Clinical TB Research Articles

Effect of diabetes on tuberculosis presentation and outcomes in Kiribati.

To determine the association between diabetes and the clinical features and treatment outcomes of TB in Kiribati. We enrolled consecutive patients with TB who presented from August 2010 to February 2012 and compared clinical features and TB treatment outcomes for patients with and without diabetes, as measured by haemoglobin A1c assay. Poor outcome was defined as death, default or treatment failure, and good outcome as treatment success or cure. Two hundred and seventy-five eligible persons with TB disease were enrolled; 101 (37%) had diabetes. TB patients with diabetes were more likely to have acid-fast bacilli (AFB) seen on sputum smear microscopy (RR: 1.3; 95% CI: 1.03-1.62). The risk of poor outcome did not differ between patients with or without diabetes (RR: 1.1; 95% CI: 0.5-2.7). TB patients with diabetes are more likely than those without to have sputum with AFB on microscopy. This could increase transmission in the community. Early detection of TB by screening patients with diabetes, and the converse, could be important public health interventions where diabetes and TB are prevalent.

Rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates using high resolution melting curve analysis

Aims and objectivesEarly detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and to initiate an effective anti-TB treatment regimen. The objective of this study is to evaluate molecular technique high resolution melting curve (HRM) assay to rapidly detect resistance-conferring mutations in rpoB and katG genes. MethodsHRM analysis was used to screen 95 Mycobacterium tuberculosis (MTB) clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R), and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. 19 MTB isolates with known drug susceptibility genotypes were used as control strains for initial validation of the assay. Polymerase chain reaction (PCR) amplicons from rpoB and katG genes were sequenced to investigate the frequency and type of mutations and to confirm HRM results. ResultsAll RIF-S and INH-S isolates generated wild-type HRM curves and were accurately identified as susceptible by this method. Similarly 19 out of 20 RIF-R and 18 out of 21 INH-R isolates correctly exhibited mutant-type HRM curves. These results were similar to those obtained by direct sequencing. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible by HRM assay. These strains were confirmed as having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. ConclusionsHRM was found to be a reliable, rapid and low-cost method to characterize drug susceptibility of clinical TB isolates in resource-limited settings.

Pyrazinamide resistance in Swedish multidrug-resistant tuberculosis 2003–2013

Aims and objectivesThe aim of this study is to determine the prevalence of pyrazinamide (PZA) resistance among multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strains isolated in Sweden between 2003 and 2013 and to study the correlation between phenotypic PZA susceptibility testing and the presence of pncA mutations. MethodsA total of 117 clinical TB isolates defined as MDR were tested for PZA resistance in the Bactec MGIT 960 system and subsequently screened for mutations within the pncA gene using Sanger sequencing. ResultsPZA is a first-line key drug in the treatment of TB, including MDR-TB susceptible to PZA, and the phenotypic test showed that 53% of the MDR isolates from Sweden were PZA resistant. Preliminary sequencing results show that 95% of the PZA-resistant strains had a mutation in the pncA gene or its putative promoter. Of the PZA-susceptible strains, 73% had a wild type pncA, a proportion which increases to 89% if the silent mutation Ser65Ser (TCC65TCT) is included in this group. Moreover, the sensitivity and specificity of pncA sequencing, using the Bactec MGIT 960 system as the gold standard, was determined to be 95% and 89%, respectively. The results also showed an increase of PZA resistance among the Swedish MDR isolates during the studied period. Between 2003 and 2008, the prevalence of PZA resistance was 44% (n=48), while in the latter period (2009–2013) the prevalence had increased to 59% (n=69). No major difference in PZA resistance was seen among MDR patients of various geographical origins. ConclusionsThe results from the last 11-year period demonstrate an increase of PZA resistance among Swedish MDR cases. Additionally, the detection of pncA gene mutations, or their absence, was confirmed as a useful method for predicting PZA resistance.

Xpert MTB/RIF Assay for Pulmonary Tuberculosis and Rifampicin Resistance in Children: a Meta-Analysis

The Xpert MTB/RIF assay has been recommended by WHO to replace conventional microscopy, culture, and drug resistance tests. It simultaneously detects both Mycobacterium tuberculosis infection (TB) and resistance to rifampicin (RIF) within two hours. The objective was to review the available research studies on the accuracy of the Xpert MTB/RIF assay for diagnosing pulmonary TB and RIF-resistance in children. A comprehensive search of Pubmed and Embase was performed up to October 28, 2014. We identified published articles estimating the diagnostic accuracy of the Xpert MTB/RIF assay in children with or without HIV using culture or culture plus clinical TB as standard reference. QUADAS-2 tool was used to evaluate the quality of the studies. A summary estimation for sensitivity, specificity, diagnostic odds ratios (DOR), and the area under the summary ROC curve (AUC) was performed. Meta-analysis was used to establish the overall accuracy. 11 diagnostic studies with 3801 patients were included in the systematic review. The overall analysis revealed a moderate sensitivity and high specificity of 65% (95% CI: 61 - 69%) and 99% (95% CI: 98 - 99%), respectively, and a pooled diagnostic odds ratio of 164.09 (95% CI: 111.89 - 240.64). The AUC value was found to be 0.94. The pooled sensitivity and specificity for paediatric rifampicin resistance were 94.0% (95% CI: 80.0 - 93.0%) and 99.0% (95% CI: 95.0 - 98.0%), respectively. Hence, the Xpert MTB/RIF assay has good diagnostic and rifampicin performance for paediatric pulmonary tuberculosis. The Xpert MTB/RIF is sensitive and specific for diagnosing paediatric pulmonary TB. It is also effective in detecting rifamnicin resistance. It can, therefore, be used as an initial diagnostic tool.

M43 The Accuracy Of Clinical Tb Diagnoses In Culture Negative Patients

Background NICE guidelines for TB diagnosis recommend that sputum is obtained for culture for all suspected cases of pulmonary TB, and biopsies for all cases extrapulmonary TB. As results can take 6 weeks, treatment initiation decisions are frequently made without microbiological confirmation. Aim This study set out to examine the accuracy of clinical diagnoses in a high incidence area, and the basis for these decisions. Methods The data entered onto the national TB database was used to obtain a list of patients for whom no culture results had been recorded. Clinic letters, laboratory records and imaging were examined to determine whether samples had been sent for culture, how diagnoses were made in the event of negative results, and if alternative diagnoses were concluded. Results Of 323 patients on the database, 7% had no samples sent for culture. There were 109 culture negative patients, of whom 13 (4% all cases) had alternative diagnoses. A combination of relevant history and imaging was the most commonly used method of diagnosis when culture was negative (47%). Histology was used in 17% patients and Mantoux or IGRA testing supported initiating treatment in 39% cases. The database was missing positive culture results for 102 patients, of which four were MDR TB. Conclusions In this study, we found accurate initial clinical diagnoses, with only 4% patients subsequently obtaining alternative diagnoses. Most diagnoses were made on the basis of relevant history and imaging. Of concern are the 7% patients for whom tissue was never sent for culture. This is likely to be an underestimate when including all patients initially suspected of TB, raising the possibility of missed diagnoses. The utility of Mantoux and IGRA testing in active disease is now disputed. It is hoped with inter-specialty education regarding the importance of culture and futility of immunological based assays, the proportion of patients with suspected TB who have sputum or tissue sent for culture increases. Accurate recording of MDR-TB on the national TB database needs to be improved, to enable efficient monitoring of intervention programmes.

Antimycobacterial activity of natural products and synthetic agents: pyrrolodiquinolines and vermelhotin as anti-tubercular leads against clinical multidrug resistant isolates of Mycobacterium tuberculosis.

Various classes of natural products and synthetic compounds were tested against reference strains and clinical multidrug resistant isolates of Mycobacterium tuberculosis. Vermelhotin (19), a natural tetramic acid from fungi, was the most active toward clinical MDR TB isolates (MIC 1.5-12.5μg/mL). Synthetic compounds (i.e. benzoxazocines, coumarins, chromenes, and pyrrolodiquinoline derivatives) were prepared by green chemistry approaches. Under microwave irradiation, a one-pot synthesis of pyrrolodiquinoline 85 was achieved by homocoupling of 1-methylquinolinium iodide; the structure of 85 was confirmed by single-crystal X-ray analysis. Compound 85 and its derivative 86 exhibited potent anti-tubercular activity (MIC 0.3-6.2μg/mL) against clinical MDR TB isolates, and they displayed weak cytotoxicity toward normal cell line. The scaffold of 85 and 86 is potential for antimycobacterial activity.

Tuberculosis and gender parity in a TB Referral Centre, South –South Nigeria

Background: Epidemiological information shows that there are differences between men and women in prevalence of TB infection, rate of progression from infection to disease, HIV and TB co-infection rate, and mortality due to pulmonary tuberculosis. Study of gender differences in prevalence and treatment outcome of patients will assist in targeting those at higher risk and ensure successful TB control planning. Aim: This study is aimed at determining gender differences in the prevalence of clinical disease, HIV and TB co-infection rate and treatment outcome amongst TB patients that attended this referral hospital between January 2003 to December 2012. Methodology: A retrospective survey was carried out on the records of all Acid Fast Bacilli (AFB) smear positive Pulmonary Tuberculosis patients who attended this hospital during the period under review. Epidemiological information was collected retrospectively from the tuberculosis laboratory and treatment registers to explore gender disparity in the prevalence of clinical disease, HIV and TB co-infection rate and treatment outcome among the TB patients. Results: A total number of 1612 HIV negative male TB patients attended the DOTS centre of this hospital as against 1013 females during the study period. Male had a higher prevalence ,the male: female ratio was 1.59. There were a total number of 156 HIV - TB co-infected female subjects constituting 9.7% of the female TB patients while 106 males were HIV-TB co-infected constituting 4.7% of the male patients. The difference was statistically significantly (P<0.00). Conclusion: Our findings bring into sharp focus the gender disparity in the TB prevalence and HIV-TB co-infection among the study subjects. TB control programme should routinely review and monitor programme reports and take appropriate measures to improve the current TB control policies and programme.

IgA Response to ESAT‐6/CFP‐10 and Rv2031 Antigens Varies in Patients With Culture‐Confirmed Pulmonary Tuberculosis, Healthy Mycobacterium tuberculosis–Infected and Non‐Infected Individuals in a Tuberculosis Endemic Setting, Ethiopia

Little attention has been given to the role of antibodies against Mycobacterium tuberculosis (Mtb) infection. We have compared the levels of IgA and IgG against ESAT-6/CFP-10 and Rv2031c antigens in sera of patients with culture-confirmed pulmonary tuberculosis (PTB), healthy Mtb-infected and non-infected individuals in endemic TB settings. Venous blood samples were collected from 166 study participants; sera were separated and assayed by an enzyme-linked immunosorbent assay (ELISA). QuantiFERON-TB Gold In-Tube (QFTGIT) assay was used for the screening of latent TB infection. The mean optical density (OD) values of IgA against ESAT-6/CFP-10 and Rv2031 were significantly higher in sera of patients with culture-confirmed PTB compared with healthy Mtb-infected and non-infected individuals (P<0.001). The mean OD values of IgG against ESAT-6/CFP-10 and Rv2031 were also significantly higher in sera of patients with culture-confirmed PTB compared with healthy Mtb-infected and non-infected individuals (P<0.05). The mean OD values of IgA against both antigens were also higher in sera of healthy Mtb-infected cases compared with non-infected individuals. There were positive correlations (P<0.05) between the level of IFN-γ induced in QFTGIT assay and the OD values of serum IgA against both antigens in healthy Mtb-infected subjects. This study shows the potential of IgA response against ESAT-6/CFP-10 and Rv2031 antigens in discriminating clinical TB from healthy Mtb-infected and non-infected cases. Nevertheless, further well-designed cohort study is needed to fully realize the full potential of this diagnostic marker.

THU0352 Trochanteric bursitis: Is there ultrasonographic evidence to suggest inflammation?

BackgroundTrochanteric bursitis (Tb) is one of the most common soft tissue abnormalities in the hip region. Tb is common; affecting 10-25% of the population.Power Doppler (PD) ultrasonography (US) is a...

Comparison of IS6110 and ‘short fragment’ devR (Rv3133c) gene targets with phenotypic methods for diagnosis of Mycobacterium tuberculosis

BackgroundTuberculosis (TB), declared a global emergency in 1993 by the WHO, remains a worldwide public health problem. Rapid diagnosis is required for treatment and prevention. This study compares genotypic methods using two gene targets [IS6110 and 'short fragment'devR (Rv3133c)] with phenotypic methods [Lowenstein Jensen (LJ) and BACTEC 460] for the diagnosis of TB while using Ziehl Neelsen (ZN) staining as the gold standard. Methods56 clinical TB samples from a tertiary care apex center along with 50 healthy control samples, excluding samples from patients already on ATT were processed by routine USP methodology. Smears were graded by ZN stain. Solid media (LJ) and liquid media (BACTEC 460) were used along with IS6110 and 'short fragment'devR (Rv3133c) specific gene amplification and comparatively analyzed. Results50/56 samples were positive by phenotypic methods, 53 by IS6110 and 45 by devR (Rv3133c) amplification. 38 samples were positive by both phenotypic and genotypic methods. IS6110 detected six and devR (Rv3133c) detected five phenotypically negative samples. Both IS6110 and devR (Rv3133c) were positive in 42 samples. 11 devR (Rv3133c) negative samples were positive by IS6110 and three IS6110 negative samples were positive by 'short fragment'devR (Rv3133c). Compared to phenotypic methods, the sensitivity, specificity, positive and negative predictive values of IS6110 was 94%, 89.29%, 88.68% and 94.34% while that of devR (Rv3133c) was 80%, 91.07%, 88.89% and 83.61% respectively. ConclusionSimultaneous use of both phenotypic and genotypic methods increases the yield of positive results.

Differential cellular recognition pattern to M. tuberculosis targets defined by IFN-γ and IL-17 production in blood from TB + patients from Honduras as compared to health care workers: TB and immune responses in patients from Honduras

BackgroundA better understanding of the quality of cellular immune responses directed against molecularly defined targets will guide the development of TB diagnostics and identification of molecularly defined, clinically relevant M.tb vaccine candidates.MethodsRecombinant proteins (n = 8) and peptide pools (n = 14) from M. tuberculosis (M.tb) targets were used to compare cellular immune responses defined by IFN-γ and IL-17 production using a Whole Blood Assay (WBA) in a cohort of 148 individuals, i.e. patients with TB + (n = 38), TB- individuals with other pulmonary diseases (n = 81) and individuals exposed to TB without evidence of clinical TB (health care workers, n = 29).ResultsM.tb antigens Rv2958c (glycosyltransferase), Rv2962c (mycolyltransferase), Rv1886c (Ag85B), Rv3804c (Ag85A), and the PPE family member Rv3347c were frequently recognized, defined by IFN-γ production, in blood from healthy individuals exposed to M.tb (health care workers). A different recognition pattern was found for IL-17 production in blood from M.tb exposed individuals responding to TB10.4 (Rv0288), Ag85B (Rv1886c) and the PPE family members Rv0978c and Rv1917c.ConclusionsThe pattern of immune target recognition is different in regard to IFN-γ and IL-17 production to defined molecular M.tb targets in PBMCs from individuals frequently exposed to M.tb. The data represent the first mapping of cellular immune responses against M.tb targets in TB patients from Honduras.

Evaluating the use of loop-mediated isothermal amplification (LAMP) method for detection of &lt;em&gt;Mycobacterium tuberculosis&lt;/em&gt; in Indonesian clinical isolates

Background: Loop-mediated isothermal amplification (LAMP) is a method already claimed as a simple technique to amplify DNA/ RNA using four to six primers as “a set” from conserved sequence of target gene. In this study we optimize the use of LAMP for detection of Mycobacterium tuberculosis in clinical isolates from Indonesia. Methods: Procedures to perform LAMP were optimized, then the method was applied to 122 archieved samples of DNA’s Mtb from clinical TB patients with Acid Fast Bacilli (AFB) smears positive. The samples were obtained in 2008 from 13 provinces in Indonesia for genotyping study, which then become collections of Center for Biomedical and Basic Technology of Health (CBBTH), NIHRD Indonesia. The optimization tests include sensitivity and specificity tests of several sets primers, which were evaluated using 10-fold serially diluted DNA of Mtb H37Rv and 12 species of Mycobacteria. Three equipments consisted of LAMP turbidimeter, heating block and water bath were compared for its ability in DNA amplification. Detection of M. tuberculosis from clinical isolates used set primers specific for gyrB gene, amplicon was detected with UV fluorescence system. Results: The results showed that the highest sensitivity was obtained using the set primers specific for 16S rRNA and gyrB which could detect 10.0 fg to 1.0 pg genomic DNA of Mtb H37Rv. The set primers specific for gyrB gene was the most specific primers. Application of LAMP using gyrB set primers on Indonesian clinical isolates showed 94.2% (114/121) positivity rate. Conclusion: LAMP method is potentially used in TB diagnosis in Indonesia. (Med J Indones. 2012;21:188-95) Keywords: Loop-mediated isothermal amplification, rim gene, 16S rRNA gene, gyrB gene, Mycobacterium tuberculosis

Long-term follow-up of contacts exposed to multidrug-resistant tuberculosis in Victoria, Australia, 1995–2010

The effectiveness of public health strategies following exposure to multidrug-resistant tuberculosis (MDR-TB) is not clear. To perform long-term follow-up of MDR-TB contacts and review individual outcomes and management approaches. Retrospective review of MDR-TB contacts identified by the Victorian Department of Health from 1995 to 2010. Health records, including personal medical and pharmacy records and statewide clinical and laboratory TB databases, were searched to identify management strategies and individual outcomes. A total of 570 contacts of 47 MDR-TB cases were identified, with a total follow-up period of 3093 person-years of observation (PYO) since exposure. Of 570 contacts, 49 (8.6%) were considered likely to have been infected with Mycobacterium tuberculosis from index cases, and 11/49 (22.5%) of these were prescribed preventive therapy tailored to isolate susceptibility. No MDR-TB cases occurred in those receiving preventive treatment, while two cases were observed in those not treated (incidence 2878/100 000 PYO during the first 2 years post exposure). The risk of MDR-TB transmission to close contacts in this low-prevalence setting highlights the potential for public health strategies involving preventive treatment.

Severe tuberculosis induces high degree of CD4 T cell differentiation (43.25)

Abstract Effector Th1 cells represent a key component of anti-TB defense. Until now it has been unclear whether active TB promotes or alters the differentiation of functionally active effector CD4 T cells. Our previous observations in mice have revealed that Mtb infection leads to the formation of highly differentiated Th1-biased effectors expressing CD27low phenotype. Here, we addressed the generation of CD27low effectors during pulmonary TB in humans. We have analyzed the percents of CD27low cells within Mtb-specific CD4 T lymphocytes (cells producing IFN-γ after stimulation with Mtb sonicate) located in the focus of lung Mtb infection or circulating in peripheral blood. In the lungs, CD27low subset formed a vast majority (&amp;gt;70%) of Mtb-specific CD4 T cells. In blood, the percents of CD27low cells varied from low (12%) to very high (92%). The major correlates for the accumulation of CD27low cells in the blood were lung tissue destruction and clinical TB severity (r&amp;gt;0.5; p&amp;lt;0.0001). Following the therapy, the content of CD27low cells declined in parallel with lung tissue repair. Compared to patients, the content of CD27low cells in healthy donors and Mtb-exposed individuals was significantly lower (p&amp;lt;0.001). Overall, there was no deficiency in the generation of CD4 effector cells during TB. Instead, active TB promoted the accumulation of highly differentiated CD27low effectors, which may serve as a valuable marker for TB severity and lung tissue destruction.

Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus

BackgroundTuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines.MethodsRecombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-γ production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus.ResultsWe identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-α and IFN-γ) in T cells from non-human primates (NHPs) after BCG vaccination.ConclusionsPBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

Development of an Antibody to Bovine IL-2 Reveals Multifunctional CD4 TEM Cells in Cattle Naturally Infected with Bovine Tuberculosis

Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2). Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ+IL-2+TNF-α+ and IFN-γ+ TNF-α+ response in naturally infected cattle. These multifunctional CD4 T cells express a CD44hiCD45RO+CD62Llo T-effector memory (TEM) phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced ‘quality of response’ as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional TEM cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future.

Lack of adherence to isoniazid chemoprophylaxis in children in contact with adults with tuberculosis in Southern Ethiopia.

SettingHawassa, Southern Region of Ethiopia.ObjectivesTo determine compliance to isoniazid (INH) preventive therapy (IPT) and its effectiveness in preventing (TB) disease in children in contact with adults with pulmonary TB (PTB).DesignThis was a prospective cohort study of children <15 years old in contact with adults with smear-positive PTB. Asymptomatic children ≤5 years were provided IPT independently of their Tuberculin Skin Test (TST) status and children >5 years old were given advice but did not receive IPT, as recommended by the National TB control programme. Compliance to IPT and incidence of clinical TB were determined monthly for six months and then quarterly for up to 30 months.ResultsOne hundred and eighty four children in contact with 83 smear-positive PTB cases were identified. Eighty two were ≤5 and 102>5 years old. Only 27 (33%) of 82 children given IPT took it for >4 months and 10 (12%) completed the 6-month course. The main reason for non-compliance was the perception that drugs were not necessary when the child was healthy. Eleven children (all except one >5 years old) developed symptoms of TB disease and initiated treatment, resulting in an incidence of 28.6 cases for all and 53.5 for children >5 years old per 1000 children-year.ConclusionCompliance to IPT in children is poor in Southern Region of Ethiopia and this was associated with the parents' perception of the low importance of chemoprophylaxis in asymptomatic children. Poor compliance might be an important barrier for the wider implementation of IPT.Trial RegistrationClinicaltrials.gov NCT00456469

Cloning and expression of pab gene of M. tuberculosis isolated from pulmonary TB patient in E.coli DH5α

Background: Mycobacterium tuberculosis antigen38 is a potent serodiagnostic agent containing two M. tuberculosis specific B-cell epitopes. The high price of imported diagnostic agents hinders realization of fast clinical TB diagnosis in developing countries. Therefore, we produced recombinant antigen38 (recAg38M) from M. tuberculosis local strain, which might be used to produce economical tuberculosis serodiagnostic kit. Methods: Pab gene that was isolated from pulmonary TB patient in Malang was cloned into a plasmid vector (pGEMTeasy) to construct pMB38. The E.coli DH5α clone carrying pMb38 was selected on X-gal medium. The expression of pab was mediated using pPRoExHTc under the control of Trc promoter and E.coli DH5α as host. Results: Alignment of the pab sequence from the white E.coli DH5α clones with that of M. tuberculosis H37Rv showed 98% homology. The recombinant protein in which the signal peptide has been deleted to prevent the protein being secreted into medium was found in the cytoplasm. Conclusion: pab gene of M. tuberculosis isolated from a TB patient could be expressed in heterologous system in E.coliDH5α. (Med J Indones 2011; 20:247-54) Keywords: Mycobacterium tuberculosis, Pab gene expression, recombinant antigen38

Rapid and Accurate Detection of Mycobacterium tuberculosis in Sputum Samples by Cepheid Xpert MTB/RIF Assay—A Clinical Validation Study

BackgroundA crucial impediment to global tuberculosis control is the lack of an accurate, rapid diagnostic test for detection of patients with active TB. A new, rapid diagnostic method, (Cepheid) Xpert MTB/RIF Assay, is an automated sample preparation and real-time PCR instrument, which was shown to have good potential as an alternative to current reference standard sputum microscopy and culture.MethodsWe performed a clinical validation study on diagnostic accuracy of the Xpert MTB/RIF Assay in a TB and HIV endemic setting. Sputum samples from 292 consecutively enrolled adults from Mbeya, Tanzania, with suspected TB were subject to analysis by the Xpert MTB/RIF Assay. The diagnostic performance of Xpert MTB/RIF Assay was compared to standard sputum smear microscopy and culture. Confirmed Mycobacterium tuberculosis in a positive culture was used as a reference standard for TB diagnosis.ResultsXpert MTB/RIF Assay achieved 88.4% (95%CI = 78.4% to 94.9%) sensitivity among patients with a positive culture and 99% (95%CI = 94.7% to 100.0%) specificity in patients who had no TB. HIV status did not affect test performance in 172 HIV-infected patients (58.9% of all participants). Seven additional cases (9.1% of 77) were detected by Xpert MTB/RIF Assay among the group of patients with clinical TB who were culture negative. Within 45 sputum samples which grew non-tuberculous mycobacteria the assay's specificity was 97.8% (95%CI = 88.2% to 99.9%).ConclusionsThe Xpert MTB/RIF Assay is a highly sensitive, specific and rapid method for diagnosing TB which has potential to complement the current reference standard of TB diagnostics and increase its overall sensitivity. Its usefulness in detecting sputum smear and culture negative patients needs further study. Further evaluation in high burden TB and HIV areas under programmatic health care settings to ascertain applicability, cost-effectiveness, robustness and local acceptance are required.

CT screening before treatment of latent tuberculous infection for the diagnosis of clinical TB among contacts

To evaluate the value of CT scan for the detection of tuberculous diseases among persons who are suspected to be infected at the contact examination. Fukujuji Hospital, Japan. Retrospective review of the medical records of 22 persons who were normal with plain chest X-ray at an outbreak at a private school, with which total 46 TB cases and 93 infected persons were detected by either symptomatic visits or contact examinations done mainly at public health centers. Among the 44 persons who visited Fukujuji Hospital, 4 persons were symptomatic visits, 3 persons were detected as TB cases by the contact examination at public health centers and 3 persons visited Fukujuji Hospital for the purpose of contact examination before examinations at the public health centers. Eight of these 10 persons were with abnormal chest plain Xray findings and the remaining two persons were normal with plain chest X-ray findings (group A). Among the 34 persons who were referred to Fukujuji Hospital from public health centers as the infected person without diseases, one person showed abnormal chest plain X-ray and 33 persons showed normal chest plain X-ray (group B). Group A persons were examined in June and Group B persons were examined in The 2 persons in the group A were with abnormal CT findings. They were not diagnosed as TB by the CT findings but followed up after sputum examinations. Both of them were diagnosed as TB by the positive TB culture. Among the 33 persons in the group B, 20 persons were tested with CT scan at the examination done before starting treatment of latent tuberuclous infection and six of these 20 persons were with abnormal findings and were judged as TB diseases. In the case of outbreak with many TB cases and infected persons, CT should be considered for the detection of TB cases among contacts.