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.

Introduction The Joint United Nations Programme on HIV/AIDS (UNAIDS) has set a target of ≥90% of people living with HIV (PLHIV) receiving antiretroviral therapy (ART) to have viral load suppression (VLS). We examined factors associated with nonsuppressed viral Load (NVL).Methods We included PLHIV receiving ART aged 15–59 years from Eswatini, Lesotho, Malawi, Zambia, and Zimbabwe. Blood samples from PLHIV were analyzed for HIV RNA and recent exposure to antiretroviral drugs (ARVs). Outcomes were NVL (viral load ≥1000 copies/mL), virologic failure (VF; ARVs present and viral load ≥1000 copies/mL), interrupted ART (ARVs absent and viral load ≥1000 copies/mL), and receiving second-line ART. We calculated odds ratios and incidence rate ratios for factors associated with NVL, VF, interrupted ART, and switching to second-line ART.Results The prevalence of NVL was 11.2%: 8.2% experienced VF, and 3.0% interrupted ART. Younger age, male gender, less education, suboptimal adherence, receiving nevirapine, HIV non-disclosure, never having married, and residing in Zimbabwe, Lesotho, or Zambia were associated with higher odds of NVL. Among people with NVL, marriage, female gender, shorter ART duration, higher CD4 count, and alcohol use were associated with higher odds for interrupted ART and lower odds for VF. Many people with VF (44.8%) had CD4 counts <200 cells/µL, but few (0.31% per year) switched to second-line ART.Conclusions Countries are approaching UNAIDS VLS targets for adults. Treatment support for people initiating ART with asymptomatic HIV infection, scale-up of viral load monitoring, and optimized ART regimens may further reduce NVL prevalence.Competing Interest StatementThe authors have declared no competing interest.Funding StatementFunding: This research has been supported by the President's Emergency Plan for AIDS Relief (PEPFAR) through the Centers for Disease Control and Prevention (CDC) under the terms of grant number U2GGH001226. ADH was supported by a Swiss National Science Foundation (SNF) Early Postdoc Mobility Fellowship (grant number: P2BEP3_178602). Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the funding agencies. Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The Eswatini Scientific and Ethics Committee, the National Health Science Research Committee Malawi, the National Health Research Ethics Committee Lesotho, the National Health Research Ethics Committee Lesotho, the Tropical Diseases Research Centre Ethics Review Committee, Zambia, the Medical Research Council of Zimbabwe, and the Institutional Review Boards at the Centers for Disease Control and Prevention (CDC; Atlanta, GA) and Columbia University Medical Center (New York, NY) approved the PHIA surveys.All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesPublic datasets for Eswatini, Malawi, and Zambia are available. Public datasets for Lesotho and Zimbabwe will be made available soon. For more information see: https://phia-data.icap.columbia.edu/ https://phia-data.icap.columbia.edu/

BACKGROUND: We examined the epidemiology and transmission potential of HIV population viral load (VL) in 12 sub-Saharan African countries. METHODS: We analyzed data from Population-based HIV Impact Assessments (PHIAs), large national household-based surveys conducted between 2015 and 2019 in Cameroon, Cote d'Ivoire, Eswatini, Kenya, Lesotho, Malawi, Namibia, Rwanda, Tanzania, Uganda, Zambia, and Zimbabwe. Blood-based biomarkers included HIV serology, recency of HIV infection, and VL. We estimated the number of people living with HIV (PLHIV) with suppressed viral load (<1,000 HIV-1 RNA copies/mL) and with unsuppressed viral load (viremic), the prevalence of unsuppressed HIV (population viremia), sex-specific HIV transmission ratios (number female incident HIV-1 infections/number unsuppressed male PLHIV per 100 persons-years [PY] and vice versa) and examined correlations between a variety of VL metrics and incident HIV. Country sample sizes ranged from 10,016 (Eswatini) to 30,637 (Rwanda); estimates were weighted and restricted to participants 15 years and older. RESULTS: The proportion of female PLHIV with viral suppression was higher than that among males in all countries, however, the number of unsuppressed females outnumbered that of unsuppressed males in all countries due to higher overall female HIV prevalence, with ratios ranging from 1.08 to 2.10 (median: 1.43). The spatial distribution of HIV seroprevalence, viremia prevalence, and number of unsuppressed adults often differed substantially within the same countries. The 1% and 5% of PLHIV with the highest VL on average accounted for 34% and 66%, respectively, of countries' total VL. HIV transmission ratios varied widely across countries and were higher for male-to-female (range: 2.3-28.3/100 PY) than for female-to-male transmission (range: 1.5-10.6/100 PY). In all countries mean log10 VL among unsuppressed males was higher than that among females. Correlations between VL measures and incident HIV varied, were weaker for VL metrics among females compared to males and were strongest for the number of unsuppressed PLHIV per 100 HIV-negative adults (R2 = 0.92). CONCLUSIONS: Despite higher proportions of viral suppression, female unsuppressed PLHIV outnumbered males in all countries examined. Unsuppressed male PLHIV have consistently higher VL and a higher risk of transmitting HIV than females. Just 5% of PLHIV account for almost two-thirds of countries' total VL. Population-level VL metrics help monitor the epidemic and highlight key programmatic gaps in these African countries.

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.

BACKGROUND: Little is known about HIV in South Sudan and even less about HIV among female sex workers (FSW). We characterized progress towards UNAIDS 90-90-90 targets among female sex workers (FSW) and sexually exploited female adolescents in Juba and Nimule, South Sudan. METHODS: We conducted a biobehavioral survey of FSW and sexually exploited female adolescents using respondent-driven sampling (RDS) in Juba (November 2015-March 2016) and in Nimule (January-March 2017) to estimate achievements toward the UNAIDS 90-90-90 targets (90% of HIV-positive individuals know their status; of these, 90% are receiving antiretroviral therapy [ART]; and of these, 90% are virally suppressed). Eligibility criteria were girls and women who were aged ≥15 years; spoke English, Juba Arabic, or Kiswahili; received money, goods, or services in exchange for sex in the past 6 months; and resided, worked, or socialized in the survey city for ≥1 month. Data were weighted for RDS methods. RESULTS: We sampled 838 FSW and sexually exploited female adolescents in Juba (HIV-positive, 333) and 409 in Nimule (HIV-positive, 108). Among HIV-positive FSW and sexually exploited female adolescents living in Juba, 74.8% self-reported being aware of their HIV status; of these, 73.3% self-reported being on ART; and of these, 62.2% were virally suppressed. In Nimule, 79.5% of FSW and sexually exploited female adolescents living with HIV self-reported being aware of their HIV status; of these, 62.9% self-reported being on ART; and of these, 75.7% were virally suppressed. CONCLUSIONS: Although awareness of HIV status is the lowest of the 90-90-90 indicators in many countries, treatment uptake and viral suppression were lowest among FSW and sexually exploited female adolescents in South Sudan. Differentiated service delivery facilitate linkage to and retention on treatment in support of attainment of viral suppression.

BACKGROUND: Control of the pediatric HIV epidemic is hampered by gaps in diagnosis and linkage to effective treatment. The 2015-2016 Malawi Population-based HIV impact assessment data were analyzed to identify gaps in pediatric HIV diagnosis, treatment, and viral load suppression. METHODS: In half of the surveyed households, children ages ≥18 months to <15 years were tested using the national HIV rapid test algorithm. Children ≤18 months reactive by the initial rapid test underwent HIV total nucleic acid polymerase chain reaction confirmatory testing. Blood from HIV-positive children was tested for viral load (VL) and presence of antiretroviral drugs. HIV diagnosis and antiretroviral treatment (ART) use were defined using guardian-reporting or antiretroviral detection. RESULTS: Of the 6166 children tested, 99 were HIV-positive for a prevalence of 1.5% (95% confidence intervals [CI]: 1.1-1.9) and 8.0% (95% CI: 5.6-10.5) among HIV-exposed children. The prevalence of 1.5% was extrapolated to a national estimate of 119,501 (95% CI: 89,028-149,974) children living with HIV (CLHIV), of whom, 30.7% (95% CI: 20.3-41.1) were previously undiagnosed. Of the 69.3% diagnosed CLHIV, 86.1% (95% CI: 76.8-95.6) were on ART and 57.9% (95% CI: 41.4-74.4) of those on ART had suppressed VL (<1000 HIV RNA copies/mL). Among all CLHIV, irrespective of HIV diagnosis or ART use, 57.7% (95% CI: 45.0-70.5) had unsuppressed VL. CONCLUSIONS: Critical gaps in HIV diagnosis in children persist in Malawi. The large proportion of CLHIV with unsuppressed VL reflects gaps in diagnosis and need for more effective first- and second-line ART regimens and adherence interventions.

OBJECTIVE: We present findings from the nationally representative Zimbabwe Population-based HIV Impact Assessment (ZIMPHIA) that characterize Zimbabwe's progress toward the Joint United Nations Programme on HIV/AIDS 90-90-90 targets. DESIGN: We conducted a cross-sectional household survey. METHODS: Consenting adults and children in the household were eligible to participate in ZIMPHIA (October 2015-August 2016). Participants completed face-to-face interviews and provided blood for HIV, CD4, viral load, and syphilis testing. VLS was defined as HIV RNA <1,000 copies/mL. HIV-positive specimens were tested for the presence of selected antiretroviral drugs. Data were weighted. Analysis was restricted to HIV-positive adults aged 15-64 years. RESULTS: We enrolled 11,098 men and 14,033 women aged 15-64 years. HIV prevalence was 14.1%. Of those living with HIV, 76.8% (95% confidence interval [CI]: 74.9-78.7) were aware of their HIV status or had detectable antiretroviral levels. Of these, 88.4% (95% CI: 87.1-89.7) were receiving ART, and of these people, 85.3% (95% CI: 83.4-87.1) had VLS. Male sex age 15-34 years and having one or more sexual partners were associated with being unaware of one's HIV-positive status. Age <50 years and not taking cotrimoxazole were associated with being less likely to be being both aware and taking ART. Male sex, age <50 years, and taking cotrimoxazole were associated with being on ART but not having VLS. CONCLUSIONS: Zimbabwe has made great strides toward epidemic control. Focusing resources on case finding, particularly among men, people aged<35 years, and sexually active individuals can help Zimbabwe attain 90-90-90 targets.

BACKGROUND: In the population-based HIV impact assessment surveys, early infant diagnosis (EID) was provided to infants <18 months without a prior diagnosis. For the Namibia population-based HIV impact assessment (NAMPHIA), the GeneXpert platform was assessed for the feasibility of near POC EID testing compared with the standard Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) platform. Quality assurance measures and turnaround time were compared to improve EID results reporting. METHODS: NAMPHIA participants were screened for HIV exposure using Determine HIV-1/2 rapid test; samples reactive on Determine received EID testing on the GeneXpert instrument and Xpert HIV-1 Qual assay using whole blood. Results were confirmed at the Namibia Institute of Pathology using dried blood spots on the Roche CAP/CTM platform per national guidelines. RESULTS: Of the 762 screened infants, 61 (8.0%) were Determine-reactive and considered HIV-exposed. Of the 61 exposed infants, 2 were found to be HIV-infected whereas 59 were negative on both GeneXpert and Roche platforms, achieving 100% concordance. Average turnaround time was 3.4 days for the Xpert HIV-1 Qual assay, and average time from collection to testing was 1.0 days for GeneXpert compared with 10.7 days for Roche. No samples failed using GeneXpert whereas 1 sample failed using Roche and was repeated. CONCLUSION: Quality POC EID testing is feasible in a national survey through extensive training and external quality assurance measures. The use of decentralized POC EID for national testing would provide rapid diagnosis and improve TATs which may prevent loss to follow-up, ensure linkage to care, and improve clinical outcomes for infants.

BACKGROUND: Conducting HIV surveys in resource-limited settings is challenging because of logistics, limited availability of trained personnel, and complexity of testing. We described the procedures and systems deemed critical to ensure high-quality laboratory data in the population-based HIV impact assessments and large-scale household surveys. METHODS: Laboratory professionals were engaged in every stage of the surveys, including protocol development, site assessments, procurement, training, quality assurance, monitoring, analysis, and reporting writing. A tiered network of household, satellite laboratories, and central laboratories, accompanied with trainings, optimized process for blood specimen collection, storage, transport, and real-time monitoring of specimen quality, and test results at each level proved critical in maintaining specimen integrity and high-quality testing. A plausibility review of aggregate merged data was conducted to confirm associations between key variables as a final quality check for quality of laboratory results. RESULTS: Overall, we conducted a hands-on training for 3355 survey staff across 13 surveys, with 160-387 personnel trained per survey on biomarker processes. Extensive training and monitoring demonstrated that overall, 99% of specimens had adequate volume and 99.8% had no hemolysis, indicating high quality. We implemented quality control and proficiency testing for testing, resolved discrepancies, verified >300 Pima CD4 instruments, and monitored user errors. Aggregate data review for plausibility further confirmed the high quality of testing. CONCLUSIONS: Ongoing engagement of laboratory personnel to oversee processes at all levels of the surveys is critical for successful national surveys. High-quality population-based HIV impact assessments laboratory data ensured reliable results and demonstrated the impact of HIV programs in 13 countries.

One component of the Joint United Nations Programme on HIV/AIDS (UNAIDS) goal to end the HIV/AIDS epidemic by 2030, is that 95% of all persons receiving antiretroviral therapy (ART) achieve viral suppression.(†) Thus, testing all HIV-positive persons for viral load (number of copies of viral RNA per mL) is a global health priority (1). CDC and other U.S. government agencies, as part of the U.S. President's Emergency Plan for AIDS Relief (PEPFAR), together with other stakeholders, have provided technical assistance and supported the cost for multiple countries in sub-Saharan Africa to expand viral load testing as the preferred monitoring strategy for clinical response to ART. The individual and population-level benefits of ART are well understood (2). Persons receiving ART who achieve and sustain an undetectable viral load do not transmit HIV to their sex partners, thereby disrupting onward transmission (2,3). Viral load testing is a cost-effective and sustainable programmatic approach for monitoring treatment success, allowing reduced frequency of health care visits for patients who are virally suppressed (4). Viral load monitoring enables early and accurate detection of treatment failure before immunologic decline. This report describes progress on the scale-up of viral load testing in eight sub-Saharan African countries from 2013 to 2018 and examines the trajectory of improvement with viral load testing scale-up that has paralleled government commitments, sustained technical assistance, and financial resources from international donors. Viral load testing in low- and middle-income countries enables monitoring of viral load suppression at the individual and population level, which is necessary to achieve global epidemic control. Although there has been substantial achievement in improving viral load coverage for all patients receiving ART, continued engagement is needed to reach global targets.

The ongoing global pandemic caused by coronavirus disease has once again demonstrated the role of the family Coronaviridae in causing human disease outbreaks. Because severe acute respiratory syndrome coronavirus 2 was first detected in December 2019, information on its tropism, host range, and clinical manifestations in animals is limited. Given the limited information, data from other coronaviruses might be useful for informing scientific inquiry, risk assessment, and decision-making. We reviewed endemic and emerging infections of alphacoronaviruses and betacoronaviruses in wildlife, livestock, and companion animals and provide information on the receptor use, known hosts, and clinical signs associated with each host for 15 coronaviruses detected in humans and animals. This information can be used to guide implementation of a One Health approach that involves human health, animal health, environmental, and other relevant partners in developing strategies for preparedness, response, and control to current and future coronavirus disease threats.

BACKGROUND: Early antiretroviral therapy (ART) is necessary for HIV epidemic control and depends on early diagnosis and successful linkage to care. Since 2014, annual household-based HIV testing and counselling (HBHTC) and linkage services have been provided through the Chókwè Health and Demographic Surveillance System (CHDSS) for residents testing HIV-positive in this high HIV-burden district. METHODS: District-wide Test and Start (T&S, ART for all people living with HIV [PLHIV]) began in August 2016, supported by systematic interventions to improve linkage to care and treatment. Annual rounds (R) of random household surveys were conducted to assess trends in population prevalence of ART use and viral load suppression (VLS; <1000 viral RNA copies/mL). RESULTS: Between R1 (April 2014-April 2015) and R5 (April 2018-Mar 2019), 46,090 (67.2%) of 68,620 residents aged 15-59 years were tested for HIV at home at least once, and 3,711 were newly diagnosed with HIV and provided linkage services. Population prevalence of current ART use among PLHIV increased from 65.0% to 87.5% between R1 and R5. ART population prevalence was lowest among men aged 25-34 (67.8%) and women 15-24 (78.0%) years, and highest among women aged 35-44 (93.6%) and 45-59 years (93.7%) in R5. VLS prevalence increased among all PLHIV aged 15-59 years from 52.0% in R1 to 78.3% in R5. DISCUSSION: Between 2014 and 2019, CHDSS residents surpassed the UNAIDS targets of 81% of PLHIV on ART and of those, ≥73% virally suppressed. This achievement supports the combination of efforts from HBHTC, support for linkage to care and treatment, and continued investments in T&S implementation.

INTRODUCTION: The global target for 2020 is that ≥90% of people living with HIV (PLHIV) receiving antiretroviral therapy (ART) will achieve viral load suppression (VLS). We examined VLS and its determinants among adults receiving ART for at least four months. METHODS: We analysed data from the population-based HIV impact assessment (PHIA) surveys in Eswatini, Lesotho, Malawi, Zambia and Zimbabwe (2015 to 2017). PHIA surveys are nationally representative, cross-sectional household surveys. Data collection included structured interviews, home-based HIV testing and laboratory testing. Blood samples from PLHIV were analysed for HIV RNA, CD4 counts and recent exposure to antiretroviral drugs (ARVs). We calculated representative estimates for the prevalence of VLS (viral load <1000 copies/mL), nonsuppressed viral load (NVL; viral load ≥1000 copies/mL), virologic failure (VF; ARVs present and viral load ≥1000 copies/mL), interrupted ART (ARVs absent and viral load ≥1000 copies/mL) and rates of switching to second-line ART (protease inhibitors present) among PLHIV aged 15 to 59 years who participated in the PHIA surveys in Eswatini, Lesotho, Malawi, Zambia and Zimbabwe, initiated ART at least four months before the survey and were receiving ART at the time of the survey (according to self-report or ARV testing). We calculated odds ratios and incidence rate ratios for factors associated with NVL, VF, interrupted ART, and switching to second-line ART. RESULTS: We included 9200 adults receiving ART of whom 88.8% had VLS and 11.2% had NVL including 8.2% who experienced VF and 3.0% who interrupted ART. Younger age, male sex, less education, suboptimal adherence, receiving nevirapine, HIV non-disclosure, never having married and residing in Zimbabwe, Lesotho or Zambia were associated with higher odds of NVL. Among people with NVL, marriage, female sex, shorter ART duration, higher CD4 count and alcohol use were associated with lower odds for VF and higher odds for interrupted ART. Many people with VF (44.8%) had CD4 counts <200 cells/µL, but few (0.31% per year) switched to second-line ART. CONCLUSIONS: Countries are approaching global VLS targets for adults. Treatment support, in particular for younger adults, and people with higher CD4 counts, and switching of people to protease inhibitor- or integrase inhibitor-based regimens may further reduce NVL prevalence.

The COVID-19 pandemic highlights the substantial public health, economic, and societal consequences of virus spillover from a wildlife reservoir. Widespread human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also presents a new set of challenges when considering viral spillover from people to naïve wildlife and other animal populations. The establishment of new wildlife reservoirs for SARS-CoV-2 would further complicate public health control measures and could lead to wildlife health and conservation impacts. Given the likely bat origin of SARS-CoV-2 and related beta-coronaviruses (β-CoVs), free-ranging bats are a key group of concern for spillover from humans back to wildlife. Here, we review the diversity and natural host range of β-CoVs in bats and examine the risk of humans inadvertently infecting free-ranging bats with SARS-CoV-2. Our review of the global distribution and host range of β-CoV evolutionary lineages suggests that 40+ species of temperate-zone North American bats could be immunologically naïve and susceptible to infection by SARS-CoV-2. We highlight an urgent need to proactively connect the wellbeing of human and wildlife health during the current pandemic and to implement new tools to continue wildlife research while avoiding potentially severe health and conservation impacts of SARS-CoV-2 "spilling back" into free-ranging bat populations.

BACKGROUND: Children living with HIV (CLHIV) receiving antiretroviral treatment (ART) in resource limited settings are susceptible to high rates of acquired HIV drug resistance (HIVDR), but few studies include children initiating age-appropriate WHO-recommended first-line regimens. We report data from a cohort of ART-naïve South African children who initiated first-line ART. METHODS: ART-eligible CLHIV aged 0-12 years were enrolled from 2012 to 2014 at five public South African facilities and followed for up to 24 months. Enrolled CLHIV received standard of care WHO-recommended first-line ART. At the final study visit, a dried blood spot sample was obtained for viral load and genotypic resistance testing. RESULTS: Among 72 successfully genotyped CLHIV, 49 (68.1%) received ABC/3TC/LPV/r, and 23 (31.9%) received ABC/3TC/EFV. All but 2 children on ABC/3TC/LPV/r were <3 years and all CLHIV on ABC/3TC/EFV were ≥3 years. Overall, 80.6% (58/72) had at least one drug resistance mutation (DRM). DRMs to NNRTIs and NRTIs were found among 65% and 51% of all CLHIV, respectively, with no statistical difference by ART regimen. More CLHIV on ABC/3TC/EFV, 47.8% (11/23), were found to have 0 or only 1 effective antiretroviral drug remaining in their current regimen compared to 8.2% (4/49) on ABC/3TC/LPV/r. CONCLUSIONS: High levels of NNRTI and NRTI DRMs among CLHIV receiving ABC/3TC/LPV/r suggests a lasting impact of failed PMTCT interventions on DRMs. However, drug susceptibility analysis, reveals that CLHIV with detectable viremia on ABC/3TC/LPV/r are more likely to have maintained at least two effective agents on their current HIV regimen than those on ABC/3TC/EFV.

INTRODUCTION: Logistical complexities of returning laboratory test results to participants have precluded most population-based HIV surveys conducted in sub-Saharan Africa from doing so. For HIV positive participants, this presents a missed opportunity for engagement into clinical care and improvement in health outcomes. The Population-based HIV Impact Assessment (PHIA) surveys, which measure HIV incidence and the prevalence of viral load (VL) suppression in selected African countries, are returning VL results to health facilities specified by each HIV positive participant within eight weeks of collection. We describe the performance of the specimen and data management systems used to return VL results to PHIA participants in Zimbabwe, Malawi and Zambia. METHODS: Consenting participants underwent home-based counseling and HIV rapid testing as per national testing guidelines; all confirmed HIV positive participants had VL measured at a central laboratory on either the Roche CAP/CTM or Abbott m2000 platform. On a bi-weekly basis, a dedicated data management team produced logs linking the VL test result with the participants' contact information and preferred health facility; project staff sent test results confidentially via project drivers, national courier systems, or electronically through an adapted short message service (SMS). Participants who provided cell phone numbers received SMS or phone call alerts regarding availability of VL results. RESULTS AND DISCUSSION: From 29,634 households across the three countries, 78,090 total participants 0 to 64 years in Zimbabwe and Malawi and 0 to 59 years in Zambia underwent blood draw and HIV testing. Of the 8391 total HIV positive participants identified, 8313 (99%) had VL tests performed and 8245 (99%) of these were returned to the selected health facilities. Of the 5979 VL results returned in Zimbabwe and Zambia, 85% were returned within the eight-week goal with a median turnaround time of 48 days (IQR: 33 to 61). In Malawi, where exact return dates were unavailable all 2266 returnable results reached the health facilities by 11 weeks. CONCLUSIONS: The first three PHIA surveys returned the vast majority of VL results to each HIV positive participant's preferred health facility within the eight-week target. Even in the absence of national VL monitoring systems, a system to return VL results from a population-based survey is feasible, but it requires developing laboratory and data management systems and dedicated staff. These are likely important requirements to strengthen return of results systems in routine clinical care.

Background: Neuraminidase (NA) inhibitors are the recommended antiviral medications for influenza treatment. However, their therapeutic efficacy can be compromised by NA changes that emerge naturally and/or following antiviral treatment. Knowledge of which molecular changes confer drug resistance of influenza A(H7N9) viruses (group 2NA) remains sparse. Methods: Fourteen amino acid substitutions were introduced into the NA of A/Shanghai/2/2013(H7N9). Recombinant N9 (recN9) proteins were expressed in a baculovirus system in insect cells and tested using the Centers for Disease Control and Prevention standardized NA inhibition (NI) assay with oseltamivir, zanamivir, peramivir, and laninamivir. The wild-type N9 crystal structure was determined in complex with oseltamivir, zanamivir, or sialic acid, and structural analysis was performed. Results: All substitutions conferred either reduced or highly reduced inhibition by at least 1 NA inhibitor; half of them caused reduced inhibition or highly reduced inhibition by all NA inhibitors. R292K conferred the highest increase in oseltamivir half-maximal inhibitory concentration (IC50), and E119D conferred the highest zanamivir IC50. Unlike N2 (another group 2NA), H274Y conferred highly reduced inhibition by oseltamivir. Additionally, R152K, a naturally occurring variation at the NA catalytic residue of A(H7N9) viruses, conferred reduced inhibition by laninamivir. Conclusions: The recNA method is a valuable tool for assessing the effect of NA changes on drug susceptibility of emerging influenza viruses.

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). 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. 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 (Cote 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 Cote 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. In 2014, 150,000 children died from HIV-related causes worldwide. 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. 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, 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.

BACKGROUND: Monitoring influenza virus susceptibility to neuraminidase (NA) inhibitors (NAIs) is vital for detecting drug-resistant variants, and is primarily assessed using NA inhibition (NI) assays, supplemented by NA sequence analysis. However, differences in NI testing methodologies between surveillance laboratories results in variability of 50% inhibitory concentration (IC50) values, which impacts data sharing, reporting and interpretation. In 2011, the Centers for Disease Control and Prevention (CDC), in collaboration with the Association for Public Health Laboratories (APHL) spearheaded efforts to standardize fluorescence-based NI assay testing in the United States (U.S.), with the goal of achieving consistency of IC50 data. METHODS: For the standardization process, three participating state public health laboratories (PHLs), designated as National Surveillance Reference Centers for Influenza (NSRC-Is), assessed the NAI susceptibility of the 2011-12 CDC reference virus panel using stepwise procedures with support from the CDC reference laboratory. Next, the NSRC-Is assessed the NAI susceptibility of season 2011-12 U.S. influenza surveillance isolates (n=940), with a large subset (n=742) tested in parallel by CDC. Subsequently, U.S. influenza surveillance isolates (n=9629) circulating during the next three influenza seasons (2012-15), were independently tested by the three NSRC-Is (n=7331) and CDC (n=2298). RESULTS: The NI assay IC50s generated by respective NSRC-Is using viruses and drugs prepared by CDC were similar to those obtained with viruses and drugs prepared in-house, and were uniform between laboratories. IC50s for U.S. surveillance isolates tested during four consecutive influenza seasons (2011-15) were consistent from season to season, within and between laboratories. CONCLUSION: These results show that the NI assay is robust enough to be standardized, marking the first time IC50 data have been normalized across multiple laboratories, and used for U.S. national NAI susceptibility surveillance.

Paramyxoviruses include many important animal and human pathogens. The Parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits the viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a mini-genome system, we identified regions in the N- and C- termini protein that inhibit viral RNA synthesis: one at the very N-terminus and the second at the C-terminus of V. Furthermore, we determined that residue L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolish the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibit viral RNA replication. The C-terminus inhibits viral RNA transcription, while the N-terminal domain enhances viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibits viral RNA synthesis of other paramyxoviruses such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV) and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in replication of these paramyxoviruses. SIGNIFICANCE: We identified two regions of V that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable rational design of vaccines and potential antiviral drugs.

Highly pathogenic avian influenza (HPAI) H5N1 is endemic in Vietnamese poultry and has caused sporadic human infection in Vietnam since 2003. Human infections with HPAI H5N1 are of concern due to a high mortality rate and the potential for the emergence of pandemic viruses with sustained human-to-human transmission. Viruses isolated from humans in southern Vietnam have been classified as clade 1 with a single genome constellation (VN3) since their earliest detection in 2003. This is consistent with detection of this clade/genotype in poultry viruses endemic to the Mekong River Delta and surrounding regions. Comparison of H5N1 viruses detected in humans from southern Vietnamese provinces during 2012 and 2013 revealed the emergence of a 2013 reassortant virus with clade 1.1.2 hemagglutinin (HA) and neuraminidase (NA) surface protein genes but internal genes derived from clade 2.3.2.1a viruses (A/Hubei/1/2010-like; VN12). Closer analysis revealed mutations in multiple genes of this novel genotype (referred to as VN49) previously associated with increased virulence in animal models and other markers of adaptation to mammalian hosts. Despite the changes identified between the 2012 and 2013 genotypes analyzed, their virulence in a ferret model was similar. Antigenically, the 2013 viruses were less cross-reactive with ferret antiserum produced to the clade 1 progenitor virus, A/Vietnam/1203/2004, but reacted with antiserum produced against a new clade 1.1.2 WHO candidate vaccine virus (A/Cambodia/W0526301/2012) with comparable hemagglutination inhibition titers as the homologous antigen. Together, these results indicate changes to both surface and internal protein genes of H5N1 viruses circulating in southern Vietnam compared to 2012 and earlier viruses.

BACKGROUND: Patients contracting influenza A(H7N9) often developed severe disease causing respiratory failure. Neuraminidase (NA) inhibitors (NAIs) are the primary option for treatment, but information on drug-resistance markers for A(H7N9) is limited. METHODS: Four NA variants of A/Taiwan/1/2013 (H7N9) virus containing a single substitution (NA-E119 V, NA-I222 K, NA-I222R or NA-R292 K), recovered from an oseltamivir-treated patient, were tested for NAI susceptibility in vitro; their replicative fitness was evaluated in cell culture, mice and ferrets. RESULTS: NA-R292 K led to highly reduced inhibition by oseltamivir and peramivir, while NA-E119 V, NA-I222 K and NA-I222R caused reduced inhibition by oseltamivir. Mice infected with any virus showed severe clinical signs with high mortality rates. NA-I222 K virus was the most virulent in mice, whereas virus lacking NA change (NA-WT) and NA-R292 K virus seemed the least virulent. Sequence analysis suggests that PB2-S714N increased virulence of the NA-I222 K virus in mice; NS1-K126R, alone or in combination with PB2-V227M, produced contrasting effects in NA-WT and NA-R292 K viruses. In ferrets, all viruses replicated to high titers in the upper respiratory tract, but produced only mild illness. NA-R292 K virus, showed reduced replicative fitness in this animal model. CONCLUSIONS: Our data highlight challenges in assessment of replicative fitness of H7N9 NA variants emerged in NAI-treated patients.

Since 2011, outbreaks caused by influenza A(H3N2) variant [A(H3N2)v] viruses have become a public health concern in the United States. The A(H3N2)v viruses share the A(H1N1)pdm09 M gene containing the marker of M2 blocker resistance, S31N, but do not contain any known molecular markers associated with resistance to neuraminidase (NA) inhibitors (NAIs). Using a fluorescent NA inhibition (NI) assay, the susceptibilities of recovered A(H3N2)v viruses (n = 168) to FDA-approved (oseltamivir and zanamivir) and other (peramivir, laninamivir, and A-315675) NAIs were assessed. All A(H3N2)v viruses tested, with the exception of a single virus strain, A/Ohio/88/2012, isolated from an untreated patient, were susceptible to the NAIs tested. The A/Ohio/88/2012 virus contained two rare substitutions, S245N and S247P, in the NA and demonstrated reduced inhibition by oseltamivir (31-fold) and zanamivir (66-fold) in the NI assay. Using recombinant NA (recNA) proteins, S247P was shown to be responsible for the observed altered NAI susceptibility, in addition to an approximately 60% reduction in NA enzymatic activity. The S247P substitution has not been previously reported as a molecular marker of reduced susceptibility to the NAIs. Using cell culture assays, the investigational antiviral drugs nitazoxanide, favipiravir, and fludase were shown to inhibit the replication of A(H3N2)v viruses, including the virus with the S247P substitution in the NA. This report demonstrates the importance of continuous monitoring of susceptibility of zoonotic influenza viruses to available and investigational antiviral drugs.

We assessed drug susceptibilities of 125 avian influenza A(H5N1) viruses isolated from poultry in Vietnam during 2009-2011. Of 25 clade 1.1 viruses, all possessed a marker of resistance to M2 blockers amantadine and rimantadine; 24 were inhibited by neuraminidase inhibitors. One clade 1.1 virus contained the R430W neuraminidase gene and reduced inhibition by oseltamivir, zanamivir, and laninamivir 12-, 73-, and 29-fold, respectively. Three of 30 clade 2.3.4 viruses contained a I223T mutation and showed 7-fold reduced inhibition by oseltamivir. One of 70 clade 2.3.2.1 viruses had the H275Y marker of oseltamivir resistance and exhibited highly reduced inhibition by oseltamivir and peramivir; antiviral agents DAS181 and favipiravir inhibited H275Y mutant virus replication in MDCK-SIAT1 cells. Replicative fitness of the H275Y mutant virus was comparable to that of wildtype virus. These findings highlight the role of drug susceptibility monitoring of H5N1 subtype viruses circulating among birds to inform antiviral stockpiling decisions for pandemic preparedness.

BACKGROUND: Assessing susceptibility of influenza viruses to neuraminidase (NA) inhibitors (NAIs) is primarily done in NA inhibition (NI) assays, supplemented by NA sequence analysis. However, two factors present challenges for NI assay data interpretation: lack of established IC50 values indicative of clinically relevant resistance and insufficient harmonization of NI testing methodologies among surveillance laboratories. In 2012, the WHO working group on influenza antiviral susceptibility (WHO-AVWG) developed criteria to facilitate consistent interpretation and reporting of NI assay data. METHODS: The WHO-AVWG classification criteria were applied in interpreting NI assay data for two FDA-licensed NAIs, oseltamivir and zanamivir, for viruses collected in the United States during the 2011-2012 winter season. RESULTS: All A (H1N1)pdm09 viruses (n = 449) exhibited normal inhibition by oseltamivir and zanamivir, with the exception of eight viruses (1.8%) with highly reduced inhibition by oseltamivir, which carried the H275Y marker of oseltamivir resistance. A (H3N2) viruses (n = 978) exhibited normal inhibition by both NAIs, except for one virus with highly reduced inhibition by zanamivir due to the cell culture-selected NA change, Q136K. Type B viruses (n = 343) exhibited normal inhibition by both drugs, except for an isolate with reduced inhibition by both NAIs that had the cell culture-selected A200T substitution. CONCLUSIONS: WHO-AVWG classification criteria allowed the detection of viruses carrying the established oseltamivir resistance marker, as well as viruses whose susceptibility was altered during propagation. These criteria were consistent with statistical-based criteria for detecting outliers and will be useful in harmonizing NI assay data among surveillance laboratories worldwide and in establishing laboratory correlates of clinically relevant resistance.

Assessment of drug susceptibility has become an integral part of influenza virus surveillance. In this study, we describe the drug resistance profile of influenza A(H3N2) virus, A/Mississippi/05/2011, collected from a patient treated with oseltamivir and detected via surveillance. An MDCK cell-grown isolate of this virus exhibited highly reduced inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir (8,005-fold), zanamivir (813-fold), peramivir (116-fold), and laninamivir (257-fold) in the NA inhibition assay. Sequence analysis of its NA gene revealed a known oseltamivir-resistance marker, the glutamic acid-to-valine substitution at position 119 (E119V), and an additional change, threonine to isoleucine at position 148 (T148I). Unlike E119V, T148I was not detected in the clinical sample but acquired during viral propagation in MDCK cells. Using recombinant proteins, T148I by itself was shown to cause only a 6-fold increase in the zanamivir 50% inhibitory concentration (IC50) and had no effect on inhibition by other drugs. The T148I substitution reduced NA activity by 50%, most likely by affecting the positioning of the 150 loop at the NA catalytic site. Using pyrosequencing, changes at T148 were detected in 35 (23%) of 150 MDCK cell-grown A(H3N2) viruses tested, which was lower than the frequency of changes at D151 (85%), an NA residue previously implicated in cell selection. We demonstrate that culturing of the A(H3N2) viruses (n = 11) at a low multiplicity of infection delayed the emergence of the NA variants with changes at position 148 and/or 151, especially when conducted in MDCK-SIAT1 cells. Our findings highlight the current challenges in monitoring susceptibility of influenza A(H3N2) viruses to the NAI class of antiviral drugs.

Propagation of influenza A(H3N2) viruses in MDCK cells has been associated with the emergence of neuraminidase (NA) variants carrying a change at residue 151. In this study, the pyrosequencing assay revealed that approximately 90% of A(H3N2) virus isolates analyzed (n=150) contained more than one amino acid variant (D/G/N) at position 151. Susceptibilities of the virus isolates to zanamivir and oseltamivir were assessed using the chemiluminescent and fluorescent NA inhibition (NI) assays. In the chemiluminescent assay, which utilizes NA-Star(R) substrate, up to 13-fold increase in zanamivir-IC50 was detected for isolates containing a high proportion (>50%) of the G151 NA variant. However, an increase in zanamivir-IC50s was not seen in the fluorescent assay, which uses MUNANA as substrate. To investigate this discrepancy, recombinant NAs (rNAs) were prepared and tested in both NI assays. Regardless of the assay used, the zanamivir-IC50 for the rNA G151 was much greater (>1500-fold) than that for rNA D151 wild-type. However, zanamivir resistance conferred by the G151 substitution was masked in preparations containing the D151 NA which had much greater activity, especially against MUNANA. In conclusion, the presence of NA D151G variants in cell culture-grown viruses interferes with drug susceptibility assessment and therefore measures need to be implemented to prevent their emergence.

The QFlu prototype bioluminescence-based neuraminidase (NA) inhibition (NI) assay kit was designed to detect NA inhibitor (NAI)-resistant influenza viruses at point-of-care. Here we evaluated its suitability for drug susceptibility assessment at a surveillance laboratory. A comprehensive panel of reference viruses (n=14) and a set of 90 seasonal influenza A and B isolates were included for testing with oseltamivir and/or zanamivir in the QFlu assay using the manufacturer recommended protocol and a modified version to attune to surveillance requirement. The generated IC50s were compared with the NI assays currently used for monitoring influenza drug susceptibility, the fluorescent (FL) and chemiluminescent (CL) assays. To provide proof-of-principal, clinical specimens (n=235) confirmed by real-time RT-PCR to contain influenza A(H1N1)pdm09 virus and pre-screened for the oseltamivir resistance marker H275Y using pyrosequencing, were subsequently tested in the QFlu assay. All three NI assays were able to discriminate the reference NA variants and their matching wild-types based on the difference in their IC50s. Unless the antigenic types were first identified, certain NA variants (e.g. H3N2 with E119V) could only be detected among seasonal viruses using the FL assays. Notably, the QFlu assay identified oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Y marker directly in clinical specimens, which is not feasible with the other two phenotypic assays that required prior virus culturing in cells. Furthermore, The QFlu assay allows detection of the influenza A and B virus isolates carrying established and potential NA inhibitor-resistance markers, and may become a useful tool for monitoring drug resistance in clinical specimens.