SARS-CoV-2 live virus culture and sample freeze-thaw stability.

The effect of freeze-thaw on SARS-CoV-2 viral viability is not well established. We isolated virus from 31 split clinical samples cultured fresh or after a 7- or 17/18-day freeze. We found that freeze-thaw did not significantly affect viral culture isolation. Therefore, frozen samples may be used to assess SARS-CoV-2 infectiousness.

High SARS-CoV-2 seroprevalence in Lagos, Nigeria with robust antibody and cellular immune responses.

Background: Early evidence suggested that the impact of the COVID-19 pandemic was less severe in Africa compared to other parts of the world. However, more recent studies indicate higher SARS-CoV-2 infection and COVID-19 mortality rates on the continent than previously documented. Research is needed to better understand SARS-CoV-2 infection and immunity in Africa. Methods: In early 2021, we studied the immune responses in healthcare workers (HCWs) at Lagos University Teaching Hospital (n = 134) and Oxford-AstraZeneca COVID-19 vaccine recipients from the general population (n = 116) across five local government areas (LGAs) in Lagos State, Nigeria. Western blots were used to simultaneously detect SARS-CoV-2 spike and nucleocapsid (N) antibodies (n = 250), and stimulation of peripheral blood mononuclear cells with N followed by an IFN-γ ELISA was used to examine T cell responses (n = 114). Results: Antibody data demonstrated high SARS-CoV-2 seroprevalence of 72·4% (97/134) in HCWs and 60·3% (70/116) in the general population. Antibodies directed to only SARS-CoV-2 N, suggesting pre-existing coronavirus immunity, were seen in 9·7% (13/134) of HCWs and 15·5% (18/116) of the general population. T cell responses against SARS-CoV-2 N (n = 114) were robust in detecting exposure to the virus, demonstrating 87·5% sensitivity and 92·9% specificity in a subset of control samples tested. T cell responses against SARS-CoV-2 N were also observed in 83.3% of individuals with N-only antibodies, further suggesting that prior non-SARS-CoV-2 coronavirus infection may provide cellular immunity to SARS-CoV-2. Conclusions: These results have important implications for understanding the paradoxically high SARS-CoV-2 infection with low mortality rate in Africa and supports the need to better understand the implications of SARS-CoV-2 cellular immunity.

Sars-Cov-2 antigen tests predict infectivity based on viral culture: comparison of antigen, PCR viral load, and viral culture testing on a large sample cohort.

OBJECTIVE: To define the relationship of SARS-CoV-2 antigen, viral load determined by RT-qPCR, and viral culture detection. Presumptively, viral culture can provide a surrogate measure for infectivity of sampled individuals and thereby inform how and where to most appropriately deploy antigen and nucleic acid amplification-based diagnostic testing modalities. METHODS: We compared the antigen testing results from three lateral flow and one microfluidics assay to viral culture detection and viral load determination performed in parallel in up to 189 nasopharyngeal swab samples positive for SARS-CoV-2. Sample viral loads, determined by RT-qPCR, were distributed across the range of viral load values observed in our testing population. RESULTS: Antigen tests were predictive of viral culture positivity, with the LumiraDx microfluidics method showing enhanced sensitivity (90%; 95% CI 83-94%) compared with the BD Veritor (74%, 95% CI 65-81%), CareStart (74%, 95% CI 65-81%) and Oscar Corona (74%, 95% CI 65-82%) lateral flow antigen tests. Antigen and viral culture positivity were also highly correlated with sample viral load, with areas under the receiver operator characteristic curves of 0.94 to 0.97 and 0.92, respectively. A viral load threshold of 100 000 copies/mL was 95% sensitive (95% CI, 90-98%) and 72% specific (95% CI, 60-81%) for predicting viral culture positivity. Adjusting for sample dilution inherent in our study design, sensitivities of antigen tests were ≥95% for detection of viral culture positive samples with viral loads >106 genome copies/mL, although specificity of antigen testing was imperfect. DISCUSSION: Antigen testing results and viral culture were correlated. For culture positive samples, the sensitivity of antigen tests was high at high viral loads that are likely associated with significant infectivity. Therefore, our data provides support for use of antigen testing in ruling out infectivity at the time of sampling.

Longitudinal evaluation of adherence, retention, and transition patterns of adolescents living with HIV in Nigeria.

INTRODUCTION: Adherence to antiretroviral therapy (ART) and retention in treatment programs are required for successful virologic suppression and treatment outcomes. As the number of adolescents living with HIV continues to increase globally, more information about adherence and retention patterns during and through transition from child- to adult-centered care is needed to ensure provision of a high level of care and inform development of targeted interventions to improve patient outcomes in this vulnerable population. In this analysis, we sought to describe long-term trends in adherence, retention, and virologic suppression in adolescents receiving ART at a pediatric HIV clinic in Nigeria through transition to the adult clinic. SETTING: The Jos University Teaching Hospital, United States President's Emergency Plan for AIDS Relief (PEPFAR)-funded HIV clinic in Jos, Plateau State, Nigeria. METHODS: We conducted a retrospective observational longitudinal evaluation of data that had been collected during the course of care in a large pediatric ART program in Nigeria. We used descriptive statistics to define our patient population and quantify retention from ART initiation through adolescence and transition to adult-centered care. Logistic regression was used to evaluate predictors of loss to follow-up. We used medication possession ratio (MPR) to quantify adherence for each year a patient was on ART. To evaluate adherence and virologic suppression, we measured the proportion of patients with ≥95% MPR and the proportion with virologic suppression (viral load ≤400 copies/mL) within each age cohort, and used bivariate analyses to examine any association between MPR and VL suppression for all person-years observed. RESULTS: A total of 476 patients received at least one dose of ART as an adolescent (ages 10-19 years). The proportions of patients lost to follow-up were: 11.9% (71/597) prior to adolescence, 19.1% (31/162) during adolescence, and 13.7% (10/73) during transition to adult-centered care. While over 80% of patients had ≥95% medication adherence in all age groups, their viral load suppression rates through adolescence and post-transition were only 55.6%-64.0%. For patients that successfully transitioned to adult-centered care, we observed 87.7% (50/57) retention at month 12 post-transition, but only 34.6% (9/26) viral load suppression. CONCLUSIONS: Our evaluation found considerable proportions of adolescents lost to follow-up throughout the ART program cascade. We also found discrepancies between the proportions of patients with ≥95% MPR and the proportions with VL suppression, suggesting that true medication adherence in this population may be poor. Significant attention and targeted interventions to improve retention and adherence focused on adolescents are needed in order for global programs to achieve 90-90-90 goals.

The role of point-of-care viral load monitoring in achieving the target of 90% suppression in HIV-infected patients in Nigeria: study protocol for a randomized controlled trial.

BACKGROUND: The Joint United Nations Programme on HIV/AIDS 90-90-90 goal envisions 90% of all people receiving antiretroviral therapy to be virally suppressed by 2020. Implied in that goal is that viral load be quantified for all patients receiving treatment, which is a challenging undertaking given the complexity and high cost of standard-of-care viral load testing methods. Recently developed point-of-care viral load testing devices offer new promise to improve access to viral load testing by bringing the test closer to the patient and also returning results faster, often same-day. While manufactures have evaluated point-of-care assays using reference panels, empiric data examining the impact of the new technology against standard-of-care monitoring in low- and middle-income settings are lacking. Our goal in this trial is to compare a point-of-care to standard-of-care viral load test on impact on various clinical outcomes as well to assess the acceptability and feasibility of using the assay in a resource-limited setting. METHODS: Using a two-arm randomized control trial design, we will enroll 794 patients from two different HIV treatment sites in Nigeria. Patients will be randomized 1:1 for point-of-care or standard-of-care viral load monitoring (397 patients per arm). Following initiation of treatment, viral load will be monitored at patients' 6- and 12-month follow-up visits using either point-of-care or standard-of-care testing methods, based on trial assignment. The monitoring schedule will follow national treatment guidelines. The primary outcome measure in this trial is proportion of patients with viral suppression at month 12 post-initiation of treatment. The secondary outcome measures encompass acceptability, feasibility, and virologic impact variables. DISCUSSION: This clinical trial will provide information on the impact of using point-of-care versus standard-of-care viral load testing on patient clinical outcomes; the study will also supply data on the acceptability and feasibility of point-of-care viral load monitoring in a resource-limited setting. If this method of testing is acceptable and feasible, and also superior to standard of care, the results of the trial and the information gathered will inform future scaled implementation and further optimization of the clinic-laboratory network that is critical for monitoring achievement of the 90-90-90 goals. TRIAL REGISTRATION: US National Institutes of Health Clinical Trials.gov: NCT03533868 . Date of Registration: 23 May 2018. Protocol Version: 10. Protocol Date: 30 March 2018.

A modified anthrax toxin-based enzyme-linked immunospot assay reveals robust T cell responses in symptomatic and asymptomatic Ebola virus exposed individuals.

BACKGROUND: Ebola virus (EBOV) caused more than 11,000 deaths during the 2013-2016 epidemic in West Africa without approved vaccines or immunotherapeutics. Despite its high lethality in some individuals, EBOV infection can produce little to no symptoms in others. A better understanding of the immune responses in individuals who experienced minimally symptomatic and asymptomatic infection could aid the development of more effective vaccines and antivirals against EBOV and related filoviruses. METHODOLOGY/PRINCIPLE FINDINGS: Between August and November 2017, blood samples were collected from 19 study participants in Lagos, Nigeria, including 3 Ebola virus disease (EVD) survivors, 10 individuals with documented close contact with symptomatic EVD patients, and 6 control healthcare workers for a cross-sectional serosurvey and T cell analysis. The Lagos samples, as well as archived serum collected from healthy individuals living in surrounding areas of the 1976 Democratic Republic of Congo (DRC) epidemic, were tested for EBOV IgG using commercial enzyme-linked immunosorbent assays (ELISAs) and Western blots. We detected antibodies in 3 out of 3 Lagos survivors and identified 2 seropositive individuals not known to have ever been infected. Of the DRC samples tested, we detected antibodies in 9 out of 71 (12.7%). To characterize the T cell responses in the Lagos samples, we developed an anthrax toxin-based enzyme-linked immunospot (ELISPOT) assay. The seropositive asymptomatic individuals had T cell responses against EBOV nucleoprotein, matrix protein, and glycoprotein 1 that were stronger in magnitude compared to the survivors. CONCLUSION/SIGNIFICANCE: Our data provide further evidence of EBOV exposure in individuals without EVD-like illness and, for the first time, demonstrate that these individuals have T cell responses that are stronger in magnitude compared to severe cases. These findings suggest that T cell immunity may protect against severe EVD, which has important implications for vaccine development.

Sustained Specific and Cross-Reactive T Cell Responses to Zika and Dengue Virus NS3 in West Africa.

Recent studies on the role of T cells in Zika virus (ZIKV) infection have shown that T cell responses to Asian ZIKV infection are important for protection, and that previous dengue virus (DENV) exposure amplifies the protective T cell response to Asian ZIKV. Human T cell responses to African ZIKV infection, however, remain unexplored. Here, we utilized the modified anthrax toxin delivery system to develop a flavivirus enzyme-linked immunosorbent spot (ELISPOT) assay. Using human ZIKV and DENV samples from Senegal, West Africa, our results demonstrate specific and cross-reactive T cell responses to nonstructural protein 3 (NS3). Specifically, we found that T cell responses to NS3 protease are ZIKV and DENV specific, but responses to NS3 helicase are cross-reactive. Sequential sample analyses revealed immune responses sustained many years after infection. These results have important implications for African ZIKV/DENV vaccine development, as well as for potential flavivirus diagnostics based on T cell responses.IMPORTANCE The recent Zika virus (ZIKV) epidemic in Latin America and the associated congenital microcephaly and Guillain-Barré syndrome have raised questions as to why we have not recognized these distinct clinical diseases in Africa. The human immunologic response to ZIKV and related flaviviruses in Africa represents a research gap that may shed light on the mechanisms contributing to protection. The goal of our study was to develop an inexpensive assay to detect and characterize the T cell response to African ZIKV and DENV. Our data show long-term specific and cross-reactive human immune responses against African ZIKV and DENV, suggesting the usefulness of a diagnostic based on the T cell response. Additionally, we show that prior flavivirus exposure influences the magnitude of the T cell response. The identification of immune responses to African ZIKV and DENV is of relevance to vaccine development.

Continued Transmission of Zika Virus in Humans in West Africa, 1992-2016.

First identified in 1947 in Uganda, Zika virus (ZIKV) has remained largely unstudied until the recent outbreak in Latin America. This study aimed to measure the prevalence of ZIKV in febrile patients in Senegal and Nigeria in samples collected from 1992 to 2016. The seroprevalence of ZIKV was 6.2% based on ZIKV immunoglobulin M and negative for dengue reactivity. ZIKV envelope was amplified from 4 samples. Phylogenetic analysis showed that the ZIKVs belonged to the African lineage, grouping with either the Nigerian or MR766 sublineages. This study provides evidence that ZIKV has been silently circulating in West Africa for 2 decades.

Long-Term Outcomes on Antiretroviral Therapy in a Large Scale-Up Program in Nigeria.

BACKGROUND: While there has been a rapid global scale-up of antiretroviral therapy programs over the past decade, there are limited data on long-term outcomes from large cohorts in resource-constrained settings. Our objective in this evaluation was to measure multiple outcomes during first-line antiretroviral therapy in a large treatment program in Nigeria. METHODS: We conducted a retrospective multi-site program evaluation of adult patients (age ≥15 years) initiating antiretroviral therapy between June 2004 and February 2012 in Nigeria. The baseline characteristics of patients were described and longitudinal analyses using primary endpoints of immunologic recovery, virologic rebound, treatment failure and long-term adherence patterns were conducted. RESULTS: Of 70,002 patients, 65.2% were female and median age was 35 (IQR: 29-41) years; 54.7% were started on a zidovudine-containing and 40% on a tenofovir-containing first-line regimen. Median CD4+ cell counts for the cohort started at 149 cells/mm3 (IQR: 78-220) and increased over duration of ART. Of the 70,002 patients, 1.8% were reported as having died, 30.1% were lost to follow-up, and 0.1% withdrew from treatment. Overall, of those patients retained and with viral load data, 85.4% achieved viral suppression, with 69.3% achieving suppression by month 6. Of 30,792 patients evaluated for virologic failure, 24.4% met criteria for failure and of 45,130 evaluated for immunologic failure, 34.0% met criteria for immunologic failure, with immunologic criteria poorly predicting virologic failure. In adjusted analyses, older age, ART regimen, lower CD4+ cell count, higher viral load, and inadequate adherence were all predictors of virologic failure. Predictors of immunologic failure differed slightly, with age no longer predictive, but female sex as protective; additionally, higher baseline CD4+ cell count was also predictive of failure. Evaluation of long-term adherence patterns revealed that the majority of patients retained through 84 months maintained ≥95% adherence. CONCLUSION: While improved access to HIV care and treatment remains a challenge in Nigeria, our study shows that a high quality of care was achieved as evidenced by strong long-term clinical, immunologic and virologic outcomes.

Tuberculosis Incidence and Risk Factors Among Human Immunodeficiency Virus (HIV)-Infected Adults Receiving Antiretroviral Therapy in a Large HIV Program in Nigeria.

Background. Despite the benefits of antiretroviral therapy (ART), tuberculosis (TB) is the leading cause of mortality among human immunodeficiency virus (HIV)-infected persons in Africa. Nigeria bears the highest TB burden in Africa and second highest HIV burden globally. This long-term multicenter study aimed to determine the incidence rate and predictors of TB in adults in the Harvard/AIDS Prevention Initiative in Nigeria (APIN) and President's Emergency Plan for AIDS Relief (PEPFAR) Nigeria ART program. Methods. This retrospective evaluation used data collected from 2004 to 2012 through the Harvard/APIN PEPFAR program. Risk factors for incident TB were determined using multivariate Cox proportional hazards regression with time-dependent covariates. Results. Of 50 320 adults enrolled from 2005 to 2010, 11 092 (22%) had laboratory-confirmed active TB disease at ART initiation, and 2021 (4%) developed active TB after commencing ART. During 78 228 total person-years (PY) of follow-up, the TB incidence rate was 25.8 cases per 1000 PY (95% confidence interval [CI], 24.7-27.0) overall, and it decreased significantly both with duration on ART and calendar year. Risk factors at ART initiation for incident TB included the following: earlier ART enrollment year, tenofovir-containing initial ART regimen, and World Health Organization clinical stage above 1. Time-updated risk factors included the following: low body mass index, low CD4(+) cell count, unsuppressed viral load, anemia, and ART adherence below 80%. Conclusions. The rate of incident TB decreased with longer duration on ART and over the program years. The strongest TB risk factors were time-updated clinical markers, reinforcing the importance of consistent clinical and laboratory monitoring of ART patients in prompt diagnosis and treatment of TB and other coinfections.

Loss to Follow-Up within the Prevention of Mother-to-Child Transmission Care Cascade in a Large ART Program in Nigeria.

BACKGROUND: The 2013 WHO guidelines incorporated simplified and more effective antiretroviral regimens for the purposes of preventing mother-to-child transmission of HIV. With ideal implementation of these recommendations, perinatal HIV transmission could be reduced to less than 2%. However, loss to follow-up (LTFU) has the potential to erode the success of programs and a number of studies report high rates of LTFU within the prevention of mother-to-child transmission (PMTCT) care cascade. We evaluated the timing and magnitude of LTFU in a large programmatic PMTCT cohort in Nigeria in order to focus future efforts to reduce loss in this high burden setting. METHODS: From 2004-2014, the APIN/Harvard PEPFAR program supported antenatal HIV screening for nearly one million pregnant women and provided PMTCT care to over 30,000 women. The care cascade for women enrolling in the PMTCT program includes antenatal, delivery, and infant follow-up services through 12-18 months of life. In this retrospective cohort analysis, we examined data collected between 2004-2014 from 31 clinical sites in Nigeria and assessed the numbers of mothers and infants enrolled and LTFU at various points along the care cascade. RESULTS: Among 31,504 women (median age 30, IQR: 27-34) entering PMTCT care during the antenatal period, 20,679 (66%) completed the entire cascade of services including antenatal, delivery, and at least one infant follow-up visit. The median gestational age at presentation for antenatal care services was 23 weeks (IQR: 17-29). The median infant age at last follow-up visit was 12 months (IQR: 5-18). The greatest loss in the PMTCT care cascade occurred prior to delivery care (21%), with a further 16% lost prior to first infant visit. Of the 38,223 women who entered at any point along the PMTCT cascade, an HIV DNA PCR was available for 20,202 (53%) of their infants. Among infants for whom DNA PCR results were available, the rate of HIV transmission for infants whose mothers received any antenatal and/or delivery care was 2.8% versus 20.0% if their mother received none. CONCLUSION: In this large cohort analysis, the proportion of women LTFU in the PMTCT care cascade was lower than that reported in previous cohort analyses. Nevertheless, this proportion remains unacceptably high and inhibits the program from maximally achieving the goals of PMTCT care. We also provide the largest analysis to date on rates of perinatal HIV transmission, with low rates among women receiving NNRTI- or PI-based regimens, approaching that reported in clinical trials. However, among mothers who received any antenatal care, infant outcomes were unknown for 48%, and women presented later in pregnancy than that recommended by current guidelines. Implementation research to evaluate ways to improve integration of services, particularly transitions from antenatal to delivery and pediatric care, are critically needed to reduce LTFU within PMTCT programs and achieve the ultimate goal of eliminating pediatric HIV infection.