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Objective:To compare the population-level impact of two World Health Organization-endorsed strategies for improving the diagnosis of tuberculosis (TB): same-day microscopy and Xpert MTB/RIF (Cepheid, USA).Methods:We created a compartmental transmission mod

Abstract Background Robust, flexible, and integrated health information (HIS) systems are essential to achieving national and international goals in health and development. Such systems are still uncommon in most low and middle income countries. This article describes a first-phase activity in Tanzania to integrate the country’s vertical health management information system with the help of an interoperability layer that enables cross-program data exchange. Methods From 2014 to 2019, the Tanzanian government and partners implemented a five-step procedure based on the “Mind the GAPS” (governance, architecture, program management, and standards) framework and using both proprietary and open-source tools. In collaboration with multiple stakeholders, the team developed the system to address major data challenges via four fully documented “use case scenarios” addressing data exchange among hospitals, between services and the supply chain, across digital data systems, and within the supply chain reporting system. This work included developing the architecture for health system data exchange, putting a middleware interoperability layer in place to facilitate the exchange, and training to support use of the system and the data it generates. Results Tanzania successfully completed the five-step procedure for all four use cases. Data exchange is currently enabled among 15 separate information systems, and has resulted in improved data availability and significant time savings. The government has adopted the health information exchange within the national strategy for health care information, and the system is being operated and managed by Tanzanian officials. Conclusion Developing an integrated HIS requires a significant time investment; but ultimately benefit both programs and patients. Tanzania’s experience may interest countries that are developing their HIS programs.

Objective To determine why health workers fail to follow integrated management of childhood illness (IMCI) guidelines for severely ill children at first-level outpatient health facilities in rural areas of the United Republic of Tanzania. Methods Retrospective and prospective case reviews of severely ill children aged < 5 years were conducted at health facilities in four districts. We ascertained treatment and examined the characteristics associated with referral, conducted follow-up interviews with parents of severely ill children, and gave health workers questionnaires and interviews. Findings In total, 502 cases were reviewed at 62 facilities. Treatment with antimalarials and antibiotics was consistent with the diagnosis given by health workers. However, of 240 children classified as having 'very severe febrile disease', none received all IMCI-recommended therapies, and only 25% of severely ill children were referred. Lethargy and anaemia diagnoses were independently associated with referral. Most (91%) health workers indicated that certain severe conditions can be managed without referral. Conclusion The health workers surveyed rarely adhered to IMCI treatment and referral guidelines for children with severe illness. They administered therapy based on narrow diagnoses rather than IMCI classifications, disagreed with referral guidelines and often considered referral unnecessary. To improve implementation of IMCI, attention should focus on the reasons for health worker non-adherence. Copyright © 2009 World Health Organization [ABSTRACT FROM AUTHOR]

A major challenge in tuberculosis (TB) therapeutics is that antibiotic exposure leads to changes in the physiologic state of M. tuberculosis ( Mtb ) which may enable the pathogen to withstand treatment. While antibiotic-treated Mtb have been evaluated in short-term in vitro experiments, it is unclear if and how long-term in vivo treatment with diverse antibiotics with varying treatment-shortening activity (sterilizing activity) affect Mtb physiologic states differently. Here, we used SEARCH-TB, a pathogen-targeted RNA-sequencing platform, to characterize the Mtb transcriptome in the BALB/c high-dose aerosol infection mouse model following 4-week treatment with three sterilizing and three non-sterilizing antibiotics. Certain transcriptional changes were concordant among most antibiotics, including decreased expression of genes associated with protein synthesis and metabolism, and the induction of certain genes associated with stress responses. However, the magnitude of this concordant response differed between antibiotics. Sterilizing antibiotics rifampin, pyrazinamide, and bedaquiline generated a more quiescent Mtb state than did non-sterilizing antibiotics isoniazid, ethambutol, and streptomycin, as indicated by decreased expression of genes associated with translation, transcription, secretion of immunogenic proteins, metabolism, and cell wall synthesis. Additionally, we identified distinguishing transcriptional effects specific to each antibiotic, indicating that different mechanisms of action induce distinct patterns of cellular injury. In addition to elucidating Mtb physiologic changes associated with antibiotic stress, this study demonstrates the value of SEARCH-TB as a highly granular pharmacodynamic assay that reveals antibiotic effects that are not apparent based on culture alone.

A major challenge for tuberculosis (TB) drug development is to prioritize promising combination regimens from a large and growing number of possibilities. This includes demonstrating individual drug contributions to the activity of higher-order combinations. A BALB/c mouse TB infection model was used to evaluate the contributions of each drug and pairwise combination in the clinically relevant Nix-TB regimen [bedaquiline-pretomanid-linezolid (BPaL)] during the first 3 weeks of treatment at human equivalent doses. The rRNA synthesis (RS) ratio, an exploratory pharmacodynamic (PD) marker of ongoing Mycobacterium tuberculosis rRNA synthesis, together with solid culture CFU counts and liquid culture time to positivity (TTP) were used as PD markers of treatment response in lung tissue; and their time-course profiles were mathematically modeled using rate equations with pharmacologically interpretable parameters. Antimicrobial interactions were quantified using Bliss independence and Isserlis formulas. Subadditive (or antagonistic) and additive effects on bacillary load, assessed by CFU and TTP, were found for bedaquiline-pretomanid and linezolid-containing pairs, respectively. In contrast, subadditive and additive effects on rRNA synthesis were found for pretomanid-linezolid and bedaquiline-containing pairs, respectively. Additionally, accurate predictions of the response to BPaL for all three PD markers were made using only the single-drug and pairwise effects together with an assumption of negligible three-way drug interactions. The results represent an experimental and PD modeling approach aimed at reducing combinatorial complexity and improving the cost-effectiveness of in vivo systems for preclinical TB regimen development., Competing Interests: N.D.W. and G.T.R. are listed as co-inventors on US patent no. 16/632,310 which pertains to the RS ratio.

Importance: A major reason that curing tuberculosis requires prolonged treatment is that drug exposure changes bacterial phenotypes. The physiologic adaptations of Mycobacterium tuberculosis that survive drug exposure in vivo have been obscure due to low sensitivity of existing methods in drug-treated animals. Using the novel SEARCH-TB RNA-seq platform, we elucidated Mycobacterium tuberculosis phenotypes in mice treated for with the global standard 4-drug regimen and compared them with the effect of the same regimen in vitro . This first view of the transcriptome of the minority Mycobacterium tuberculosis population that withstands treatment in vivo reveals adaptation of a broad range of cellular processes, including a shift in metabolism and cell wall modification. Surprisingly, the change in gene expression induced by treatment in vivo and in vitro was largely similar. This apparent "portability" from in vitro to the mouse provides important new context for in vitro transcriptional analyses that may support early preclinical drug evaluation., Competing Interests: The authors declare no conflict of interest.

Tuberculosis lung lesions are complex and harbor heterogeneous microenvironments that influence antibiotic effectiveness. Major strides have been made recently in understanding drug pharmacokinetics in pulmonary lesions, but the bacterial phenotypes that arise under these conditions and their contribution to drug tolerance are poorly understood. A pharmacodynamic marker called the RS ratio ® quantifies ongoing rRNA synthesis based on the abundance of newly synthesized precursor rRNA relative to mature structural rRNA. Application of the RS ratio in the C3HeB/FeJ mouse model demonstrated that Mycobacterium tuberculosis populations residing in different tissue microenvironments are phenotypically distinct and respond differently to drug treatment with rifampin, isoniazid, or bedaquiline. This work provides a foundational basis required to address how anatomic and pathologic microenvironmental niches may contribute to long treatment duration and drug tolerance during the treatment of human tuberculosis., Competing Interests: N.W.D., M.I.V., and G.T.R. are listed as co-inventors on US patent No. 16/632,310 that pertains to the RS ratio. The remaining authors have no conflicts of interest to declare.

The sigmoid E max model was used to describe the rRNA synthesis ratio (RS ratio) response of Mycobacterium tuberculosis to antimicrobial concentration. RS-E max measures the maximal ability of a drug to inhibit the RS ratio and can be used to rank-order drugs based on their RS ratio effect. RS-EC 90 is the concentration needed to achieve 90% of the RS-E max , which may guide dose selection to achieve a maximal RS ratio effect in vivo .

Murine tuberculosis drug efficacy studies have historically monitored bacterial burden based on CFU of Mycobacterium tuberculosis in lung homogenate. In an alternative approach, a recently described molecular pharmacodynamic marker called the RS ratio quantifies drug effect on a fundamental cellular process, ongoing rRNA synthesis. Here, we evaluated the ability of different pharmacodynamic markers to distinguish between treatments in three BALB/c mouse experiments at two institutions. We confirmed that different pharmacodynamic markers measure distinct biological responses. We found that a combination of pharmacodynamic markers distinguishes between treatments better than any single marker. The combination of the RS ratio with CFU showed the greatest ability to recapitulate the rank order of regimen treatment-shortening activity, providing proof of concept that simultaneous assessment of pharmacodynamic markers measuring different properties will enhance insight gained from animal models and accelerate development of new combination regimens. These results suggest potential for a new era in which antimicrobial therapies are evaluated not only on culture-based measures of bacterial burden but also on molecular assays that indicate how drugs impact the physiological state of the pathogen.

Droplet digital PCR is a particularly valuable tool for ratiometric assays because it provides simultaneous absolute quantification of two target sequences in a single assay. This manuscript addresses a challenge in establishing a new ratiometric droplet digital PCR assay for use in sputum, the rRNA synthesis ratio. In principle, the methods established to evaluate precision and determine the limit of quantification for a single measurand cannot be applied to a ratiometric assay. The precision of a ratio depends on precision in both the numerator and denominator. Here, we evaluated the MOVER approximated coefficient of variation as indicator of assay precision that does not require technical replicates. We estimated the MOVER approximated coefficient of variation in dilution series and routine assays and evaluated its agreement with the traditional coefficient of variation. We found that the MOVER approximated coefficient of variation was able to recapitulate the traditional coefficient of variation without the requirement for replicate assays. We also demonstrated that the MOVER approximated coefficient of variation threshold can be used to define the limit of quantification of the rRNA synthesis Ratio. In conclusion, the MOVER approximated coefficient of variation may be useful not only for the rRNA synthesis ratio but for other assays that measure ratios via droplet digital PCR., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

There is a critical need for improved pharmacodynamic markers for use in human tuberculosis (TB) drug trials. Pharmacodynamic monitoring in TB has conventionally used culture or molecular methods to enumerate the burden of Mycobacterium tuberculosis organisms in sputum. A recently proposed assay called the rRNA synthesis (RS) ratio measures a fundamentally novel property, how drugs impact ongoing bacterial rRNA synthesis. Here, we evaluated RS ratio as a potential pharmacodynamic monitoring tool by testing pretreatment sputa from 38 Ugandan adults with drug-susceptible pulmonary TB. We quantified the RS ratio in paired pretreatment sputa and evaluated the relationship between the RS ratio and microbiologic and molecular markers of M. tuberculosis burden. We found that the RS ratio was highly repeatable and reproducible in sputum samples. The RS ratio was independent of M. tuberculosis burden, confirming that it measures a distinct new property. In contrast, markers of M. tuberculosis burden were strongly associated with each other. These results indicate that the RS ratio is repeatable and reproducible and provides a distinct type of information from markers of M. tuberculosis burden. IMPORTANCE This study takes a major next step toward practical application of a novel pharmacodynamic marker that we believe will have transformative implications for tuberculosis. This article follows our recent report in Nature Communications that an assay called the rRNA synthesis (RS) ratio indicates the treatment-shortening of drugs and regimens. Distinct from traditional measures of bacterial burden, the RS ratio measures a fundamentally novel property, how drugs impact ongoing bacterial rRNA synthesis.

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.

The objective of this prospective cross-sectional study, conducted at a national referral hospital in Kampala, Uganda, was to determine diagnostic performance of serum C-reactive protein (CRP) as a triage test for tuberculosis (TB) among HIV-seronegative inpatients. We calculated the sensitivity, specificity, positive and negative likelihood ratios, and positive and negative predictive values to determine the diagnostic performance of a CRP enzyme-linked immunosorbent assay (ELISA) (Eurolyser) in comparison to that of a reference standard of Mycobacterium tuberculosis culture on two sputum samples. We constructed receiver operating curves and reported performance in reference to the manufacturer's cutoff and also to a threshold chosen to achieve sensitivity of >90%, in accordance with the WHO's target-product profile for a triage test. Among 119 HIV-seronegative inpatients, 46 (39%) had culture-positive pulmonary TB. In reference to M. tuberculosis culture, CRP had a sensitivity of 78% (95% confidence interval [CI], 64 to 89%) and a specificity of 52% (95% CI, 40 to 64%) at the manufacturer's threshold of 10 mg/liter. At a threshold of 1.5 mg/liter, the sensitivity was 91% (95% CI, 79 to 98%) but the specificity was only 21% (95% CI, 12 to 32%). Performance did not differ when stratified by illness severity at either threshold. In conclusion, among HIV-seronegative inpatients, CRP testing performed substantially below targets for a TB triage test. Additional studies among HIV-seronegative individuals in clinics and community settings are needed to assess the utility of CRP for TB screening., (Copyright © 2020 American Society for Microbiology.)

Background: HIV infection and opportunistic infections cause oxidative stress (OS), which is associated with tissue damage. Anti-retroviral therapy (ART) is used to treat HIV and decrease the risk of opportunistic infections, but it is unclear whether ART reduces OS. Association of ART with OS was investigated., Methods: We stratified a convenience sample of frozen serum or plasma from HIV-infected, ART-naïve (n=21); HIV-infected, ART-treated (n=14); HIV and PTB co-infected, ART-naïve (n=21); HIV and PTB co-infected, ART-treated (n=25) patients. Controls (n=21) were HIV-negative adults without TB symptoms. Concentration of OS markers namely: transaminases (ALT and AST), gamma glutamyl transpeptidase (GGT), albumin, total protein, malondialdehyde (MDA), vitamin C, and total anti-oxidant status (TAS) were determined., Results: AST (p<0.001), GGT (p<0.001), total protein (p=0.001) and MDA (p<0.001) were higher in HIV patients compared to controls. Vitamin C (P<0.0001) and albumin (p<0.01) were lower in HIV-patients relative to controls. ART was only associated with higher albumin (p=0.001), higher GGT (p=0.02) and lower vitamin C (p=0.009). HIV and PTB co-infection was only significantly associated with higher GGT (p=0.01) and AST (p=0.03)., Conclusion: We identified severe OS among HIV-patients. ART was associated with both increased and reduced markers of OS hence suggesting that ART may not attenuate OS.

Background: According to the traditional tuberculosis (TB) treatment paradigm, the initial doses of treatment rapidly kill most Mycobacterium tuberculosis (Mtb) bacilli in sputum, yet many more months of daily treatment are required to eliminate a small, residual subpopulation of drug-tolerant bacilli. This paradigm has recently been challenged following the discovery that up to 90% of Mtb bacilli in sputum are culturable only with growth-factor supplementation. These "differentially culturable" bacilli are hypothesized to be more drug-tolerant than routinely culturable bacilli. This hypothesis implies an alternative paradigm in which TB treatment does not rapidly reduce the total Mtb population but only the small, routinely culturable subpopulation. To evaluate these competing paradigms, we developed a culture-independent method for quantifying the viable fraction of Mtb bacilli in sputum during treatment., Methods: We used GeneXpert MTB/RIF to quantify Mtb DNA in sputa collected longitudinally from Ugandan adults taking standard 4-drug treatment for drug-susceptible pulmonary TB. We modeled GeneXpert cycle thresholds over time using nonlinear mixed-effects regression. We adjusted these models for clearance of DNA from killed-but-not-yet-degraded bacilli, assuming clearance half-lives ranging from 0 to 1.25 days. We used a convolution integral to quantify DNA from viable bacilli only, and converted cycle thresholds to Mtb genomic equivalents. We replicated our results in a South African cohort., Results: We enrolled 41 TB patients in Uganda. Assuming a DNA-clearance half-life of 0 days, genomic equivalents of viable sputum bacilli decreased by 0.22 log/day until 8.8 days, then by 0.07 log/day afterwards. Assuming a DNA-clearance half-life of 1.25 days, genomic equivalents of viable bacilli decreased by 0.36 log/day until 5.0 days, then by 0.06 log/day afterwards. By day 7, viable Mtb had decreased by 97.2-98.8%. We found similar results for 19 TB patients in South Africa., Discussion: Using a culture-independent method, we found that TB treatment rapidly eliminates most viable Mtb in sputum. These findings are incompatible with the hypothesis that differentially culturable bacilli are drug-tolerant., Conclusions: A culture-independent method for measuring viable Mtb in sputum during treatment corroborates the traditional TB treatment paradigm in which a rapid bactericidal phase precedes slow, elimination of a small, residual bacillary subpopulation.

A growing body of research suggests bacterial metabolism and membrane bioenergetics affect the lethality of a broad spectrum of antibiotics. Electrochemical gradients spanning energy-transducing membranes are the foundation of the chemiosmotic hypothesis and are essential for life; accordingly, their dysfunction appears to be a critical factor in bacterial death. Proton flux across energy-transducing membranes is central for cellular homeostasis as vectorial proton translocation generates a proton motive force used for ATP synthesis, pH homeostasis, and maintenance of solute gradients. Our recent investigations indicate that maintenance of pH homeostasis is a critical factor in antibiotic killing and suggest an imbalance in proton flux initiates disruptions in chemiosmotic gradients that lead to cell death. The complex and interconnected relationships between electron transport systems, central carbon metabolism, oxidative stress generation, pH homeostasis, and electrochemical gradients provide challenging obstacles to deciphering the roles for each of these processes in antibiotic lethality. In this chapter, we will present evidence for the pH homeostasis hypothesis of antibiotic lethality that bactericidal activity flows from disruption of cellular energetics and loss of chemiosmotic homeostasis. A holistic understanding of the interconnection of energetic processes and antibiotic activity may direct future research toward the development of more effective therapeutic interventions., (© 2018 Elsevier Ltd. All rights reserved.)

Mycobacterium africanum lineage (L) 6 is an important pathogen in West Africa, causing up to 40% of pulmonary tuberculosis (TB). The biology underlying the clinical differences between M. africanum and M. tuberculosis sensu stricto remains poorly understood. We performed ex vivo expression of 2179 genes of the most geographically dispersed cause of human TB, M. tuberculosis L4 and the geographically restricted, M. africanum L6 directly from sputa of 11 HIV-negative TB patients from The Gambia who had not started treatment. The DosR regulon was the most significantly decreased category in L6 relative to L4. Further, we identified nonsynonymous mutations in major DosR regulon genes of 44 L6 genomes of TB patients from The Gambia and Ghana. Using Lebek's test, we assessed differences in oxygen requirements for growth. L4 grew only at the aerobic surface while L6 grew throughout the medium. In the host, the DosR regulon is critical for M. tuberculosis in adaptation to oxygen limitation. However, M. africanum L6 appears to have adapted to growth under hypoxic conditions or to different biological niches. The observed under expression of DosR in L6 fits with the genomic changes in DosR genes, microaerobic growth and the association with extrapulmonary disease., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mycobacterium avium subsp. hominissuis is an environmentally acquired bacterium known to cause pulmonary and soft tissue infections, lymphadenitis, and disseminated disease in humans. We report here the complete genome sequence of strain H87, isolated from an indoor water sample, as a single circular chromosome of 5,626,623 bp with a G+C content of 68.8%., (Copyright © 2017 Zhao et al.)

Treatment of non-tuberculous mycobacterial lung disease (NTM-LD) is challenging for several reasons including the relative resistance of NTM to currently available drugs and the difficulty in tolerating prolonged treatment with multiple drugs. Yet-to-be-done, large, multicenter, prospective randomized studies to establish the best regimens will also be arduous because multiple NTM species are known to cause human lung disease, differences in virulence and response to treatment between different species and strains within a species will make randomization more difficult, the need to distinguish relapse from a new infection, and the difficulty in adhering to the prescribed treatment due to intolerance, toxicity, and/or drug-drug interactions, often necessitating modification of therapeutic regimens. Furthermore, the out-of-state resident status of many patients seen at the relatively few centers that care for large number of NTM-LD patients pose logistical issues in monitoring response to treatment. Thus, current treatment regimens for NTM-LD is largely based on small case series, retrospective analyses, and guidelines based on expert opinions. It has been nearly 10 years since the publication of a consensus guideline for the treatment of NTM-LD. This review is a summary of the available evidence on the treatment of the major NTM-LD until more definitive studies and guidelines become available., Competing Interests: Conflict of Interest Dr. Julie V. Philley, Dr. Jennifer R. Honda, Dr. Michael M. Chan, Dr. Shannon Kasperbauer, Dr. Nicholas D. Walter, and Dr. Edward D. Chan declare that they have no conflict of interest. Dr. Mary Ann DeGroote is a co-PI for the pre-clinical evaluation of new therapeutic entities for NTM therapeutics with Crestonepharma Inc. This is an SBIR phase II grant. There is no overlap with antimicrobial agents described in this review.

Background: It is unknown whether immunosuppression influences the physiologic state of Mycobacterium tuberculosis in vivo. We evaluated the impact of host immunity by comparing M. tuberculosis and human gene transcription in sputum between human immunodeficiency virus (HIV)-infected and uninfected patients with tuberculosis., Methods: We collected sputum specimens before treatment from Gambians and Ugandans with pulmonary tuberculosis, revealed by positive results of acid-fast bacillus smears. We quantified expression of 2179 M. tuberculosis genes and 234 human immune genes via quantitative reverse transcription-polymerase chain reaction. We summarized genes from key functional categories with significantly increased or decreased expression., Results: A total of 24 of 65 patients with tuberculosis were HIV infected. M. tuberculosis DosR regulon genes were less highly expressed among HIV-infected patients with tuberculosis than among HIV-uninfected patients with tuberculosis (Gambia, P < .0001; Uganda, P = .037). In profiling of human genes from the same sputa, HIV-infected patients had 3.4-fold lower expression of IFNG (P = .005), 4.9-fold higher expression of ARG1 (P = .0006), and 3.4-fold higher expression of IL10 (P = .0002) than in HIV-uninfected patients with tuberculosis., Conclusions: M. tuberculosis in HIV-infected patients had lower expression of the DosR regulon, a critical metabolic and immunomodulatory switch induced by NO, carbon monoxide, and hypoxia. Our human data suggest that decreased DosR expression may result from alternative pathway activation of macrophages, with consequent decreased NO expression and/or by poor granuloma formation with consequent decreased hypoxic stress., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Pathogen-targeted transcriptional profiling in human sputum may elucidate the physiologic state of Mycobacterium tuberculosis (M. tuberculosis) during infection and treatment. However, whether M. tuberculosis transcription in sputum recapitulates transcription in the lung is uncertain. We therefore compared M. tuberculosis transcription in human sputum and bronchoalveolar lavage (BAL) samples from 11 HIV-negative South African patients with pulmonary tuberculosis. We additionally compared these clinical samples with in vitro log phase aerobic growth and hypoxic non-replicating persistence (NRP-2). Of 2179 M. tuberculosis transcripts assayed in sputum and BAL via multiplex RT-PCR, 194 (8.9%) had a p-value <0.05, but none were significant after correction for multiple testing. Categorical enrichment analysis indicated that expression of the hypoxia-responsive DosR regulon was higher in BAL than in sputum. M. tuberculosis transcription in BAL and sputum was distinct from both aerobic growth and NRP-2, with a range of 396-1020 transcripts significantly differentially expressed after multiple testing correction. Collectively, our results indicate that M. tuberculosis transcription in sputum approximates M. tuberculosis transcription in the lung. Minor differences between M. tuberculosis transcription in BAL and sputum suggested lower oxygen concentrations or higher nitric oxide concentrations in BAL. M. tuberculosis-targeted transcriptional profiling of sputa may be a powerful tool for understanding M. tuberculosis pathogenesis and monitoring treatment responses in vivo., Competing Interests: Authors declare no conflicts of interest., (Published by Elsevier Ltd.)

Rationale: In 2007, World Health Organization (WHO) issued emergency recommendations on empiric treatment of sputum acid-fast bacillus smear-negative patients with possible tuberculosis (TB) in HIV-prevalent areas, and called for operational research to evaluate their effectiveness. We sought to determine if early, empiric TB treatment of possible TB patients with abnormal chest radiography or severe illness as suggested by the 2007 WHO guidelines, is associated with improved survival., Methods: We prospectively enrolled consecutive HIV-seropositive inpatients at Mulago Hospital in Kampala, Uganda, from 2007 to 2011 with cough for ≥2 weeks. We retrospectively examined the effect of empiric TB treatment before discharge on 8-week survival among those with and without a WHO-defined "danger sign," including fever >39°C, tachycardia >120 beats per minute, or tachypnea >30 breaths per minute. We modeled the interaction between empiric TB treatment and danger signs, and their combined effect on 8-week survival, and adjusted for relevant covariates., Results: Among 631 sputum smear-negative patients, 322 (51%) had danger signs. Cumulative 8-week survival of patients with danger signs was significantly higher with empiric TB treatment (80%) than without treatment (64%, P < 0.001). After adjusting for duration of cough and concurrent hypoxemia, patients with danger signs who received empiric TB treatment had a 44% reduction in 8-week mortality (risk ratio 0.56, 95% confidence interval: 0.34-0.91, P = 0.020)., Conclusions: Empiric TB treatment of HIV-seropositive, smear-negative, presumed pulmonary TB patients with 1 or more danger signs is associated with improved 8-week survival. Enhanced implementation of the 2007 WHO empiric treatment recommendations should be encouraged whenever and wherever rapid and highly sensitive diagnostic tests for TB are unavailable.

Unlabelled: Blood transcriptional signatures are promising for tuberculosis (TB) diagnosis but have not been evaluated among U.S., Patients: To be used clinically, transcriptional classifiers need reproducible accuracy in diverse populations that vary in genetic composition, disease spectrum and severity, and comorbidities. In a prospective case-control study, we identified novel transcriptional classifiers for active TB among U.S. patients and systematically compared their accuracy to classifiers from published studies. Blood samples from HIV-uninfected U.S. adults with active TB, pneumonia, or latent TB infection underwent whole-transcriptome microarray. We used support vector machines to classify disease state based on transcriptional patterns. We externally validated our classifiers using data from sub-Saharan African cohorts and evaluated previously published transcriptional classifiers in our population. Our classifier distinguishing active TB from pneumonia had an area under the concentration-time curve (AUC) of 96.5% (95.4% to 97.6%) among U.S. patients, but the AUC was lower (90.6% [89.6% to 91.7%]) in HIV-uninfected Sub-Saharan Africans. Previously published comparable classifiers had AUC values of 90.0% (87.7% to 92.3%) and 82.9% (80.8% to 85.1%) when tested in U.S., Patients: Our classifier distinguishing active TB from latent TB had AUC values of 95.9% (95.2% to 96.6%) among U.S. patients and 95.3% (94.7% to 96.0%) among Sub-Saharan Africans. Previously published comparable classifiers had AUC values of 98.0% (97.4% to 98.7%) and 94.8% (92.9% to 96.8%) when tested in U.S., Patients: Blood transcriptional classifiers accurately detected active TB among U.S. adults. The accuracy of classifiers for active TB versus that of other diseases decreased when tested in new populations with different disease controls, suggesting additional studies are required to enhance generalizability. Classifiers that distinguish active TB from latent TB are accurate and generalizable across populations and can be explored as screening assays., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Treatment initiation rapidly kills most drug-susceptible Mycobacterium tuberculosis, but a bacterial subpopulation tolerates prolonged drug exposure. We evaluated drug-tolerant bacilli in human sputum by comparing messenger RNA (mRNA) expression of drug-tolerant bacilli that survive the early bactericidal phase with treatment-naive bacilli., Methods: M. tuberculosis gene expression was quantified via reverse-transcription polymerase chain reaction in serial sputa from 17 Ugandans treated for drug-susceptible pulmonary tuberculosis., Results: Within 4 days, bacterial mRNA abundance declined >98%, indicating rapid killing. Thereafter, the rate of decline slowed >94%, indicating drug tolerance. After 14 days, 16S ribosomal RNA transcripts/genome declined 96%, indicating slow growth. Drug-tolerant bacilli displayed marked downregulation of genes associated with growth, metabolism, and lipid synthesis and upregulation in stress responses and key regulatory categories-including stress-associated sigma factors, transcription factors, and toxin-antitoxin genes. Drug efflux pumps were upregulated. The isoniazid stress signature was induced by initial drug exposure, then disappeared after 4 days., Conclusions: Transcriptional patterns suggest that drug-tolerant bacilli in sputum are in a slow-growing, metabolically and synthetically downregulated state. Absence of the isoniazid stress signature in drug-tolerant bacilli indicates that physiological state influences drug responsiveness in vivo. These results identify novel drug targets that should aid in development of novel shorter tuberculosis treatment regimens., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objective: Pre-immigration tuberculosis (TB) screening, followed by post-arrival rescreening during the first year, is critical to reducing TB among foreign-born people in the United States. However, existing U.S. public health surveillance is inadequate to monitor TB among immigrants during subsequent years. We developed and tested a novel method for ascertaining post-U.S.-arrival TB outcomes among high-TB-risk immigrant cohorts to improve surveillance., Methods: We used a probabilistic record linkage program to link pre-immigration screening records from U.S.-bound immigrants from the Philippines (n=422,593) and Vietnam (n=214,401) with the California TB registry during 2000-2010. We estimated sensitivity using Monte Carlo simulations to account for uncertainty in key inputs. Specificity was evaluated by using a time-stratified approach, which defined false-positives as TB records linked to pre-immigration screening records dated after the person had arrived in the United States., Results: TB was reported in 4,382 and 2,830 people born in the Philippines and Vietnam, respectively, in California during the study period. Of these TB cases, records for 973 and 452 cases of people born in the Philippines and Vietnam, respectively, were linked to pre-immigration screening records. Sensitivity and specificity of linkage were 89% (90% credible interval [CrI] 83, 97) and 100%, respectively, for the Philippines, and 90% (90% CrI 83, 98) and 99.9%, respectively, for Vietnam., Conclusion: Electronic linkage of pre-immigration screening records to a domestic TB registry was feasible, sensitive, and highly specific in two high-priority immigrant cohorts. Transnational record linkage can be used for program evaluation and routine monitoring of post-U.S.-arrival TB risk among immigrants, but requires interagency data sharing and collaboration.

Advances in multiplex qRT-PCR have enabled increasingly accurate and robust quantification of RNA, even at lower concentrations, facilitating RNA expression profiling in clinical and environmental samples. Here we describe a data-driven qRT-PCR normalization method, the minimum variance method, and evaluate it on clinically derived Mycobacterium tuberculosis samples with variable transcript detection percentages. For moderate to significant amounts of nondetection (∼50%), our minimum variance method consistently produces the lowest false discovery rates compared to commonly used data-driven normalization methods., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Current guidelines limit latent tuberculosis infection (LTBI) evaluation to persons in the United States less than or equal to 5 years based on the assumption that high TB rates among recent entrants are attributable to high LTBI reactivation risk, which declines over time. We hypothesized that high postarrival TB rates may instead be caused by imported active TB., Objectives: Estimate reactivation and imported TB in an immigrant cohort., Methods: We linked preimmigration records from a cohort of California-bound Filipino immigrants during 2001-2010 with subsequent TB reports. TB was likely LTBI reactivation if the immigrant had no evidence of active TB at preimmigration examination, likely imported if preimmigration radiograph was abnormal and TB was reported less than or equal to 6 months after arrival, and likely reactivation of inactive TB if radiograph was abnormal but TB was reported more than 6 months after arrival., Measurements and Main Results: Among 123,114 immigrants, 793 TB cases were reported. Within 1 year of preimmigration examination, 85% of TB was imported; 6 and 9% were reactivation of LTBI and inactive TB, respectively. Conversely, during Years 2-9 after U.S. entry, 76 and 24% were reactivation of LTBI and inactive TB, respectively. The rate of LTBI reactivation (32 per 100,000) did not decline during Years 1-9., Conclusions: High postarrival TB rates were caused by detection of imported TB through active postarrival surveillance. Among immigrants without active TB at baseline, reported TB did not decline over 9 years, indicating sustained high risk of LTBI reactivation. Revised guidelines should support LTBI screening and treatment more than 5 years after U.S. arrival.

Adithya Cattamanchi and colleagues reflect on recent research by Michael Levin and coworkers into the use of whole blood mRNA expression signatures to detect tuberculosis. The authors highlight challenges faced in getting this promising technology into clinics in low-resource settings. Please see later in the article for the Editors' Summary.

Objective: To compare the population-level impact of two World Health Organization-endorsed strategies for improving the diagnosis of tuberculosis (TB): same-day microscopy and Xpert MTB/RIF (Cepheid, USA)., Methods: We created a compartmental transmission model of TB in a representative African community, fit to the regional incidence and mortality of TB and HIV. We compared the population-level reduction in TB burden over ten years achievable with implementation over two years of same-day microscopy, Xpert MTB/RIF testing, and the combination of both approaches., Findings: Same-day microscopy averted an estimated 11.0% of TB incidence over ten years (95% uncertainty range, UR: 3.3%-22.5%), and prevented 11.8% of all TB deaths (95% UR: 7.7%-27.1%). Scaling up Xpert MTB/RIF to all centralized laboratories to achieve 75% population coverage had similar impact on incidence (9.3% reduction, 95% UR: 1.9%-21.5%) and greater effect on mortality (23.8% reduction, 95% UR: 8.6%-33.4%). Combining the two strategies (i.e., same-day microscopy plus Xpert MTB/RIF) generated synergistic effects: an 18.7% reduction in incidence (95% UR: 5.6%-39.2%) and 33.1% reduction in TB mortality (95% UR: 18.1%-50.2%). By the end of year ten, combining same-day microscopy and Xpert MTB/RIF could reduce annual TB mortality by 44% relative to the current standard of care., Conclusion: Scaling up novel diagnostic tests for TB and optimizing existing ones are complementary strategies that, when combined, may have substantial impact on TB epidemics in Africa.

Background: Cryptococcal infection occurs in HIV-seropositive patients and is associated with high mortality. However, limited information is available on the prevalence and outcomes of cryptococcal antigenemia among hospitalized HIV-seropositive patients in sub-Saharan Africa., Objectives: To determine the prevalence of and risk factors for cryptococcal antigenemia among HIV-seropositive patients presenting to Mulago Hospital (Kampala, Uganda) with unexplained cough ≥2 weeks and suspected tuberculosis (TB) and also to determine if antigenemia is associated with an increased mortality., Methods: Between September 2009 and September 2010, we enrolled consecutive HIV-seropositive adults hospitalized at Mulago Hospital with cough ≥2 weeks and suspected TB. Banked serum was tested for cryptococcal antigen. We compared demographic and clinical characteristics, and 2-month mortality in patients with and without cryptococcal antigenemia., Results: Of 563 HIV-seropositive patients, 32 (5.7%) were cryptococcal antigen (CrAg) positive. None had Cryptococcus neoformans detected on fungal culture of bronchoalveolar lavage fluid (n = 116). CrAg-positive patients had a lower median CD4 count compared with CrAg-negative patients (25 vs. 55 cells/μL, P = 0.02), and a substantial proportion of CrAg-positive patients also had concurrent TB (31%). A positive CrAg test was not associated with increased mortality during the 2-month follow-up period (hazard ratio: 0.99, 95% confidence interval: 0.63 to 1.54, P = 0.95) after adjusting for CD4 count and antiretroviral therapy use at enrollment and/or follow-up., Conclusions: Occult cryptococcal antigenemia occurs commonly among hospitalized HIV-seropositive patients with suspected TB. CrAg testing should be considered in hospitalized HIV-seropositive patients with CD4 count <50 cells/μL, coupled with longer follow-up to evaluate the diagnostic value of CrAg and therapeutic interventions in patients with asymptomatic cryptococcal antigenemia.

Multidrug (MDR)- and extensively drug-resistant (XDR) tuberculosis (TB) impose a heavy toll of human suffering and social costs. Controlling drug-resistant TB is a complex global public health challenge. Basic science advances including elucidation of the genetic basis of resistance have enabled development of new assays that are transforming the diagnosis of MDR-TB. Molecular epidemiological approaches have provided new insights into the natural history of TB with important implications for drug resistance. In the future, progress in understanding Mycobacterium tuberculosis strain-specific human immune responses, integration of systems biology approaches with traditional epidemiology and insight into the biology of mycobacterial persistence have potential to be translated into new tools for diagnosis and treatment of MDR- and XDR-TB. We review recent basic sciences developments that have contributed or may contribute to improved public health response., (© 2012 The Authors. Respirology © 2012 Asian Pacific Society of Respirology.)

Background: Although seasonal variation in tuberculosis incidence has been described in several recent studies, the mechanism underlying this seasonality remains unknown. Seasonality of tuberculosis disease may indicate the presence of season-specific risk factors that could potentially be controlled if they were better understood. We conducted this study to determine whether tuberculosis is seasonal in the United States and to describe patterns of seasonality in specific populations., Methods: We performed a time series decomposition analysis of tuberculosis cases reported to the Centers for Disease Control and Prevention from 1993 through 2008. Seasonal amplitude of tuberculosis disease (the difference between the months with the highest and lowest mean case counts), was calculated for the population as a whole and for populations with select demographic, clinical, and epidemiologic characteristics., Results: A total of 243 432 laboratory-confirmed tuberculosis cases were reported over a period of 16 years. A mean of 21.4% more cases were diagnosed in March, the peak month, compared with November, the trough month. The magnitude of seasonality did not vary with latitude. The greatest seasonal amplitude was found among children aged <5 years and in cases associated with disease clusters., Conclusions: Tuberculosis is a seasonal disease in the United States, with a peak in spring and trough in late fall. The latitude independence of seasonality suggests that reduced winter sunlight exposure may not be a strong contributor to tuberculosis risk. Increased seasonality among young children and clustered cases suggests that disease that is the result of recent transmission is more influenced by season than disease resulting from activation of latent infection.

Inflammatory oncotaxis, the phenomenon in which mechanically injured tissues are predisposed to cancer metastases, has been reported for a number of tumor types, but not previously for histologically proven lung cancer. We review clinical and experimental evidence and mechanisms that may underlie inflammatory oncotaxis, and provide illustrative examples of two patients with squamous cell carcinoma of the lung who developed distant, localized metastatic disease at sites of recent physical trauma. Trauma may predispose to metastasis through two distinct, but not mutually exclusive, mechanisms: (1) physical trauma induces tissue damage and local inflammation, creating a favorable environment that is permissive for seeding of metastatic cells from distant sites; and/or (2) micrometastatic foci are already present at the time of physical injury, and trauma initiates changes in the microenvironment that stimulate the proliferation of the metastatic cells. Further exploration of post-traumatic inflammatory oncotaxis may elucidate fundamental mechanisms of metastasis and could provide novel strategies to prevent cancer metastasis.

Background: Animal models and data from influenza pandemics suggest that influenza infection predisposes individuals to pneumococcal pneumonia. Influenza may contribute to high winter rates of pneumococcal pneumonia during non-pandemic periods, but the magnitude of this effect is unknown. With use of United States surveillance data during 1995-2006, we estimated the association between influenza circulation and invasive pneumococcal pneumonia rates., Methods: Weekly invasive pneumococcal pneumonia incidence, defined by isolation of pneumococci from normally sterile sites in persons with clinical or radiographic pneumonia, was estimated from active population-based surveillance in 3 regions of the United States. We used influenza virus data collected by World Health Organization collaborating laboratories in the same 3 regions in seasonally adjusted negative binomial regression models to estimate the influenza-associated fraction of pneumococcal pneumonia., Results: During approximately 185 million person-years of surveillance, we observed 21,239 episodes of invasive pneumococcal pneumonia; 485,691 specimens were tested for influenza. Influenza circulation was associated with 11%-14% of pneumococcal pneumonia during periods of influenza circulation and 5%-6% overall. In 2 of 3 regions, the association was strongest when influenza circulation data were lagged by 1 week., Conclusions: During recent seasonal influenza epidemics in the United States, a modest but potentially preventable fraction of invasive pneumococcal pneumonia was associated with influenza circulation.

Background: Mycoplasma pneumoniae continues to be a significant cause of community-acquired pneumonia (CAP). A more definitive methodology for reliable detection of M. pneumoniae is needed to identify outbreaks and to prevent potentially fatal extrapulmonary complications., Methods: We analyzed 2 outbreaks of CAP due to M. pneumoniae. Nasopharyngeal and/or oropharyngeal swab specimens and serum samples were obtained from persons with clinically defined cases, household contacts, and asymptomatic individuals. Real-time polymerase chain reaction (PCR) for M. pneumoniae was performed on all swab specimens, and the diagnostic utility was compared with that of 2 commercially available serologic test kits., Results: For cases, 21% yielded positive results with real-time PCR, whereas 81% and 54% yielded positive results with the immunoglobulin M and immunoglobulin G/immunoglobulin M serologic tests, respectively. For noncases, 1.8% yielded positive results with real-time PCR, whereas 63% and 79% yielded serologically positive results with the immunoglobulin M and immunoglobulin G/immunoglobulin M kits, respectively. The sensitivity of real-time PCR decreased as the duration between symptom onset and sample collection increased, with a peak sensitivity of 48% at 0-21 days. A specificity of 43% for the immunoglobulin M antibody detection assay was observed for persons aged 10-18 years, but the sensitivity increased to 82% for persons aged 19 years., Discussion: Thorough data analysis indicated that no single available test was reliable for the identification of an outbreak of CAP due to M. pneumoniae. A combination of testing methodologies proved to be the most reliable approach for identification of outbreaks of CAP due to M. pneumoniae, especially in the absence of other suspected respiratory pathogens.

Background: We investigated an outbreak of severe neurologic disease and pneumonia that occurred among students at 4 schools in Rhode Island., Methods: We identified cases of encephalitis, encephalomyelitis, and pneumonia that occurred among schoolchildren from 1 September 2006 through 9 February 2007, and we performed serologic tests, polymerase chain reaction (PCR) analysis, and culture for the detection of multiple pathogens in oropharyngeal and nasopharyngeal specimens. Students with positive results of M. pneumoniae IgM serologic testing and no alternative diagnosis were considered to be infected with M. pneumoniae. At school A, we used questionnaires to identify students and their household contacts who made visits to physicians for pneumonia and cough. We compared observed and expected rates of pneumonia., Results: Rates of pneumonia among elementary students (122 cases/1000 student-years) were > 5-fold higher than expected. Three students had encephalitis or encephalomyelitis, and 76 had pneumonia. Of these 2 groups of students, 2 (66%) and 57 students (75%), respectively, had M. pneumoniae infection. M. pneumoniae was detected by PCR in 10 students with pneumonia; 5 of these specimens were cultured, and M. pneumoniae was isolated in 4. Of 202 households of students attending school A, 20 (10%) accounted for 61% of visits to physicians for pneumonia or cough. Of 19 household contacts of students with pneumonia, 8 (42%) developed pneumonia and 6 (32%) reported visits for cough., Conclusions: M. pneumoniae caused a community-wide outbreak of cough illness and pneumonia and was associated with the development of life-threatening neurologic disease. Although M. pneumoniae was detected in schools, its transmission in households amplified the outbreak. Interrupting household transmission should be a priority during future outbreaks.

Analysis of whether assiduous implementation of American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America guidelines for targeted testing and treatment of latent tuberculosis infection could have prevented any of 223 cases of active tuberculosis in foreign-born persons in San Francisco during the period 2002-2003. We report that 62% of these cases were not preventable and conclude that a further reduction in the incidence of tuberculosis among foreign-born persons will be modest without modification of current guidelines.