A molecular epidemiological and genetic diversity study of tuberculosis in Ibadan, Nnewi and Abuja, Nigeria

BACKGROUND: Nigeria has the tenth highest burden of tuberculosis (TB) among the 22 TB high-burden countries in the world. This study describes the biodiversity and epidemiology of drug-susceptible and drug-resistant TB in Ibadan, Nnewi and Abuja, using 409 DNAs extracted from culture positive TB isolates. METHODOLOGY/PRINCIPAL FINDINGS: DNAs extracted from clinical isolates of Mycobacterium tuberculosis complex were studied by spoligotyping and 24 VNTR typing. The Cameroon clade (CAM) was predominant followed by the M. africanum (West African 1) and T (mainly T2) clades. By using a smooth definition of clusters, 32 likely epi-linked clusters related to the Cameroon genotype family and 15 likely epi-linked clusters related to other "modern" genotypes were detected. Eight clusters concerned M. africanum West African 1. The recent transmission rate of TB was 38%. This large study shows that the recent transmission of TB in Nigeria is high, without major regional differences, with MDR-TB clusters. Improvement in the TB control programme is imperative to address the TB control problem in Nigeria.     

Comparison of the mycobacteria growth indicator tube with radiometric and solid culture for isolation of mycobacteria from clinical specimens and susceptibility testing of Mycobacterium tuberculosis

We compared the mycobacteria growth indicator tube (MGIT) system with the BACTEC 460 TB and Loewenstein-Jensen (LJ) systems for the recovery of mycobacteria (acid-fast bacilli [AFB]) from 600 clinical specimens. A total of 50 AFB (32 Mycobacterium tuberculosis complex, 10 M. avium complex, 3 M. gordonae, 3 M. xenopi, 1 M. terrae and 1 M. fortuitum) were detected. MGIT recovered 50 isolates of AFB (100% sensitivity), and BACTEC 460 TB and LJ recovered 49 (98% sensitivity) and 19 (38% sensitivity) AFB isolates, respectively. The mean times to detect mycobacteria were 10, 10 and 25 days for MGIT, BACTEC 460, and LJ slants. All isolates of M. tuberculosis complex were tested for susceptibility to streptomycin, isoniazid, rifampin, and ethambutol with the MGIT and BACTEC 460 TB. Both systems yielded identical susceptibility data with different mean times to report (5.38 days for MGIT versus 7.33 days for BACTEC 460 TB, P<0.05). The results suggest that MGIT is equivalent to BACTEC 460 TB in its ability to support the growth of mycobacteria, but significantly more efficient than LJ. MGIT may also be used for susceptibility testing of primary antituberculosis drugs.     

A two-step strategy for molecular typing of multidrug-resistant Mycobacterium tuberculosis clinical isolates from Poland

Tuberculosis (TB) continues to be one of the most challenging public health problems in the world. An important contributor to the global burden of the disease is the emergence and spread of drug-resistant and particularly multidrug-resistant Mycobacterium tuberculosis strains (MDR), defined as being resistant to at least isoniazid and rifampicin. In recent years, the introduction of different DNA-based molecular typing methods has substantially improved the knowledge of the epidemiology of TB. The purpose of this study was to employ a combination of two PCR-based genotyping methods, namely spoligotyping and IS6110-Mtb1/Mtb2 PCR to investigate the clonal relatedness of MDR M. tuberculosis clinical isolates recovered from pulmonary TB patients from Poland. Among the 50 isolates examined, 28 (56%) were clustered by spoligotyping, whereas IS6110-Mtb1/Mtb2 PCR resulted in 16 (32%) clustered isolates. The isolates that clustered in both typing methods were assumed to be clonally related. A two-step strategy consisting of spoligotyping as a first-line test, performed on the entire pool of isolates, and IS6110-Mtb1/Mtb2 PCR typing as a confirmatory subtyping method, performed only within spoligotype-defined clusters, is an efficient approach for determining clonal relatedness among M. tuberculosis clinical isolates.     

Direct rapid diagnosis of rifampicin-resistant M. tuberculosis infection in clinical samples by line probe assay (INNO LiPA Rif-TB)

M. tuberculosis is one of the leading causes of death worldwide and Multi Drug Resistant Tuberculosis (MDR-TB) is associated with a high case-fatality rate. Rapid identification of resistant strains is crucial to institute prompt appropriate therapy, and prevent the development of further resistance and spreading of MDR strains. The INNO-LiPA Rif. TB is a commercial reverse hybridisation line probe assay designed for rapid detection of rpoB gene mutations in clinical isolates. We applied this test directly to 44 smear-positive and 45 smear-negative clinical specimens collected from patients suspected of active TB. The capability of this technique to correctly identify local MDR-TB strains was tested on 50 MDR strains isolated in Italy. Results of the test were compared to conventional antibiogram performed on isolated strains. The concordance rate of the LiPA test results on clinical specimens with those obtained with "in vitro" sensitivity was 100%. These results show that the LiPA test can be useful in rapid detection and prompt management of tuberculosis when MDR disease is suspected.     

Xpert MTB/RIF在人类免疫缺陷病毒感染/艾滋病患者中诊断结核病的价值

为探讨利福平耐药结核分枝杆菌实时荧光定量核酸扩增检测技术(Xpert Mycobacterium tuberculosis/rifampicin,Xpert MTB/RIF)在人类免疫缺陷病毒感染/艾滋病(human immunodeficiency virus infection/acquired immunodeficiency syndrome,HIV/AIDS)患者中诊断结核病的价值,本研究回顾性分析了2018年1月1日—2020年12月31日复旦大学附属公共卫生临床中心感染与免疫科收治的801例HIV/AIDS合并疑似结核病患者的临床资料。801例患者中,657例进行了Xpert MTB/RIF、外周血结核感染T细胞斑点试验(tuberculosis T cell spot test,T-SPOT.TB)、抗酸染色涂片镜检和BACTEC MGIT 960液体培养等检测。以液体培养及菌型鉴定结果作为结核病诊断的“金标准”,确诊结核病92例,Xpert MTB/RIF、T-SPOT.TB、抗酸染色涂片镜检在HIV/AIDS患者中诊断结核病(包括肺结核和肺外结核)的灵敏度分别为72.8%、55.4%和69.6%,特异度分别为96.8%、90.3%和84.4%,与“金标准”行一致性检验,Kappa值分别为0.719 (P<0.01)、0.430(P<0.01)和0.424(P<0.01)。Xpert MTB/RIF检测502份呼吸道样本,结果显示其诊断肺结核的灵敏度和特异度分别为66.7%和96.0%;在痰涂片阳性和阴性的患者中,Xpert MTB/RIF诊断肺结核的灵敏度分别为77.4%和35.2%,特异度分别为87.7%和 97.8%。采用Xpert MTB/RIF检测343份肺外标本,结果显示其诊断肺外结核的灵敏度和特异度分别为63.3%和95.2%。以上结果提示,Xpert MTB/RIF在HIV/AIDS患者中诊断结核病(包括肺结核和肺外结核)具有较高的灵敏度和特异度,诊断肺结核的灵敏度高于肺外结核,因此推荐将其作为HIV/AIDS患者疑似结核病的首选检测方法。     

Automatized PCR evaluation of Mycobacterium tuberculosis complex in respiratory and nonrespiratory specimens

In this study, Mycobacterium tuberculosis complex isolates recovered from respiratory and nonrespiratory specimens with culture were evaluated using an automatized PCR method. Specimens with suspected tuberculous disease were decontaminated and concentrated using the standard N-acetyl-L-cysteine NaOH method and were inoculated onto glycerol-supplemented L?wenstein-Jensen media and BACTEC B12 vials. Forty-one specimens with typical colonies on solid media and 127 specimens identified as M. tuberculosis complex in a BACTEC system were selected as the study group. As the control group, 46 specimens without growth on either culture media were selected. The PCR results were positive in 33 (80.5%) and 87 (68.5%) samples that were culture-positive on solid and liquid media, respectively. All (100%) culture-negative specimens within the control group were also negative in the COBAS AMPLICOR Mycobacterium tuberculosis (MTB) PCR method. In conclusion, although it is a fast method for identifying M. tuberculosis complex isolates from clinical specimens, the COBAS AMPLICOR MTB PCR method is found to be less sensitive than culture techniques, we propose therefore that it should only be used in combination with culture results in the clinical diagnosis of tuberculosis.     

Zoonotic transmission of tuberculosis between pastoralists and their livestock in South-East Ethiopia

Despite huge global efforts in tuberculosis (TB) control, pastoral areas remain under-investigated. During two years sputum and fine needle aspirate (FNA) specimens were collected from 260 Ethiopian pastoralists of Oromia and Somali Regional States with suspected pulmonary TB and from 32 cases with suspected TB lymphadenitis. In parallel, 207 suspected tuberculous lesions were collected from cattle, camels and goats at abattoirs. All specimens were processed and cultured for mycobacteria; samples with acid-fast stained bacilli (AFB) were further characterized by molecular methods including genus and deletion typing as well as spoligotyping. Non-tuberculous mycobacteria (NTM) were sequenced at the 16S rDNA locus. Culturing of AFB from human sputum and FNA samples gave a yield of 174 (67%) and 9 (28%) isolates, respectively. Molecular typing was performed on 173 of these isolates and 160 were confirmed as Mycobacterium tuberculosis, three as M. bovis, and the remaining 10 were typed as NTMs. Similarly, 48 AFB isolates (23%) yielded from tuberculous lesions of livestock, of which 39 were molecular typed, including 24 M. bovis and 4 NTMs from cattle, 1 M. tuberculosis and 1 NTM from camels and 9 NTMs from goats. Isolation of M. bovis from humans and M. tuberculosis from livestock suggests transmission between livestock and humans in the pastoral areas of South-East Ethiopia.     

Detection of mycobacteria in clinical specimen using the mycobacteria growth indicator tube (MGIT) and the Lowenstein Jensen medium.

The recovery rates of mycobacteria strains isolated from 1200 clinical specimens using the mycobacteria growth indicator tube (MGIT) system and the conventional Lowenstein Jensen medium (LJ) were assessed. Of the 87 mycobacterial isolates recovered, 54 belonged to the M. tuberculosis complex (MTB) and 33 to the non-tuberculosis complex (NTM). MGIT recovered 78 (89.65%) mycobacteria isolates (51 MTB (94.44%) and 27 NTM (81.81%) and LJ recovered 70 (80.46%) mycobacteria isolates (49 MTB (90.74%) and 21 NTM (63.63%). Sixty one (70.1%) of the total mycobacteria isolates were recovered with both systems (46 (85.2%) MTB and 15 (45.5%) NTM). No significant difference was found between MGIT and LJ (p > 0.05) in both MTB and NTM recoveries. The average detection time for MTB was significantly shorter with MGIT than with LJ, in both the smear-positive specimens (8 vs 30 days: p < 0.0001) and smear-negative specimens (15 vs 30 days: p < 0.001). The average detection time of NTM was also shorter for MGIT (15 vs 30 days: p < 0.0001). However, the contamination rate was higher in MGIT (8.5%) than in LJ (3%). The results suggest that the use of MGIT contributes to a more rapid and effective diagnosis of mycobacterial infections particularly when combined with the classical LJ.     

Molecular identification and characterization of rifampicin-resistant Mycobacterium tuberculosis isolates by line probe assay: an approach for rapid diagnosis of multidrug-resistant tuberculosis

Aim:  Early identification and characterization of rifampicin-resistant (R^r) Mycobacterium tuberculosis isolates recovered from the samples of tuberculosis (TB) patients in the Aegean (West Anatolian) Region was intended.
Methods and Results:  Sixty isolates [47 (78·3%) multidrug-resistant (MDR)], which were identified as M. tuberculosis complex and phenotypically resistant to rifampicin by both BACTEC mycobacteria growth indicator tube (MGIT) 960 and 460 systems were analysed by a commercial line probe assay (INNO-LiPA Rif TB). The concordance of LiPA with the in vitro susceptibility test was found as 98·3%. Among the isolates, S531L (R5 pattern; 46·7%) and L511P/R, S512T, Q513L/K (ΔS1 pattern; 11·7%) were the most frequent mutation patterns. As compared with the BACTEC systems and conventional techniques for cultivation, identification and in vitro susceptibility testing, INNO-LiPA Rif TB after cultivation in BACTEC MGIT 960 system provided an average of 20 days early diagnosis of R^r M. tuberculosis isolates.
Conclusions:  Rapid molecular identification and characterization of R^r M. tuberculosis isolates after BACTEC MGIT 960 cultivation would be useful for faster diagnosis, infection control and planning of accurate treatment in MDR-TB patients.
Significance and Impact of the Study:  Patients with MDR-TB need a specified treatment and efficient follow-up strategies. Rapid and practical methodologies to diagnose and follow these patients should be applied in routine use.     

Molecular Epidemiology and Clinical Characteristics of Drug-Resistant Mycobacterium tuberculosis in a Tuberculosis Referral Hospital in China

Background

Despite the large number of drug-resistant tuberculosis (TB) cases in China, few studies have comprehensively analyzed the drug resistance-associated gene mutations and genotypes in relation to the clinical characteristics of M. tuberculosis (Mtb) isolates.

Methodology/Principal Findings

We thus analyzed the phenotypic and genotypic drug resistance profiles of 115 Mtb clinical isolates recovered from a tuberculosis referral hospital in Beijing, China. We also performed genotyping by 28 loci MIRU-VNTR analysis. Socio-demographic and clinical data were retrieved from medical records and analyzed. In total, 78 types of mutations (including 42 previously reported and 36 newly identified ones) were identified in 115 Mtb clinical isolates. There was significant correlation between phenotypic and genotypic drug resistance rates for first-line anti-TB drugs (P<0.001). Genotyping revealed 101 MIRU-VNTR types, with 20 isolates (17.4%) being clustered and 95 isolates (82.6%) having unique genotypes. Higher proportion of re-treatment cases was observed among patients with clustered isolates than those with unique MIRU-VNTR genotypes (75.0% vs. 41.1%). Moreover, clinical epidemiological links were identified among patients infected by Mtb strains belonging to the same clusters, suggesting a potential of transmission among patients.

Conclusions/Significance

Our study provided information on novel potential drug resistance-associated mutations in Mtb. In addition, the genotyping data from our study suggested that enforcement of the implementation of genotyping in diagnostic routines would provide important information for better monitor and control of TB transmission.     

Genotypic,Phenotypic and Clinical Validation of GeneXpert in Extra-Pulmonary and Pulmonary Tuberculosis in India

Background

Newer molecular diagnostics have brought paradigm shift in early diagnosis of tuberculosis [TB]. WHO recommended use of GeneXpert MTB/RIF [Xpert] for Extra-pulmonary [EP] TB; critics have since questioned its efficiency.

Methods

The present study was designed to assess the performance of GeneXpert in 761 extra-pulmonary and 384 pulmonary specimens from patients clinically suspected of TB and compare with Phenotypic, Genotypic and Composite reference standards [CRS].

Results

Comparison of GeneXpert results to CRS, demonstrated sensitivity of 100% and 90.68%, specificity of 100% and 99.62% for pulmonary and extra-pulmonary samples. On comparison with culture, sensitivity for Rifampicin [Rif] resistance detection was 87.5% and 81.82% respectively, while specificity was 100% for both pulmonary and extra-pulmonary TB. On comparison to sequencing of rpoB gene [Rif resistance determining region, RRDR], sensitivity was respectively 93.33% and 90% while specificity was 100% in both pulmonary and extra-pulmonary TB. GeneXpert assay missed 533CCG mutation in one sputum and dual mutation [517 & 519] in one pus sample, detected by sequencing. Sequencing picked dual mutation [529, 530] in a sputum sample sensitive to Rif, demonstrating, not all RRDR mutations lead to resistance.

Conclusions

Current study reports observations in a patient care setting in a high burden region, from a large collection of pulmonary and extra-pulmonary samples and puts to rest questions regarding sensitivity, specificity, detection of infrequent mutations and mutations responsible for low-level Rif resistance by GeneXpert. Improvements in the assay could offer further improvement in sensitivity of detection in different patient samples; nevertheless it may be difficult to improve sensitivity of Rif resistance detection if only one gene is targeted. Assay specificity was high both for TB detection and Rif resistance detection. Despite a few misses, the assay offers major boost to early diagnosis of TB and MDR-TB, in difficult to diagnose pauci-bacillary TB.     

miRNA Signatures in Sera of Patients with Active Pulmonary Tuberculosis

Several studies showed that assessing levels of specific circulating microRNAs (miRNAs) is a non-invasive, rapid, and accurate method for diagnosing diseases or detecting alterations in physiological conditions. We aimed to identify a serum miRNA signature to be used for the diagnosis of tuberculosis (TB). To account for variations due to the genetic makeup, we enrolled adults from two study settings in Europe and Africa. The following categories of subjects were considered: healthy (H), active pulmonary TB (PTB), active pulmonary TB, HIV co-infected (PTB/HIV), latent TB infection (LTBI), other pulmonary infections (OPI), and active extra-pulmonary TB (EPTB). Sera from 10 subjects of the same category were pooled and, after total RNA extraction, screened for miRNA levels by TaqMan low-density arrays. After identification of “relevant miRNAs”, we refined the serum miRNA signature discriminating between H and PTB on individual subjects. Signatures were analyzed for their diagnostic performances using a multivariate logistic model and a Relevance Vector Machine (RVM) model. A leave-one-out-cross-validation (LOOCV) approach was adopted for assessing how both models could perform in practice. The analysis on pooled specimens identified selected miRNAs as discriminatory for the categories analyzed. On individual serum samples, we showed that 15 miRNAs serve as signature for H and PTB categories with a diagnostic accuracy of 82% (CI 70.2–90.0), and 77% (CI 64.2–85.9) in a RVM and a logistic classification model, respectively. Considering the different ethnicity, by selecting the specific signature for the European group (10 miRNAs) the diagnostic accuracy increased up to 83% (CI 68.1–92.1), and 81% (65.0–90.3), respectively. The African-specific signature (12 miRNAs) increased the diagnostic accuracy up to 95% (CI 76.4–99.1), and 100% (83.9–100.0), respectively. Serum miRNA signatures represent an interesting source of biomarkers for TB disease with the potential to discriminate between PTB and LTBI, but also among the other categories.     

IS6110 restriction fragment length polymorphism typing of Mycobacterium tuberculosis isolates from East Azerbaijan Province of Iran

To investigate the genetic variation among Mycobacterium tuberculosis isolates in the East Azerbaijan Province of Iran and to evaluate the level of and risk factors for recent transmission of tuberculosis (TB), we performed IS6110-based restriction fragment length polymorphism analysis of strains, isolated from 105 patients during the period of September 2002 to March 2003 in TB centers and university hospitals of the province. Among 105 isolates, 81 different IS6110 patterns were found, of which 70 were observed only once and 11 were shared by two to eight isolates. Ninety-six isolates (91.4%) were found to have more than five copies of IS6110 and together with high patterns polymorphism, shows that IS6110-RFLP typing could be useful for studying the epidemiology of TB in Azerbaijan. The minimum estimated rate of recent transmission was 23%, suggesting that the degree of recent transmission in East Azerbaijan Province is relatively low. Clustering was not associated with age, sex or site of infection of TB but drug-resistant isolates were less likely to be clustered than sensitive isolates (p < 0.05).     

IS6110 restriction fragment length polymorphism of Mycobacterium tuberculosis isolated from patients with pulmonary tuberculosis in Campinas,Brazil: evidence of intercontinental distribution of strains

Tuberculosis (TB) is a major concern in developing countries. In Brazil, few genotyping studies have been conducted to verify the number of IS6110 copies present in local prevalent strains of Mycobacterium tuberculosis, the distribution and clustering of strains. IS6110 DNA fingerprinting was performed on a sample of M. tuberculosis isolates from patients with AFB smear-positive pulmonary TB, at a hospital in Brazil. The IS6110 profiles were analyzed and compared to a M. tuberculosis database of the Houston Tuberculosis Initiative, Houston, US. Seventy-six fingerprints were obtained from 98 patients. All M. tuberculosis strains had an IS6110 copy number between 5-21 allowing for differentiation of the isolates. Human immunodeficiency virus infection was confirmed in nearly half the patients of whom data was available. Fifty-eight strains had unique patterns, while 17 strains were grouped in 7 clusters (2 to 6 strains). When compared to the HTI database, 6 strains matched isolates from El Paso, Ciudad de Juarez, Houston, and New York. Recently acquired infections were documented in 19% of cases. The community transmission of infection is intense, since some clustered strains were recovered during the four-year study period. The intercontinental dissemination of M. tuberculosis strains is suspected by demonstration of identical fingerprints in a distant country.     

Cytokine Kinetics in the First Week of Tuberculosis Therapy as a Tool to Confirm a Clinical Diagnosis and Guide Therapy

Background

Many patients treated for tuberculosis (TB) in low and middle income countries are treated based on clinical suspicion without bacteriological confirmation. This is often due to lack of rapid simple accurate diagnostics and low healthcare provider confidence in the predictive value of current tests. We previously reported in an animal TB model that levels of host markers rapidly change in response to treatment initiation.

Methods

We assessed the potential of host biomarker kinetics of TB patients during the first two weeks of therapy to identify patients responding to treatment. Adult patients clinically diagnosed with and treated for TB, 29 in Nigeria and 24 in Nepal, were analyzed.

Results

Changes in concentrations of non-specific host biomarkers, particularly IP-10, in response to the first week of anti-TB therapy were strongly associated with bacteriological confirmation of TB. A decrease in IP-10 level of >300pg/ml between 0 and 7 days of treatment identified 75% of both smear-positive and smear-negative culture positive patients and correctly excluded TB in all nine culture negative patients.

Conclusions

Monitoring of early IP-10 responses to treatment could form the basis of a simplified assay and could help identify patients who were erroneously clinically diagnosed with TB or those infected with drug resistant strains on inappropriate treatment. We believe this approach may be particularly appropriate for difficult to diagnose patients, e.g. smear-negative HIV-positive, or those with extra-pulmonary TB, often treated without bacterial confirmation.     

Polymorphisms of SP110 Are Associated with both Pulmonary and Extra-Pulmonary Tuberculosis among the Vietnamese

Background

Tuberculosis (TB) is an infectious disease that remains a major cause of morbidity and mortality worldwide, yet the reasons why only 10% of people infected with Mycobacterium tuberculosis go on to develop clinical disease are poorly understood. Genetically determined variation in the host immune response is one factor influencing the response to M. tuberculosis. SP110 is an interferon-responsive nuclear body protein with critical roles in cell cycling, apoptosis and immunity to infection. However association studies of the gene with clinical TB in different populations have produced conflicting results.

Methods

To examine the importance of the SP110 gene in immunity to TB in the Vietnamese we conducted a case-control genetic association study of 24 SP110 variants, in 663 patients with microbiologically proven TB and 566 unaffected control subjects from three tertiary hospitals in northern Vietnam.

Results

Five SNPs within SP110 were associated with all forms of TB, including four SNPs at the C terminus (rs10208770, rs10498244, rs16826860, rs11678451) under a dominant model and one SNP under a recessive model, rs7601176. Two of these SNPs were associated with pulmonary TB (rs10208770 and rs16826860) and one with extra-pulmonary TB (rs10498244).

Conclusion

SP110 variants were associated with increased susceptibility to both pulmonary and extra-pulmonary TB in the Vietnamese. Genetic variants in SP110 may influence macrophage signaling responses and apoptosis during M. tuberculosis infection, however further research is required to establish the mechanism by which SP110 influences immunity to tuberculosis infection.     

Childhood Tuberculosis in a Sub-Saharan Tertiary Facility: Epidemiology and Factors Associated with Treatment Outcome

Childhood tuberculosis (TB) is a diagnostic challenge in developing countries, and patient outcome can be influenced by certain factors. We report the disease course, clinical profile and factors associated with treatment outcome in a tertiary facility of Kinshasa. Documentary and analytical studies were conducted using clinical and exploratory data for children aged up to 15 years who were admitted to the University Clinics of Kinshasa for TB. Data are presented as frequencies and averages, and binary and logistic regression analyses were performed. Of 283 children with TB, 82 (29.0%) had smear-negative TB, 40 (14.1%) had smear-positive TB, 159 (56.1%) had extra-pulmonary TB (EPTB), 2 (0.7%) had multidrug-resistant TB (MDR-TB), 167 (59.0%) completed treatment, 30 (10.6%) were cured, 7 (2.5%) failed treatment, 4 (1.4%) died, 55 (19.4%) were transferred to health centers nearest their home, and 20 (7.0%) were defaulters. In the binary analysis, reported TB contacts (p = 0.048), type of TB (p = 0.000), HIV status (p = 0.050), Ziehl-Nielsen test result (p = 0.000), Lowenstein culture (p = 0.004) and chest X-ray (p = 0.057) were associated with outcome. In the logistic regression, none of these factors was a significant predictor of outcome. Tertiary level care facilities must improve the diagnosis and care of patients with childhood TB, which justifies the development of alternative diagnostic techniques and the assessment of other factors that potentially affect outcome.     

TRICORE, a novel bead-based specimen concentration method for the culturing of Mycobacterium tuberculosis

Centrifugation is a necessary concentrating step for the detection of Mycobacterium tuberculosis in a liquid culture. However, centrifugation is biologically hazardous and presents an obstacle in the development of an automated culture system. A bead-based bacterial concentration method, TRICORE, was recently developed by Genetein Co., Ltd. We compared the efficacy of TRICORE and conventional centrifugation for concentrating M. tuberculosis in clinical sputum specimens by using liquid and solid culture systems. Among 90 pretreated clinical sputum specimens, 51 (57.3%) and 55 (61.8%) M. tuberculosis isolates were recovered by the MGIT culture system by using the centrifugation and TRICORE methods, respectively (chi-square test, p=0.5413). The detection time for the centrifugation method was 359.3±117.0 h, while that for the bead-based concentration method was 377.6±162.3 h (p=0.5637). However, the number of colonies recovered on solid media were significantly higher with the TRICORE method (p=0.003). In particular, among the smear-negative specimens, culture positivity of the TRICORE method was 39.6%, while that of the centrifugation method was 15.1%. The TRICORE bead-based concentration method was considered equivalent to centrifugation and enabled efficient collection of paucibacillary specimens in solution. Thus, the new noncentrifugation concentration method could yield more positive culture results.     

Childhood Tuberculosis in Household Contacts of Newly Diagnosed TB Patients

Introduction

Childhood tuberculosis (TB), although estimated to account for a major proportion of the global TB disease burden, has a lower public health priority. Reliable research and surveillance data on childhood TB is limited in most regions of the world. This study was conducted to assess the burden of childhood TB among the household contacts of new TB patients in Karachi, Pakistan.

Methods

A retrospective analysis of children (<15 years) who were household contacts of new adult TB patients presenting to Marie Adelaide Leprosy Center (MALC) clinics in Karachi during the period of 2008 to 2010 was conducted.

Results

Of the household children contacts (n = 6613) screened, 317 were suspected and 121(1.8%) diagnosed with TB. These included 89 (73.6%) with pulmonary and 32 (26.4%) with extra-pulmonary disease. Smear positivity rate in pulmonary cases was 32.6%. Mean age of children diagnosed with TB was 11.7 (±2.8) years. Within the child-contacts screened, disease was found to be significantly higher among females (2.3%) in comparison to males (1.2%) (p-value <0.01). The commonest relationship of source cases to diagnosed children was the mother (n = 51, 42.1%). The source case was a female for 66.1% (n = 76) of the children.

Conclusion

A smear positivity rate of 32.6% amongst pulmonary cases suggests their potential to spread disease and emphasizes a need to review the contribution of children in transmission of TB within communities. Greater vulnerability of the female child and considerable role of mother in disease transmission highlights a need to increase focus on females in TB control programs in Pakistan.     

Diagnostic Performance of Interferon-Gamma Releasing Assay in HIV-Infected Patients in China

Background

Active tuberculosis infection represents a very common and significant threat to HIV-infected patients. But measures to accurately detect it are limited.

Objective

To compare and analyze the diagnostic efficacy of T-SPOT.TB alone and in combination with TST in HIV-infected patients in China.

Method

TST (tuberculin skin test) and T-SPOT.TB were performed on 131 HIV-infected patients admitted in Beijing You’an Hospital and Beijing Ditan Hospital between Oct, 2010 and Jul, 2012, who were initially diagnosed as suspected ATB (active TB). The patients were further categorized into ATB and Not ATB based on clinical and cultural evidences. The performance of TST and T-SPOT.TB were analyzed and compared.

Results

The sensitivity and specificity of T-SPOT.TB were 41.3% and 94.6%, respectively, both higher than TST (12.9% and 91.8%). By combining T-SPOT.TB and TST, the sensitivity did not increase, but specificity was elevated to 100%. TST, T-SPOT.TB and their combinations all performed better in patients with extra-pulmonary diseases than with pulmonary disorders. False-positive T-SPOT.TB results were found to be associated with history of prior TB. In addition, concomitant bacterial infections and low CD4 counts were associated with increased ATB risk.

Conclusions

T-SPOT.TB is superior in screening ATB in HIV-infected patients in China over traditional TST. Additional TST would help to confirm a positive T-SPOT.TB result. Both tests work better for patients with extra-pulmonary conditions.