Shriprakash Kalantri

New tools and emerging technologies for the diagnosis of tuberculosis: Part I. Latent tuberculosis.

Nearly a third of the world’s population is estimated to be infected with Mycobacterium tuberculosis. This enormous pool of latently infected individuals poses a major hurdle for global tuberculosis (TB) control. Currently, diagnosis of latent TB infection (LTBI) relies on the tuberculin skin test (TST), a century-old test with known limitations. In this review, the first of a two-part series on new tools for TB diagnosis, recent advances in the diagnosis of LTBI are described. The biggest advance in recent years has been the development of in vitro T-cell-based interferon-γ release assays (IGRAs) that use antigens more specific to M. tuberculosis than the purified protein derivative used in the TST. Available research evidence on IGRAs suggests they have higher specificity than TST, better correlation with surrogate markers of exposure to M. tuberculosis in low-incidence settings, and less cross-reactivity due to BCG vaccination than the TST. IGRAs also appear to be at least as sensitive as the purified protein derivative-based TST for active TB. In the absence of a gold standard for LTBI, sensitivity and specificity for LTBI are not well defined. Besides high specificity, other potential advantages of IGRAs include logistical convenience, avoidance of poorly reproducible measurements, such as skin induration, need for fewer patient visits and the ability to perform serial testing without inducing the boosting phenomenon. Overall, due to its high specificity, IGRAs may be useful in low-endemic, high-income settings where cross-reactivity due to BCG might adversely impact the utility of TST. However, despite the growing evidence supporting the use of IGRAs, several unresolved and unexplained issues remain. The review concludes by highlighting areas where evidence is lacking, and provides an agenda for future research. Active TB and drug resistance are discussed in Part II; 423–432 of this issue.

New tools and emerging technologies for the diagnosis of tuberculosis: Part II. Active tuberculosis and drug resistance

Tuberculosis (TB) is one of the world’s most important infectious causes of morbidity and mortality among adults. Between 8 and 9 million develop TB disease, and approximately 2 million die from TB each year. Despite this enormous global burden, case detection rates are low, posing major hurdles for TB control. Conventional TB diagnosis continues to rely on smear microscopy, culture and chest radiography. These tests have known limitations. Conventional tests for detection of drug resistance are slow, tedious and difficult to perform in field conditions. This second half of a two-part review series on new tools for TB diagnosis describes recent advances and emerging technologies in the diagnosis of active disease, and detection of drug resistance. For diagnosis, new tools include newer versions of nucleic acid amplification tests, immune-based assays, skin patch test and rapid culture systems. For drug resistance, new tools include line-probe assays, bacteriophage-based assays, molecular beacons and microscopic observation drug susceptibility assay. Although the ideal test for TB is still not in sight, substantial progress has been made in the past decade. With the resurgence of interest in the development of new tools for TB control, it is likely that the next decade will see greater progress and tangible benefits. However, the challenge will be to ensure that new tools undergo rigorous evaluations in field conditions, and also to make sure that benefits of promising new tools actually reach the populations in developing countries that need them most. Latent TB is discussed in Part I; 413–422 of this issue.

Persistently elevated T cell interferon-γ responses after treatment for latent tuberculosis infection among health care workers in India: a preliminary report

BackgroundT cell-based interferon-γ (IFN-γ) release assays (IGRAs) are novel tests for latent tuberculosis infection (LTBI). It has been suggested that T cell responses may be correlated with bacterial burden and, therefore, IGRAs may have a role in monitoring treatment response. We investigated IFN-γ responses to specific TB antigens among Indian health care workers (HCWs) before, and after LTBI preventive therapy.MethodsIn 2004, we established a cohort of HCWs who underwent tuberculin skin testing (TST) and a whole-blood IGRA (QuantiFERON-TB-Gold In-Tube [QFT-G], Cellestis Ltd, Victoria, Australia) at a rural hospital in India. HCWs positive by either test were offered 6 months of isoniazid (INH) preventive therapy. Among the HCWs who underwent therapy, we prospectively followed-up 10 nursing students who were positive by both tests at baseline. The QFT-G assay was repeated 4 and 10 months after INH treatment completion (i.e. approximately 12 months and 18 months after the initial testing). IFN-γ responses to ESAT-6, CFP-10 and TB7.7 peptides were measured using ELISA, and IFN-γ ≥0.35 IU/mL was used to define a positive QFT-G test result.ResultsAll participants (N = 10) reported direct contact with smear-positive TB patients at baseline, during and after LTBI treatment. All participants except one started treatment with high baseline IFN-γ responses (median 10.0 IU/mL). The second QFT-G was positive in 9 of 10 participants, but IFN-γ responses had declined (median 5.0 IU/mL); however, this difference was not significant (P = 0.10). The third QFT-G assay continued to be positive in 9 of 10 participants, with persistently elevated IFN-γ responses (median 7.9 IU/mL; P = 0.32 for difference against baseline average).ConclusionIn an environment with ongoing, intensive nosocomial exposure, HCWs had strong IFN-γ responses at baseline, and continued to have persistently elevated responses, despite LTBI treatment. It is plausible that persistence of infection and/or re-infection might account for this phenomenon. Our preliminary findings need confirmation in larger studies in high transmission settings. Specifically, research is needed to study T cell kinetics during LTBI treatment, and determine the effect of recurrent exposures on host cellular immune responses.

T-Cell Assays for Tuberculosis Infection: Deriving Cut-Offs for Conversions Using Reproducibility Data

Background Although interferon-gamma release assays (IGRA) are promising alternatives to the tuberculin skin test, interpretation of repeated testing results is hampered by lack of evidence on optimal cut-offs for conversions and reversions. A logical start is to determine the within-person variability of T-cell responses during serial testing. Methodology/Principal Findings We performed a pilot study in India, to evaluate the short-term reproducibility of QuantiFERON-TB Gold In Tube assay (QFT) among 14 healthcare workers (HCWs) who underwent 4 serial QFT tests on day 0, 3, 9 and 12. QFT ELISA was repeated twice on the same sets of specimens. We assessed two types of reproducibility: 1) test-retest reproducibility (between-test variability), and 2) within-person reproducibility over time. Test-retest reproducibility: with dichotomous test results, extremely high concordance was noticed between two tests performed on the same sets of specimens: of the 56 samples, the test and re-test results agreed for all but 2 individuals (κ = 0.94). Discordance was noted in subjects who had IFN-γ values around the cut-off point, with both increases and decreases noted. With continuous IFN-γ results, re-test results tended to produce higher estimates of IFN-γ than the original test. Within-person reproducibility: when continuous IFN-γ data were analyzed, the within-person reproducibility was moderate to high. While persons with negative QFT results generally stayed negative, positive results tended to vary over time. Our data showed that increases of more than 16% in the IFN-γ levels are statistically improbable in the short-term. Conclusions Conservatively assuming that long-term variability might be at least twice higher than short-term, we hypothesize that a QFT conversion requires two conditions to be met: 1) change from negative to positive result, and 2) at least 30% increase in the baseline IFN-γ response. Larger studies are needed to confirm our preliminary findings, and determine the conversion thresholds for IGRAs.

Sensitivity of a Whole-Blood Interferon-Gamma Assay Among Patients with Pulmonary Tuberculosis and Variations in T-Cell Responses During Anti-Tuberculosis Treatment

Background:Interferon-γ (IFN-γ) assays are new tests for tuberculosis (TB) infection, and T-cell responses may be correlated with antigen burden. However, it is unclear if IFN-γ assays can be used to monitor response to TB treatment.Materials and Methods:We measured T-cell responses to TB specific antigens in 60 Indian patients with microbiologically confirmed active pulmonary tuberculosis, before, during, and after TB treatment. Most patients were hospitalized and had moderate to advanced disease. IFN-γ responses were measured using the commercial whole-blood Quanti-FERON-TB Gold In Tube (QFT-G) assay at three time-points: at diagnosis (N = 60), after 2 months of intensive treatment (N = 47), and at 6 months (treatment completion) (N = 39).Results:At baseline, 44 of 60 (73%) patients were positive by QFT-G. At the second time-point, 38 of 47 (81%) patients were positive. At treatment completion, 31 of 39 (79%) patients were positive. Changes in IFN-γ responses over time were highly inconsistent - some individuals showed increases, while others showed decreases or no changes. Although the average IFN-γ levels decreased slightly during treatment (not significant), the QFT-G sensitivity remained mostly unchanged during therapy.Conclusions:Our data suggest that the QFT-G assay has modest sensitivity in patients with moderate to advanced pulmonary disease, but our results do not show a clear correlation between antigen burden and T-cell responses. Further research is needed to understand the kinetics of Tcell responses during TB treatment.

Impact of round-the-clock, rapid oral fluid HIV testing of women in labor in rural India.

Background Testing pregnant women for HIV at the time of labor and delivery is the last opportunity for prevention of mother-to-child HIV transmission (PMTCT) measures, particularly in settings where women do not receive adequate antenatal care. However, HIV testing and counseling of pregnant women in labor is a challenge, especially in resource-constrained settings. In India, many rural women present for delivery without any prior antenatal care. Those who do get antenatal care are not always tested for HIV, because of deficiencies in the provision of HIV testing and counseling services. In this context, we investigated the impact of introducing round-the-clock, rapid, point-of-care HIV testing and counseling in a busy labor ward at a tertiary care hospital in rural India. Methods and Findings After they provided written informed consent, women admitted to the labor ward of a rural teaching hospital in India were offered two rapid tests on oral fluid and finger-stick specimens (OraQuick Rapid HIV-1/HIV-2 tests, OraSure Technologies). Simultaneously, venous blood was drawn for conventional HIV ELISA testing. Western blot tests were performed for confirmatory testing if women were positive by both rapid tests and dual ELISA, or where test results were discordant. Round-the-clock (24 h, 7 d/wk) abbreviated prepartum and extended postpartum counseling sessions were offered as part of the testing strategy. HIV-positive women were administered PMTCT interventions. Of 1,252 eligible women (age range 18 y to 38 y) approached for consent over a 9 mo period in 2006, 1,222 (98%) accepted HIV testing in the labor ward. Of these, 1,003 (82%) women presented with either no reports or incomplete reports of prior HIV testing results at the time of admission to the labor ward. Of 1,222 women, 15 were diagnosed as HIV-positive (on the basis of two rapid tests, dual ELISA and Western blot), yielding a seroprevalence of 1.23% (95% confidence interval [CI] 0.61%–1.8%). Of the 15 HIV test–positive women, four (27%) had presented with reported HIV status, and 11 (73%) new cases of HIV infection were detected due to rapid testing in the labor room. Thus, 11 HIV-positive women received PMTCT interventions on account of round-the-clock rapid HIV testing and counseling in the labor room. While both OraQuick tests (oral and finger-stick) were 100% specific, one false-negative result was documented (with both oral fluid and finger-stick specimens). Of the 15 HIV-infected women who delivered, 13 infants were HIV seronegative at birth and at 1 and 4 mo after delivery; two HIV-positive infants died within a month of delivery. Conclusions In a busy rural labor ward setting in India, we demonstrated that it is feasible to introduce a program of round-the-clock rapid HIV testing, including prepartum and extended postpartum counseling sessions. Our data suggest that the availability of round-the-clock rapid HIV testing resulted in successful documentation of HIV serostatus in a large proportion (82%) of rural women who were unaware of their HIV status when admitted to the labor room. In addition, 11 (73%) of a total of 15 HIV-positive women received PMTCT interventions because of round-the-clock rapid testing in the labor ward. These findings are relevant for PMTCT programs in developing countries.

Evaluation of Diagnostic Accuracy, Feasibility and Client Preference for Rapid Oral Fluid-Based Diagnosis of HIV Infection in Rural India

Background Oral fluid-based rapid tests are promising for improving HIV diagnosis and screening. However, recent reports from the United States of false-positive results with the oral OraQuick® ADVANCE HIV1/2 test have raised concerns about their performance in routine practice. We report a field evaluation of the diagnostic accuracy, client preference, and feasibility for the oral fluid-based OraQuick® Rapid HIV1/2 test in a rural hospital in India. Methodology/Principal Findings A cross-sectional, hospital-based study was conducted in 450 consenting participants with suspected HIV infection in rural India. The objectives were to evaluate performance, client preference and feasibility of the OraQuick® Rapid HIV-1/2 tests. Two Oraquick® Rapid HIV1/2 tests (oral fluid and finger stick) were administered in parallel with confirmatory ELISA/Western Blot (reference standard). Pre- and post-test counseling and face to face interviews were conducted to determine client preference. Of the 450 participants, 146 were deemed to be HIV sero-positive using the reference standard (seropositivity rate of 32% (95% confidence interval [CI] 28%, 37%)). The OraQuick test on oral fluid specimens had better performance with a sensitivity of 100% (95% CI 98, 100) and a specificity of 100% (95% CI 99, 100), as compared to the OraQuick test on finger stick specimens with a sensitivity of 100% (95% CI 98, 100), and a specificity of 99.7% (95% CI 98.4, 99.9). The OraQuick oral fluid-based test was preferred by 87% of the participants for first time testing and 60% of the participants for repeat testing. Conclusion/Significance In a rural Indian hospital setting, the OraQuick® Rapid- HIV1/2 test was found to be highly accurate. The oral fluid-based test performed marginally better than the finger stick test. The oral OraQuick test was highly preferred by participants. In the context of global efforts to scale-up HIV testing, our data suggest that oral fluid-based rapid HIV testing may work well in rural, resource-limited settings.

Accuracy of physical signs for detecting meningitis: a hospital-based diagnostic accuracy study.

OBJECTIVES To evaluate accuracy of physical signs for detecting meningitis. PATIENTS AND METHODS We enrolled patients aged 12 years or more, admitted with acute encephalitis syndrome (fever, headache, altered mental status, vomiting, seizures, neurodeficit) to a rural teaching hospital. The design was a double-blind, cross-sectional analysis of consecutive patients, independently comparing signs of meningeal inflammation (nuchal rigidity, head jolt accentuation of headache, Kernigs sign and Brudzinskis sign) elicited by internal medicine residents against an established reference standard (cerebrospinal fluid white cell count >5 white cells/μL). Diagnostic accuracy was measured by computing sensitivity, specificity and likelihood ratios (LRs) and their 95% confidence interval (CI) values. RESULTS Of 190 patients (119 men, 71 women; ages 13-81 years; mean 38(SD 18) years) CSF analysis identified meningitis in 99 (52%; 95% CI 44, 59%) patients. No physical sign of meningeal irritation could accurately distinguish those with and without meningitis: nuchal rigidity (LR+ 1.33 (0.89, 1.98) and LR- 0.86 (0.70, 1.06)), head jolt accentuation of headache (LR+ 5.52 (0.67, 44.9) and LR- 0.95(0.89, 1.00)), Kernigs sign (LR+ 1.84 (0.77, 4.35) and LR- 0.93(0.84, 1.03)) and Brudzinskis sign (LR+ 1.69 (0.65, 4.37) and LR- 0.95 (0.87, 1.04)). CONCLUSION Physical signs of meningeal inflammation do not help clinicians rule in or rule out meningitis accurately. Patients suspected to have meningitis should undergo a lumbar puncture regardless of the presence or absence of physical signs.

Prevalence of Abnormal Radiological Findings in Health Care Workers with Latent Tuberculosis Infection and Correlations with T Cell Immune Response

Background More than half of all health care workers (HCWs) in high TB-incidence, low and middle income countries are latently infected with tuberculosis (TB). We determined radiological lesions in a cohort of HCWs with latent TB infection (LTBI) in India, and determined their association with demographic, occupational and T-cell immune response variables. Methodology We obtained chest radiographs of HCWs who had undergone tuberculin skin test (TST) and QuantiFERON-TB Gold In Tube (QFT), an interferon-γ release assay, in a previous cross-sectional study, and were diagnosed to have LTBI because they were positive by either TST or QFT, but had no evidence of clinical disease. Two observers independently interpreted these radiographs using a standardized data form and any discordance between them resolved by a third observer. The radiological diagnostic categories (normal, suggestive of inactive TB, and suggestive of active TB) were compared with results of TST, QFT assay, demographic, and occupational covariates. Results A total of 330 HCWs with positive TST or QFT underwent standard chest radiography. Of these 330, 113 radiographs (34.2%) were finally classified as normal, 206 (62.4%) had lesions suggestive of inactive TB, and 11 (3.4%) had features suggestive of active TB. The mean TST indurations and interferon-γ levels in the HCWs in these three categories were not significantly different. None of the demographic or occupational covariates was associated with prevalence of inactive TB lesions on chest radiography. Conclusion/Significance In a high TB incidence setting, nearly two-thirds of HCWs with latent TB infection had abnormal radiographic findings, and these findings had no clear correlation with T cell immune responses. Further studies are needed to verify these findings and to identify the causes and prognosis of radiologic abnormalities in health care workers.

Nosocomial Tuberculosis in India

India is well positioned to address the problem of nosocomial tuberculosis transmission.