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RATIONALE: Tuberculosis (TB) remains a major cause of mortality. A better understanding of the immune responses to mycobacterial antigens may be helpful to develop improved vaccines and diagnostics. OBJECTIVES: The mycobacterial antigen heparin-binding hemagglutinin (HBHA) induces strong IFN-[gamma] responses by circulating lymphocytes from subjects latently infected with Mycobacterium tuberculosis, and low responses associated with CD4(+) regulatory T (Treg) cells in patients with TB. Here, we investigated HBHA-specific IFN-[gamma] responses at the site of the TB disease. METHODS: Bronchoalveolar lavages, pleural fluids, and blood were prospectively collected from 61 patients with a possible diagnosis of pulmonary or pleural TB. HBHA-specific IFN-[gamma] production was analyzed by flow cytometry and ELISA. The suppressive effect of pleural Treg cells was investigated by depletion experiments. MEASUREMENTS AND MAIN RESULTS: The percentages of HBHA-induced IFN-[gamma](+) alveolar and pleural lymphocytes were higher for pulmonary (P < 0.0001) and for pleural (P < 0.01) TB than for non-TB controls. Local CD4(+) and CD8(+) T cells produced the HBHA-specific IFN-[gamma]. This local secretion was not suppressed by Treg lymphocytes, contrasting with previously reported data on circulating lymphocytes. CONCLUSIONS: Patients with TB display differential effector and regulatory T-cell responses to HBHA in local and circulating lymphocytes with a predominant effector CD4(+) and CD8(+) response locally, compared with a predominant Treg response among circulating lymphocytes. These findings may be helpful for the design of new vaccines against TB, and the detection of HBHA-specific T cells at the site of the infection may be a promising tool for the rapid diagnosis of active TB. [ABSTRACT FROM AUTHOR]

Abstract Background Paratuberculosis remains today a major global problem in animal health, especially for dairy cattle. However, the diagnosis of its etiologic agent, Mycobacterium avium subsp. paratuberculosis (Map), still lacks sensitivity because of the lack of available antigens. Little is known about the virulence factors for this pathogen. In this study we have developed a method to produce and purify the heparin-binding hemagglutinin (HBHA), a major adhesin of Mycobacteria, from a culture of Map. Findings For this extremely slow-growing Mycobacterium, a culture was established in a 3-liter bioreactor. Using the bioreactor the amount of the Map biomass was increased 5-fold compared to a classical culture in flasks. The map-HBHA was purified from a Map lysate by heparin-Sepharose chromatography on HiTrap columns. Binding of map-HBHA onto heparin-Sepharose can be reduced in the presence of salt. Consequently, all steps of sample preparation and column equilibration were carried out in 20 mM Tris–HCl (pH 7.2). The map-HBHA was eluted by a linear NaCl gradient. High resolution mass spectrometry analyses revealed that the native form of map-HBHA has posttranslational modifications, including the removal of the initiation methionine, acetylation of the alanine residue at the N-terminal extremity and the presence of methylated lysines in the C-terminal domain of the protein. Conclusions An optimized culture of Map in a bioreactor was established to purify the native map-HBHA from a Map lysate by heparin-Sepharose chromatography. The availability of this antigen offers the possibility to study the structure of the protein and to examine its role in pathogenicity, in particular to better understand the specific interactions of Map with the intestinal tissue. The map-HBHA obtained in its native immunogenic form may also be useful to improve the diagnostic test, especially for the development of a new T-cell-based interferon gamma release assays.

The early period of the COVID-19 pandemic limited access to HIV services for children and adolescents living with HIV (C/ALHIV). To determine progress in providing care and treatment services, we describe viral load coverage (VLC) and suppression (VLS) (<1000 copies/ mL) rates during the COVID-19 pandemic in 12 United States President's Emergency Plan for AIDS Relief (PEPFAR)-supported countries. Data for children (0-9 years) and adolescents (10-19 years) on VLC and VLS were analyzed for 12 sub-Saharan African (SSA) countries between 2019 (pre-COVID-19) and 2020 (during COVID-19). We report the number of viral load (VL) tests, and percent change in VLC and VLS for patients on ART. For 12 countries, 181,192 children had a VL test during the pre-COVID-19 period compared with 177,683 December 2020 during COVID-19. VLC decreased from 68.8% to 68.3% overall. However, 9 countries experienced an increase ranging from a 0.7%-point increase for Tanzania and Zimbabwe to a 15.3%-point increase for Nigeria. VLS increased for all countries from 71.2% to 77.7%. For adolescents the number with a VL test increased from 377,342 to 402,792. VLC decreased from 77.4% to 77.1%. However, 7 countries experienced an increase ranging from 1.8% for Mozambique to 13.8% for Cameroon. VLS increased for all countries from 76.8% to 83.8%. This analysis shows variation in HIV VLC across 12 SSA countries. VLS consistently improved across all countries demonstrating resilience of countries during 2020. Countries should continue to improve clinical outcomes from C/ALHIV despite service disruptions that may occur during pandemic response., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

As antiretroviral treatment (ART) coverage for people living with HIV (PLHIV) increases, HIV programmes require up-to-date information about evolving HIV risk behaviour and transmission risk, including those with low-level viremia (LLV; >50 to ≤1000 copies/mL), to guide prevention priorities. We aimed to assess differences in sexual risk behaviours, distribution of viral load (VL) and proportion of transmission across PLHIV subgroups. We analysed data from Population-based HIV Impact Assessment surveys in 14 sub-Saharan African countries during 2015-2019. We estimated adjusted prevalence ratios (aPR) of self-reported HIV high-risk behaviour (multiple partners and condomless sex) across cascade stages via generalised estimation equations. We modelled the proportions of transmission from each subgroup using relative self-reported sexual risk, a Hill function for transmission rate by VL, and proportions within cascade stages from surveys and UNAIDS country estimates for 2010-2020. Compared to PLHIV with undetectable VL (≤50 copies/mL), undiagnosed PLHIV (aPR women: 1.28 [95% CI: 1.08-1.52]; men: 1.61 [1.33-1.95]) and men diagnosed but untreated (2.06 [1.52-2.78]) were more likely to self-report high-risk sex. High-risk behaviour was not significantly associated with LLV. Mean VL was similar among undiagnosed, diagnosed but untreated, and on ART but non-suppressed sub-groups. Across surveys, undiagnosed and diagnosed but untreated contributed most to transmission (40-91% and 1-41%, respectively), with less than 1% from those with LLV. Between 2010 and 2020, the proportion of transmission from individuals on ART but non-suppressed increased. In settings with high ART coverage, effective HIV testing, ART linkage, and retention remain priorities to reduce HIV transmission. Persons with LLV are an increasing share of PLHIV but their contribution to HIV transmission was small. Improving suppression among PLHIV on ART with VL ≥1000 copies/mL will become increasingly important., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Jeffrey W. Imai-Eaton has received grants/contracts from NIH and WHO, consulting fees from BAO Systems, support for attending meetings from UNAIDS, SACEMA and the International AIDS Society and is a member of the editorial board for PLOS Global Public Health. Olanrewaju Edun has received consulting fees from University of Cape Town and WHO and support for attending meetings from UNAIDS. All other authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Cholesterol is a crucial component in Mycobacterium tuberculosis virulence as it is required for phagocytosis of mycobacteria by macrophages. In addition, the tubercle bacilli can grow using cholesterol as the sole carbon source. Thus, cholesterol catabolism represents a valuable target for the development of new antitubercular drugs. However, the molecular partners of cholesterol catabolism remain elusive in mycobacteria. Here, we focused on HsaC and HsaD, enzymes involved in two consecutive steps of cholesterol ring degradation and identified putative partners, using a BirA-based proximity-dependent biotin identification (BioID) approach in Mycobacterium smegmatis. In rich medium, the fusion protein BirA-HsaD was able to fish the endogenous cognate HsaC, thus validating this approach to study protein-protein interactions and to infer metabolic channeling of cholesterol ring degradation. In chemically defined medium, both HsaC and HsaD interacted with four proteins, BkdA, BkdB, BkdC, and MSMEG&#95;1634. BkdA, BkdB, and BkdC are enzymes that participate in the degradation of branched-chain amino acids. As cholesterol and branched-chain amino acid catabolism both generate propionyl-CoA, which is a toxic metabolite for mycobacteria, this interconnection suggests a compartmentalization to avoid dissemination of propionyl-CoA into the mycobacterial cytosol. Moreover, the BioID approach allowed us to decipher the interactome of MSMEG&#95;1634 and MSMEG&#95;6518, two proteins of unknown function, which are proximal to the enzymes involved in cholesterol and branched-chain amino acid catabolism. In conclusion, BioID is a powerful tool to characterize protein-protein interactions and to decipher the interconnections between different metabolic pathways, thereby facilitating the identification of new mycobacterial targets., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Introduction: We assessed progress in HIV viral load (VL) scale up across seven sub-Saharan African (SSA) countries and discussed challenges and strategies for improving VL coverage among patients on anti-retroviral therapy (ART)., Methods: A retrospective review of VL testing was conducted in Côte d'Ivoire, Kenya, Lesotho, Malawi, Namibia, Tanzania, and Uganda from January 2016 through June 2018. Data were collected and included the cumulative number of ART patients, number of patients with ≥ 1 VL test result (within the preceding 12 months), the percent of VL test results indicating viral suppression, and the mean turnaround time for VL testing., Results: Between 2016 and 2018, the proportion of PLHIV on ART in all 7 countries increased (range 5.7%-50.2%). During the same time period, the cumulative number of patients with one or more VL test increased from 22,996 to 917,980. Overall, viral suppression rates exceeded 85% for all countries except for Côte d'Ivoire at 78% by June 2018. Reported turnaround times for VL testing results improved in 5 out of 7 countries by between 5.4 days and 27.5 days., Conclusions: These data demonstrate that remarkable progress has been made in the scale-up of HIV VL testing in the seven SSA countries., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The US President's Emergency Plan for AIDS Relief (PEPFAR) supports molecular HIV and tuberculosis diagnostic networks and information management systems in low- and middle-income countries. We describe how national programs leveraged these PEPFAR-supported laboratory resources for SARS-CoV-2 testing during the COVID-19 pandemic. We sent a spreadsheet template consisting of 46 indicators for assessing the use of PEPFAR-supported diagnostic networks for COVID-19 pandemic response activities during April 1, 2020, to March 31, 2021, to 27 PEPFAR-supported countries or regions. A total of 109 PEPFAR-supported centralized HIV viral load and early infant diagnosis laboratories and 138 decentralized HIV and TB sites reported performing SARS-CoV-2 testing in 16 countries. Together, these sites contributed to >3.4 million SARS-CoV-2 tests during the 1-year period. Our findings illustrate that PEPFAR-supported diagnostic networks provided a wide range of resources to respond to emergency COVID-19 diagnostic testing in 16 low- and middle-income countries.

Introduction: Achieving optimal HIV outcomes, as measured by global 90-90-90 targets, that is awareness of HIV-positive status, receipt of antiretroviral (ARV) therapy among aware and viral load (VL) suppression among those on ARVs, respectively, is critical. However, few data from sub-Saharan Africa (SSA) are available on older people (50+) living with HIV (OPLWH). We examined 90-90-90 progress by age, 15-49 (as a comparison) and 50+ years, with further analyses among 50+ (55-59, 60-64, 65+ vs. 50-54), in 13 countries (Cameroon, Cote d'Ivoire, Eswatini, Ethiopia, Kenya, Lesotho, Malawi, Namibia, Rwanda, Tanzania, Uganda, Zambia and Zimbabwe)., Methods: Using data from nationally representative Population-based HIV Impact Assessments, conducted between 2015and 2019, participants from randomly selected households provided demographic and clinical information and whole blood specimens for HIV serology, VL and ARV testing. Survey weighted outcomes were estimated for 90-90-90 targets. Country-specific Poisson regression models examined 90-90-90 variation among OPLWH age strata., Results: Analyses included 24,826 HIV-positive individuals (15-49 years: 20,170; 50+ years: 4656). The first, second and third 90 outcomes were achieved in 1, 10 and 5 countries, respectively, by those aged 15-49, while OPLWH achieved outcomes in 3, 13 and 12 countries, respectively. Among those aged 15-49, women were more likely to achieve 90-90-90 targets than men; however, among OPLWH, men were more likely to achieve first and third 90 targets than women, with second 90 achievement being equivalent. Country-specific 90-90-90 regression models among OPLWH demonstrated minimal variation by age stratum across 13 countries. Among OLPWH, no first 90 target differences were noted by age strata; three countries varied in the second 90 by older age strata but not in a consistent direction; one country showed higher achievement of the third 90 in an older age stratum., Conclusions: While OPLWH in these 13 countries were slightly more likely than younger people to be aware of their HIV-positive status (first 90), this target was not achieved in most countries. However, OPLWH achieved treatment (second 90) and VL suppression (third 90) targets in more countries than PLWH <50. Findings support expanded HIV testing, prevention and treatment services to meet ongoing OPLWH health needs in SSA., (© 2022 The Authors. Journal of the International AIDS Society published by John Wiley & Sons Ltd on behalf of the International AIDS Society.)

The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane β barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TpsB transporter FhaC. This revealed substantial, spontaneous conformational changes on a slow time scale, with parts of the POTRA2 domain approaching the lipid bilayer and the protein's surface loops. Specifically, our data indicate that an amphipathic POTRA2 β hairpin can insert into the β barrel. We propose that these motions enlarge the channel and initiate substrate secretion. Our data propose a solution to the conundrum how TpsB transporters mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sicoli, Konijnenberg, Guérin, Hessmann, Del Nero, Hernandez-Alba, Lecher, Rouaut, Müggenburg, Vezin, Cianférani, Sobott, Schneider and Jacob-Dubuisson.)

Repeating the BinaxNOW antigen test for severe acute respiratory syndrome coronavirus 2 using 2 groups of readers within 30 minutes resulted in high concordance (98.9%) in 2110 encounters. Same-day repeat antigen testing did not significantly improve test sensitivity (77.2% to 81.4%) while specificity remained high (99.6%)., (Published by Oxford University Press for the Infectious Diseases Society of America 2021.)

The two-component system BvgAS controls the virulence regulon in Bordetella pertussis BvgS is the prototype of a family of sensor histidine-kinases harboring periplasmic Venus flytrap (VFT) domains. The VFT domains are connected to the cytoplasmic kinase moiety by helical linkers separated by a Per-ARNT-Sim (PAS) domain. Antagonism between the two linkers, as one forms a coiled coil when the other is dynamic and vice versa, regulates BvgS activity. Here we solved the structure of the intervening PAS domain by X-ray crystallography. Two forms were obtained that notably differ by the connections between the PAS core domain and the flanking helical linkers. Structure-guided mutagenesis indicated that those connections participate in the regulation of BvgS activity. The PAS domain thus appears to function as a switch-facilitator module whose conformation determines the output of the system. As many BvgS homologs have similar architectures, the mechanisms unveiled here are likely to generally apply to the regulation of sensor-histidine kinases of that family. IMPORTANCE The whooping cough agent Bordetella pertussis colonizes the human respiratory tract using virulence factors co-regulated by the sensory transduction system BvgAS. BvgS is a model for a family of sensor-kinase proteins, some of which are found in important bacterial pathogens. BvgS functions as a kinase or a phosphatase depending on external signals, which determines if B. pertussis is virulent or avirulent. Deciphering its mode of action might thus lead to new ways of fighting infections. Here we used X-ray crystallography to solve the three-dimensional structure of the domain that precedes the enzymatic moiety and identified features that regulate BvgS activity. As many sensor-kinases of the BvgS family harbor homologous domains, the mechanism unveiled here might be of general relevance., (Copyright © 2021 American Society for Microbiology.)

T cell epitopes are mostly nonmodified peptides, although posttranslationally modified peptide epitopes have been described, but they originated from viral or self-proteins. In this study, we provide evidence of a bacterial methylated T cell peptide epitope. The mycobacterial heparin-binding hemagglutinin (HBHA) is a protein Ag with a complex C-terminal methylation pattern and is recognized by T cells from humans latently infected with Mycobacterium tuberculosis By comparing native HBHA with recombinant HBHA produced in Mycobacterium smegmatis (rHBHA- Ms ), we could link antigenic differences to differences in the methylation profile. Peptide scan analyses led to the discovery of a peptide containing methyl lysines recognized by a mAb that binds to native HBHA ∼100-fold better than to rHBHA- Ms This peptide was also recognized by T cells from latently infected humans, as evidenced by IFN-γ release upon peptide stimulation. The nonmethylated peptide did not induce IFN-γ, arguing that the methyl lysines are part of the T cell epitope., (Copyright © 2020 by The American Association of Immunologists, Inc.)

The heparin-binding hemagglutinin (HBHA) is a multifunctional protein involved in adherence of Mycobacterium tuberculosis to non-phagocytic cells and in the formation of intracytosolic lipid inclusions. We demonstrate that the expression of hbhA is regulated by a transcriptional repressor, named HbhR, in Mycobacterium marinum. The hbhR gene, located upstream of hbhA, was identified by screening a transposon insertion library and detailed analysis of a mutant overproducing HBHA. HbhR was found to repress both hbhA and hbhR transcription by binding to the promoter regions of both genes. Complementation restored production of HBHA. RNA-seq analyses comparing the mutant and parental strains uncovered 27 genes, including hbhA, that were repressed and 20 genes activated by HbhR. Among the former, the entire locus of genes coding for a type-VII secretion system, including esxA, esxB and pe-ppe paralogs, as well as the gene coding for PspA, present in intracellular lipid vesicles, was identified, as was katG, a gene involved in the sensitivity to isoniazid. The latter category contains genes that play a role in diverse functions, such as metabolism and resistance to oxidative conditions. Thus, HbhR appears to be a master regulator, linking the transcriptional regulation of virulence, metabolic and antibiotic sensitivity genes in M. marinum., (© 2019 John Wiley & Sons Ltd.)

Pertussis is a severe respiratory disease mainly caused by Bordetella pertussis Despite wide global vaccination coverage with efficacious pertussis vaccines, it remains one of the least well-controlled vaccine-preventable diseases, illustrating the shortcomings of the current vaccines. We have developed the live attenuated nasal pertussis vaccine BPZE1, currently undergoing clinical evaluation in human phase 2 trials. We have previously shown that in mice, BPZE1 provides strong and long-lasting protection against B. pertussis challenge by inducing potent Ab and T cell responses as well as secretory IgA and IL-17-producing resident memory T lymphocytes in the nasal cavity. In this study, we show that BPZE1 induces protection in mice against B. pertussis within days after vaccination, at a time when Ab and T cell responses were not detectable. Early protection was independent of T and B cell responses, as demonstrated by the use of SCID mice. Instead, it was due to TLR4-dependent signaling through the MyD88-dependent pathway of the innate immune response, as demonstrated in experiments with TLR4-deficient and MyD88-knockout mice. TLR2-dependent signaling did not play a major role in early protection. In addition, this study also shows that even at high doses, BPZE1 is safe in the severely immunocompromised MyD88-deficient mice, whereas virulent B. pertussis caused a severe pathological condition and death in these mice, even at a low dose. Finally, coadministration of virulent B. pertussis with BPZE1 did not cause exacerbated outgrowth of the virulent strain, thereby adding to the safety profile of this live vaccine candidate., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Introduction: HIV testing serves as an entry point for HIV care services for those who test HIV positive, and prevention services for those who test HIV negative. The Centers for Disease Control and Prevention recommends routine testing of adults and adolescents in healthcare settings. To identify missed opportunities for HIV testing at U.S. physicians' offices, data from the National Ambulatory Care Surveys from 2009 to 2012 were analyzed., Methods: The mean annual number and percentage of visits with an HIV test among HIV-uninfected nonpregnant females and males aged 15-65 years was estimated using weighted survey data. Factors associated with HIV testing at visits to physicians' offices were identified., Results: The mean annual number of U.S. physicians' office visits with an HIV test conducted was 1,396,736 (0.4% of all visits) among nonpregnant females and 986,891 (0.5% of all visits) among males. For both nonpregnant females and males, HIV testing prevalence was highest among those aged 20-29 years (1.3% of all visits by nonpregnant females; 1.7% of all visits by males) and non-Hispanic blacks (1.1% of all visits by nonpregnant females; 1.0% of all visits by males). An HIV test was not conducted at 98.5% of visits at which venipuncture was performed for both nonpregnant females and males., Conclusions: Important opportunities exist to increase HIV testing coverage at U.S. physicians' offices. Structural interventions, such as routine opt-out testing policies, electronic medical record notifications, and use of non-clinical staff for testing could be implemented to increase HIV testing in these settings., (Published by Elsevier Inc.)

Evidence suggests that the resurgence of pertussis in many industrialized countries may result from the failure of current vaccines to prevent nasopharyngeal colonization by Bordetella pertussis, the principal causative agent of whooping cough. Here, we used a baboon model to test the protective potential of the novel, live attenuated pertussis vaccine candidate BPZE1. A single intranasal/intratracheal inoculation of juvenile baboons with BPZE1 resulted in transient nasopharyngeal colonization and induction of immunoglobulin G and immunoglobulin A to all antigens tested, while causing no adverse symptoms or leukocytosis. When BPZE1-vaccinated baboons were challenged with a high dose of a highly virulent B. pertussis isolate, they were fully protected against disease, whereas naive baboons developed illness (with 1 death) and leukocytosis. Total postchallenge nasopharyngeal virulent bacterial burden of vaccinated animals was substantially reduced (0.002%) compared to naive controls, providing promising evidence in nonhuman primates that BPZE1 protects against both pertussis disease and B. pertussis infection., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

The World Health Organization (WHO) recommends viral load testing as the preferred method for monitoring the clinical response of patients with human immunodeficiency virus (HIV) infection to antiretroviral therapy (ART) (1). Viral load monitoring of patients on ART helps ensure early diagnosis and confirmation of ART failure and enables clinicians to take an appropriate course of action for patient management. When viral suppression is achieved and maintained, HIV transmission is substantially decreased, as is HIV-associated morbidity and mortality (2). CDC and other U.S. government agencies and international partners are supporting multiple countries in sub-Saharan Africa to provide viral load testing of persons with HIV who are on ART. This report examines current capacity for viral load testing based on equipment provided by manufacturers and progress with viral load monitoring of patients on ART in seven sub-Saharan countries (Côte d'Ivoire, Kenya, Malawi, Namibia, South Africa, Tanzania, and Uganda) during January 2015-June 2016. By June 2016, based on the target numbers for viral load testing set by each country, adequate equipment capacity existed in all but one country. During 2015, two countries tested >85% of patients on ART (Namibia [91%] and South Africa [87%]); four countries tested <25% of patients on ART. In 2015, viral suppression was >80% among those patients who received a viral load test in all countries except Côte d'Ivoire. Sustained country commitment and a coordinated global effort is needed to reach the goal for viral load monitoring of all persons with HIV on ART.

Pediatric human immunodeficiency virus (HIV) infection remains an important public health issue in resource-limited settings. In 2015, 1.4 million children aged <15 years were estimated to be living with HIV (including 170,000 infants born in 2015), with the vast majority living in sub-Saharan Africa (1). In 2014, 150,000 children died from HIV-related causes worldwide (2). Access to timely HIV diagnosis and treatment for HIV-infected infants reduces HIV-associated mortality, which is approximately 50% by age 2 years without treatment (3). Since 2011, the annual number of HIV-infected children has declined by 50%. Despite this gain, in 2014, only 42% of HIV-exposed infants received a diagnostic test for HIV (2), and in 2015, only 51% of children living with HIV received antiretroviral therapy (1). Access to services for early infant diagnosis of HIV (which includes access to testing for HIV-exposed infants and clinical diagnosis of HIV-infected infants) is critical for reducing HIV-associated mortality in children aged <15 years. Using data collected from seven countries supported by the U.S. President's Emergency Plan for AIDS Relief (PEPFAR), progress in the provision of HIV testing services for early infant diagnosis was assessed. During 2011-2015, the total number of HIV diagnostic tests performed among HIV-exposed infants within 6 weeks after birth (tests for early infant diagnosis of HIV), as recommended by the World Health Organization (WHO) increased in all seven countries (Cote d'Ivoire, the Democratic Republic of the Congo, Haiti, Malawi, South Africa, Uganda, and Zambia); however, in 2015, the rate of testing for early infant diagnosis among HIV-exposed infants was <50% in five countries. HIV positivity among those tested declined in all seven countries, with three countries (Cote d'Ivoire, the Democratic Republic of the Congo, and Uganda) reporting >50% decline. The most common challenges for access to testing for early infant diagnosis included difficulties in specimen transport, long turnaround time between specimen collection and receipt of results, and limitations in supply chain management. Further reductions in HIV mortality in children can be achieved through continued expansion and improvement of services for early infant diagnosis in PEPFAR-supported countries, including initiatives targeted to reach HIV-exposed infants, ensure access to programs for early infant diagnosis of HIV, and facilitate prompt linkage to treatment for children diagnosed with HIV infection.

To achieve global targets for universal treatment set forth by the Joint United Nations Programme on human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) (UNAIDS), viral load monitoring for HIV-infected persons receiving antiretroviral therapy (ART) must become the standard of care in low- and middle-income countries (LMIC) (1). CDC and other U.S. government agencies, as part of the President's Emergency Plan for AIDS Relief, are supporting multiple countries in sub-Saharan Africa to change from the use of CD4 cell counts for monitoring of clinical response to ART to the use of viral load monitoring, which is the standard of care in developed countries. Viral load monitoring is the preferred method for immunologic monitoring because it enables earlier and more accurate detection of treatment failure before immunologic decline. This report highlights the initial successes and challenges of viral load monitoring in seven countries that have chosen to scale up viral load testing as a national monitoring strategy for patients on ART in response to World Health Organization (WHO) recommendations. Countries initiating viral load scale-up in 2014 observed increases in coverage after scale-up, and countries initiating in 2015 are anticipating similar trends. However, in six of the seven countries, viral load testing coverage in 2015 remained below target levels. Inefficient specimen transport, need for training, delays in procurement and distribution, and limited financial resources to support scale-up hindered progress. Country commitment and effective partnerships are essential to address the financial, operational, technical, and policy challenges of the rising demand for viral load monitoring.

Mycobacterium tuberculosis strains of the Beijing lineage are globally distributed and are associated with the massive spread of multidrug-resistant (MDR) tuberculosis in Eurasia. Here we reconstructed the biogeographical structure and evolutionary history of this lineage by genetic analysis of 4,987 isolates from 99 countries and whole-genome sequencing of 110 representative isolates. We show that this lineage initially originated in the Far East, from where it radiated worldwide in several waves. We detected successive increases in population size for this pathogen over the last 200 years, practically coinciding with the Industrial Revolution, the First World War and HIV epidemics. Two MDR clones of this lineage started to spread throughout central Asia and Russia concomitantly with the collapse of the public health system in the former Soviet Union. Mutations identified in genes putatively under positive selection and associated with virulence might have favored the expansion of the most successful branches of the lineage.

Almost one century after the discovery of the BCG vaccine, tuberculosis remains a major cause of global mortality and morbidity, emphasizing the urgent need to design more efficient vaccines. The heparin-binding haemagglutinin (HBHA) appears to be a promising vaccine candidate, as it was shown to afford protection to mice against a challenge infection with Mycobacterium tuberculosis when combined with the strong adjuvant DDA/MPL (dimethyldioctadecyl-ammonium bromide/monophosphoryl lipid A), a TLR4 ligand. In this study, we investigated the immunological response and protection of mice immunized with HBHA formulated in lipid-containing nanoparticles and adjuvanted with CpG, a TLR9 ligand. Subcutaneous immunization with this HBHA formulation led to a marked Th1 response, characterized by high IFN-γ levels, but no significant IL-17 production, both in spleen and lung, in contrast to DDA/MPL MPL-formulated HBHA, which induced both IFN-γ and IL-17. This cytokine profile was also observed in BCG-primed mice and persisted after M. tuberculosis infection. No significant protection was obtained against challenge infection after vaccination with the nanoparticle-CpG formulation, and this was associated with a failure to mount a memory immune response. These results suggest the importance of both Th1 and Th17 immune responses for vaccine-induced immunity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: To test the feasibility of offering rapid point-of-care human immunodeficiency virus (HIV) testing at community pharmacies and retail clinics., Design: Pilot program to determine how to implement confidential HIV testing services in community pharmacies and retail clinics., Setting: 21 community pharmacies and retail clinics serving urban and rural patients in the United States, from August 2011 to July 2013., Participants: 106 community pharmacy and retail clinic staff members., Intervention: A model was developed to implement confidential HIV counseling and testing services using community pharmacy and retail clinic staff as certified testing providers, or through collaborations with organizations that provide HIV testing. Training materials were developed and sites selected that serve patients from urban and rural areas to pilot test the model. Each site established a relationship with its local health department for HIV testing policies, developed referral lists for confirmatory HIV testing/care, secured a CLIA Certificate of Waiver, and advertised the service. Staff were trained to perform a rapid point-of-care HIV test on oral fluid, and provide patients with confidential test results and information on HIV. Patients with a preliminary positive result were referred to a physician or health department for confirmatory testing and, if needed, HIV clinical care., Main Outcome Measures: Number of HIV tests completed and amount of time required to conduct testing., Results: The 21 participating sites administered 1,540 HIV tests, with 1,087 conducted onsite by staff during regular working hours and 453 conducted at 37 different HIV testing events (e.g., local health fairs). The median amount of time required for pretest counseling/consent, waiting for test results, and posttest counseling was 4, 23, and 3 minutes, respectively. A majority of the sites (17) said they planned to continue HIV testing after the project period ended and would seek assistance or support from the local health department, a community-based organization, or an AIDS service organization., Conclusion: This pilot project established HIV testing in several community pharmacies and retail clinics to be a feasible model for offering rapid, point-of-care HIV testing. It also demonstrated the willingness and ability of staff at community pharmacies and retail clinics to provide confidential HIV testing to patients. Expanding this model to additional sites and evaluating its feasibility and effectiveness may serve unmet needs in urban and rural settings.

Background: Preventing unintended pregnancies among HIV-positive women through family planning (FP) reduces pregnancy-related morbidity and mortality, decreases the number of pediatric HIV infections, and has also proven to be a cost-effective way to prevent mother-to-child HIV transmission. A key element of a comprehensive HIV prevention agenda, aimed at avoiding unintended pregnancies, is recognizing the attitudes towards FP among HIV-positive women and their spouse or partner. In this study, we analyze FP attitudes among HIV-infected pregnant women enrolled in a PMTCT clinical trial in Western Kenya., Methods and Findings: Baseline data were collected on 522 HIV-positive pregnant women using structured questionnaires. Associations between demographic variables and the future intention to use FP were examined using Fisher's exact tests and permutation tests. Most participants (87%) indicated that they intended to use FP. However, only 8% indicated condoms as a preferred FP method, and 59% of current pregnancies were unintended. Factors associated with positive intentions to use FP were: marital status (p = 0.04), having talked to their spouse or partner about FP (p<0.001), perceived spouse or partner approval of FP (p<0.001), previous use of a FP method (p = 0.006), attitude toward the current pregnancy (p = 0.02), disclosure of a sexually transmitted infection (STI) diagnosis (p = 0.03) and ethnic group (p = 0.03)., Conclusion: A significant gap exists between future FP intentions and current FP practices. Support and approval by the spouse or partner are key elements of FP intentions. Counseling services should be offered to both members of a couple to increase FP use, especially given the high number of unplanned pregnancies among HIV-positive women. Condoms should be promoted as part of a dual use method for HIV and STI prevention and for contraception. Integration of individual and couple FP services into routine HIV care, treatment and support services is needed in order to avoid unintended pregnancies and to prevent mother-to-child HIV transmission.

Background: Patients with end-stage renal disease (ESRD) and latently infected with Mycobacterium tuberculosis (LTBI) are at higher risk to develop tuberculosis (TB) than healthy subjects. Interferon-gamma release assays (IGRAs) were reported to be more sensitive than tuberculin skin tests for the detection of infected individuals in dialysis patients., Methods: On 143 dialysis patients prospectively enrolled, we compared the results from the QuantiFERON®-TB Gold assay (QFT), to those of an IGRA in response to in vitro stimulation of circulating mononuclear cells with the mycobacterial latency antigen Heparin-Binding Haemagglutinin purified from Mycobacterium bovis BCG (native HBHA, nHBHA)., Results: Seven patients had a past history of active TB and 1 had an undetermined result with both IGRAs. Among the other 135 patients, 94 had concordant results with the QFT and nHBHA-IGRA, 40.0% being negative and therefore not latently infected, and 29.6% being positive and thus LTBI. Discrepant results between these tests were found for 36 patients positive only with the nHBHA-IGRA and 5 only with the QFT., Conclusions: The nHBHA-IGRA is more sensitive than the QFT for the detection of LTBI dialysis patients, and follow-up of the patients will allow us to define the clinical significance of discrepant results between the nHBHA-IGRA and the QFT.

Heparin-binding haemagglutinin (HBHA)-specific immune responses have been linked to protection against tuberculosis (TB). We investigated the hypothesis that BCG vaccination of human infants primes an HBHA-specific response, using multiplex to measure secreted cytokines and chemokines following HBHA and Mycobacterium tuberculosis purified protein derivative (PPD) stimulation of diluted whole blood samples from BCG-vaccinated or -unvaccinated infants. Of 42 analytes measured, 24 and 32 significant, BCG-associated increases were detected in response to HBHA and PPD, respectively. Both response profiles included Th-1, Th-2, Th-17 and inflammatory cytokines and chemokines (e.g. IFN-γ, TNF-α, IL-5, IL-10, IL-13, IL-17, MIP-1α and MIP-1β). We also found that six of the seven responses most closely correlated with IFN-γ were common to both HBHA and PPD. Notably, all HBHA-specific secretion of cytokines and chemokines from infant samples was dependent on previous BCG vaccination. Also, long-term persistence of HBHA-specific responses was found in adolescents with evidence of infant BCG vaccination. This study demonstrates for the first time BCG priming of an HBHA-specific immune response in infants that is characterised by a broad cytokine and chemokine signature. It also suggests a number of BCG vaccination associated, HBHA-induced responses that should be useful for future studies of biomarkers of protection against TB., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The President's Emergency Plan for AIDS Relief (PEPFAR) programme for the Caribbean Region was established in 2008 to address health system challenges, including fragile laboratory services and systems. The laboratory component of this programme consisted of several phases: assessment of laboratory needs of all 12 countries engaged in the programme; addressing gaps identified during the assessment; and monitoring and evaluation of the progress achieved. After one year of PEPFAR collaboration with national governments and other partners, laboratory services and systems greatly improved. Some of the milestones include: (1) the accreditation of a public laboratory; (2) improved access to HIV diagnosis with faster turnaround time; (3) establishment of capacity for platforms for DNA PCR, viral load and HIV drug resistance; (4) development of the laboratory workforce; and (5) establishment of a framework for implementation of sustainable quality management systems for laboratory accreditation. The progress recorded in strengthening laboratory health systems after one year of initiating this collaboration shows that with a rigorous initial assessment, programme design and intervention and strategic partnership, national laboratory health systems can be greatly enhanced to support programme implementation. Continued collaboration and country leadership is critical to create an integrated and sustainable laboratory network in the Caribbean.

Background: Most individuals infected with Mycobacterium tuberculosis develop latent tuberculosis infection (LTBI). Some may progress to active disease and would benefit from preventive treatment yet no means currently exists to predict who will reactivate. Here, we provide an approach to stratify LTBI based on IFN-γ responses to two antigens, the recombinant Early-Secreted Antigen Target-6 (rESAT-6) and the latency antigen Heparin-Binding Haemagglutinin (HBHA)., Methods: We retrospectively analyzed results from in-house IFN-γ-release assays with HBHA (HBHA-IGRA) and rESAT-6 (rESAT-6-IGRA) performed during a 12-year period on serial blood samples (3 to 9) collected from 23 LTBI subjects in a low-TB incidence country. Both the kinetics of the absolute IFN-γ concentrations secreted in response to each antigen and the dynamics of HBHA/rESAT-6-induced IFN-γ concentrations ratios were examined., Results: This analysis allowed the identification among the LTBI subjects of three major groups. Group A featured stable HBHA and rESAT-6-IGRA profiles with an HBHA/rESAT-6 ratio persistently higher than 1, and with high HBHA- and usually negative rESAT-6-IGRA responses throughout the study. Group B had changing HBHA/rESAT-6 ratios fluctuating from 0.0001 to 10,000, with both HBHA and rESAT-6 responses varying over time at least once during the follow-up. Group C was characterized by a progressive disappearance of all responses., Conclusions: By combining the measures of IFN-γ concentrations secreted in response to an early and a latency antigens, LTBI subjects can be stratified into different risk groups. We propose that disappearing responses indicate cure, that persistent responses to HBHA with HBHA/rESAT-6 ratios ≥ 1 represent stable LTBI subjects, whereas subjects with ratios varying from >1 to <1 should be closely monitored as they may represent the highest-risk group, as illustrated by a case report, and should therefore be prioritized for preventive treatment.

Pulmonary tuberculosis remains a major health problem. Effective vaccination strategies are urgently needed. It was previously demonstrated that purified Mycobacterium bovis BCG Heparin Binding Haemagglutinin (HBHA) is able to induce in BALB/c mice protection levels against a Mycobacterium tuberculosis infection that are similar to those offered by BCG. Here we developed a heterologous prime/boost immunisation approach using a combination of BCG and HBHA in order to increase the protective immune response. We show that the time period between BCG priming and HBHA boosting strongly influences the efficacy of the boost. The optimized vaccine protocol consisting of a BCG administration followed 8 months later by boosting with HBHA resulted in an increase in the level of protection of up to 0.7log when compared to BCG alone. These results suggest an immunisation strategy where BCG is administered to neonates and is followed by subcutaneous HBHA boosting in young adults.

DctP6 and DctP7 are two Bordetella pertussis proteins which belong to the extracytoplasmic solute receptors (ESR) superfamily. ESRs are involved in the transport of substrates from the periplasm to the cytosol of Gram-negative bacteria. DctP6 and DctP7 have been crystallized and diffraction data were collected using a synchrotron-radiation source. DctP6 crystallized in space group P4(1)2(1)2, with unit-cell parameters a = 108.39, b = 108.39, c = 63.09 A, while selenomethionyl-derivatized DctP7 crystallized in space group P2(1)2(1)2(1), with unit-cell parameters a = 64.87, b = 149.83, c = 170.65 A. The three-dimensional structure of DctP7 will be determined by single-wavelength anomalous diffraction, while the DctP6 structure will be solved by molecular-replacement methods.

Complete and continuous documentation in patient records is an important condition for adequate communication with patients, between the professions concerned and to ensure the quality of the following working steps in care provision. Part of a German research project concerning the interprofessional communication in hospital was therefore to analyse the use of the documentation system. 54 users were asked about practical aspects of their documentation system and 450 patient records were evaluated. The analysis focused on the medical and nursing documentation of admission, process and discharge. Deficits that need to be improved appeared first of all in the practical aspects of the documentation system, the flow of information between the professions, in specific gaps of medical and nursing admission, documentation of process and discharge. Quality management is asked to improve and develop the documentation in collaboration with the users and to consider specific problems when introducing computer based records.