Contribution of PEPFAR-Supported HIV and TB Molecular Diagnostic Networks to COVID-19 Testing Preparedness in 16 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.

Cross-Reactivity of Two SARS-CoV-2 Serological Assays in a Setting Where Malaria Is Endemic.

Accurate SARS-CoV-2 serological assays are critical for COVID-19 serosurveillance. However, previous studies have indicated possible cross-reactivity of these assays, including in areas where malaria is endemic. We tested 213 well-characterized prepandemic samples from Nigeria using two SARS-CoV-2 serological assays, Abbott Architect IgG and Euroimmun NCP IgG assay, both targeting SARS-CoV-2 nucleocapsid protein. To assess antibody binding strength, an avidity assay was performed on these samples and on plasma from SARS-CoV-2 PCR-positive persons. Thirteen (6.1%) of 212 samples run on the Abbott assay and 38 (17.8%) of 213 run on the Euroimmun assay were positive. Anti-Plasmodium IgG levels were significantly higher among false positives for both Abbott and Euroimmun; no association was found with active Plasmodium falciparum infection. An avidity assay using various concentrations of urea wash in the Euroimmun assay reduced loosely bound IgG: of 37 positive/borderline prepandemic samples, 46%, 86%, 89%, and 97% became negative using 2 M, 4 M, 5 M, and 8 M urea washes, respectively. The wash slightly reduced avidity of antibodies from SARS-CoV-2 patients within 28 days of PCR confirmation; thereafter, avidity increased for all urea concentrations except 8 M. This validation found moderate to substantial cross-reactivity on two SARS-CoV-2 serological assays using samples from a setting where malaria is endemic. A simple urea wash appeared to alleviate issues of cross-reactivity.

In vivo efficacy of sulphadoxine-pyrimethamine for the treatment of asymptomatic parasitaemia in pregnant women in Machinga District, Malawi.

BACKGROUND: The effectiveness of sulphadoxine-pyrimethamine (SP) intermittent preventive treatment of malaria in pregnancy (IPTp) might be compromised by high prevalence of resistance-associated Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutations. As a proxy for IPTp-SP effectiveness, the in vivo efficacy of SP to clear parasitaemia and prevent reinfection in asymptomatic parasitaemic pregnant women in an area with high SP resistance prevalence was assessed. METHODS: Pregnant women 16-26 weeks' gestation with asymptomatic parasitaemia presenting for antenatal care were given IPTp-SP and followed for 42 days. The primary outcome was polymerase chain reaction (PCR) uncorrected 42-day survival rate; the per cent of patients without recrudescence or reinfection by day 42. PCR was used to distinguish recrudescence from reinfection. DNA was sequenced to detect resistance-associated dhfr and dhps mutations. RESULTS: Of 245 pregnant women included in the intention-to-treat analysis, 93.9% cleared their parasitaemia by day 7. The day 42 PCR-uncorrected survival rate was 58.1% (95% confidence interval (CI) 51.5-65.7) and day 42 PCR-corrected survival was 68.7% (CI 61.4-76.0). Recrudescence was more common among primi- than among multigravid women; recrudescence rate 33.3% (CI 25.1-42.4%) versus 21.4% (CI 15.0-29.0%) (log rank test p-value 0.006). The quintuple mutant was present in nearly all samples (95%), while 2% were sextuple mutants with an additional mutation at dhps A581G. CONCLUSIONS: SP efficacy for acute malaria treatment has been compromised by resistance, but SP retains partial activity among pregnant women with asymptomatic parasitaemia, and thus might be useful for IPTp. Nonetheless, research on non-SP IPTp regimens should continue. TRIAL REGISTRATION: ClinicalTrials.gov NCT01120145 .

Genetic diversity of Plasmodium falciparum parasite by microsatellite markers after scale-up of insecticide-treated bed nets in western Kenya.

BACKGROUND: An initial study of genetic diversity of Plasmodium falciparum in Asembo, western Kenya showed that the parasite maintained overall genetic stability 5 years after insecticide-treated bed net (ITN) introduction in 1997. This study investigates further the genetic diversity of P. falciparum 10 years after initial ITN introduction in the same study area and compares this with two other neighbouring areas, where ITNs were introduced in 1998 (Gem) and 2004 (Karemo). METHODS: From a cross-sectional survey conducted in 2007, 235 smear-positive blood samples collected from children ≤15-year-old in the original study area and two comparison areas were genotyped employing eight neutral microsatellites. Differences in multiple infections, allele frequency, parasite genetic diversity and parasite population structure between the three areas were assessed. Further, molecular data reported previously (1996 and 2001) were compared to the 2007 results in the original study area Asembo. RESULTS: Overall proportion of multiple infections (MA) declined with time in the original study area Asembo (from 95.9 %-2001 to 87.7 %-2007). In the neighbouring areas, MA was lower in the site where ITNs were introduced in 1998 (Gem 83.7 %) compared to where they were introduced in 2004 (Karemo 96.7 %) in 2007. Overall mean allele count (MAC ~ 2.65) and overall unbiased heterozygosity (H e  ~ 0.77) remained unchanged in 1996, 2001 and 2007 in Asembo and was the same level across the two neighbouring areas in 2007. Overall parasite population differentiation remained low over time and in the three areas at FST < 0.04. Both pairwise and multilocus linkage disequilibrium showed limited to no significant association between alleles in Asembo (1996, 2001 and 2007) and between three areas. CONCLUSIONS: This study showed the P. falciparum high genetic diversity and parasite population resilience on samples collected 10 years apart and in different areas in western Kenya. The results highlight the need for long-term molecular monitoring after implementation and use of combined and intensive prevention and intervention measures in the region.

Schistosomiasis Seroprevalence among Children Aged 0-14 Years in Nigeria, 2018.

The first nationally representative, population-based study of schistosomiasis seroprevalence in Nigeria was conducted using blood samples and risk-factor data collected during the 2018 Nigeria HIV/AIDS Indicator and Impact Survey (NAIIS). Schistosomiasis seroprevalence was estimated by analyzing samples for reactivity to schistosome soluble egg antigen (SEA) in a multiplex bead assay; NAIIS survey data were assessed to identify potential risk factors for seropositivity. The SEA antibody data were available for 31,459 children aged 0 to 14 years. Overall seroprevalence was 17.2% (95% CI: 16.3-18.1%). Seropositive children were identified in every age group, including children < 5 years, and seroprevalence increased with increasing age (P < 0.0001). Several factors were associated with increased odds of seropositivity, including being a boy (odds ratio [OR] = 1.34, 95% CI: 1.24-1.45), living in a rural area (OR = 2.2, 95% CI: 1.9-2.5), and animal ownership (OR = 1.67, 95% CI: 1.52-1.85). Access to improved sanitation and drinking water sources were associated with decreased odds of seropositivity (OR = 0.52, 95% CI: 0.47-0.58 and OR = 0.53, 95% CI: 0.47-0.60, respectively) regardless of whether the child lived in a rural (sanitation: adjusted odds ratio [aOR] = 0.7, 95% CI: 0.6-0.8; drinking water: aOR = 0.7, 95% CI: 0.6-0.8) or urban area (sanitation: aOR = 0.6, 95% CI: 0.5-0.7; drinking water: aOR = 0.5, 95% CI: 0.4-0.6), highlighting the importance of these factors for schistosomiasis prevention and control. These results identified additional risk populations (children < 5 years) and a new risk factor (animal ownership) and could be used to monitor the impact of control programs.

Treponemal Antibody Seroprevalence Using a Multiplex Bead Assay from Samples Collected during the 2018 Nigeria HIV/AIDS Indicator and Impact Survey: Searching for Yaws in Nigeria.

Yaws is a chronic, relapsing disease of skin, bone, and cartilage caused by Treponema pallidum subsp. pertenue. Yaws was last reported in Nigeria in 1996, although neighboring countries have recently reported cases. We investigated serological evidence for yaws among children aged 0-14 years in Nigeria by measuring antibodies to the treponemal antigens rp17 and TmpA in blood specimens from a 2018 nationally representative HIV survey using a multiplex bead assay. The presence of antibodies to both antigens ("double positive") likely reflects current or recent treponemal infection. Overall, 1.9% (610/31,549) of children had anti-TmpA antibodies, 1.5% (476/31,549) had anti-rp17 antibodies, and 0.1% (39/31,549) were double positive. Among households, 0.5% (84/18,021) had a double-positive child, with a clustering of double-positive children. Although numbers are low, identification of antibodies to both TmpA and rp17 may warrant investigation, including more granular epidemiologic and clinical data, to assess the potential for continuing yaws transmission in Nigerian children.

Non-falciparum malaria infection and IgG seroprevalence among children under 15 years in Nigeria, 2018.

Plasmodium falciparum (Pf) is the dominant malaria parasite in Nigeria though P. vivax (Pv), P. ovale (Po), and P. malariae (Pm) are also endemic. Blood samples (n = 31,234) were collected from children aged 0-14 years during a 2018 nationwide HIV survey and assayed for Plasmodium antigenemia, Plasmodium DNA, and IgG against Plasmodium MSP1-19 antigens. Of all children, 6.6% were estimated to have Pm infection and 1.4% Po infection with no Pv infections detected. The highest household wealth quintile was strongly protective against infection with Pm (aOR: 0.11, 95% CI: 0.05-0.22) or Po (aOR= 0.01, 0.00-0.10). Overall Pm seroprevalence was 34.2% (95% CI: 33.3-35.2) with lower estimates for Po (12.1%, 11.6-12.5) and Pv (6.3%, 6.0-6.7). Pm seropositivity was detected throughout the country with several local government areas showing >50% seroprevalence. Serological and DNA indicators show widespread exposure of Nigerian children to Pm with lower rates to Po and Pv.

Dynamics of IgG antibody response against Plasmodium antigens among Nigerian infants and young children.

Background: Plasmodium falciparum malaria is a leading cause of child mortality in Nigeria. Neonates are born with maternal antibodies from placental transfer which may protect against malaria infection in the first months of life. The IgG dynamics of the transition from passively transferred antimalarial antibodies to actively acquired IgG from natural exposure have not been well elucidated. Methods: Blood samples collected during a 2018 Nigeria nationwide HIV/AIDS household survey were available for 9,443 children under 5 years of age, with a subset of infants under 2 months of age having maternal samples available (n=41). Samples were assayed for the P. falciparum HRP2 antigen and anti-malarial IgG antibodies. LOESS regression examined the dynamics in IgG response in the first 5 years of life. Correlation with maternal IgG levels was assessed for mother/child pairs. Results: Consistent decreases were observed in median IgG levels against all Plasmodium spp. antigen targets for the first months of life. At a population level, P. falciparum apical membrane antigen-1 (AMA1) and merozoite surface protein-1 19kD (PfMSP1) IgG decreased during the first 12 months of life before reaching a nadir, whereas IgGs to other targets only declined for the first 4 months of life. Seropositivity showed a similar decline with the lowest seropositivity against AMA1 and PfMSP1 at 10-12 months, though remaining above 50% during the first 2 years of life in higher transmission areas. No protective association was observed between IgG positivity and P. falciparum infection in infants. Maternal antibody levels showed a strong positive correlation with infant antibody levels for all P. falciparum antigens from birth to 2 months of age, but this correlation was lost by 6 months of age. Discussion: Maternally transferred anti-malarial IgG antibodies rapidly decline during the first 6 months of life, with variations among specific antigens and malaria transmission intensity. From 3-23 months of age, there was a wide range in IgG levels for the blood-stage antigens indicating high individual variation in antibody production as children are infected with malaria. Non-falciparum species-specific antigens showed similar patterns in waning immunity and correlation with paired mother's IgG levels compared to P. falciparum antigens.

Plasmodium falciparum infection prevalence among children aged 6-59 months from independent DHS and HIV surveys: Nigeria, 2018.

Prevalence estimates are critical for malaria programming efforts but generating these from non-malaria surveys is not standard practice. Malaria prevalence estimates for 6-59-month-old Nigerian children were compared between two national household surveys performed simultaneously in 2018: a Demographic and Health Survey (DHS) and the Nigeria HIV/AIDS Indicator and Impact Survey (NAIIS). DHS tested via microscopy (n = 8298) and HRP2-based rapid diagnostic test (RDT, n = 11,351), and NAIIS collected dried blood spots (DBS) which were later tested for histidine-rich protein 2 (HRP2) antigen (n = 8029). National Plasmodium falciparum prevalence was 22.6% (95% CI 21.2- 24.1%) via microscopy and 36.2% (34.6- 37.8%) via RDT according to DHS, and HRP2 antigenemia was 38.3% (36.7-39.9%) by NAIIS DBS. Between the two surveys, significant rank-order correlation occurred for state-level malaria prevalence for RDT (Rho = 0.80, p < 0.001) and microscopy (Rho = 0.75, p < 0.001) versus HRP2. RDT versus HRP2 positivity showed 24 states (64.9%) with overlapping 95% confidence intervals from the two independent surveys. P. falciparum prevalence estimates among 6-59-month-olds in Nigeria were highly concordant from two simultaneous, independently conducted household surveys, regardless of malaria test utilized. This provides evidence for the value of post-hoc laboratory HRP2 detection to leverage non-malaria surveys with similar sampling designs to obtain accurate P. falciparum estimates.

Seroprevalence of poliovirus antibodies in Nigeria: refining strategies to sustain the eradication effort.

Introduction: in 2016, a switch from trivalent oral poliovirus vaccine (OPV) (containing serotypes 1,2,3) to bivalent OPV (types 1,3) was implemented globally. We assessed the seroprevalence of poliovirus antibody levels in selected Nigerian states, before and after the switch, documented poliovirus type2 outbreak responses conducted and ascertained factors associated with immunity gaps based on seroprevalence rates. Methods: we conducted a secondary analysis of stored serum samples from the 2018 Nigeria National HIV/AIDS Indicator and Impact Survey. Serum from 1,185 children aged 0-119 months residing in one southern and four northern states were tested for serotype-specific PV neutralizing antibodies; seropositivity was a reciprocal titer ≥8. We conducted regression analysis to determine sociodemographic risk factors associated with low seroprevalence using SAS 9.4. Results: children aged 24-119 months (pre-switch cohort) had seroprevalence against PV1, PV2, and PV3, of 97.3% (95% CI:96.4-98.2), 93.8% (95% CI:92.2-95.5), and 91.3% (95% CI:89.2-93.4), while children aged <24 months (post-switch) had seroprevalence of 86.0% (95% CI:81.2-90.8), 55.6% (95% CI: 47.7-63.4), and 77.2% (95% CI:71.0-83.4) respectively. Regression analysis showed age <24 months was associated with lower seroprevalence against all PV serotypes, (p<0.0001); females had lower seroprevalence against PV1 (p=0.0184) and PV2 (p=0.0354); northern states lower seroprevalence against PV1 (p=0.0039), while well-water source lower seroprevalence against PV3 (p=0.0288). Conclusion: this study showed high seroprevalence rates against PV 1, 2, and 3 in pre-switch children (aged 24-119 months). However, post-switch children (<24 months) had low immunity against PV2 despite outbreak responses. Strategies to increase routine immunization coverage and high-quality polio campaigns can increase immunity against polio virus.

Comparison of one single-antigen assay and three multi-antigen SARS-CoV-2 IgG assays in Nigeria.

Objectives: Determining an accurate estimate of SARS-CoV-2 seroprevalence has been challenging in African countries where malaria and other pathogens are endemic. We compared the performance of one single-antigen assay and three multi-antigen SARS-CoV-2 IgG assays in a Nigerian population endemic for malaria. Methods: De-identified plasma specimens from SARS-CoV-2 RT-PCR positive, dried blood spot (DBS) SARS-CoV-2 RT-PCR positive, and pre-pandemic negatives were used to evaluate the performance of the four SARS-CoV-2 assays (Tetracore, SARS2MBA, RightSign, xMAP). Results: Results showed higher sensitivity with the multi-antigen (81% (Tetracore), 96% (SARS2MBA), 85% (xMAP)) versus the single-antigen (RightSign (64%)) SARS-CoV-2 assay. The overall specificities were 98% (Tetracore), 100% (SARS2MBA and RightSign), and 99% (xMAP). When stratified based on <15 days to ≥15 days post-RT-PCR confirmation, the sensitivities increased from 75% to 88.2% for Tetracore; from 93% to 100% for the SARS2MBA; from 58% to 73% for RightSign; and from 83% to 88% for xMAP. With DBS, there was no positive increase after 15-28 days for the three assays (Tetracore, SARS2MBA, and xMAP). Conclusion: Multi-antigen assays performed well in Nigeria, even with samples with known malaria reactivity, and might provide more accurate measures of COVID-19 seroprevalence and vaccine efficacy.

Temporal trends of sulphadoxine-pyrimethamine (SP) drug-resistance molecular markers in Plasmodium falciparum parasites from pregnant women in western Kenya.

BACKGROUND: Resistance to sulphadoxine-pyrimethamine (SP) in Plasmodium falciparum parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes and has spread worldwide. SP remains the recommended drug for intermittent preventive treatment for malaria in pregnancy (IPTp) and information on population prevalence of the SP resistance molecular markers in pregnant women is limited. METHODS: Temporal trends of SP resistance molecular markers were investigated in 489 parasite samples collected from pregnant women at delivery from three different observational studies between 1996 and 2009 in Kenya, where SP was adopted for both IPTp and case treatment policies in 1998. Using real-time polymerase chain reaction, pyrosequencing and direct sequencing, 10 single-nucleotide polymorphisms (SNPs) of SP resistance molecular markers were assayed. RESULTS: The prevalence of quintuple mutant (dhfr N51I/C59R/S108N and dhps A437G/K540E combined genotype) increased from 7% in the first study (1996-2000) to 88% in the third study (2008-2009). When further stratified by sample collection year and adoption of IPTp policy, the prevalence of the quintuple mutant increased from 2.4% in 1998 to 44.4% three years after IPTp policy adoption, seemingly in parallel with the increase in percentage of SP use in pregnancy. However, in the 1996-2000 study, more mutations in the combined dhfr/dhps genotype were associated with SP use during pregnancy only in univariable analysis and no associations were detected in the 2002-2008 and 2008-2009 studies. In addition, in the 2008-2009 study, 5.3% of the parasite samples carried the dhps triple mutant (A437G/K540E/A581G). There were no differences in the prevalence of SP mutant genotypes between the parasite samples from HIV + and HIV- women over time and between paired peripheral and placental samples. CONCLUSIONS: There was a significant increase in dhfr/dhps quintuple mutant and the emergence of new genotype containing dhps 581 in the parasites from pregnant women in western Kenya over 13 years. IPTp adoption and SP use in pregnancy only played a minor role in the increased drug-resistant parasites in the pregnant women over time. Most likely, other major factors, such as the high prevalence of resistant parasites selected by the use of SP for case management in large non-pregnant population, might have contributed to the temporally increased prevalence of SP resistant parasites in pregnant women. Further investigations are needed to determine the linkage between SP drug resistance markers and efficacy of IPTp-SP.

Improving the quality of HIV rapid testing in Ghana using the dried tube specimen-based proficiency testing program.

BACKGROUND: The introduction of human immunodeficiency virus (HIV) antibody rapid testing (RT) in resource-limited settings has proven to be a successful intervention to increase access to prevention measures and improve timely linkage to care. However, the quality of testing has not always kept pace with the scale-up of this testing strategy. To monitor the accuracy of HIV RT test results, a national proficiency testing (PT) program was rolled out at selected testing sites in Ghana using the dried tube specimen (DTS) approach. METHODS: Between 2015 and 2018, 635 HIV testing sites, located in five regions and supported by the U.S. President's Emergency Plan for AIDS Relief (PEPFAR), were enrolled in the HIV PT program of the Ghana Health Service National AIDS/STI Control Programme. These sites offered various services: HIV Testing and Counselling (HTC), prevention of mother-to-child transmission (PMTCT) and Antiretroviral Treatment (ART). The PT panels, composed of six DTS, were prepared by two regional laboratories, using fully characterized plasma obtained from the regional blood banks and distributed to the testing sites. The results were scored by the PT providers according to the predefined acceptable performance criteria which was set at ≥ 95%. RESULTS: Seven rounds of PT panels were completed successfully over three years. The number of sites enrolled increased from 205 in round 1 (June 2015) to 635 in round 7 (December 2018), with a noticeable increase in Greater Accra and Eastern regions. The average participation rates of enrolled sites ranged from 88.0% to 98.0% across the PT rounds. By round 7, HTC (257/635 (40.5%)) and PMTCT (237/635 (37.3%)) had a larger number of sites that participated in the PT program than laboratory (106/635 (16.7%)) and ART (12/635 (1.9%)) sites. The average testing performance rate improved significantly from 27% in round 1 to 80% in round 7 (p < 0.001). The highest performance rate was observed for ART (100%), HTC (92%), ANC/PMTCT (90%) and Laboratory (89%) in round 5. CONCLUSION: The DTS PT program showed a significant increase in the participation and performance rates during this period. Sub-optimal performances observed was attributed to non-compliance to the national testing algorithm and testing technique. However, the implementation of review meetings, peer-initiated corrective action, supportive supervisory training, and mentorship proved impactful. The decentralized approach to preparing the PT panels ensured ownership by the region and districts.

Performance of SARS-CoV-2 Antigens in a Multiplex Bead Assay for Integrated Serological Surveillance of Neglected Tropical and Other Diseases.

Serosurveillance can provide estimates of population-level exposure to infectious pathogens and has been used extensively during the COVID-19 pandemic. Simultaneous, serological testing for multiple pathogens can be done using bead-based immunoassays to add value to disease-specific serosurveys. We conducted a validation of four SARS-CoV-2 antigens-full-length spike protein, two receptor binding domain proteins, and the nucleocapsid protein-on our existing multiplex bead assay (MBA) for enteric diseases, malaria, and vaccine preventable diseases. After determining the optimal conditions for coupling the antigens to microsphere beads, the sensitivity and specificity of the assay were determined on two instruments (Luminex-200 and MAGPIX) when testing singly (monoplex) versus combined (multiplex). Sensitivity was assessed using plasma from 87 real-time reverse transcription polymerase chain reaction (rRT-PCR) positive persons collected in March-May of 2020 and ranged from 94.3% to 96.6% for the different testing conditions. Specificity was assessed using 98 plasma specimens collected prior to December 2019 and plasma from 19 rRT-PCR negative persons and ranged from 97.4% to 100%. The positive percent agreement was 93.8% to 97.9% using 48 specimens collected > 21 days post-symptom onset, while the negative percent agreement was ≥ 99% for all antigens. Test performance was similar using monoplex or multiplex testing. Integrating SARS-CoV-2 serology with other diseases of public health interest could add significant value to public health programs that have suffered severe programmatic setbacks during the COVID-19 pandemic.

Performance of HIV rapid testing algorithm in Nigeria: Findings from a household-based Nigeria HIV/AIDS Indicator and Impact Survey (NAIIS).

BACKGROUND: The Nigeria AIDS Indicator and Impact Survey (NAIIS), a cross-sectional household survey, was conducted in 2018 with primary objectives to estimate HIV prevalence, HIV-1 incidence, and status of UNAIDS 90-90-90 cascade. We conducted retrospective analysis of the performance of HIV rapid tests and the national HIV testing algorithm used in Nigeria. METHODS: The national algorithm included Determine HIV-1/2 as test 1 (T1), Unigold HIV-1/2 as test 2 (T2), and StatPak HIV-1/2 as the tie-breaker test (T3). Individuals reactive with T1 and either T2 or T3 were considered HIV-positive. HIV-positive specimens from the algorithm were further confirmed for the survey using supplemental test Geenius HIV-1/2. If Geenius did not confirm HIV-positive status, HIV-1 Western blot was performed. We calculated the concordance between tests and positive predictive value (PPV) of the algorithm on unweighted data. RESULTS: Of 204,930 participants (ages ≥18 months) 5,103 (2.5%) were reactive on T1. Serial testing of T1 reactive specimens with T2 or if needed by tiebreaker T3 identified 2958 (1.44%) persons as HIV-positive. Supplemental testing confirmed 2,800 (95%) as HIV-positive (HIV-1 = 2,767 [98.8%]; HIV-2 = 5 [0.2%]; dual infections = 22 [0.8%]). Concordance between T1 and T2 was 56.6% while PPV of the national algorithm was 94.5%. CONCLUSIONS: Our results show high discordant rates and poor PPV of the national algorithm with a false-positive rate of about 5.5% in the NAIIS survey. Considering our findings have major implications for HIV diagnosis in routine HIV testing services, additional evaluation of testing algorithm is warranted in Nigeria.

Validation of xMAP SARS-CoV-2 Multi-Antigen IgG assay in Nigeria.

OBJECTIVE: There is a need for reliable serological assays to determine accurate estimates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence. Most single target antigen assays have shown some limitations in Africa. To assess the performance of a multi-antigen assay, we evaluated a commercially available SARS-CoV-2 Multi-Antigen IgG assay for human coronavirus disease 2019 (COVID-19) in Nigeria. METHODS: Validation of the xMAP SARS-CoV-2 Multi-Antigen IgG assay was carried out using well-characterized SARS-CoV-2 reverse transcription polymerase chain reactive positive (97) and pre-COVID-19 pandemic (86) plasma panels. Cross-reactivity was assessed using pre-COVID-19 pandemic plasma specimens (213) from the 2018 Nigeria HIV/AIDS Indicator and Impact Survey (NAIIS). RESULTS: The overall sensitivity of the xMAP SARS-CoV-2 Multi-Antigen IgG assay was 75.3% [95% CI: 65.8%- 82.8%] and specificity was 99.0% [95% CI: 96.8%- 99.7%]. The sensitivity estimate increased to 83.3% [95% CI: 70.4%- 91.3%] for specimens >14 days post-confirmation of diagnosis. However, using the NAIIS pre-pandemic specimens, the false positivity rate was 1.4% (3/213). CONCLUSIONS: Our results showed overall lower sensitivity and a comparable specificity with the manufacturer's validation. There appears to be less cross-reactivity with NAIIS pre-pandemic COVID-19 specimens using the xMAP SARS-CoV-2 Multi-Antigen IgG assay. In-country SARS-CoV-2 serology assay validation can help guide the best choice of assays in Africa.

Seroprevalence of SARS-CoV-2 in four states of Nigeria in October 2020: A population-based household survey.

The observed epidemiology of SARS-CoV-2 in sub-Saharan Africa has varied greatly from that in Europe and the United States, with much lower reported incidence. Population-based studies are needed to estimate true cumulative incidence of SARS-CoV-2 to inform public health interventions. This study estimated SARS-CoV-2 seroprevalence in four selected states in Nigeria in October 2020. We implemented a two-stage cluster sample household survey in four Nigerian states (Enugu, Gombe, Lagos, and Nasarawa) to estimate age-stratified prevalence of SARS-CoV-2 antibodies. All individuals in sampled households were eligible for interview, blood draw, and nasal/oropharyngeal swab collection. We additionally tested participants for current/recent malaria infection. Seroprevalence estimates were calculated accounting for the complex survey design. Across all four states, 10,629 (96·5%) of 11,015 interviewed individuals provided blood samples. The seroprevalence of SARS-CoV-2 antibodies was 25·2% (95% CI 21·8-28·6) in Enugu State, 9·3% (95% CI 7·0-11·5) in Gombe State, 23·3% (95% CI 20·5-26·4) in Lagos State, and 18·0% (95% CI 14·4-21·6) in Nasarawa State. Prevalence of current/recent malaria infection ranged from 2·8% in Lagos to 45·8% in Gombe and was not significantly related to SARS-CoV-2 seroprevalence. The prevalence of active SARS-CoV-2 infection in the four states during the survey period was 0·2% (95% CI 0·1-0·4). Approximately eight months after the first reported COVID-19 case in Nigeria, seroprevalence indicated infection levels 194 times higher than the 24,198 officially reported COVID-19 cases across the four states; however, most of the population remained susceptible to COVID-19 in October 2020.

Evaluation of the Nigeria national HIV rapid testing algorithm.

Human Immunodeficiency Virus (HIV) diagnosis remains the gateway to HIV care and treatment. However, due to changes in HIV prevalence and testing coverage across different geopolitical zones, it is crucial to evaluate the national HIV testing algorithm as false positivity due to low prevalence could be detrimental to both the client and the service delivery. Therefore, we evaluated the performance of the national HIV rapid testing algorithm using specimens collected from multiple HIV testing services (HTS) sites and compared the results from different HIV prevalence levels across the six geopolitical zones of Nigeria. The evaluation employed a dual approach, retrospective, and prospective. The retrospective evaluation focused on a desktop review of program data (n = 492,880) collated from patients attending routine HTS from six geopolitical zones of Nigeria between January 2017 and December 2019. The prospective component utilized samples (n = 2,895) collected from the field at the HTS and tested using the current national serial HIV rapid testing algorithm. These samples were transported to the National Reference Laboratory (NRL), Abuja, and were re-tested using the national HIV rapid testing algorithm and HIV-1/2 supplementary assays (Geenius to confirm positives and resolve discordance and multiplex assay). The retrospective component of the study revealed that the overall proportion of HIV positives, based on the selected areas, was 5.7% (28,319/492,880) within the study period, and the discordant rate between tests 1 and 2 was 1.1%. The prospective component of the study indicated no significant differences between the test performed at the field using the national HIV rapid testing algorithm and the re-testing performed at the NRL. The comparison between the test performed at the field using the national HIV rapid testing algorithm and Geenius HIV-1/2 supplementary assay showed an agreement rate of 95.2%, while that of the NRL was 99.3%. In addition, the comparison of the field results with HIV multiplex assay indicated a sensitivity of 96.6%, the specificity of 98.2%, PPV of 97.0%, and Kappa Statistic of 0.95, and that of the NRL with HIV multiplex assay was 99.2%, 99.4%, 99.0%, and 0.99, respectively. Results show that the Nigeria national serial HIV rapid testing algorithm performed very well across the target settings. However, the algorithm's performance in the field was lower than the performance outcomes under a controlled environment in the NRL. There is a need to target testers in the field for routine continuous quality improvement implementation, including refresher trainings as necessary.

Validation of Commercial SARS-CoV-2 Immunoassays in a Nigerian Population.

Validated assays are essential for reliable serosurveys; however, most SARS-CoV-2 immunoassays have been validated using specimens from China, Europe, or U.S. populations. We evaluated the performance of five commercial SARS-CoV-2 immunoassays to inform their use in serosurveys in Nigeria. Four semiquantitative enzyme-linked immunosorbent assays (ELISAs) (Euroimmun anti-SARS-CoV-2 nucleocapsid protein [NCP] immunoglobulin G [IgG], Euroimmun spike SARS-CoV-2 IgG, Mologic Omega COVID-19 IgG, Bio-Rad Platelia SARS-CoV-2 Total Ab) and one chemiluminescent microparticle immunoassay (Abbott Architect SARS-CoV-2 IgG) were evaluated. We estimated the analytical performance characteristics using plasma from 100 SARS-CoV-2 PCR-positive patients from varied time points post-PCR confirmation and 100 prepandemic samples (50 HIV positive and 50 hepatitis B positive). The Bio-Rad assay failed the manufacturer-specified validation steps. The Euroimmun NCP, Euroimmun spike, and Mologic assays had sensitivities of 73.7%, 74.4%, and 76.9%, respectively, on samples taken 15 to 58 days after PCR confirmation and specificities of 97%, 100%, and 83.8%, respectively. The Abbott assay had 71.3% sensitivity and 100% specificity on the same panel. Parallel or serial algorithms combining two tests did not substantially improve the sensitivity or specificity. Our results showed lower sensitivity and, for one immunoassay, lower specificity compared to the manufacturers' results and other reported validations. Seroprevalence estimates using these assays might need to be interpreted with caution in Nigeria and similar settings. These findings highlight the importance of in-country validations of SARS-CoV-2 serological assays prior to use to ensure that accurate results are available for public health decision-making to control the COVID-19 pandemic in Africa. IMPORTANCE This study used positive and negative sample panels from Nigeria to test the performance of several commercially available SARS-CoV-2 serological assays. Using these prepandemic and SARS-CoV-2-positive samples, we found much lower levels of sensitivity in four commercially available assays than most assay manufacturer reports and independent evaluations. The use of these assays with suboptimal sensitivity and specificity in Nigeria or countries with population exposure to similar endemic pathogens could lead to a biased estimate of the seroprevalence, over- or underestimating the true disease prevalence, and limit efforts to stop the spread of SARS-CoV-2. It is important to conduct in-country validations of serological SARS-CoV-2 assays prior to their widespread use, especially in countries with limited representation in published assay validations.

Considerations for quality assurance of multiplex malaria antigen detection assays with large sample sets.

Multiplex assays for malaria antigen detection can gather data from large sample sets, but considerations for the consistency and quality assurance (QA) of mass testing lack evaluation. We present a QA framework for a study occurring November 2019 to March 2020 involving 504 assay plates detecting four Plasmodium antigens: pan-Plasmodium aldolase and lactate dehydrogenase (LDH), histidine-rich protein 2 (HRP2), P. vivax LDH (PvLDH). Controls on each plate included buffer blank, antigen negative blood, and 4-point positive dilution curve. The blank and negative blood provided consistently low signal for all targets except for pAldolase, which showed variability. Positive curve signals decreased throughout the 5-month study duration but retained a coefficient of variation (CV) of < 5%, with the exception of HRP2 in month 5 (CV of 11%). Regression fittings for inter-plate control signals provided mean and standard deviations (SDs), and of 504 assay plates, 6 (1.2%) violated the acceptable deviation limits and were repeated. For the 40,272 human blood samples assayed in this study, of 161,088 potential data points (each sample × 4 antigens), 160,641 (99.7%) successfully passed quality checks. The QA framework presented here can be utilized to ensure quality of laboratory antigen detection for large sample sets.