Accurate mapping of mutations of pyrazinamide-resistant Mycobacterium tuberculosis strains with a scanning-frame oligonucleotide microarray

The increasing emergence of drug-resistant Mycobacterium tuberculosis poses significant threat to the treatment of tuberculosis. Conventional susceptibility testing for the front-line tuberculosis drug pyrazinamide (PZA) is difficult, because of the requirement for acid pH for the drug to show activity. Resistance to PZA in M. tuberculosis is caused by mutations in the pncA gene, and detection of pncA mutations can be an indicator of PZA resistance. In this study, we examined the feasibility of a microarray-based approach exploiting short overlapping oligonucleotides (sliding-frame array) to rapidly detect pncA mutations (substitutions, deletions, and insertions) in multiple strains of PZA-resistant M. tuberculosis. The genetic mapping of these mutations is necessary to link the gene sequence to the protein function defined by mutant phenotype. Microarray analysis was performed in a blind manner using 57 isolates of M. tuberculosis for which the sequence of the pncA gene was previously determined. Our results showed that all mutations could be unambiguously detected, suggesting that microarray can be a routine and valuable tool for rapid identification of drug-resistant M. tuberculosis isolates. We expect that mutation mapping with a sliding-frame microarray will accelerate the molecular analysis of drug-resistant M. tuberculosis bacteria and the microorganism populations.     

Sarcoidosis during treatment of pulmonary tuberculosis: a rare case report and review of the literature

The coexistence of pulmonary tuberculosis and pulmonary sarcoidosis is rare. Further, the morphological features of pulmonary tuberculosis with comorbid pulmonary sarcoidosis are similar to those of tuberculosis alone. There are obvious clinical, histological, and radiological similarities between sarcoidosis and tuberculosis, which makes differential diagnosis very challenging, particularly in countries with a high burden of tuberculosis. Here, a rare case of computed tomography (CT) findings of sarcoidosis that developed during tuberculosis treatment is reported. The 46-year-old male patient had no significant symptoms and was undergoing treatment for Mycobacterium tuberculosis infection. Chest CT revealed enlargement of multiple lymph nodes, without cystic or necrotic changes, in the mediastinum and both hili, and post-infectious changes consistent with the sequelae of tuberculosis infection in the left upper lobe. Chest radiographic evidence was accompanied by compatible clinical features and noncaseating granulomas on biopsy. As the patient was clinically stable, corticosteroid treatment was not initiated. To date, the patient remains without specific symptoms and outpatient follow-ups continue. Although rare, sarcoidosis may occur during treatment of pulmonary tuberculosis, and requires attention for diagnosis and treatment. The present case draws a radiological picture of how tuberculosis evolved to sarcoidosis.     

一种新型结核分枝杆菌感染快速检测方法的应用

目的探讨酶联免疫斑点试验（ELISPOT）快速诊断结核病的价值。方法选取15例确诊为活动性肺结核的患者和100例入境人员的外周抗凝全血标本,采用ELISPOT检测试剂盒（TSPOT-TB）测定结核杆菌特异抗原刺激反应的效应T淋巴细胞数量。结果TSPOT-TB检测结核病的敏感性为93．3％,特异性为100．0％,阳性预测值为100．0％,阴性预测值为81．8％。采用该方法检测的结核潜伏感染率与病例来源呈密切相关,而与年龄和性别无相关性。结论TSPOT-TB的敏感性和特异性均较高,可用于结核感染的快速检测,但无法判断结核的活动性或潜伏感染。     

Pursuing a diagnosis in a Caribbean man

A case study of an HIV-infected Caribbean male with extrapulmonary tuberculosis details his diagnosis, treatment regimens, and follow-up. His presenting symptoms included epigastric pain and fever. Endoscopy and gastric biopsy showed gastritis and helicobacter infection, which were treated symptomatically, and TMP-SMX was given for possible salmonellosis. Serologic tests for common opportunistic infections were negative. After all other expected conditions were ruled out, concurrent symptoms were diagnosed as extrapulmonary tuberculosis, and multi-drug treatment was successfully conducted. The problem of interactions between protease inhibitors and anti-tuberculosis drugs in treating HIV and tuberculosis concurrently is discussed. Three options are addressed: (1) discontinue (or delay starting) the protease inhibitor until at least 6 months of a standard rifampin-containing tuberculosis regimen is completed; (2) discontinue (or delay starting) the protease inhibitor until 2 months of a standard rifampin-containing regimen are completed; and (3) use of rifabutin rather than rifampin.     

Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program

An estimated two billion persons are latently infected with Mycobacterium tuberculosis. The host factors that initiate and maintain this latent state and the mechanisms by which M. tuberculosis survives within latent lesions are compelling but unanswered questions. One such host factor may be nitric oxide (NO), a product of activated macrophages that exhibits antimycobacterial properties. Evidence for the possible significance of NO comes from murine models of tuberculosis showing progressive infection in animals unable to produce the inducible isoform of NO synthase and in animals treated with a NO synthase inhibitor. Here, we show that O2 and low, nontoxic concentrations of NO competitively modulate the expression of a 48-gene regulon, which is expressed in vivo and prepares bacilli for survival during long periods of in vitro dormancy. NO was found to reversibly inhibit aerobic respiration and growth. A heme-containing enzyme, possibly the terminal oxidase in the respiratory pathway, likely senses and integrates NO and O2 levels and signals the regulon. These data lead to a model postulating that, within granulomas, inhibition of respiration by NO production and O2 limitation constrains M. tuberculosis replication rates in persons with latent tuberculosis.     

Inactive pulmonary lesions: a potent risk factor of tuberculosis.

Case histories and previous chest X-ray films of 142 patients with bacteriologically confirmed active pulmonary tuberculosis from eastern Finland were re-examined. In 18% of the series the disease was a reactivation of previously diagnosed and treated tuberculosis. In 70% of the patients with first clinical attack of tuberculosis, previous RP-films revealed fibrotic lesions in the lungs. Only 23% of the whole series had no previous abnormality on chest X-ray examination or previous clinical tuberculosis. Fibrotic pulmonary lesions are therefore a significant risk factor in the epidemiology of pulmonary tuberculosis, which has been given too little attention.     

Increased incidence of the outbreak strain of Mycobacterium tuberculosis in the surrounding community after an outbreak in a jail

BACKGROUND: Between 1995 and 1997, a tuberculosis outbreak occurred in a large, urban jail. We investigated whether the outbreak strain of Mycobacterium tuberculosis (M. tuberculosis) was circulating in the surrounding community after that outbreak. METHODS: We performed a retrospective cohort study of people with tuberculosis in Shelby County, TN, from January 1998 through August 1999, with molecular fingerprinting of M. tuberculosis strains. RESULTS: From January 1998 through August 1999, 23% of cases in the community involved a strain of M. tuberculosis that was indistinguishable from the previous jail outbreak strain. Twelve people (63%) with that strain had no history of recent incarceration. CONCLUSION: Two years after a tuberculosis outbreak in the jail, the outbreak strain was more prevalent in the surrounding community than it was during the jail outbreak. Jails can be important reservoirs of tuberculosis, which may subsequently circulate outside the institution. If efforts to eliminate tuberculosis are to be successful, the disease must be controlled successfully in such high-risk populations.     

Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis

The inhibition of apoptosis of infected host cells is a well-known but poorly understood function of pathogenic mycobacteria. We show that inactivation of the secA2 gene in Mycobacterium tuberculosis, which encodes a component of a virulence-associated protein secretion system, enhanced the apoptosis of infected macrophages by diminishing secretion of mycobacterial superoxide dismutase. Deletion of secA2 markedly increased priming of antigen-specific CD8(+) T cells in vivo, and vaccination of mice and guinea pigs with a secA2 mutant significantly increased resistance to M. tuberculosis challenge compared with standard M. bovis bacille Calmette-Guérin vaccination. Our results define a mechanism for a key immune evasion strategy of M. tuberculosis and provide what we believe to be a novel approach for improving mycobacterial vaccines.     

Identifying genetic susceptibility factors for tuberculosis in Africans: a combined approach using a candidate gene study and a genome-wide screen

There is convincing evidence that host genes affect the outcome of infection in human tuberculosis. Two complementary strategies were used to identify the genes involved. A linkage-based genome-wide screen was carried out to locate the positions of genes exerting a major population-wide effect on tuberculosis susceptibility. A candidate-gene-based case-control study was used to examine the effects of genes that may exert a more moderate effect on risk of clinical tuberculosis. The genome screen was conducted in two stages. In the first stage 299 microsatellite markers, spanning all 23 chromosomes, were typed in 92 independent sib-pairs, and seven regions showed some evidence of co-segregation with the disease. These seven regions were examined in a second set of 81 sib-pairs, and markers on chromosomes 15q and Xq showed evidence of linkage to tuberculosis. An X chromosome susceptibility gene may contribute to the excess of males with tuberculosis observed in many populations. The candidate gene approach compared the frequency of polymorphisms in several genes in over 400 subjects with smear-positive pulmonary tuberculosis and 400 ethnically matched healthy controls. Polymorphisms in genes encoding natural-resistance-associated macrophage protein, vitamin D receptor and mannose-binding lectin were associated with tuberculosis. These results suggest that many genes may be involved in determining host susceptibility to tuberculosis, and highlight the importance of using several different study methods to locate them.     

淋巴结结核并发丘疹坏死性结核疹一例

丘疹坏死性结核疹(PNT)是身体对其他部位(皮肤)结核分枝杆菌感染的一种免疫、过敏反应,其早期极易与变应性血管炎混淆,可依据病理活组织检查(活检)鉴别,也可行PCR检测明确诊断。该文报道1例淋巴结结核、药物性肝损害合并PNT患者,以双足皮疹为首发表现,予脱敏、对症治疗效果欠佳,追问患者既往病史并行病理活检后临床确诊。调整抗结核药及对症治疗,门诊随访未见新发皮疹。该例提示,对疑有结核分枝杆菌感染并发皮疹的患者,应警惕并发各型皮肤结核及其鉴别诊断,对无法耐受常规治疗的患者应合理调整药物配伍方案。     

Effect of a publicly funded tuberculosis service on the diagnosis of tuberculosis at a teaching hospital

The speed and accuracy of diagnosis of tuberculosis on general medical teaching services were retrospectively compared to that on a designated publicly funded tuberculosis service and to recent discouraging reports in the literature. The diagnosis was confirmed in all patients on the designated service and in 78.8% of patients not on the designated service within one week. No patient was discharged undiagnosed. Acid-fast smears done by the hospital laboratory showed a sensitivity of 82.5% and a specificity of 98.4%. Radiologic reports indicated the presence of tuberculosis or cavities in 85% of chest x-ray films in patients with pulmonary disease. These results may indicate that a publicly funded tuberculosis service, by providing emphasis on tuberculosis, allows more rapid and accurate diagnosis of tuberculosis in all patients. The impact of such training on future health care delivery should be recognized when decisions regarding allocation of public funds are made.     

安全敏感的涂片查抗酸杆菌方法的评价

建立了一种安全，简便、敏感涂片查抗酸杆菌方法。痰标本加等体积１０％８４消毒液和０．５ｍｌ的三甲苯，室温下，充分混合１５４分种，痰液化后，室温静置１５分层。用接种环取液相与二甲苯层之间的魄粘末层物质涂片，进行染色或金胺Ｏ荧光染色。此法消毒效果可靠，敏感性较直接涂片法高。     

Comparison of tuberculin skin test and a specific T-cell-based test, T-Spot.TB, for the diagnosis of latent tuberculosis infection

There is substantial evidence that the detection of T cells specific for the proteins ESAT-6 and CFP-10 using the ex vivo enzyme-linked immunospot technique is a marked improvement on the existing tuberculin skin test (TST). This new technique, which detects gamma-interferon-producing T cells, is now available as the commercial assay, T-Spot.TB. We compared the T-Spot.TB test with the TST for the diagnosis of latent tuberculosis infection (LTBI) in different groups of subjects. Significant accordance (85.7%) between the tests was found in subjects with active lung tuberculosis and in tuberculosis clinic and laboratory personnel (63.6%) but accordance was lowest and not significant amongst house contacts of tuberculosis patients (53.6%). We conclude that, in countries where vaccination is routinely performed, the T-Spot.TB test is a useful diagnostic test for LTBI in high-risk groups when carried out either together with the TST and/or to confirm the TST result.     

Therapeutic effect of DNA vaccines combined with chemotherapy in a latent infection model after aerosol infection of mice with Mycobacterium tuberculosis

The prevention of Mycobacterium tuberculosis (M. tuberculosis) reactivation would greatly reduce the incidence of the disease, particularly among the elderly. Here, we evaluated the efficacy of DNA vaccine in combination with a conventional TB chemotherapy on the prevention of M. tuberculosis reactivation. Mice were treated with isoniazid and pyrazinamide for 3 months from 4 weeks after aerosol infection with M. tuberculosis H37Rv. During this period of chemotherapy, DNA immunization was performed three times monthly with an antigen 85A (Ag85A) DNA or an IL-12 mutant (IL-12N220L) DNA, which is known to lead to a reduction in the secretion of the p40 subunit, but not of a bioactive IL-12p70. The reactivation of M. tuberculosis was dramatically reduced in mice treated with either Ag85A DNA (P<0.01) or IL-12N220L DNA (P<0.05) in combination with chemotherapy, compared with control mice receiving only chemotherapy. Ag85A DNA vaccine showed higher IFN-gamma responses to Ag85A protein, but a lower response to culture filtrate than IL-12N220L DNA vaccine. In addition, Ag85A DNA vaccine prevented the reactivation of M. tuberculosis more efficiently than IL-12N220L DNA vaccine, indicating that Ag85A-specific IFN-gamma response might correlate with M. tuberculosis control. This study suggests that immunotherapy using Ag85A or IL-12N220L DNA vaccine combined with conventional chemotherapy might be effective clinically for the prevention of tuberculosis reactivation and may offer a more effective cure for humans than chemotherapy alone.     

Characteristic resistance mechanism of Mycobacterium tuberculosis to DC-159a, a new respiratory quinolone

A G88C mutation in GyrA is one of the key alterations by which Mycobacterium tuberculosis mutants acquire DC-159a resistance in vitro. A novel double mutation in GyrA, G88C D94H, conferred high DC-159a resistance. Different mutation patterns in GyrA were demonstrated for DC-159a-resistant mutants and quinolone-resistant multidrug-resistant (QR-MDR) M. tuberculosis isolates, with a mutation either at position 90 or 94 and double mutations at 90 and 91 or at 90 and 94. DC-159a might be promising for QR M. tuberculosis treatment.     

结核病免疫学实验室检测技术新进展

每年有数百万人罹患结核病,早期诊断和治疗可以避免大量患者死亡。当前结核病的诊断方法存在许多局限性。对于所有形式的结核病,即肺结核、肺外结核、结核病-艾滋病联合感染和潜伏结核感染等,仍然缺乏可靠和准确的快速诊断技术。随着基础免疫学的发展和多学科诊断技术的交融,具有简单、快速和低成本等优势的结核病免疫学实验室检测技术不断出现。该文主要回顾近几年国内外结核病免疫学实验室检测新技术,包括以结核感染T细胞斑点试验(T-SPOT.TB)为代表的体外免疫检测技术、重组蛋白体内皮试试验为代表的体内免疫检测技术以及采用多学科交融手段检测结核分枝杆菌特异性抗原(TBAg)的体液免疫检测技术等。     

Controversies in classification of peritoneal tuberculosis and a proposal for clinico-radiological classification

Introduction: Peritoneal tuberculosis is a common type of abdominal tuberculosis. The most commonly used classification divides peritoneal tuberculosis into wet-ascitic type, dry-plastic type, and fixed-fibrotic type.

Areas covered: We performed a systematic literature search on the definitions of existing classification of peritoneal tuberculosis. The literature search identified confusion in the classification of peritoneal tuberculosis. The classification system also fails to classify some patterns of peritoneal tuberculosis like an abdominal cocoon and a substantial overlap in various categories was found. The impact of the present classification on clinical management is unclear. Lack of prospective studies and the presence of heterogeneity in reporting add to the confusion.

Expert opinion: We suggest that a uniform system which better classifies peritoneal tuberculosis and helps in clinical management should be used in future studies. We propose a simple, clinico-radiological classification of peritoneal tuberculosis into two types: distension-dominant and/or pain-obstruction dominant based on the clinical presentation. This approach will be relevant to clinicians as patients with the pain-obstruction dominant presentation are more likely to receive surgical interventions and may benefit from additional measures aimed to reduce fibrosis-like addition of steroids to ATT. Future studies should aim to validate the proposed clinico-radiological classification in patients with peritoneal tuberculosis.     

慢性肾脏病合并结核感染诊治进展

慢性肾脏病（Chronic kidney disease ,CKD）和结核病是危及人类健康的慢性疾病,慢性肾脏病患者感染结核的风险增加,与CKD的进展有关,且肺外结核发生率高,临床症状不典型,结核相关筛查实验阳性率低,CKD患者抗结核药物副作用较普通人群高且严重,预后更差,早期诊断及及时治疗对改善预后有重要意义。     

Diagnosis of MDR-TB: a developing world problem on a developed world budget

The resurgence of tuberculosis worldwide has been accompanied by an increase in the incidence of multidrug-resistant tuberculosis on all continents. While significant advances have been made in the rapid and accurate diagnosis of Mycobacterium tuberculosis, the molecular biology methods used in the research laboratory to elucidate the mechanisms of drug resistance cannot be transferred to the centers delivering patient care. These methods require skilled operators, cumbersome protocols and extravagant expense. A number of companies that already have a large investment in M. tuberculosis diagnostics are adapting their high-throughput technology to drug susceptibility testing. These methodologies are not applicable to the developing world not only because of the costs involved but through a lack of infrastructure that is required to operate these machines and deliver specimens to the point of testing. Alternative technologies for drug susceptibility testing that do not rely on an investment in expensive hardware are presented and their potential use in the field is discussed. Though still relatively expensive to perform, these newer innovations may lead to the development of less intricate technologies that have universal application and begin to move away from our obsession with molecular-based diagnostics to produce on all encompassing gold standard.     

面颈部结核病及误诊病例的临床分析

目的：回顾分析面颈部结核病病例，以探讨面颈部结核病的临床特点及误诊的原因。方法：上海第九人民医院自1995年5月～2003年5月8年间经细胞学或组织学确诊的62例面颈部结核病病例进行临床总结和分析。结果；淋巴结结核55例，其中颈淋巴结结核39例，腮腺淋巴结结核17例，结外型结核6例，3例发生于颊黏膜和牙龈，3例病变位于下颌骨。23例发生误诊，误诊率为37．1％。结论：面颈部结核的发病率低，症状缺乏特异性，容易误诊。可疑结核病者，有必要进行穿刺检查或病理检查，以明确诊断。