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Several tuberculosis vaccines have been shown to reduce the risk of disease and death due to tuberculosis in humans, but only one is used in global immunization programs: Mycobacterium bovis bacillus Calmette-Guerin (BCG). The development of new tuberculosis vaccines has been informed by evolving data on the natural history of tuberculosis, by new data on the immunology of infection with and immunization against Mycobacterium tuberculosis, by reanalysis of the role of nontuberculous mycobacteria (NTM) in protection against tuberculosis, by a clearer understanding of the benefits and risks of BCG, and by molecular techniques that have permitted identification of immunodominant antigens of M. tuberculosis and new methods of antigen delivery. This chapter talks about tuberculosis in children, older children, adults, elderly, and immunocompromised hosts, health care providers and other International travelers. At least partial immune protection from tuberculosis disease results from prior mycobacterial infection, whether naturally acquired or vaccine induced. The chapter discusses mechanisms of immune control of tuberculosis, and talks about BCG vaccine. Immunization with BCG induces a mild systemic infection in healthy hosts. It has been shown that immune responses to BCG are impaired in infants with human immunodeficiency virus (HIV) infection. The efficacy of BCG against tuberculosis is described. The favorable and unfavorable characteristics of BCG are summarized. There is wide consensus that additional tuberculosis vaccine development should focus on identifying a booster vaccine to follow BCG immunization, or on developing a completely novel two-vaccine booster regimen.

Background SRL172 prevented disease due to Mycobacterium tuberculosis in a Phase 3 trial. DAR-901 represents a scalable manufacturing process for SRL172. We sought to determine if DAR-901 would prevent infection with M. tuberculosis among BCG-primed adolescents age 13–15 years in Tanzania. Methods Adolescents with a negative T- SPOT.TBR interferon gamma release assay (IGRA) were randomized 1:1 to three intradermal injections of DAR-901 or saline placebo at 0, 2 and 4 months. Repeat IGRAs were performed at 2 months, and at 1, 2, and 3 years. The primary efficacy outcome was time to new TB infection (IGRA conversion to positive); the secondary outcome was time to persistent TB infection (IGRA conversion with repeat positive IGRA). Results Among 936 participants screened 667 were eligible and randomized to their first dose of vaccine or placebo (safety cohort). At 2 months, 625 participants remained IGRA-negative and were scheduled for the additional two doses (efficacy cohort). DAR-901 was safe and well-tolerated. One DAR-901 recipient developed a vaccine site abscess. Neither the primary nor secondary endpoints differed between the two treatment arms (p = 0.90 and p = 0.20, respectively). DAR-901 IGRA converters had median responses to ESAT-6 of 50.1 spot-forming cells (SFCs) vs. 19.6 SFCs in placebo IGRA converters (p = 0.03). Conclusions A three-dose series of 1 mg DAR-901 was safe and well-tolerated but did not prevent initial or persistent IGRA conversion. DAR-901 recipients with IGRA conversion demonstrated enhanced immune responses to ESAT-6. Since protection against disease may require different immunologic responses than protection against infection a trial of DAR-901 to prevent TB disease is warranted. Trial Registration. The trial is registered at ClinicalTrials.gov as NCT02712424.

Background Ratios of different immune cell populations (i.e., monocyte-to-lymphocyte, neutrophil-to-lymphocyte, and platelet-to-lymphocyte ratios) have been studied as a means of predicting future tuberculosis (TB) disease risk or to assist in the diagnosis of incident TB disease. No studies to-date, however, have evaluated the potential of these ratios to predict or assist in the diagnosis of incident TB infection - the first step in the natural history of TB disease. Methods In this prospective study, we evaluated the complete blood count (CBC)-derived metrics of monocyte-to-lymphocyte ratio (MLR), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) as predictors of future TB infection risk or aids in the diagnosis of TB infection among 145 Tanzanian adolescents enrolled in the DAR-901 vaccine trial, using paired CBCs and interferon-gamma release assays (IGRAs) obtained at 0, 60 and 720 days after study enrollment. Results At baseline, there were no significant differences between study participants who remained persistently IGRA negative throughout the study period and those who subsequently converted to IGRA positive with respect to MLR (0.18 vs 0.17, p = 0.10), NLR (0.88 vs 1.02, p = 0.08), or PLR (115 vs 120, p = 0.28). Similarly, no significant differences were noted with respect to MLR, NLR, and PLR between IGRA converters and time-matched negative controls at the time of IGRA conversion. With respect to other blood cell measures, however, there were modest but significant differences between IGRA negatives and IGRA converters with respect to red blood cell count (4.8 vs 4.6 × 106 cells/mcL, p = 0.008), hemoglobin (12.6 vs 12.3 g/dL, p = 0.01), and hematocrit (38.8 vs 37.8%, p = 0.005). Conclusions In contrast to prior studies that have suggested that the ratios of different immune cell populations are associated with development of TB disease, our present findings do not demonstrate an association between these ratios and the development of TB infection. However, decreased red blood cell measures were associated with the subsequent development of TB infection, suggesting either that dysregulation of iron metabolism may play a role in TB pathogenesis or that following TB infection, iron dysregulation may precede IGRA positivity. Trial registration Clinicaltrials.gov NCT02712424. Date of registration: March 14, 2016.

BackgroundProspective studies of interferon-gamma release assays (IGRA) on healthy subjects in tuberculosis-endemic regions have not examined the long-term variability of serial assays. This issue is relevant to the interpretation of tuberculosis (TB) vaccine trials based on prevention of infection.MethodsT-SPOT.TB assays were performed manually on healthy adolescents during a tuberculosis vaccine trial in Tanzania at 5 intervals over 3 years. Assay results were defined as negative, positive, borderline or invalid. Subsequently, microtiter plates were analyzed by an automated reader to obtain quantitative counts of spot forming cells (SFCs) for the present analysis.Results3387 T-SPOT.TB samples were analyzed from 928 adolescents; manual and automated assay results were 97% concordant. Based on the quantitative results 143 (15%) participants were prevalent IGRA-positives at baseline, were ineligible for further study. Among the remaining IGRA-negative participants, the annual rate of IGRA conversion was 2·9%. Among 43 IGRA converters with repeat assays 12 (28%) were persistent converters, 16 (37%) were transient converters, and 15 (35%) comprised a new category defined as irregular converters (≥2 different subsequent results). ESAT-6 and CFP-10 responses were higher in prevalent than incident positives: 53 vs 36 for CFP-10 (p< 0·007); 44 vs 34 for ESAT-6 (p= 0·12).ConclusionsDefinitions of IGRA conversion, reversion, and persistence depend critically on the frequency of testing. Multiple shifts in categories among adolescents in a TB-endemic country may represent multiple infections, variable host responses in subclinical infection, or assay variation. These findings should to be considered in the design and interpretation of TB vaccine trials based on prevention of infection. Household contact studies could determine whether even transient IGRA conversion might represent exposure to an active case ofM.tuberculosisdisease.

An amendment to this paper has been published and can be accessed via the original article.

Background Tuberculosis (TB) is the most deadly infectious disease globally and is highly prevalent in the developing world. For individuals infected with both Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV), the risk of active TB is 10% or more annually. Previously, we identified in a genome-wide association study (GWAS) a region on chromosome 5 associated with resistance to TB, which included epigenetic marks that could influence gene regulation. We hypothesized that HIV-infected individuals exposed to Mtb who remain disease free carry epigenetic changes that strongly protect them from active TB. Methods We conducted a methylome-wide study in HIV-infected, TB-exposed cohorts from Uganda and Tanzania and integrated data from our GWAS. Results We identified 3 regions of interest that included markers that were differentially methylated between TB cases and controls with latent TB infection: chromosome 1 (RNF220, P = 4 × 10–5), chromosome 2 (between COPS8 and COL6A3, P = 2.7 × 10–5), and chromosome 5 (CEP72, P = 1.3 × 10–5). These methylation results co-localized with associated single-nucleotide polymorphisms (SNPs), methylation QTLs, and methylation × SNP interaction effects. These markers were in regions with regulatory markers for cells involved in TB immunity and/or lung. Conclusions Epigenetic regulation is a potential biologic factor underlying resistance to TB in immunocompromised individuals that can act in conjunction with genetic variants.

Background: Ratios of different immune cell populations (i.e., monocyte-to-lymphocyte, neutrophil-to-lymphocyte, and platelet-to-lymphocyte ratios) have been studied as a means of predicting future tuberculosis (TB) disease risk or to assist in the diagnosis of incident TB disease. No studies to-date, however, have evaluated the potential of these ratios to predict or assist in the diagnosis of incident TB infection - the first step in the natural history of TB disease. Methods: In this prospective study, we evaluated the complete blood count (CBC)-derived metrics of monocyte-to-lymphocyte ratio (MLR), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) as predictors of future TB infection risk or aids in the diagnosis of TB infection among 145 Tanzanian adolescents enrolled in the DAR-901 vaccine trial, using paired CBCs and interferon-gamma release assays (IGRAs) obtained at 0, 60 and 720 days after study enrollment.Results: At baseline, there were no significant differences between study participants who remained persistently IGRA negative throughout the study period and those who subsequently converted to IGRA positive with respect to MLR (0.18 vs 0.17, p=0.10), NLR (0.88 vs 1.02, p=0.08), or PLR (115 vs 120, p=0.28). Similarly, no significant differences were noted with respect to MLR, NLR, and PLR between IGRA converters and time-matched negative controls at the time of IGRA conversion. With respect to other blood cell measures, however, there were modest but significant differences between IGRA negatives and IGRA converters with respect to red blood cell count (4.8 vs 4.6 × 106 cells/mcL, p=0.008), hemoglobin (12.6 vs 12.3 g/dL, p=0.01), and hematocrit (38.8 vs 37.8 %, p=0.005).Conclusions: In contrast to prior studies that have suggested that the ratios of different immune cell populations are associated with development of TB disease, our present findings do not demonstrate an association between these ratios and the development of TB infection. However, decreased red blood cell measures were associated with the subsequent development of TB infection, suggesting either that dysregulation of iron metabolism may play a role in TB pathogenesis or that following TB infection, iron dysregulation may precede IGRA positivity. Trial Registration: Clinicaltrials.gov NCT02712424. Date of registration: March 14, 2016.

BackgroundTuberculosis (TB) is the most deadly infectious disease globally and highly prevalent in the developing world, especially sub-Saharan Africa. Even though a third of humans are exposed to Myocbacterium tuberculosis (Mtb), most infected immunocompetent individuals do not develop active TB. In contrast, for individuals infected with both TB and the human immunodeficiency virus (HIV), the risk of active disease is 10% or more per year. Previously, we identified in a genome-wide association study a region on chromosome 5 that was associated with resistance to TB. This region included epigenetic marks that could influence gene regulation so we hypothesized that HIV-infected individuals exposed to Mtb, who remain disease free, carry epigenetic changes that strongly protect them from active TB. To test this hypothesis, we conducted a methylome-wide study in HIV-infected, TB-exposed cohorts from Uganda and Tanzania.ResultsIn 221 HIV-infected adults from Uganda and Tanzania, we identified 3 regions of interest that included markers that were differentially methylated between TB cases and LTBI controls, that also included methylation QTLs and associated SNPs: chromosome 1 (RNF220, p=4×10−5), chromosome 2 (between COPS8 and COL6A3 genes, p=2.7×10−5), and chromosome 5 (CEP72, p=1.3×10−5). These methylation results colocalized with associated SNPs, methylation QTLs, and methylation × SNP interaction effects. These markers were in regions with regulatory markers for cells involved in TB immunity and/or lung.ConclusionEpigenetic regulation is a potential biologic factor underlying resistance to TB in immunocompromised individuals that can act in conjunction with genetic variants.

Background In accordance with international guidance for tuberculosis (TB) prevention, the Tanzanian Ministry of Health recommends isoniazid preventive therapy (IPT) for children aged 12 months and older who are living with HIV. Concerns about tolerability, adherence, and potential mistreatment of undiagnosed TB with monotherapy have limited uptake of IPT globally, especially among children, in whom diagnostic confirmation is challenging. We assessed IPT implementation and adherence at a pediatric HIV clinic in Tanzania. Methods In this prospective cohort study, eligible children living with HIV aged 1–15 years receiving care at the DarDar Pediatric Program in Dar es Salaam who screened negative for TB disease were offered a 6-month regimen of daily isoniazid. Patients could choose to receive IPT via facility- or community-based care. Parents/caregivers and children provided informed consent and verbal assent respectively. Isoniazid was dispensed with the child’s antiretroviral therapy every 1–3 months. IPT adherence and treatment completion was determined by pill counts, appointment attendance, and self-report. Patients underwent TB symptom screening at every visit. Results We enrolled 66 children between July and December 2017. No patients/caregivers declined IPT. Most participants were female (n = 43, 65.1%) and the median age was 11 years (interquartile range [IQR] 8, 13). 63 (95.5%) participants chose the facility-based model; due to the small number of participants who chose the community-based model, valid comparisons between the two groups could not be made. Forty-nine participants (74.2%) completed IPT within 10 months. Among the remaining 17, 11 had IPT discontinued by their provider due to adverse drug reactions, 5 lacked documentation of completion, and 1 had unknown outcomes due to missing paperwork. Of those who completed IPT, the average monthly adherence was 98.0%. None of the participants were diagnosed with TB while taking IPT or during a median of 4 months of follow-up. Conclusions High adherence and treatment completion rates can be achieved when IPT is integrated into routine, self-selected facility-based pediatric HIV care. Improved record-keeping may yield even higher completion rates. IPT was well tolerated and no cases of TB were detected. IPT for children living with HIV is feasible and should be implemented throughout Tanzania.

Objectives: The efficacy of childhood BCG vaccination in the prevention of adult pulmonary tuberculosis is not universally accepted. Methods: We reviewed the published literature and summarized studies of BCG vaccination reporting long-term protection against pulmonary TB. Results: We identified 15 papers reporting prospective studies and their long-term follow up, retrospective studies or systematic reviews. Conclusions: Good quality evidence supports the efficacy of BCG vaccination in the prevention of adult pulmonary tuberculosis. Keywords: tuberculosis, BCG, vaccine

Background DAR-901 is an inactivated whole cell tuberculosis booster vaccine, prepared using a new scalable, broth-grown method from the master cell bank of SRL172, a vaccine previously shown to prevent tuberculosis. This study examined whether DAR-901 (a) induces CD4+ T cell cytokine profiles previously proposed as correlates of protection and (b) has a specific vaccine-induced immunological signature compared to BCG or placebo. Methods We analysed CD4+ T cell cytokine immune responses from 10 DAR-901 recipients, 9 BCG recipients and 9 placebo recipients from the Phase I DAR-901 MDES trial. In that study, HIV-negative, IGRA-negative participants with prior BCG immunization were randomized (double-blind) to receive three intradermal injections of DAR-901 or saline placebo or two injections of saline placebo followed by an intradermal injection of BCG. Antigen-specific functional and phenotypic CD4+ T cell responses along with effector phenotype of responder cells were measured by intracellular cytokine staining. Results DAR-901 recipients exhibited increased DAR-901 antigen-specific polyfunctional or bifunctional T cell responses compared to baseline. Vaccine specific CD4+ IFNγ, IL2, TNFα and any cytokine responses peaked at 7 days post-dose 3. Th1 responses predominated, with most responder cells exhibiting a polyfunctional effector memory phenotype. BCG induced greater CD4+ T cell responses than placebo while the more modest DAR-901 responses did not differ from placebo. Neither DAR-901 nor BCG induced substantial or sustained Th17 /Th22 cytokine responses. Conclusion DAR-901, a TB booster vaccine grown from the master cell bank of SRL 172 which was shown to prevent TB, induced low magnitude polyfunctional effector memory CD4+ T cell responses. DAR-901 responses were lower than those induced by BCG, a vaccine that has been shown ineffective as a booster to prevent tuberculosis disease. These results suggest that induction of higher levels of CD4+ cytokine stimulation may not be a critical or pre-requisite characteristic for candidate TB vaccine boosters. Trial registration ClinicalTrials.gov NCT02063555.

Objective To quantify total sialic acid in milk from HIV-positive Tanzanian mothers and to determine the impact of maternal diet on milk sialic acid levels. Design Milk samples were analyzed from 74 HIV-positive, Tanzanian women enrolled in a randomized, controlled clinical study of a dietary macronutrient supplement. Women were provided with a daily protein-calorie supplement and a micronutrient supplement or micronutrient supplement only during the last trimester of pregnancy and up to the first 6 months of breastfeeding. Methods Milk samples were collected at approximately 2 weeks and at least 3 months postpartum and assayed for total sialic acid. Milk sialic acid was assessed relative to maternal macronutrient intake, age, BMI, CD4+ cell count and infant birth weight. Results The mean concentration of milk sialic acid was highest in the first 2 weeks postpartum (6.89 ± 2.79 mmol/l) and declined rapidly by 3 months (2.49 ± 0.60 mmol/l). Sialic acid content in milk was similar between both treatment arms of the study, and did not correlate with maternal macronutrient intake. No correlation was found between maternal age, BMI, CD4+ cell count or infant birth weight and total milk sialic acid concentration. Conclusion Milk sialic acid levels in HIV-positive, Tanzanian women without malnutrition are comparable with reported values for women of European descent and show a similar temporal decline during early lactation. These findings suggest that total milk sialic acid is maintained despite macronutrient deficiencies in maternal diet and support a conserved role for milk sialic acid in neonatal development.

OBJECTIVES In a cross-sectional study of human immunodeficiency virus (HIV)-infected adults, the authors showed lower distortion product otoacoustic emissions (DPOAEs) in HIV+ individuals compared with controls as well as findings consistent with a central auditory processing deficit in HIV+ adults on antiretroviral therapy. The authors hypothesized that HIV+ children would also have a higher prevalence of abnormal central and peripheral hearing test results compared with HIV- controls. DESIGN Pure-tone thresholds, DPOAEs, and tympanometry were performed on 244 subjects (131 HIV+ and 113 HIV- subjects). Thirty-five of the HIV+, and 3 of the HIV- subjects had a history of tuberculosis treatment. Gap detection results were available for 18 HIV- and 44 HIV+ children. Auditory brainstem response results were available for 72 HIV- and 72 HIV+ children. Data from ears with abnormal tympanograms were excluded. RESULTS HIV+ subjects were significantly more likely to have abnormal tympanograms, histories of ear drainage, tuberculosis, or dizziness. All audiometric results were compared between groups using a two-way ANOVA with HIV status and ear drainage history as grouping variables. Mean audiometric thresholds, gap detection thresholds, and auditory brainstem response latencies did not differ between groups, although the HIV+ group had a higher proportion of individuals with a hearing loss >25 dB HL in the better ear. The HIV+ group had reduced DPOAE levels (p < 0.05) at multiple frequencies compared with HIV- subjects. No relationships were found between treatment regimens or delay in starting treatment and audiological parameters. CONCLUSIONS As expected, children with HIV+ were more likely to have a history of ear drainage, and to have abnormal tympanograms. Similar to the adult findings, the HIV+ group did not show significantly reduced audiometric thresholds, but did have significantly lower DPOAE magnitudes. These data suggest that (1) HIV+ children often have middle ear damage which complicates understanding the direct effects of HIV on the hearing system, and (2) even when corrected for confounders DPOAEs were lower in the HIV+ group. Previous studies suggest ototoxicity from antiretroviral drugs is an unlikely cause of the reduced DPOAE magnitudes. Other possibilities include effects on efferent pathways connecting to outer hair cells or a direct effect of HIV on the cochlea.

Background The current vaccine against tuberculosis, BCG, is effective when given in most TB-endemic countries at birth but has diminished efficacy against pulmonary TB after 15–20 years. As a result, new booster vaccines for adolescents and adults are being developed to realize the World Health Organization target of global elimination of TB by 2035. Multiple TB candidates thus are in active clinical development. Methods One of these, DAR-901, is advancing in human clinical trials. These clinical trials are conducted in BCG immunized adults with and without HIV infection in order to assess safety and efficacy among the people most in need of a new vaccine. A Phase I dose escalation trial of DAR-901 in BCG-immunized adults with or without HIV infection was conducted between 2014 and 2016. This offered an unusual opportunity to qualitatively examine why foreign-born adults living in the United States – a poorly studied population – decide to participate, or not, in clinical trials. Results We conducted a qualitative study of individuals who were recruited to participate in this Phase I vaccine trial, interviewing those who agreed and declined to participate. We found diverse motivations for participation or refusal; varied understandings of tuberculosis and vaccines; and complex views about how ‘informed consent’ can be at odds with cultural understandings of power, authority, and medical decision-making. These dynamics included: knowledge (direct or indirect) of tuberculosis, a desire to be altruistic and simultaneous hopes for personal gain as well as concerns over what remuneration for participation could mean, the importance of personal relationships with care providers in shaping volunteerism, concerns over privacy, and evidence of how culture and history shape medical decision-making. Conclusions This US-based trial, aimed at addressing a crucible global health issue, raises productive questions about the interface between altruism and scepticism regarding clinical research participation. Trial registration NCT02063555.

Major advances in donor identification, antigen probe design, and experimental methods to clone pathogen-specific antibodies have led to an exponential growth in the number of newly characterized broadly neutralizing antibodies (bnAbs) that recognize the HIV-1 envelope glycoprotein. Characterization of these bnAbs has defined new epitopes and novel modes of recognition that can result in potent neutralization of HIV-1. However, the translation of envelope recognition profiles in biophysical assays into an understanding of in vivo activity has lagged behind, and identification of subjects and mAbs with potent antiviral activity has remained reliant on empirical evaluation of neutralization potency and breadth. To begin to address this discrepancy between recombinant protein recognition and virus neutralization, we studied the fine epitope specificity of a panel of CD4-binding site (CD4bs) antibodies to define the molecular recognition features of functionally potent humoral responses targeting the HIV-1 envelope site bound by CD4. Whereas previous studies have used neutralization data and machine-learning methods to provide epitope maps, here, this approach was reversed, demonstrating that simple binding assays of fine epitope specificity can prospectively identify broadly neutralizing CD4bs–specific mAbs. Building on this result, we show that epitope mapping and prediction of neutralization breadth can also be accomplished in the assessment of polyclonal serum responses. Thus, this study identifies a set of CD4bs bnAb signature amino acid residues and demonstrates that sensitivity to mutations at signature positions is sufficient to predict neutralization breadth of polyclonal sera with a high degree of accuracy across cohorts and across clades., Antibody fine epitope specificity can serve as a powerful tool in neutralization prediction for both mAbs and polyclonal sera.

We compared macronutrient intake food insecurity and anthropometrics in breastfeeding women: 40 HIV-positive women not yet on antiretroviral therapy and 40 HIV-negative women. Calculated deficits at 2 weeks were 517 kcal per day for HIV-positive women vs 87 kcal per day surplus for HIV-negative women (P = 0.01) and 29 g protein per day for HIV-positive women vs 16 g protein per day for HIV-negative women (P = 0.04). Food insecurity scores were 11.3 for HIV-positive women vs 7.8 for HIV-negative women (P < 0.01). Enhanced dietary education together with macronutrient supplementation may be required to improve health outcomes in HIV-positive women and their infants.

Background Development of a tuberculosis vaccine to boost BCG is a major international health priority. SRL172, an inactivated whole cell booster derived from a non-tuberculous mycobacterium, is the only new vaccine against tuberculosis to have demonstrated efficacy in a Phase 3 trial. In the present study we sought to determine if a three-dose series of DAR-901 manufactured from the SRL172 master cell bank by a new, scalable method was safe and immunogenic. Methods We performed a single site, randomized, double-blind, controlled, Phase 1 dose escalation trial of DAR-901 at Dartmouth-Hitchcock Medical Center in the United States. Healthy adult subjects age 18–65 with prior BCG immunization and a negative interferon-gamma release assay (IGRA) were enrolled in cohorts of 16 subjects and randomized to three injections of DAR-901 (n = 10 per cohort), or saline placebo (n = 3 per cohort), or two injections of saline followed by an injection of BCG (n = 3 per cohort; 1–8 x 106 CFU). Three successive cohorts were enrolled representing DAR-901 at 0.1, 0.3, and 1 mg per dose. Randomization was performed centrally and treatments were masked from staff and volunteers. Subsequent open label cohorts of HIV-negative/IGRA-positive subjects (n = 5) and HIV-positive subjects (n = 6) received three doses of 1 mg DAR-901. All subjects received three immunizations at 0, 2 and 4 months administered as 0.1 mL injections over the deltoid muscle alternating between right and left arms. The primary outcomes were safety and immunogenicity. Subjects were followed for 6 months after dose 3 for safety and had phlebotomy performed for safety studies and immune assays before and after each injection. Immune assays using peripheral blood mononuclear cells included cell-mediated IFN-γ responses to DAR-901 lysate and to Mycobacterium tuberculosis (MTB) lysate; serum antibody to M. tuberculosis lipoarabinomannan was assayed by ELISA. Results DAR-901 had an acceptable safety profile and was well-tolerated at all dose levels in all treated subjects. No serious adverse events were reported. Median (range) 7-day erythema and induration at the injection site for 1 mg DAR-901 were 10 (4–20) mm and 10 (4–16) mm, respectively, and for BCG, 30 (10–107) mm and 38 (15–55) mm, respectively. Three mild AEs, all headaches, were considered possibly related to DAR-901. No laboratory or vital signs abnormalities were related to immunization. Compared to pre-vaccination responses, three 1 mg doses of DAR-901 induced statistically significant increases in IFN-γ response to DAR-901 lysate and MTB lysate, and in antibody responses to M. tuberculosis lipoarabinomannan. Ten subjects who received 1 mg DAR-901 remained IFN-γ release assay (IGRA) negative after three doses of vaccine. Conclusions A three-injection series of DAR-901 was well-tolerated, had an acceptable safety profile, and induced cellular and humoral immune responses to mycobacterial antigens. DAR-901 is advancing to efficacy trials. Trial registration ClinicalTrials.gov NCT02063555

Herpes simplex virus (HSV) oral shedding has not been studied among HIV-positive children in Africa. We sought to evaluate longitudinal oral HSV reactivation in HIV-positive and -negative children. Twenty HIV-positive antiretroviral-naive and 10 HIV-negative children aged 3–12 years in Tanzania were followed prospectively for 14 days. Oral swabs were collected daily and submitted for HSV DNA PCR analysis. Clinical data were collected via chart review and daily diaries. HSV DNA was detected in 10 (50%) of HIV-positive and 4 (40%) of HIV-negative children. Children who shed HSV had virus detected in a median of 21.4% of samples; shedding was intermittent. Median CD4 count among HIV-infected children was 667 cells/µL in those with positive HSV DNA and 886 cells/µL in those who were negative ( p = 0.6). Of the HIV-positive children reporting prior sores, five (83%) had positive HSV swabs, whereas the one HIV-negative child with prior sores did not have a PCR-positive swab. HSV is detected frequently in children with and without HIV. HIV-infected children reporting oral sores have a high rate of HSV detection. Given the proven strong interactions between HIV and HSV, further study of co-infection with these viruses is warranted in children.

Heritable interferon (IFN) γ signaling deficiencies are associated with heightened susceptibility to tuberculosis [1] and deficient IFN-γ responses probably contribute to the high vulnerability to tuberculosis of persons with human immunodeficiency virus (HIV) infection [2]. Among HIV-infected and BCG-immunized adults, we have shown that detectable IFN-γ responses to single and multiple mycobacterial antigens are associated with protection from HIV-associated tuberculosis [3, 4]. To our knowledge, however, there have been no prospective studies evaluating whether IFN-γ responses reduce the tuberculosis bacillary load when active tuberculosis occurs. To address this question, we correlated baseline IFN-γ responses to mycobacterial antigens with the bacillary burden of tuberculosis disease among HIV-infected adults who subsequently developed culture-confirmed tuberculosis in the DarDar phase III tuberculosis vaccine trial in Tanzania.

Active tuberculosis (TB) among HIV-infected patients, even when successfully treated, may be associated with excess mortality. We conducted a prospective cohort study nested in a randomized TB vaccine trial to compare mortality between HIV-infected patients diagnosed and treated for TB (TB, n = 77) and HIV-infected patients within the same CD4 range, who were not diagnosed with or treated for active TB (non-TB, n = 308) in the period 2001-2008. Only twenty four subjects (6%) were on antiretroviral therapy at the beginning of this study. After accounting for covariate effects including use of antiretroviral therapy, isoniazid preventive therapy, and receipt of vaccine, we found a four-fold increase in mortality in TB patients compared with non-TB patients (adjusted Hazard Ratio 4.61; 95% Confidence Interval (CI): 1.63, 13.05). These findings suggest that treatment for TB alone is not sufficient to avert the excess mortality associated with HIV-related TB and that prevention of TB may provide a mortality benefit.

Tuberculosis infects millions of people worldwide and remains a leading global killer despite widespread neonatal administration of the tuberculosis vaccine, bacillus Calmette-Guérin (BCG). BCG has clear and sustained efficacy, but after 10 years, its efficacy appears to wane, at least in some populations. Fortunately, there are many new tuberculosis vaccines in development today, some in advanced stages of clinical trial testing. Here we review the epidemiological need for tuberculosis vaccination, including evolving standards for administration to at risk individuals in developing countries. We also examine proven sources of immune protection from tuberculosis, which to date have exclusively involved natural or vaccine exposure to whole cell mycobacteria. After summarizing evidence for the use and efficacy of BCG, we detail the most promising new candidate vaccines against tuberculosis. The global need for a new tuberculosis vaccine is acute and huge, but clinical trials to be completed in the coming few years are likely either to identify a new tuberculosis vaccine or to substantially reframe how we understand immune protection from this historical scourge.

One in three people has been infected with Mycobacterium tuberculosis (MTB), and the risk for MTB infection in HIV-infected individuals is even higher. We hypothesized that HIV-positive individuals living in tuberculosis-endemic regions who do not get infected by Mycobacterium tuberculosis are genetically resistant. Using an “experiment of nature” design that proved successful in our previous work, we performed a genome-wide association study of tuberculin skin test positivity using 469 HIV-positive patients from prospective study cohorts of tuberculosis from Tanzania and Uganda to identify genetic loci associated with MTB infection in the context of HIV-infection. Among these individuals, 244 tested were tuberculin skin test (TST) positive either at enrollment or during the >8 year follow up, while 225 were not. We identified a genome-wide significant association between a dominant model of rs877356 and binary TST status in the combined cohort (Odds ratio = 0.2671, p = 1.22x10-8). Association was replicated with similar significance when examining TST induration as a continuous trait. The variant lies in the 5q31.1 region, 57kb downstream from IL9. Two-locus analyses of association of variants near rs877356 showed a haplotype comprised of rs877356 and an IL9 missense variant, rs2069885, had the most significant association (p = 1.59x10-12). We also replicated previously linked loci on chromosomes 2, 5, and 11. IL9 is a cytokine produced by mast cells and TH2 cells during inflammatory responses, providing a possible link between airway inflammation and protection from MTB infection. Our results indicate that studying uninfected, HIV-positive participants with extensive exposure increases the power to detect associations in complex infectious disease., Author summary Approximately one-third of the world’s population has been exposed to Mycobacterium tuberculosis, the bacterium that causes tuberculosis. A small number of those infected develop active disease; however, there is a substantial portion of exposed people who do not even show evidence of an immunological response. These people who appear to resist infection, as measured by a negative tuberculin skin test, represent a subpopulation from which we can learn about resistance. We used a genome-wide approach to study the genetic basis of this resistance in unique cohorts of hypervulnerable, HIV-positive individuals from Uganda and Tanzania, in which exposure was virtually assured. We identified one locus that was highly significantly associated and conferred more than 70% protection from infection. The most significant variant, rs8773656, was near IL9 and SLC25A48, and a haplotype including this variant and a missense mutation in IL9 was even more significantly associated with negative skin tests. Although it is impossible based solely on our data to determine the causal variant or genes, IL9 is an attractive candidate as its product has previously been associated with bronchial hyperresponsiveness, thereby providing a possible link between inflammation and protection from Mycobacterium tuberculosis infection.

Background: Poor nutrition has been associated with impaired immunity and accelerated disease progression in HIV-infected children. The aim of this study was to quantify the levels of nutrient intake in HIV-infected children and compare these to standard recommendations. Methods: We surveyed HIV-infected Tanzanian children enrolled in a pediatric care program that provided routine nutritional counseling and vitamin supplementation. We obtained anthropometric measurements and determined 24-hour macronutrient and micronutrient intakes and food insecurity. Values were compared to recommended nutrient intakes based on age and gender. Results: We interviewed 48 pairs of children and their caregiver(s). The age of the child ranged from 2-14 years; median age 6 and 60% female. The median weight-for-height z-score for children ≤ 5 years was 0.69 and BMI-for-age z-scores for children >5 was -0.84. Macronutrient evaluation showed that 29 (60%) children were deficient in dietary intake of energy; deficiency was more common in older children (p=0.004). Micronutrient evaluation shows that over half of study subjects were deficient in dietary intake of vitamin A, vitamin D, vitamin E, thiamine, riboflavin, niacin, folate, vitamin B12, and calcium. Food insecurity was reported by 20 (58%) caregivers. Conclusions and Public Health Implications: The diets of many HIV-infected children at a specialized treatment center in Tanzania do not meet recommended levels of macro-and micro-nutrients. Food insecurity was a contributory factor. Enhanced dietary counseling and provision of macro- and micro-nutrient supplements will be necessary to achieve optimal nutrition for most HIV-infected children in resource-poor regions.

Immunosuppression resulting from HIV infection increases the risk of progression to active tuberculosis (TB) both in individuals newly exposed to Mycobacterium tuberculosis (MTB) and in those with latent infections. We hypothesized that HIV-positive individuals who do not develop TB, despite living in areas where it is hyperendemic, provide a model of natural resistance. We performed a genome-wide association study of TB resistance by using 581 HIV-positive Ugandans and Tanzanians enrolled in prospective cohort studies of TB; 267 of these individuals developed active TB, and 314 did not. A common variant, rs4921437 at 5q33.3, was significantly associated with TB (odds ratio = 0.37, p = 2.11 × 10(-8)). This variant lies within a genomic region that includes IL12B and is embedded in an H3K27Ac histone mark. The locus also displays consistent patterns of linkage disequilibrium across African populations and has signals of strong selection in populations from equatorial Africa. Along with prior studies demonstrating that therapy with IL-12 (the cytokine encoded in part by IL12B, associated with longer survival following MTB infection in mice deficient in CD4 T cells), our results suggest that this pathway might be an excellent target for the development of new modalities for treating TB, especially for HIV-positive individuals. Our results also indicate that studying extreme disease resistance in the face of extensive exposure can increase the power to detect associations in complex infectious disease.

Background. T-SPOT.TBis an interferon gamma release assay for detectingMycobacterium tuberculosisinfection. The requirement to process within 8 hours is constraining, deters use, and leads to invalid results. Addition of T CellXtendreagent may allow delayed processing, but has not been extensively field tested.Design. Consecutive AFB smear positive adult tuberculosis patients were prospectively recruited in Dar es Salaam, Tanzania. Patients provided a medical history, 1–3 sputum samples for culture and 1 blood sample which was transported to the laboratory under temperature-controlled conditions. After overnight storage, 25 μL of T CellXtendreagent was added per mL of blood, and the sample was tested using T-SPOT.TB.Results. 143 patients were enrolled: 57 patients were excluded because temperature control was not maintained, 19 patients were excluded due to red blood cell contamination, and one did not provide a sputum sample for culture. Among 66 evaluable patients, overall agreement between T-SPOT.TBand culture was 95.4% (95%CI; 87.1–99.0%) with Kappa value 0.548. Sensitivity of T-SPOT.TBwhen using T CellXtendreagent was 96.8% (95%CI; 88.8–99.6%).Conclusions. When T CellXtendreagent is added to specimens held overnight at recommended temperatures, T-SPOT.TBis as sensitive as the standard assay in patients with tuberculosis.

Background A positive tuberculin skin test (TST) may indicate cross-reacting immunity to non-tuberculous mycobacteria (NTM) and not latent tuberculosis infection (LTBI). Objectives To assess misclassification of LTBI, as assessed by skin testing with Mycobacterium avium sensitin (MaS), and to determine how this misclassification affects the analysis of risk factors for LTBI. Methods In a population-based survey, participants underwent skin testing with M. tuberculosis purified protein derivative (PPD) and MaS. A PPD-dominant skin test was a reaction that was ≥ 3 mm larger than the MaS reaction; a MaS-dominant skin test was a reaction that was ≥ 3 mm larger than the PPD reaction. Results Of 447 randomly selected persons, 135 (30%) had a positive PPD test. Of these, 21 (16%) were MaS- dominant, and were therefore attributable to NTM and misclassified as LTBI. PPD reactions of 5-14 mm were more likely to be misclassified than those ≥ 15 mm (OR = 5.0, 95%CI 1.9-13.2). Adjusting for misclassification had only a small impact on the analysis of risk factors for LTBI. Conclusions A substantial number of individuals who are diagnosed with LTBI are actually sensitized to NTM. Using dual skin testing would reduce misdiagnosis and prevent unnecessary treatment.

Peripheral tuberculous lymphadenitis accounts for ~10% of tuberculosis cases in the United States. Epidemiologic characteristics include a 1.4:1 female-to-male ratio, a peak age range of 30-40 years, and dominant foreign birth, especially East Asian. Patients present with a 1-2 month history of painless swelling of a single group of cervical lymph nodes. Definitive diagnosis is by culture or nucleic amplification of Mycobacterium tuberculosis; demonstration of acid fast bacilli and granulomatous inflammation may be helpful. Excisional biopsy has the highest sensitivity at 80%, but fine-needle aspiration is less invasive and may be useful, especially in immunocompromised hosts and in resource-limited settings. Antimycobacterial therapy remains the cornerstone of treatment, but response is slower than with pulmonary tuberculosis; persistent pain and swelling are common, and paradoxical upgrading reactions may occur in 20% of patients. The role of steroids is controversial. Initial excisional biopsy deserves consideration for both optimal diagnosis and management of the otherwise slow response to therapy.

Preventive immunization with whole inactivated Mycobacterium vaccae (MV) confers protection against HIV-associated tuberculosis (TB) in BCG-immunized adults with CD4 counts ≥ 200 cells/ul. We evaluated the immunogenicity of MV in the 2,013 subjects of the phase III DarDarTrial using an interferon gamma (IFN-γ) enzyme linked immunosorbent assay (ELISA), tritiated thymidine lymphocyte proliferation assay (LPA) and an ELISA for antibodies to the TB glycolipid lipoarabinomannan (LAM). MV immunization boosts IFN-γ and LPA responses to MV sonicate, and antibody responses to LAM. Post-immunization immune responses to MV correlated with baseline clinical factors, but the responses did not predict protection from HIV-associated TB.

The tuberculin skin test is limited by its inability to distinguish between infection with Mycobacterium tuberculosis and non-tuberculous mycobacteria (NTM). Newer interferon-gamma release assays using ESAT-6 and CFP-10 antigens should have a higher specificity for tuberculosis but have not been widely tested in adults with pulmonary disease due to NTM. In this study, we tested the T-SPOT.TB Test in patients with pulmonary disease due to Mycobacterium avium complex (MAC), the most common disease-causing NTM. Fourteen patients with prior culture-confirmed pulmonary disease due to MAC, 10 patients with prior culture-confirmed tuberculosis and 4 healthy controls were interviewed and tested with the T-SPOT.TB Test. 13 patients with MAC disease and 4 healthy subjects (negative controls) had non-reactive T-SPOT.TB results and 10 patients with prior tuberculosis (positive controls) had reactive results. One patient with MAC disease had a minimally reactive result on initial testing and a non-reactive result on re-testing. The T-SPOT.TB Test had a specificity of 94% for distinguishing between patients with prior MAC disease and prior tuberculosis disease, and will be useful in low tuberculosis prevalence settings where most mycobacterial infections are due to MAC. Reactions to the T-SPOT.TB Test may persist months to years after treatment of tuberculosis.

Diagnostic Criteria of Nontuberculous Mycobacterial Lung Disease Key Laboratory Features of NTM Health Careand Hygiene-associated Disease Prevention Prophylaxis and Treatment of NTM Disease Introduction Methods Taxonomy Epidemiology Pathogenesis Host Defense and Immune Defects Pulmonary Disease Body Morphotype Tumor Necrosis Factor Inhibition Laboratory Procedures Collection, Digestion, Decontamination, and Staining of Specimens Respiratory Specimens Body Fluids, Abscesses, and Tissues Blood Specimen Processing Smear Microscopy Culture Techniques Incubation of NTM Cultures NTM Identification Antimicrobial Susceptibility Testing for NTM Molecular Typing Methods of NTM Clinical Presentations and Diagnostic Criteria Pulmonary Disease Cystic Fibrosis Hypersensitivity-like Disease Transplant Recipients Disseminated Disease Lymphatic Disease Skin, Soft Tissue, and Bone Disease

Symptomatic disease by nontuberculous mycobacteria has been linked to potable water from institutional and domestic potable water systems. Potable water samples were collected from homes and institutions of patients with AIDS. Colonization of potable water with nontuberculous mycobacteria was demonstrated in 230 (15%) of 1489 samples collected from domestic and institutional water systems of patients with HIV infection in the United States and Finland.Mycobacterium aviumwas the most common species and colonization was favored at temperatures of 40–50°C in recirculating hot water systems. Such systems are a plausible source of human infection and disease.

Infection with Mycobacterium avium complex is acquired from the environment, but risk factors for M. avium complex infection and disease are poorly understood. To identify risk factors for infection, the authors performed a 1998-2000 cross-sectional study in western Palm Beach County, Florida, using a population-based random household survey. M. avium complex infection was identified by use of the M. avium sensitin skin test. Of 447 participants, 147 (32.9%) had a positive test reaction, 186 (41.6%) had a negative test reaction, and, for 114 (25.5%), test results were indeterminate. Among the 333 participants with positive or negative M. avium sensitin skin tests, age-adjusted independent predictors of M. avium complex infection in a multivariate model included Black race (odds ratio = 3.8, 95% confidence interval: 2.2, 6.6), birth outside the United States (odds ratio = 2.1, 95% confidence interval: 1.1, 3.9), and more than 6 years' cumulative occupational exposure to soil (odds ratio = 2.7, 95% confidence interval: 1.3, 6.0). Exposure to water, food, or pets was not associated with infection. Results indicate that soil is a reservoir for M. avium complex associated with human infection and that persons whose occupations involve prolonged soil exposure are at increased risk of M. avium complex infection.