A retrospective genomic analysis of drug-resistant strains of M. tuberculosis in a high-burden setting, with an emphasis on comparative diagnostics and reactivation and reinfection status.

BACKGROUND: Recurrence of drug-resistant tuberculosis (DR-TB) after treatment occurs through relapse of the initial infection or reinfection by a new drug-resistant strain. Outbreaks of DR-TB in high burden regions present unique challenges in determining recurrence status for effective disease management and treatment. In the Republic of Moldova the burden of DR-TB is exceptionally high, with many cases presenting as recurrent. METHODS: We performed a retrospective analysis of Mycobacterium tuberculosis from Moldova to better understand the genomic basis of drug resistance and its effect on the determination of recurrence status in a high DR-burden environment. To do this we analyzed genomes from 278 isolates collected from 189 patients, including 87 patients with longitudinal samples. These pathogen genomes were sequenced using Illumina technology, and SNP panels were generated for each sample for use in phylogenetic and network analysis. Discordance between genomic resistance profiles and clinical drug-resistance test results was examined in detail to assess the possibility of mixed infection. RESULTS: There were clusters of multiple patients with 10 or fewer differences among DR-TB samples, which is evidence of person-to-person transmission of DR-TB. Analysis of longitudinally collected isolates revealed that many infections exhibited little change over time, though 35 patients demonstrated reinfection by divergent (number of differences > 10) lineages. Additionally, several same-lineage sample pairs were found to be more divergent than expected for a relapsed infection. Network analysis of the H3/4.2.1 clade found very close relationships among 61 of these samples, making differentiation of reactivation and reinfection difficult. There was discordance between genomic profile and clinical drug sensitivity test results in twelve samples, and four of these had low level (but not statistically significant) variation at DR SNPs suggesting low-level mixed infections. CONCLUSIONS: Whole-genome sequencing provided a detailed view of the genealogical structure of the DR-TB epidemic in Moldova, showing that reinfection may be more prevalent than currently recognized. We also found increased evidence of mixed infection, which could be more robustly characterized with deeper levels of genomic sequencing.

The TB Portals: an Open-Access, Web-Based Platform for Global Drug-Resistant-Tuberculosis Data Sharing and Analysis.

The TB Portals program is an international consortium of physicians, radiologists, and microbiologists from countries with a heavy burden of drug-resistant tuberculosis working with data scientists and information technology professionals. Together, we have built the TB Portals, a repository of socioeconomic/geographic, clinical, laboratory, radiological, and genomic data from patient cases of drug-resistant tuberculosis backed by shareable, physical samples. Currently, there are 1,299 total cases from five country sites (Azerbaijan, Belarus, Moldova, Georgia, and Romania), 976 (75.1%) of which are multidrug or extensively drug resistant and 38.2%, 51.9%, and 36.3% of which contain X-ray, computed tomography (CT) scan, and genomic data, respectively. The top Mycobacterium tuberculosis lineages represented among collected samples are Beijing, T1, and H3, and single nucleotide polymorphisms (SNPs) that confer resistance to isoniazid, rifampin, ofloxacin, and moxifloxacin occur the most frequently. These data and samples have promoted drug discovery efforts and research into genomics and quantitative image analysis to improve diagnostics while also serving as a valuable resource for researchers and clinical providers. The TB Portals database and associated projects are continually growing, and we invite new partners and collaborations to our initiative. The TB Portals data and their associated analytical and statistical tools are freely available at https://tbportals.niaid.nih.gov/.

Limited HIV infection of central memory and stem cell memory CD4+ T cells is associated with lack of progression in viremic individuals.

A rare subset of HIV-infected individuals, designated viremic non-progressors (VNP), remain asymptomatic and maintain normal levels of CD4+ T-cells despite persistently high viremia. To identify mechanisms potentially responsible for the VNP phenotype, we compared VNPs (average >9 years of HIV infection) to HIV-infected individuals who have similar CD4+ T-cell counts and viral load, but who are likely to progress if left untreated ("putative progressors", PP), thus avoiding the confounding effect of differences related to substantial CD4+ T cell depletion. We found that VNPs, compared to PPs, had preserved levels of CD4+ stem cell memory cells (TSCM (p<0.0001), which was associated with decreased HIV infection of these cells in VNPs (r = -0.649, p = 0.019). In addition, VNPs had decreased HIV infection in CD4+ central memory (TCM) cells (p = 0.035), and the total number of TCM cells was associated with increased proliferation of memory CD4+ T cells (r = 0.733, p = 0.01). Our results suggest that, in HIV-infected VNPs, decreased infection of CD4+ TCM and TSCM, cells are involved in preservation of CD4+ T cell homeostasis and lack of disease progression despite high viremia.

The use of cash transfers for HIV prevention--are we there yet?

Poverty and social inequality are significant drivers of the HIV epidemic and are risk factors for acquiring HIV. As such, many individuals worldwide are at risk for new HIV infection, especially young women in East and Southern Africa. By addressing these drivers, social protection programmes may mitigate the impact of poverty and social inequality on HIV risk. There is reason to believe that social protection can be used successfully for HIV prevention; social protection programmes, including cash transfers, have led to positive health outcomes and behaviour in other contexts, and they have been used successfully to promote education and increased income and employment opportunities. Furthermore, cash transfers have influenced sexual behaviour of young women and girls, thereby decreasing sexual risk factors for HIV infection. When HIV outcomes have been measured, several randomised controlled trials have shown that indirectly, cash transfers have led to reduced HIV prevalence and incidence. In these studies, school attendance and safer sexual health were directly incentivised through the cash transfer, yet there was a positive effect on HIV outcomes. In this review, we discuss the growth of social protection programmes, their benefits and impact on health, education and economic potential, and how these outcomes may affect HIV risk. We also review the studies that have shown that cash transfers can lead to reduced HIV infection, including study limitations and what questions still remain with regard to using cash transfers for HIV prevention.

Increased stability and limited proliferation of CD4+ central memory T cells differentiate nonprogressive simian immunodeficiency virus (SIV) infection of sooty mangabeys from progressive SIV infection of rhesus macaques.

UNLABELLED: Depletion of CD4(+) central memory T (TCM) cells dictates the tempo of progression to AIDS in simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) both in the natural history of infection and in the context of vaccination. CD4(+) TCM cells of sooty mangabeys (SMs), a natural host for SIV in which infection is nonpathogenic, are less susceptible to SIV infection than CD4(+) TCM cells of RMs. Whether this relative protection from infection translates into increased stability of CD4(+) TCM cells in natural versus nonnatural hosts has not yet been determined. Here we compared, both cross-sectionally and longitudinally, the levels of CD4(+) TCM cells in a large cohort of SMs and RMs and the association between CD4(+) TCM levels and the main virologic and immunologic markers of disease progression. Consistent with their lower susceptibility to infection, CD4(+) TCM cells of SIV-infected SMs are lost with kinetics 20 times slower than those of SIV-infected RMs. Remarkably, the estimated length of time of SIV infection needed for CD4(+) TCM cells to fall to half of their initial levels is <16 months for RMs but >17 years for SMs. Furthermore, the fraction of proliferating CD4(+) TCM cells is significantly lower in SIV-infected SMs than in SIV-infected RMs, and the extent of CD4(+) TCM cell proliferation is associated positively with CD4(+) T cell levels in SIV-infected SMs but negatively with CD4(+) T cell levels in SIV-infected RMs. Collectively, these findings identify increased stability and maintenance of the prohomeostatic role of CD4(+) TCM cells as features distinguishing nonprogressive from progressive SIV infections and support the hypothesis of a direct mechanistic link between the loss of CD4(+) TCM cells and disease progression. IMPORTANCE: Comparison of the immunologic effects of simian immunodeficiency virus (SIV) infection on rhesus macaques (RMs), a species characterized by progression to AIDS, and natural host sooty mangabeys (SMs), a species which remains AIDS free, has become a useful tool for identifying mechanisms of human immunodeficiency virus (HIV) disease progression. One such distinguishing feature is that CD4(+) central memory T (TCM) cells in SIV-infected SMs are less infected than the same cells in RMs. Here we investigated whether lower levels of infection in SMs translate into a better-preserved CD4(+) TCM compartment. We found that the CD4(+) TCM compartment is significantly more stable in SIV-infected SMs. Likely to compensate for this cell loss, we also found that CD4(+) TCM cells increase their level of proliferation upon SIV infection in RMs but not in SMs, which mechanistically supports their preferential infectivity. Our study provides new insights into the importance of long-term maintenance of CD4(+) TCM homeostasis during HIV/SIV infection.

The economics, financing and implementation of HIV treatment as prevention: what will it take to get there?

The 2013 Lancet Commission Report, Global Health 2035, rightly pointed out that we are at a unique place in history where a "grand convergence" of health initiatives to reduce both infectious diseases, and child and maternal mortality--diseases that still plague low income countries--would yield good returns in terms of development and health outcomes. This would also be a good economic investment. Such investments would support achieving health goals of reducing under-five (U5) mortality to 16 per 1000 live births, reducing deaths due to HIV/AIDS to 8 per 100,000 population, and reducing annual TB deaths to 4 per 100,000 population. Treatment as prevention (TasP) holds enormous potential in reducing HIV transmission, and morbidity and mortality associated with HIV/AIDS--and therefore contributing to Global Health 2035 goals. However, TasP requires large financial investments and poses significant implementation challenges. In this review, we discuss the potential effectiveness, financing and implementation of TasP. Overall, we conclude that TasP shows great promise as a cost-effective intervention to address the dual aims of reducing new HIV infections and reducing the global burden of HIV-related disease. Successful implementation will be no easy feat, though. The dramatic increases in the numbers of persons who need antiretroviral therapy (ART) under a TasP approach will pose enormous challenges at all stages of the HIV treatment cascade: HIV diagnosis, antiretroviral (ARV) initiation, ARV adherence and retention, and increased drug resistance with long-term enrolment on ART. Overcoming these implementation challenges will require targeted implementation, not focusing exclusively on TasP, most-at-risk population (MARP)-friendly services for key populations, integrating services, task shifting, more efficient programme management, balancing supply and demand, integration into universal health coverage efforts, demand creation, improved ART retention and adherence strategies, the use of incentives to improve HIV treatment outcomes and reduce unit costs, continued operational research and tapping into technological innovations.

One Health activities to reinforce intersectoral coordination at local levels in India.

India's dense human and animal populations, agricultural economy, changing environment, and social dynamics support conditions for emergence/re-emergence of zoonotic diseases that necessitate a One Health (OH) approach for control. In addition to OH national level frameworks, effective OH driven strategies that promote local intersectoral coordination and collaboration are needed to truly address zoonotic diseases in India. We conducted a literature review to assess the landscape of OH activities at local levels in India that featured intersectoral coordination and collaboration and supplemented it with our own experience conducting OH related activities with local partners. We identified key themes and examples in local OH activities. Our landscape assessment demonstrated that intersectoral collaboration primarily occurs through specific research activities and during outbreaks, however, there is limited formal coordination among veterinary, medical, and environmental professionals on the day-to-day prevention and detection of zoonotic diseases at district/sub-district levels in India. Examples of local OH driven intersectoral coordination include the essential role of veterinarians in COVID-19 diagnostics, testing of human samples in veterinary labs for Brucella and leptospirosis in Punjab and Tamil Nadu, respectively, and implementation of OH education targeted to school children and farmers in rural communities. There is an opportunity to strengthen local intersectoral coordination between animal, human and environmental health sectors by building on these activities and formalizing the existing collaborative networks. As India moves forward with broad OH initiatives, OH networks and experience at the local level from previous or ongoing activities can support implementation from the ground up.

A novel CCR5 mutation common in sooty mangabeys reveals SIVsmm infection of CCR5-null natural hosts and efficient alternative coreceptor use in vivo.

In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species.

CCR5 blockade is well tolerated and induces changes in the tissue distribution of CCR5+ and CD25+ T cells in healthy, SIV-uninfected rhesus macaques.

BACKGROUND: CCR5 is a main co-receptor for HIV, but also homes lymphocytes to sites of inflammation. We hypothesized that inhibition of CCR5 signaling would reduce HIV-associated chronic immune activation. METHODS: To test this hypothesis, we administered an antagonistic anti-CCR5 monoclonal antibody (HGS101) to five uninfected rhesus macaques (RMs) and monitored lymphocyte dynamics in blood and tissue. RESULTS: CCR5 blockade resulted in decreased levels of CCR5+ T cells in blood and, at later timepoints, in lymph nodes. Additionally, the levels of CD25+ T cells increased in lymph nodes, but decreased in blood, bone marrow, and rectal mucosa. Finally, a profile of gene expression from HGS101-treated RMs revealed a subtle, but consistent, in vivo signature of CCR5 blockade that suggests a mild immune-modulatory effect. CONCLUSIONS: Treatment with anti-CCR5 antibody induces changes in the tissue distribution of CCR5+ and CD25+ T cells that may impact on the overall levels of immune activation during HIV and SIV infection.

A five-year longitudinal analysis of sooty mangabeys naturally infected with simian immunodeficiency virus reveals a slow but progressive decline in CD4+ T-cell count whose magnitude is not predicted by viral load or immune activation.

Natural simian immunodeficiency virus (SIV) infection in sooty mangabeys (SMs) typically does not result in AIDS, despite high-level viremia and significant depletion of mucosal CD4(+) T cells. Here, we report the results of the first longitudinal study of a large cohort of SMs naturally infected with SIV (n = 78) housed at the Yerkes National Primate Research Center from which samples were obtained three times over a 5-year period. In this study, we observed (i) no signs of simian AIDS, (ii) stable SIV loads, (iii) a slow but progressive decline in CD4(+) T-cell counts (from a mean of 1,067.0 cells/mm(3) at time point 1 to 764.8 cells/mm(3) at time point 3) and increases in the numbers of animals with CD4(+) T-cell levels below 500 and 200 cells/mm(3) (from 8 to 28 of 78 and from 1 to 4 of 78, respectively), (iv) progressive declines in percentages of naïve CD4(+) and CD8(+) T cells (from 37.7 to 24.8% and from 21.0 to 13.0%, respectively), and (v) stably low levels of activated/proliferating T cells as well as CD4(+) CCR5(+) T cells. Since the level of total CD4(+) T cells and the fraction of naïve T cells in SIV-uninfected SMs also declined, it is possible that some of these observations are related to aging, as the SIV-infected animals were significantly older than the uninfected animals. In contrast to the decline in CD4(+) T cell counts in individuals infected with human immunodeficiency virus (HIV), the decline in CD4(+) T cell counts in SMs naturally infected with SIV over a 5-year period was not predicted by either plasma viremia or levels of T-cell activation. Taken together, these results confirm that natural SIV infection is nonprogressive from a clinical, virological, and immunological point of view and that stable levels of viremia associated with persistently low-level immune activation represent key differences from the natural course of HIV infection in humans.

A primate model of severe malarial anaemia: a comparative pathogenesis study.

Severe malarial anaemia (SMA) is the most common life-threatening complication of Plasmodium falciparum infection in African children. SMA is characterised by haemolysis and inadequate erythropoiesis, and is associated with dysregulated inflammatory responses and reduced complement regulatory protein levels (including CD35). However, a deeper mechanistic understanding of the pathogenesis requires improved animal models. In this comparative study of two closely related macaque species, we interrogated potential causal factors for their differential and temporal relationships to onset of SMA. We found that rhesus macaques inoculated with blood-stage Plasmodium coatneyi developed SMA within 2 weeks, with no other severe outcomes, whereas infected cynomolgus macaques experienced only mild/ moderate anaemia. The abrupt drop in haematocrit in rhesus was accompanied by consumption of haptoglobin (haemolysis) and poor reticulocyte production. Rhesus developed a greater inflammatory response than cynomolgus macaques, and had lower baseline levels of CD35 on red blood cells (RBCs) leading to a significant reduction in the proportion of CD35+ RBCs during infection. Overall, severe anaemia in rhesus macaques infected with P. coatneyi has similar features to SMA in children. Our comparisons are consistent with an association of low baseline CD35 levels on RBCs and of early inflammatory responses with the pathogenesis of SMA.

Mobilising a global response to hepatitis: Lessons learned from the HIV movement.

Hepatitis caused by hepatitis B and C virus is increasingly becoming a significant global health threat, with widespread prevalence that may have severe disease and economic impacts in the future. Yet, preventative measures are not implemented universally and high costs of medicines limits treatment efforts. The global response to HIV/AIDS faced similar issues, but overcame them through a global movement that brought attention to the crisis and ultimately resulted in the creation and implementation of and access to better tools for HIV prevention and treatment. This also included effective policies and programmes behind and supporting the movement. Such could be done for hepatitis, specifically using lessons from the HIV response. Here, we will discuss the current and potentially severe future burden of hepatitis globally, the challenges in addressing this epidemic, and how principles applied from the global HIV response can facilitate a successful and similar hepatitis movement.

The Sustainable Development Goals and the Global Health Security Agenda: exploring synergies for a sustainable and resilient world.

Both the Sustainable Development Goals (SDGs) and the Global Health Security Agenda (GHSA) represent bold initiatives to address systematically gaps in previous efforts to assure that societies can be resilient when confronted with potentially overwhelming threats to health. Despite their obvious differences, and differing criticisms of both, they shift away from vertical (problem- or disease-specific) to horizontal (comprehensive) solutions. Despite the comprehensiveness of the SDGs, they lack a specific target for global health security. The GHSA focuses primarily on infectious diseases and neglects non-communicable diseases and socioeconomic drivers of health. Even though each agenda has limitations and unique challenges, they are complementary. We discuss ways to understand and implement the two agendas synergistically to hasten progress toward a more sustainable and resilient world.

Financial incentives to improve progression through the HIV treatment cascade.

PURPOSE OF REVIEW: We reviewed recent literature on conditional and unconditional financial incentives for their impact on improving movement through the HIV care cascade and HIV prevention. RECENT FINDINGS: Concepts from behavioral economics may help improve engagement in HIV care by addressing upstream structural risk factors for HIV, such as poverty, or providing conditional rewards for immediate, measurable outcomes related to HIV care. Incentives have been shown to increase uptake of HIV testing. Yet, few studies to date focus on linkage to care: one large USA-based randomized trial failed to show an effect of incentives; and a smaller trial showed improved linkage to care among drug users, but no difference in virologic suppression. Several small USA-based studies have shown an impact of financial incentives on antiretroviral therapy adherence, but without durability beyond the incentive period. HIV prevention has the most robust evidence for decreasing HIV risk-taking behavior among adolescents and may serve as a model for research on the care cascade. SUMMARY: Financial incentives show promise for improving engagement in HIV testing, care, and prevention. Understanding the durability, scalability, ease of implementation, and cost-effectiveness of these different approaches will be critical for maximizing the impact of incentives in curtailing the HIV epidemic.

Heterologous HA DNA vaccine prime--inactivated influenza vaccine boost is more effective than using DNA or inactivated vaccine alone in eliciting antibody responses against H1 or H3 serotype influenza viruses.

The trivalent inactivated vaccine (TIV) is used to prevent seasonal influenza virus infection in humans, however, the immunogenicity of this vaccine may be influenced by the priming effect of previous influenza vaccinations or exposure to antigenically related influenza viruses. The current study examines the immunogenicity of a clinically licensed TIV in rabbits naïve to influenza antigens. Animals were immunized with either the licensed TIV, a bivalent (H1 and H3) HA DNA vaccine or the combination of both. Temporal and peak level serum anti-influenza virus IgG responses were determined by enzyme-linked immunosorbent assay (ELISA). Functional antibody responses were measured by hemagglutination inhibition and microneutralization against either A/NewCaledonia//20/99 (H1N1) or A/Panama/2007/99 (H3N2) influenza viruses. Our results demonstrate that the immunogenicity of the TIV is low in sero-negative animals. More significantly, the heterologous DNA prime-TIV boost regimen was more immunogenic than the homologous prime-boost using either TIV or DNA vaccines alone. This finding justifies further investigation of HA DNA vaccines as a priming immunogen for the next generation of vaccines against seasonal or pandemic influenza virus infections.

Hemagglutinin (HA) proteins from H1 and H3 serotypes of influenza A viruses require different antigen designs for the induction of optimal protective antibody responses as studied by codon-optimized HA DNA vaccines.

Effective antibody responses provide crucial immunity against influenza virus infection. The hemagglutinin (HA) protein is the major target of protective antibody responses induced by viral infection and by vaccination with both inactivated and live-attenuated flu vaccines, but knowledge about the optimal designs of protective HA antigens from different flu serotypes is still limited. In this study, we have significantly improved the immunogenicity of HA-expressing DNA vaccines by using codon-optimized HA sequences for either an H1 serotype (A/NewCal/20/99) or an H3 serotype (A/Panama/2007/99) human influenza A virus and then used these constructs as model antigens to identify the optimal HA antigen designs to elicit high-level protective antibody responses. Two forms of HA antigen, a wild-type, full-length HA and a secreted form with transmembrane (TM) domain-truncated HA, were produced. Both forms of HA DNA vaccines, from either H1 or H3 serotypes, were able to elicit high levels of HA-specific immunoglobulin G responses in immunized rabbits as measured by enzyme-linked immunosorbent assay. Interestingly, the abilities of H1 HA and H3 HA antigens to elicit hemagglutination inhibition (HI) and neutralizing antibody (NAb) responses differ. For the H1 HA antigens, the full-length HA induced significantly higher HI and NAb responses than did the TM-truncated HA. For the H3 HA antigen, both the full-length HA and TM-truncated HA induced high levels of HI and NAb responses. These data indicate that H1 and H3 antigens have different expression requirements for the induction of an optimal protective antibody response and that the structure integrity of HA antigens is critical for eliciting type-specific protective antibody responses. Our findings will have an important impact on future subunit-based flu vaccine development.