Incidence of tuberculosis and the predictive value of ELISPOT and Mantoux tests in Gambian case contacts

Background

Studies of Tuberculosis (TB) case contacts are increasingly being utilised for understanding the relationship between M. tuberculosis and the human host and for assessing new interventions and diagnostic tests. We aimed to identify the incidence rate of new TB cases among TB contacts and to relate this to their initial Mantoux and ELISPOT test results.

Methods and Findings

After initial Mantoux and ELISPOT tests and exclusion of co-prevalent TB cases, we followed 2348 household contacts of sputum smear positive TB cases. We visited them at 3 months, 6 months, 12 months, 18 months and 24 months, and investigated those with symptoms consistent with TB. Those who were diagnosed separately at a government clinic had a chest x-ray. Twenty six contacts were diagnosed with definite TB over 4312 person years of follow-up (Incidence rate 603/100,000 person years; 95% Confidence Interval, 370–830). Nine index and secondary case pairs had cultured isolates available for genotyping. Of these, 6 pairs were concordant and 3 were discordant. 2.5% of non-progressors were HIV positive compared to 12% of progressors (HR 6.2; 95% CI 1.7–22.5; p = 0.010). 25 secondary cases had initial Mantoux results, 14 (56%) were positive ; 21 had initial ELISPOT results, 11 (52%) were positive; 15 (71%) of 21 tested were positive by one or the other test. Of the 6 contacts who had concordant isolates with their respective index case, 4 (67%) were Mantoux positive at recruitment, 3 (50%) were ELISPOT positive; 5 (83%) were positive by one or other of the two tests. ELISPOT positive contacts, and those with discordant results, had a similar rate of progression to those who were Mantoux positive. Those negative on either or both tests had the lowest rate of progression.

Conclusions

The incidence rate of TB disease in Gambian TB case contacts, after screening for co-prevalent cases, was 603/100,000 person years. Since initial ELISPOT test and Mantoux tests were each positive in only just over half of cases, but 71% were positive by one or other test, positivity by either might be the best indication for preventive treatment. These data do not support the replacement of the Mantoux test by an ELISPOT test in The Gambia or similar settings.     

Separating true positive predicted residue contacts from false positive ones in mainly alpha proteins, using constrained Metropolis MC simulations

We present a method that significantly improves the accuracy of predicted proximal residue pairs in protein molecules. Computational methods for predicting pairs of amino acids that are distant in the protein sequence but close in the protein 3D structure can benefit attempts to in silico recognize the fold of a protein molecule. Unfortunately, currently available methods suffer from low predictive accuracy. In this work, we use Monte Carlo simulations to fold protein molecules with proximal pair predictions used as additional energy constraints. To test our methods, we study molecules with known tertiary structures. With Monte Carlo, we generate ensembles of structures for each set of residues constraints. The distribution of the root mean square deviation of the folded structures from the known native structure reveals clear information about the accuracy of the constraint sets used. With recursive substitutions of constraints, false positive predictions are identified and filtered out and significant improvements in accuracy are observed.     

Revisiting the false positive rate in detecting recent positive selection

There is increasing interest in studying the molecular mechanisms of recent adaptations caused by positive selection in the genomics era. Such endeavors to detect recent positive selection, however, have been severely handicapped by false positives due to the confounding impact of demography and the population structure. To reduce false positives, it is critical to conduct a functional analysis to identify the true candidate genes/mutations from those that are filtered through neutrality tests. However, the extremely high cost of such functional analysis may restrict studies within a small number of model species. In particular, when the false positive rate of neutrality tests is high, the efficiency of the functional analysis will also be very low. Therefore, although the recent improvements have been made in the (joint) inference of demography and selection, our ultimate goal, which is to understand the mechanism of adaptation generally in a wide variety of natural populations, may not be achieved using the currently available approaches. More attention should thus be spent on the development of more reliable tests that could not only free themselves from the confounding impact of demography and the population structure but also have reasonable power to detect selection.     

Use of a molecular diagnostic test in AFB smear positive tuberculosis suspects greatly reduces time to detection of multidrug resistant tuberculosis

Background

The WHO has recommended the implementation of rapid diagnostic tests to detect and help combat M/XDR tuberculosis (TB). There are limited data on the performance and impact of these tests in field settings.

Methods

The performance of the commercially available Genotype MTBDRplus molecular assay was compared to conventional methods including AFB smear, culture and drug susceptibility testing (DST) using both an absolute concentration method on Löwenstein-Jensen media and broth-based method using the MGIT 960 system. Sputum specimens were obtained from TB suspects in the country of Georgia who received care through the National TB Program.

Results

Among 500 AFB smear-positive sputum specimens, 458 (91.6%) had both a positive sputum culture for Mycobacterium tuberculosis and a valid MTBDRplus assay result. The MTBDRplus assay detected isoniazid (INH) resistance directly from the sputum specimen in 159 (89.8%) of 177 specimens and MDR-TB in 109 (95.6%) of 114 specimens compared to conventional methods. There was high agreement between the MTBDRplus assay and conventional DST results in detecting MDR-TB (kappa = 0.95, p<0.01). The most prevalent INH resistance mutation was S315T (78%) in the katG codon and the most common rifampicin resistance mutation was S531L (68%) in the rpoB codon. Among 13 specimens from TB suspects with negative sputum cultures, 7 had a positive MTBDRplus assay (3 with MDR-TB). The time to detection of MDR-TB was significantly less using the MTBDRplus assay (4.2 days) compared to the use of standard phenotypic tests (67.3 days with solid media and 21.6 days with broth-based media).

Conclusions

Compared to conventional methods, the MTBDRplus assay had high accuracy and significantly reduced time to detection of MDR-TB in an area with high MDR-TB prevalence. The use of rapid molecular diagnostic tests for TB and drug resistance should increase the proportion of patients promptly placed on appropriate therapy.     

The presence of arginine may be a source of false positive results in the Ames test

An increase in the number of revertant colonies in the Ames test is generally taken as a strong indication of mutagenic activity of a test compound. However, irrelevant positive findings may constitute a major problem in regulatory drug testing. In this study, mixtures containing only amino acids such as glycine, lysine, arginine and isoleucine, routinely used as peptide preservatives in polypeptide pharmaceutical products, were investigated for mutagenesis in the Ames Salmonella typhimurium test. The results demonstrated that in the presence of metabolic activation, all the solutions containing arginine induced an increase in the number of revertant colonies in strains TA98, TA100 and TA1535 compared with the solvent control. More specifically, for strain TA98, all arginine doses tested, i.e. from 0.4 to 8 mg/plate induced a statistically significant increase in the number of revertants. This increase was biologically significant from 1.2 to 8 mg/plate. For strain TA100, the five highest test doses, i.e. from 1.2 to 8 mg/plate, induced statistically and biologically significant increases in the number of revertants. A statistically significant increase in colony number was also observed in strain TA1535, but only at the maximal test dose of 8 mg/plate arginine. These increases were observed with arginine from two different sources, suggesting that the observed effect would not be due to the presence of potential impurities in the type of arginine used. Our findings show that a functional metabolic activation system was required to induce an increase in the number of colonies. The presence of vitamin C inhibited the arginine-induced increase in the number of revertant colonies in S. typhimurium strain TA98, suggesting a potential involvement of oxidative stress.     

Serial QuantiFERON-TB Gold In-Tube assay and tuberculin skin test to diagnose latent tuberculosis in household Mexican contacts: conversion and reversion rates and associated factors using conventional and borderline zone definitions

A cohort of 123 adult contacts was followed for 18‐24 months (86 completed the follow-up) to compare conversion and reversion rates based on two serial measures of QuantiFERON (QFT) and tuberculin skin test (TST) (PPD from TUBERSOL, Aventis Pasteur, Canada) for diagnosing latent tuberculosis (TB) in household contacts of TB patients using conventional (C) and borderline zone (BZ) definitions. Questionnaires were used to obtain information regarding TB exposure, TB risk factors and socio-demographic data. QFT (IU/mL) conversion was defined as <0.35 to ≥0.35 (C) or <0.35 to >0.70 (BZ) and reversion was defined as ≥0.35 to <0.35 (C) or ≥0.35 to <0.20 (BZ); TST (mm) conversion was defined as <5 to ≥5 (C) or <5 to >10 (BZ) and reversion was defined as ≥5 to <5 (C). The QFT conversion and reversion rates were 10.5% and 7% with C and 8.1% and 4.7% with the BZ definitions, respectively. The TST rates were higher compared with QFT, especially with the C definitions (conversion 23.3%, reversion 9.3%). The QFT conversion and reversion rates were higher for TST ≥5; for TST, both rates were lower for QFT <0.35. No risk factors were associated with the probability of converting or reverting. The inconsistency and apparent randomness of serial testing is confusing and adds to the limitations of these tests and definitions to follow-up close TB contacts.     

Estimating false positive and false negative error rates in cervical cell classification.

The performance of a cell recognition system on unknown data is often estimated in terms of its error rates on a test set. This paper investigates methods for producing estimates of error rates in cervical cell classification. Classification performance curves calculated using these methods are given for several classification schemes used to classify 1500 cervical cells.     

Rotavirus epidemiology adjusted pattern in a tropical setting: mathematical correction for false positive problem relating to primary immunochromatography test surveillance

Background: Rotaviruses are the most common cause of acute gastroenteritis in neonates and young children worldwide. Human rotaviruses are the leading cause of acute gastroenteritis in neonates and young children worldwide. The immunochromatography test is frequently used in clinical practice to detect rotavirus infection. When the immunochromatography test is incorrectly positive, there may be a discrepancy between the two tests, the immunochromatography test and the nucleic acid test. As a result, when interpreting the findings of basic rotavirus monitoring in a system based on immunochromatography tests, we must made adjustments to address the issue of accuracy. Methods: The findings on the expected pattern of rotavirus epidemiology in a tropical setting was presented. The modified rotavirus pattern was created to address the issue of false positives. To solve the false positive issue, the modified rotavirus pattern derived from mathematical model-based correction by extracting false positivity was predicted. Results: We demonstrated an altered rotavirus epidemiology pattern in the setting studied in this study. Rotavirus has been detected in up to 19.3% of patients with rotavirus-like symptoms, with G4P[8] accounting for 6% of those infected. Conclusion: As a result, the rotavirus remains an important problem that must be addressed in the framework of this study.     

Using fluorescent nanoparticles and SYBR Green I based two-color flow cytometry to determine Mycobacterium tuberculosis avoiding false positives

A method using an improved two-color flow cytometric analysis by a combination of bioconjugated fluorescent silica nanoparticles and SYBR Green I (FSiNP@SG-FCM) has been developed for detection of pathogenic Mycobacterium tuberculosis. Antibody-conjugated nanoparticles were prepared by oriented immobilization of the anti-M. tuberculosis antibody onto Tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate-doped fluorescent silica nanoparticles (RuBpy-doped FSiNPs) through Protein A. M. tuberculosis was specially labeled with antibody-conjugated RuBpy-doped FSiNPs, then stained with a nucleic acid dye SYBR Green I to exclude background detrital particles, followed by multiparameter determination with flow cytometry. With this method, false positives caused by aggregates of nanoparticle-bioconjugates and nonspecific binding of nanoparticle-bioconjugates to background debris could be significantly decreased. This assay allowed for detection of as low as 3.5 x 10(3) and 3.0 x 10(4)cellsml(-1)M. tuberculosis in buffer and spiked urine respectively, with higher sensitivities than the FITC-based conventional flow cytometry. The total assay time including sample pretreatment was within 2h. This proposed FSiNP@SG-FCM method will be promising for rapid detection of M. tuberculosis or other pathogenic bacteria in clinical samples.     

Occurrence of fungal spores in the respiratory tract and homes of patients with positive skin test to fungi

The occurrence of fungal spores in the upper respiratory tract and in the homes of 86 patients with allergic manifestations and positive skin tests for fungi has been investigated.Alternaria tenuis andCladosporium herbarum have been more frequently found to give positive reactions, followed byAspergillus mix,Candida albicans andPenicillium notatum. Cladosporium andAlternaria represented also the most common moulds isolated from nasal specimens. The prevalent fungi isolated from patients' environments wereCladosporium, Penicillium andAlternaria, occurring respectively in 93%, 78% and 72% of the homes sampled. An examination of individual cases have revealed a surprisingly close correlation, in 47 out of 86 patients, between positive skin test response and presence in high amount of the same fungi in patient's respiratory tract and home. In the majority of cases the fungi involved wereAlternaria and/orCladosporium.     

Whole blood interferon-gamma responses to mycobacterium tuberculosis antigens in young household contacts of persons with tuberculosis in Uganda

Background

Due to immunologic immaturity, IFN-γ-producing T cell responses may be decreased in young children compared to adults, thus we hypothesized that IFN-γ responses to mycobacterial antigens in household contacts exposed to Mycobacterium tuberculosis (Mtb) would be impaired in young children relative to adults. The objective of this study was to compare whole blood IFN-γ production in response to mycobacterial antigens between children and adults in Uganda.

Methodology/Principal Findings

We studied household contacts of persons with culture-positive pulmonary tuberculosis (TB) enrolled in a cohort study conducted in Kampala, Uganda. Whole blood IFN-γ production in response to Mtb culture-filtrate antigens was measured by ELISA and compared between infants (<2 years old, n = 80), young children (2 <5 years old, n = 216), older children (5 <15 years old, n = 443) and adults (≥15 years old, n = 528). We evaluated the relationship between IFN-γ responses and the tuberculin skin test (TST), and between IFN-γ responses and epidemiologic factors that reflect exposure to Mtb, and the effect of prior BCG vaccination on IFN-γ responses. Young household contacts demonstrated robust IFN-γ responses comparable to those of adults that were associated with TST and known risk factors for infection. There was no effect of prior BCG immunization on the IFN-γ response.

Conclusions/Significance

Young children in a TB endemic setting can mount robust IFN-γ responses generally comparable to those of adults, and as in adults, these responses correlated with the TST and known epidemiologic risk factors for Mtb infection.     

Occurrence of fungal spores in the respiratory tract and homes of patients with positive skin test to fungi

The occurrence of fungal spores in the upper respiratory tract and in the homes of 86 patients with allergic manifestations and positive skin tests for fungi has been investigated.Alternaria tenuis andCladosporium herbarum have been more frequently found to give positive reactions, followed byAspergillus mix,Candida albicans andPenicillium notatum. Cladosporium andAlternaria represented also the most common moulds isolated from nasal specimens. The prevalent fungi isolated from patients' environments wereCladosporium, Penicillium andAlternaria, occurring respectively in 93%, 78% and 72% of the homes sampled. An examination of individual cases have revealed a surprisingly close correlation, in 47 out of 86 patients, between positive skin test response and presence in high amount of the same fungi in patient's respiratory tract and home. In the majority of cases the fungi involved wereAlternaria and/orCladosporium.     

运动试验假阴性而冠状动脉造影阳性冠心病的特点分析

目的:探讨平板运动试验(TET)假阴性而冠状动脉造影(CAG)阳性冠心病病例的特点.方法:对同期内(相距时间1周)TET阴性而有心前区不适者进行CAG的冠心病患者60例(男性42例,女性18例,平均年龄66±10.岁)进行回顾性分析.TET以运动中及恢复期R波为主的导联出现ST段水平型下移0.05mV为阴性标准.CAG以冠状动脉狭窄直径≥50%为阳性标准.结果:60例患者CAG共累计病变106处,其中左主干2例2处、左前降支21例30处、左回旋支7例13处、右冠状动脉27例36处、对角支7例10处、间隔支3例3处、钝缘支3例3处、后降支6例6处、中间支2例3处.病变范围在在50%～74%占65处、病变范围在75%～89%占30处、病变范围在90%～95%占11处.冠状动脉单支病变40例(占66.7%),以右冠状动脉及左前降支为多,占70.0%(28/40),双支病变14例(占23.3%),左前降支+右冠状动脉较多,占57.1%(8/14),三支病变6例(占10.0%).60例患者中有侧支循环者31例(51.7%).有16 例(26.7%)试验运动量不足,男性明显多于女性(P<0.05).结论:冠心痛患者TET假阴性的因为可能为冠脉病变小、狭窄程度轻、狭窄病变供血心肌建立了良好的侧支循环以及试验运动量不够.对于有临床症状而TET阴性的患者应行CAG检查,以明确诊断.