control.
Assuntos
Técnicas de Tipagem Bacteriana/tendências , Mycobacterium tuberculosis/classificação , Tuberculose/diagnóstico , Técnicas de Tipagem Bacteriana/métodos , Resistência Microbiana a Medicamentos/genética , Resistência a Múltiplos Medicamentos/genética , Doenças Endêmicas , Saúde Global , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Sensibilidade e Especificidade , Tuberculose Resistente a Múltiplos Medicamentos/diagnósticoRESUMO
Questions have recently been raised regarding the potency of purified protein derivative (PPD) RT-23, the tuberculin most widely used world-wide for skin testing. We performed simultaneous testing with PPD RT-23 and Tubersol to compare these reagents during a tuberculin survey of 202 adult health care workers at a large teaching hospital in 1997. Individuals were tested with 2 tuberculin units (TU) PPD RT-23 and 5 TU Tubersol using the Mantoux method. Using a 10 mm cut-point for a positive PPD test, 39% of the health care workers were positive when tested with Tubersol and 35% were positive when tested with PPD RT-23. The median PPD size with Tubersol was 4 mm (interquartile range = 0-12 mm) compared to 2 mm (interquartile range = 0-12 mm) with PPD RT-23. PPD skin test reactivity with the two reagents was highly correlated (Spearman's rank coefficient, rho = 0.92; P = 0.01, two tailed). Concordance between the skin test reaction size with the two antigens did not differ based on age, sex or prior BCG vaccination. Mantoux skin test reactivity in health care workers in an area with a high prevalence of tuberculosis was comparable with PPD RT-23 and Tubersol.
Assuntos
Recursos Humanos em Hospital , Teste Tuberculínico/métodos , Tuberculina , Adolescente , Adulto , Brasil , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Pele/patologiaRESUMO
SETTING: Urban public teaching and referral hospital in Espirito Santo, Brazil. OBJECTIVE: To assess whether rates of infection and progression to active tuberculosis (TB) differed between household contacts of patients with multidrug-resistant (MDR) and drug susceptible (DS) pulmonary tuberculosis. DESIGN: Household contacts were assessed for evidence of TB infection and disease by purified protein derivative (PPD) skin testing, physical examination, chest X-ray, and sputum smear and culture. RESULTS: Among 133 close contacts of patients with MDR-TB, 44% were PPD-positive (> or =10 mm) compared to 37% of 231 contacts of the DS-TB cases (P = 0.18, chi2 test, OR 1.2, 95%CI 0.8-2). In a multivariate logistic regression analysis, after allowance for between-household variation in PPD responses, PPD positivity among household contacts of patients with MDR-TB remained comparable to PPD positivity in contacts of patients with DS-TB (OR 2.1, 95%CI 0.7-6.5). Respectively six (4%) and 11 (4%) contacts of the MDR- and DS-TB cases were found to have active TB at the time of initial evaluation or during follow-up (P = 0.78, chi2 test). Five of six contacts of MDR-TB cases and nine of nine contacts of DS-TB cases who developed TB, and for whom drug susceptibility test results were available, had the same bacterial susceptibility profiles as their index cases. DNA fingerprinting analysis of Mycobacterium tuberculosis isolates was identical between household contacts with active TB and the index MDR or DS-TB case for all 14 pairs compared. CONCLUSION: Our data suggest that the prevalence of tuberculous infection and progression to active TB among household contacts exposed to DS and MDR-TB cases is comparable, despite a longer duration of exposure of contacts to the index case in patients with MDR-TB.
Assuntos
Transmissão de Doença Infecciosa/estatística & dados numéricos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/transmissão , Adolescente , Adulto , Distribuição por Idade , Brasil/epidemiologia , Estudos de Coortes , Intervalos de Confiança , Progressão da Doença , Características da Família , Feminino , Hospitais de Ensino , Humanos , Controle de Infecções , Masculino , Radiografia Pulmonar de Massa , Pessoa de Meia-Idade , Razão de Chances , Exame Físico , Prevalência , Fatores de Risco , Distribuição por Sexo , Escarro/microbiologia , Teste Tuberculínico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/transmissão , População UrbanaRESUMO
BACKGROUND: The Special Programme for Research and Training in Tropical Diseases established a specimen bank in 1999 to support the development and evaluation of new tuberculosis (TB) diagnostic tools. OBJECTIVE: To provide a narrative of the bank's development and discuss lessons learned, the bank's limitations and potential future applications. RESULTS: Collection sites were selected in high- and low-prevalence settings. Patients with TB symptoms, consenting to participate and to undergo human immunodeficiency virus testing were enrolled and diagnosed. Serum, sputum, saliva and urine samples were collected and sent to the bank's repositories. The bank has stocked 41,437 samples from 2524 patients at 11 sites worldwide. Ninety-five requests for specimens have been reviewed and 67 sets have been approved. Approved applicants have received sets of 20 or 200 samples. The bank allowed an evaluation of 19 commercial lateral flow tests and showed that none of them had broad global utility for TB diagnosis. CONCLUSIONS: The establishment and development of the specimen bank have provided a wealth of experience. It is fulfilling a need to provide quality specimens, but the type and number of samples may not fulfil the demands of future end-users. Plans are underway to review the mechanisms of specimen collection and distribution to maximise their impact on product development.
Assuntos
Bancos de Espécimes Biológicos/organização & administração , Manejo de Espécimes/métodos , Tuberculose/diagnóstico , Humanos , Escarro/microbiologia , Medicina Tropical/métodos , Tuberculose/epidemiologiaRESUMO
We evaluated the diagnostic performance of the Diagnos TB AG immunoassay in 171 Tanzanians with suspected pulmonary tuberculosis (TB). The sensitivity and specificity, and positive and negative predictive values of the rapid test for the detection of pulmonary TB in this population were respectively 60.0%, 33.3%, 40.3% and 52.6%. In its current configuration, this test will not help overcome difficulties in the rapid diagnosis of TB.
Assuntos
Antígenos de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/diagnóstico , Adulto , Feminino , Humanos , Imunoensaio/métodos , Masculino , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Tanzânia , Fatores de Tempo , Tuberculose Pulmonar/imunologiaRESUMO
Latex agglutination (LA) of capsular polysaccharide bacterial antigen is a frequently performed laboratory procedure, but its use is controversial. To assess the clinical utility of this test, we reviewed all LA tests performed over a 10-month period at two sites, a major university-based referral center and a private specialty pediatric hospital. Samples were assayed either individually or as a panel for the group B streptococcus, Streptococcus pneumoniae, Haemophilus influenzae, and three sets of Neisseria meningitidis serogroups (A and Y, C and W135, and B and Escherichia coli K1). Of 5,169 assays performed on 1,268 clinical samples (786 urine and 478 cerebrospinal fluid, 3 pleural fluid, and 1 synovial fluid sample), 57 (1.1%) were positive, including 1.7% of urine and 0.3% of cerebrospinal fluid samples. All LA true-positive cerebrospinal fluid samples showed the causative microorganisms by Gram stain. Detailed chart review of these 57 positive samples showed that the LA result was false-positive in 31 (54%), true-positive in 22 (38%), and indeterminate in 4 (7%) samples. Therapy was not altered on the basis of any of the true-positive LA results. The 31 false-positive results led to additional cost, prolonged hospitalization, and some clinical complications. Total patient charges were $175,000 ($7,954 per true-positive), with no detectable clinical benefit. Our retrospective study does not support the current use of LA for rapid antigen detection. What, if any, specific indications exist for this test remain to be elucidated.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico , Testes de Fixação do Látex , Adolescente , Adulto , Infecções Bacterianas/microbiologia , Criança , Pré-Escolar , Reações Cruzadas , Estudos de Avaliação como Assunto , Reações Falso-Positivas , Infecções por Haemophilus/diagnóstico , Haemophilus influenzae/imunologia , Humanos , Lactente , Recém-Nascido , Testes de Fixação do Látex/estatística & dados numéricos , Infecções Meningocócicas/diagnóstico , Neisseria meningitidis/imunologia , Infecções Pneumocócicas/diagnóstico , Estudos Retrospectivos , Infecções Estreptocócicas/diagnóstico , Streptococcus/imunologia , Streptococcus pneumoniae/imunologiaRESUMO
A mouse model of respiratory syncytial virus (RSV) infection is described. A high-titered, large-volume inoculum results in replication of RSV to a high titer in lungs of BALB/c mice. Mice older than 15 weeks of age are more susceptible to RSV infection. Titers up to 10(6.9) plaque-forming units (pfu)/gram lung can be attained in 32-week-old mice. Older mice experience a clinical illness manifested by ruffled fur, reduced activity, and weight loss. Lung histology of older mice infected with RSV shows bronchiolitis and increased number of lymphocytes and macrophages in alveolar spaces compared with that of mice less than 8 weeks old. This model will serve as the basis for investigating immunodeterminants of recovery and protection from RSV infection.
Assuntos
Pulmão/microbiologia , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Infecções por Respirovirus/microbiologia , Fatores Etários , Animais , Anticorpos Antivirais/biossíntese , Modelos Animais de Doenças , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Cavidade Nasal/microbiologia , Testes de Neutralização , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/fisiologia , Infecções por Respirovirus/patologia , Ensaio de Placa Viral , Replicação ViralRESUMO
Reverse transcriptase PCR (RT-PCR) is an important tool for Mycobacterium tuberculosis research and diagnostics. A standard procedure using N-acetyl-L-cysteine (NALC) and NaOH has been widely adopted for digestion and decontamination of sputum specimens for mycobacterial culture. The objective of this study was to determine the compatibility of this method with the recovery of RNA for RT-PCR assays. Nineteen sputum specimens were collected from smear-positive, pretreatment tuberculosis patients. After homogenization with NALC and glass beads, specimens were further processed by the addition of either NaOH, as per the standard decontamination protocol, or phosphate buffer. RNA was prepared by using a modified guanidine-phenol extraction method developed specifically for sputum sediments. DNA was isolated from the same specimens. Reverse transcriptions of alpha antigen (85B protein) mRNA and 16S rRNA were performed together, and aliquots were removed for separate PCRs. In all specimens, the 85B mRNA target was greatly diminished by treatment with NaOH; however, the 16S rRNA target remained unaffected. Storing sputum specimens for 48 h at 4 degrees C before processing did not seem to affect the integrity or yield of RNA; however, some degradation occurred by 72 h. Data suggest that the NaOH-NALC method for processing sputum samples is not suitable for detecting mRNA targets in RT-PCR assays.
Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA Bacteriano/análise , RNA Mensageiro/análise , Escarro/microbiologia , Álcalis , DNA Bacteriano/análise , DNA Bacteriano/genética , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Sensibilidade e EspecificidadeRESUMO
We report a case of Mycobacterium xenopi infection of the spine in a 70-year-old woman. The findings of our case and of five other published reports of bone or joint infection with M. xenopi illustrate the problems with diagnosis and management of infection with this organism. Detection of M. xenopi in clinical specimens may require prolonged incubation at 37 degrees C or incubation at higher temperatures. Furthermore, M. xenopi may be misidentified as Mycobacterium avium-Mycobacterium intracellulare complex. For these reasons, human infection due to M. xenopi may be underrecognized. The optimal therapy for human infection with M. xenopi is unknown.
Assuntos
Infecções por Mycobacterium não Tuberculosas/complicações , Tuberculose da Coluna Vertebral/microbiologia , Idoso , Antibacterianos , Antituberculosos/uso terapêutico , Quimioterapia Combinada/uso terapêutico , Feminino , Humanos , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/isolamento & purificação , Radiografia , Vértebras Torácicas/diagnóstico por imagem , Vértebras Torácicas/microbiologia , Tuberculose da Coluna Vertebral/diagnóstico por imagem , Tuberculose da Coluna Vertebral/tratamento farmacológicoRESUMO
Capillary electrophoresis (CE) is a separation technique particularly suited to the analysis of pharmaceutical compounds. This review offers a detailed discussion of the four common modes of detection coupled to CE-UV absorption, fluorescence, electrochemical, and mass spectrometry-and gives examples of the use of these methods in pharmaceutical analyses. Sample preparation and pretreatment techniques used for CE separations are described, as well as methods of preconcentration including hydrophobic retention, affinity concentration, sample stacking, and isotachophoresis. The use of affinity CE, chiral CE, and capillary gel electrophoresis for analysis of pharmaceuticals is covered in detail, and recent advances in capillary electrochromatography and CE on a chip are also discussed.
Assuntos
Eletroforese Capilar , Preparações Farmacêuticas/análise , Preparações Farmacêuticas/isolamento & purificação , Análise EspectralRESUMO
The attenuated bacille Calmette-Guérin (BCG) vaccine is administered to prevent tuberculosis. Complications of vaccination are uncommon. We report a new case of disseminated BCG disease and review 27 additional cases identified from a review of > 5,000 reports published between 1980 and 1996. Twenty-four of the 28 total cases were associated with an immune deficiency, including nine cases of AIDS. Seventy-one percent of the cases occurred in children younger than 2 years old. Sixty-eight percent of the patients were male. About one-half of the patients were vaccinated in a developed nation, but 85% of the cases were reported from a developed nation. Response to therapy was poor, with an overall mortality rate of 71%. We made two new observations. Disseminated BCG disease has historically been a disease of infants, but cases now occur in adults and older children coinfected with human immunodeficiency virus. Cases also occur after revaccination of individuals who were anergic following the initial administration of BCG vaccine. Disseminated BCG disease is an uncommon but devastating complication of vaccination that should be considered in the appropriate clinical setting. Immunocompromised infants and patients with late-stage AIDS are at greatest risk and respond poorly to standard therapies.
Assuntos
Vacina BCG/efeitos adversos , Vacinação/efeitos adversos , Feminino , Infecções por HIV/complicações , Humanos , LactenteRESUMO
Our objectives were to determine how the prevalences of caries in elementary school children vary between geochemically defined regions of the state of Missouri and to compare this variation with that found for prehistoric Missouri inhabitants (Hildebolt et al.: Am. J. Phys. Anthropol. 75:1-14, 1988). Caries data on 6,584 school children were used in the study of second and sixth graders drinking optimally and suboptimally fluoridated water. Geochemical regions were based on maps recently published by the United States Geological Survey. Differences in mean caries scores and proportions of children with caries were tested by analysis of covariance, analysis of variance, Student t, and chi-squared tests. We found that caries prevalences do vary between the geochemical regions of the state. In the total sample, however, there were no significant differences between those children drinking optimally fluoridated water and those drinking suboptimally fluoridated water. We conclude that there is variation in caries rates among geochemically defined regions of the state and that geochemical factors associated with young parent materials may be antagonizing the action of fluoride.
Assuntos
Cárie Dentária/epidemiologia , Fluoretação , Geologia , Criança , Fenômenos Geológicos , Humanos , MissouriRESUMO
Mycobacterium tuberculosis can persist in sputum for long periods of time after the initiation of antituberculosis chemotherapy. The purpose of this study was to determine whether quantitative estimates of M. tuberculosis DNA in sputum correlate with the numbers of viable bacilli and thus measure the therapeutic response of patients during treatment. Two methods of M. tuberculosis DNA quantification were examined by using DNA isolated from sputum specimens serially collected during the course of chemotherapy. A competitive PCR assay was compared to an automated system of real-time quantification with the ABI Prism 7700 Sequence Detection System (TaqMan). The ABI 7700 system uses standard PCR in conjunction with a fluorogenic probe in which the intensity of fluorescence is proportional to the amount of target DNA present. The results showed that both PCR systems are reproducible and accurate. The amounts of M. tuberculosis DNA quantified in sputum corresponded well with the numbers of acid-fast bacilli (AFB) counted by microscopy. Before initiation of antituberculosis therapy, measures of AFB, M. tuberculosis DNA, and cultivable bacilli were similar, suggesting that quantification of DNA is a good method for measuring the initial bacillary load. However, the rate of disappearance of both AFB and M. tuberculosis DNA did not correlate with the decline in cultivable bacilli in the specimen; therefore, these tests are not appropriate for monitoring treatment efficacy.
Assuntos
Elementos de DNA Transponíveis , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Escarro/microbiologia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia , Antituberculosos/uso terapêutico , Técnicas Bacteriológicas , Sequência de Bases , Ligação Competitiva , Contagem de Colônia Microbiana , Primers do DNA , DNA Bacteriano/análise , Quimioterapia Combinada , Humanos , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Fatores de TempoRESUMO
Numerous assays have been described for the detection of DNA and rRNA sequences that are specific for the Mycobacterium tuberculosis complex. Although beneficial to initial diagnosis, such assays have proven unsuitable for monitoring therapeutic efficacy owing to the persistence of these nucleic acid targets long after conversion of smears and cultures to negative. However, prokaryotic mRNA has a typical half-life of only a few minutes and we have previously shown that the presence of mRNA is a good indicator of bacterial viability. The purpose of the present study was to develop a novel reverse transcriptase-strand displacement amplification system for the detection of M. tuberculosis alpha-antigen (85B protein) mRNA and to demonstrate the use of this assay in assessing chemotherapeutic efficacy in patients with pulmonary tuberculosis. The assay was applied to sequential, noninduced sputum specimens collected from four patients: 10 of 11 samples (91%) collected prior to the start of therapy were positive for alpha-antigen mRNA, compared with 1 of 8 (13%), 2 of 8 (25%), 2 of 8 (25%), and 0 of 8 collected on days 2, 4, 7, and 14 of treatment, respectively. In contrast, 39 of 44 samples (89%) collected on or before day 14 were positive for alpha-antigen DNA. The loss of detectable mRNA corresponded to a rapid drop over the first 4 days of treatment in the number of viable organisms present in each sputum sample, equivalent to a mean fall of 0.43 log10 CFU/ml/day. Analysis of mRNA is a potentially useful method for monitoring therapeutic efficacy and for rapid in vitro determination of drug susceptibility.
Assuntos
Aciltransferases , Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , RNA Mensageiro/genética , Escarro/microbiologia , Tuberculose/diagnóstico , Antígenos de Bactérias/genética , Antituberculosos/uso terapêutico , Proteínas de Bactérias/genética , Sequência de Bases , Primers do DNA , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Meia-Vida , Humanos , Mycobacterium tuberculosis/genética , RNA Mensageiro/isolamento & purificação , Fatores de Tempo , Tuberculose/tratamento farmacológicoRESUMO
Effective treatment regimens for pulmonary tuberculosis are difficult to assess because of the slow growth rate of Mycobacterium tuberculosis in culture and its protracted clearance from sputum. A rapid method that reflects effective antimicrobial activity would markedly advance evaluation of treatment and promote the assessment of new antituberculosis drugs. Conventional methods measure the progressive reduction of numbers of acid-fast bacilli in the sputum smear and the clearance of organisms in sputum culture. In this study, we measured levels of M. tuberculosis 85B (alpha antigen) messenger RNA (mRNA), 16S ribosomal RNA (rRNA), and IS6110 DNA in patients' sputa to ascertain whether they could serve as potential surrogate markers of response to chemotherapy. Sputum specimens were sequentially collected for up to a year from 19 smear-positive pulmonary tuberculosis patients receiving an optimal drug treatment regimen. Nucleic acids were isolated from these specimens, and two M. tuberculosis molecular targets (mRNA, DNA) were quantified, using the ABI Prism 7700 Sequence Detection System. The Mycobacterium genus-specific 16S rRNA was quantified with a limiting dilution RT-PCR assay. Results show that levels of 85B mRNA declined after initiation of therapy, as did viable M. tuberculosis colony counts, with 90% of patients becoming negative for both markers after 2 mo of treatment. The rapid disappearance of M. tuberculosis mRNA from sputum suggests that it is a good indicator of microbial viability and a useful marker for rapid assessment of response to chemotherapy.