Detection of a SARS-CoV-2 variant of concern in South Africa.

Continued uncontrolled transmission of SARS-CoV-2 in many parts of the world is creating conditions for substantial evolutionary changes to the virus1,2. Here we describe a newly arisen lineage of SARS-CoV-2 (designated 501Y.V2; also known as B.1.351 or 20H) that is defined by eight mutations in the spike protein, including three substitutions (K417N, E484K and N501Y) at residues in its receptor-binding domain that may have functional importance3-5. This lineage was identified in South Africa after the first wave of the epidemic in a severely affected metropolitan area (Nelson Mandela Bay) that is located on the coast of the Eastern Cape province. This lineage spread rapidly, and became dominant in Eastern Cape, Western Cape and KwaZulu-Natal provinces within weeks. Although the full import of the mutations is yet to be determined, the genomic data-which show rapid expansion and displacement of other lineages in several regions-suggest that this lineage is associated with a selection advantage that most plausibly results from increased transmissibility or immune escape6-8.

Insights to HIV-1 coreceptor usage by estimating HLA adaptation with Bayesian generalized linear mixed models.

The mechanisms triggering the human immunodeficiency virus type I (HIV-1) to switch the coreceptor usage from CCR5 to CXCR4 during the course of infection are not entirely understood. While low CD4+ T cell counts are associated with CXCR4 usage, a predominance of CXCR4 usage with still high CD4+ T cell counts remains puzzling. Here, we explore the hypothesis that viral adaptation to the human leukocyte antigen (HLA) complex, especially to the HLA class II alleles, contributes to the coreceptor switch. To this end, we sequence the viral gag and env protein with corresponding HLA class I and II alleles of a new cohort of 312 treatment-naive, subtype C, chronically-infected HIV-1 patients from South Africa. To estimate HLA adaptation, we develop a novel computational approach using Bayesian generalized linear mixed models (GLMMs). Our model allows to consider the entire HLA repertoire without restricting the model to pre-learned HLA-polymorphisms. In addition, we correct for phylogenetic relatedness of the viruses within the model itself to account for founder effects. Using our model, we observe that CXCR4-using variants are more adapted than CCR5-using variants (p-value = 1.34e-2). Additionally, adapted CCR5-using variants have a significantly lower predicted false positive rate (FPR) by the geno2pheno[coreceptor] tool compared to the non-adapted CCR5-using variants (p-value = 2.21e-2), where a low FPR is associated with CXCR4 usage. Consequently, estimating HLA adaptation can be an asset in predicting not only coreceptor usage, but also an approaching coreceptor switch in CCR5-using variants. We propose the usage of Bayesian GLMMs for modeling virus-host adaptation in general.

Persistent Severe Acute Respiratory Syndrome Coronavirus 2 Infection With accumulation of mutations in a patient with poorly controlled Human Immunodeficiency Virus infection.

A 22-year-old woman with uncontrolled advanced human immunodeficiency virus (HIV) infection was persistently infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) beta variant for 9 months, the virus accumulating >20 additional mutations. Antiretroviral therapy suppressed HIV and cleared SARS-CoV-2 within 6 to 9 weeks. Increased vigilance is warranted to benefit affected individuals and prevent the emergence of novel SARS-CoV-2 variants.

SARS-CoV-2 infection and pulmonary tuberculosis in children and adolescents: a case-control study.

BACKGROUND: The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pandemic has had an impact on the global tuberculosis (TB) epidemic but evidence on the possible interaction between SARS-CoV-2 and TB, especially in children and adolescents, remains limited. We aimed to evaluate the relationship between previous infection with SARS-CoV-2 and the risk of TB in children and adolescents. METHODS: An unmatched case-control study was conducted using SARS-CoV-2 unvaccinated children and adolescents recruited into two observational TB studies (Teen TB and Umoya), between November 2020 and November 2021, in Cape Town, South Africa. Sixty-four individuals with pulmonary TB (aged < 20 years) and 99 individuals without pulmonary TB (aged < 20 years) were included. Demographics and clinical data were obtained. Serum samples collected at enrolment underwent quantitative SARS-CoV-2 anti-spike immunoglobulin G (IgG) testing using the Abbott SARS-CoV-2 IgG II Quant assay. Odds ratios (ORs) for TB were estimated using unconditional logistic regression. RESULTS: There was no statistically significant difference in the odds of having pulmonary TB between those who were SARS-CoV-2 IgG seropositive and those who were seronegative (adjusted OR 0.51; 95% CI: 0.23-1.11; n = 163; p = 0.09). Of those with positive SARS-CoV-2 serology indicating prior infection, baseline IgG titres were higher in individuals with TB compared to those without TB (p = 0.04) and individuals with IgG titres in the highest tertile were more likely to have pulmonary TB compared to those with IgG levels in the lowest tertile (OR: 4.00; 95%CI: 1.13- 14.21; p = 0.03). CONCLUSIONS: Our study did not find convincing evidence that SARS-CoV-2 seropositivity was associated with subsequent pulmonary TB disease; however, the association between magnitude of SARS-CoV-2 IgG response and pulmonary TB warrants further investigation. Future prospective studies, evaluating the effects of sex, age and puberty on host immune responses to M. tuberculosis and SARS-CoV-2, will also provide more clarity on the interplay between these two infections.

Delineating the Spread and Prevalence of SARS-CoV-2 Omicron Sublineages (BA.1-BA.5) and Deltacron Using Wastewater in the Western Cape, South Africa.

This study was one of the first to detect Omicron sublineages BA.4 and BA.5 in wastewater from South Africa. Spearman rank correlation analysis confirmed a strong positive correlation between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA in wastewater samples and clinical cases (r = 0.7749, P < .0001). SARS-CoV-2 viral load detected in wastewater, resulting from the Delta-driven third wave, was significantly higher than during the Omicron-driven fourth wave. Whole-genome sequencing confirmed presence of Omicron lineage defining mutations in wastewater with the first occurrence reported 23 November 2021 (BA.1 predominant). The variant spread rapidly, with prevalence of Omicron-positive wastewater samples rising to >80% by 10 January 2022 with BA.2 as the predominant sublineage by 10 March 2022, whilst on 18 April 2022 BA.4 and BA.5 were detected in selected wastewater sites. These findings demonstrate the value of wastewater-based epidemiology to monitor the spatiotemporal spread and potential origin of new Omicron sublineages.

Prevalence and patterns of HIV drug resistance in patients with suspected virological failure in North-Western Tanzania.

BACKGROUND: More than 15 million people in sub-Saharan Africa receive ART. Treatment failure is common, but the role of HIV drug resistance in treatment failure is largely unknown because drug resistance testing is not routinely done. This study determined the prevalence and patterns of HIV drug resistance in patients with suspected virological failure. MATERIALS AND METHODS: A single high viral load of >1000 viral RNA copies/mL of plasma at any point during ART was considered as suspected virological failure. HIV-1 RNA was extracted from plasma samples of these patients using the QIAamp Viral RNA kit. The protease and part of the RT regions of the HIV pol gene were characterized. RESULTS: Viral load was determined in 317 patients; 64 (20.2%) had suspected virological failure. We successfully genotyped 56 samples; 48 (85.7%) had at least one major resistance-associated mutation (RAM). Common mutations in RT were M184V (75%), T215Y (41.1%), K103N (39.3%), M41L (32.1%), D67DN (30.3%), G190A (28.6%) and A98G (26.8%). No RAMs were detected in ART regimens based on a ritonavir-boosted PI. CONCLUSIONS: The Tanzanian national guidelines define 'virological failure' as two consecutive viral load measurement results, at 3 month intervals, above the WHO threshold (1000 copies/mL). Here, we show that a single viral load above the WHO threshold is associated with high rates of RAMs. This suggests that a single high viral load measurement could be used to predict virological failure and avoid delays in switching patients from first-line to higher genetic barrier second-line regimens.

Primary resistance against integrase strand transfer inhibitors in integrase strand transfer inhibitor-naive patients failing first- and second-line ART in Tanzania.

INTRODUCTION: Sub-Saharan African countries are introducing integrase strand transfer inhibitors (INSTIs) in their ART programmes as the preferred first-line regimen, and dolutegravir is the INSTI of choice due to its potency, tolerability and high genetic barrier to resistance. Dolutegravir was introduced into the first-line ART regimen in Tanzania in 2019. However, there is a paucity of data on the occurrence of mutations in HIV lineages circulating in Tanzania. This study aimed to determine the prevalence of INSTI primary resistance mutations in Tanzanian patients exposed to ART but not INSTIs. METHODS: Plasma samples from 50 INSTI-naive patients failing first- or second-line ART [median (IQR) age: 40 (21.93-46.41) years; 68% women] were subjected to Sanger sequencing of the HIV integrase gene. Participants had been on ART for a median (IQR) duration of 7.32 (4.73-9.29) years, with 80% and 20% failing first- and second-line ART, respectively. RESULTS: No major INSTI mutations were found, but 2 (4%) participants had the accessory mutation T97A. Using the REGA HIV-1 subtyping tool, HIV subtype A1 (53.1%) was found to be dominant, followed by subtypes C (30.6%) and D (16.3%). CONCLUSIONS: This study found no current evidence for transmitted resistance against INSTIs among unexposed patients failing ART and supports the scale-up of INSTI-based regimens. However, the presence of accessory mutations calls for the surveillance of INSTI resistance mutations to ensure that the anticipated long-term desired outcomes are achieved.

Outcomes of laboratory-confirmed SARS-CoV-2 infection in the Omicron-driven fourth wave compared with previous waves in the Western Cape Province, South Africa.

OBJECTIVES: The objective was to compare COVID-19 outcomes in the Omicron-driven fourth wave with prior waves in the Western Cape, assess the contribution of undiagnosed prior infection to differences in outcomes in a context of high seroprevalence due to prior infection and determine whether protection against severe disease conferred by prior infection and/or vaccination was maintained. METHODS: In this cohort study, we included public sector patients aged ≥20 years with a laboratory-confirmed COVID-19 diagnosis between 14 November and 11 December 2021 (wave four) and equivalent prior wave periods. We compared the risk between waves of the following outcomes using Cox regression: death, severe hospitalisation or death and any hospitalisation or death (all ≤14 days after diagnosis) adjusted for age, sex, comorbidities, geography, vaccination and prior infection. RESULTS: We included 5144 patients from wave four and 11,609 from prior waves. The risk of all outcomes was lower in wave four compared to the Delta-driven wave three (adjusted hazard ratio (aHR) [95% confidence interval (CI)] for death 0.27 [0.19; 0.38]. Risk reduction was lower when adjusting for vaccination and prior diagnosed infection (aHR: 0.41, 95% CI: 0.29; 0.59) and reduced further when accounting for unascertained prior infections (aHR: 0.72). Vaccine protection was maintained in wave four (aHR for outcome of death: 0.24; 95% CI: 0.10; 0.58). CONCLUSIONS: In the Omicron-driven wave, severe COVID-19 outcomes were reduced mostly due to protection conferred by prior infection and/or vaccination, but intrinsically reduced virulence may account for a modest reduction in risk of severe hospitalisation or death compared to the Delta-driven wave.

From Easing Lockdowns to Scaling Up Community-based Coronavirus Disease 2019 Screening, Testing, and Contact Tracing in Africa-Shared Approaches, Innovations, and Challenges to Minimize Morbidity and Mortality.

The arrival of coronavirus disease 2019 (COVID-19) on the African continent resulted in a range of lockdown measures that curtailed the spread of the infection but caused economic hardship. African countries now face difficult choices regarding easing of lockdowns and sustaining effective public health control measures and surveillance. Pandemic control will require efficient community screening, testing, and contact tracing; behavioral change interventions; adequate resources; and well-supported, community-based teams of trained, protected personnel. We discuss COVID-19 control approaches in selected African countries and the need for shared, affordable, innovative methods to overcome challenges and minimize mortality. This crisis presents a unique opportunity to align COVID-19 services with those already in place for human immunodeficiency virus, tuberculosis, malaria, and non communicable diseases through mobilization of Africa's interprofessional healthcare workforce. By addressing the challenges, the detrimental effect of the COVID-19 pandemic on African citizens can be minimized.

Viral hepatitis associated hepatocellular carcinoma on the African continent, the past, present, and future: a systematic review.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths in Africa. In Africa, the major causes of HCC include chronic infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV). Knowledge of the changes in the incidence of viral hepatitis-associated HCC over time and the factors responsible for such changes is key in informing policies for the prevention of viral hepatitis-associated HCC in Africa. AIM: The study aimed to systematically summarize the changes in the prevalence of viral hepatitis among HCC patients and the overall effect of the prevalence of viral hepatitis on the incidence of HCC over the past four decades in Africa (1980-2019). METHODS: A literature search was conducted in MEDLINE (PubMed), Google Scholar, Science Direct, Scopus, Web of Science, and African wide web for articles published on viral hepatitis-associated HCC in Africa from 1980 to 2019. The abstracts of the articles were screened for eligibility and those meeting the inclusion criteria were retrieved and reviewed. RESULTS: A total of 272 studies were included in the analysis. Viral hepatitis-related HCC incidence changed by 1.17% (95% confidence interval (CI): 0.63-1.71, p < 0.001), 0.82% (95% CI: 0.45-1.18, p < 0.001), and 3.34% (95% CI: 2.44-4.25, p < 0.001) for every 1% change in the prevalence of HBV, HCV, and hepatitis D virus (HDV) respectively, per decade. The incidence of HBV-related HCC decreased by - 0.50% (95% CI: - 0.74 - - 0.25, p < 0.001) over the last 40 years, while HCV-related HCC increased. CONCLUSION: Overall, the incidence of viral hepatitis-associated HCC has not declined, mainly due to no decline in the prevalence of HCV, HDV, and the high number of chronic hepatitis B carriers on the African continent. There is an urgent need for the allocation of resources for the implementation of treatment and preventive programs for HBV, HCV, HDV, and HCC in Africa. This systematic review is registered with PROSPERO®, number CRD42020169723.

Prevalence and risks of hepatitis E virus infection in blood donors from the Western Cape, South Africa.

BACKGROUND: Transfusion-transmitted hepatitis E virus (HEV) infection is a potential risk to recipients of blood transfusions. Infection with HEV poses a high risk to immunocompromised recipients with an increased likelihood of developing chronic infection. The aims of this study were to determine the prevalence of past and active HEV infections in donors from the Western Cape and to identify the risk factors associated with infection. MATERIALS AND METHODS: We prospectively tested 10 250 blood donors for HEV infection. A risk factor sub-study investigated 250 donors who completed a questionnaire, and plasma samples were tested for HEV IgG antibodies and pooled for HEV RNA detection. The demographic and risk factors associated with HEV infection were assessed. The molecular study tested 10 000 individual donations using a commercial assay to detect viraemia. HEV viral load and genotype were also determined. RESULTS: The overall anti-HEV IgG seroprevalence was 42·8% (107/250) among donors participating in the risk factor sub-study. The likelihood of past HEV infection was higher with an increase in age. Of the 10 000 donor samples individually tested for HEV RNA, one sample was positive with a viral load of 7·9 x 104 IU/ml and belonged to HEV genotype 3. CONCLUSION: We found a high seroprevalence of anti-HEV IgG but a low HEV RNA prevalence among donors in the Western Cape, South Africa. The study provides evidence for a potential risk of HEV contamination in the blood supply in South Africa. A cost-benefit analysis is needed before considering the introduction of routine donor screening in our setting.

Hepatitis B virus-associated hepatocellular carcinoma in South Africa in the era of HIV.

BACKGROUND: Patients co-infected with hepatitis B virus (HBV) and the human immunodeficiency virus (HIV) are at risk of developing hepatocellular carcinoma (HCC). In sub-Saharan Africa, the overlap between high HIV and HBV prevalence may increase the incidence of HCC. This study investigated the impact of HBV/HIV co-infection on age at presentation and survival of HCC. METHODS: Ethical approval was obtained to recruit, following informed written consent, patients diagnosed with HCC at oncology units at four South African hospitals. Between December 2012 and August 2015, patients newly diagnosed with HCC were recruited and provided demographic and clinical data and blood specimens. Patients were tested for HBV, hepatitis C virus (HCV) and HIV. Survival data was available for a subset of patients. RESULTS: Of 107 HCC cases, 83 (78%) were male. Median age was 46 years (range 18 to 90 years), 68/106 (64%) were HBsAg-positive, and 22/100 (22%) were HIV infected. Among HBV surface antigen (HBsAg)-positive HCC cases, 18/66 (27%) were HIV-infected compared to 3/34 (9%) among those that were HBsAg-negative (p = 0.04). A greater proportion of HBV/HIV co-infected cases were female than HBV mono-infected (6/18, 33% vs 6/47, 13%; p = 0.005). In addition, HBV/HIV co-infected females presented at a younger mean age (36.8 years) than HBV mono-infected women (50.5 years) (p = 0.09). Median survival was 82 days among the HIV-infected HCC patients compared to 181 days among those without HIV (p = 0.15). CONCLUSIONS: HCC is an important complication in the HIV/HBV infected patient. HIV-positive patients presented with HCC at a younger age than HIV-negative patients, this effect appears to be greater in women. These data provide more evidence supporting the call to address. HCC as a cause of morbidity and mortality in the HBV/HIV co-infected patient population. (281 words).

Lessons in diagnostic virology: expected and unexpected sources of error.

Viral diagnostics have shown continued innovation, with serological and molecular diagnostic assays pushing the limits of sensitivity. Technology has provided new automated shared diagnostic platforms that reduce hands-on time, while with globalisation of the diagnostic market, commercial assays are applied across epidemiologically diverse settings on different patient and viral populations. However, with these novel developments, new and often unexpected sources of diagnostic error emerge. In this review we will reflect on case studies that highlight these often underappreciated or unexpected diagnostic errors spanning pre-analytical, analytical, and post-analytic processes. We will also suggest approaches that could help identify error and reduce the impact on patient management.

Prevalence of chronic HBV infection in pregnant woman attending antenatal care in a tertiary hospital in Mwanza, Tanzania: a cross-sectional study.

BACKGROUND: Tanzania has a high prevalence (7.17%) of chronic hepatitis B infection. Mother to Child transmission is very common, resulting in high rate of chronic infections. Currently, there is no screening program for HBV in pregnant women. This study investigated the prevalence and risk factors for chronic HBV infection in pregnant women in a tertiary hospital in Mwanza, Tanzania. METHODS: Seven hundred and forty-three women attending antenatal care and/or delivering at the Bugando Medical Centre were enrolled. All answered a questionnaire on sociodemographic and other risk factors and were tested for HBsAg using a rapid test. In HBsAg positive mothers, maternal blood and umbilical cord blood samples collected after delivery were analyzed for serological (HBsAg, HBeAg and anti-HBe) and virologic (HBV-DNA viral load and genotype) markers. All their babies were vaccinated within 24 h of delivery. The children were followed up at 3 years of age. Data was analyzed using the Mann-Whitney U-test, independent sample T-test and logistic regression. RESULTS: Of the 743 participants, 22 (3%) were positive for HBsAg, and 2 (9%) had detectable HBe-antigen. Low condom use was the only statistically significant risk factor for chronic HBV infection (OR = 3.514, 95%CI = 1.4-8.0). Of 14 maternal blood samples genotyped, 10 (71%) were genotype A and 4 (29%) were genotype D. HBV-DNA was detected in 21/22 samples, with a median of 241 IU/ml (range: 27.4-25.9 × 107 IU/ml). Five (33%) of 15 available cord blood samples were positive for HBsAg and 10 (67%) were negative. At follow-up, one child showed chronic HBV infection characteristics, one had anti-HBs level of 7 mIU/ml and 5/7(71%) had protective anti-HBs levels (> 10 mIU/ml). CONCLUSION: This cohort of pregnant women showed a lower-intermediate prevalence of HBV of 3%. In the 3 years follow-up only 1 out of 7 children showed evidence of chronic HBV infection. The child's mother with high viral load (25.9 × 107 IU/ml), was positive for HBeAg with a high degree of sequence similarity suggesting vertical transmission. These results highlight a need for improved diagnosis and treatment of HBV infection in pregnant women in Tanzania, in order to prevent vertical transmission.

Viral hepatitis B and C in HIV-exposed South African infants.

BACKGROUND: Whilst much attention is given to eliminating HIV mother-to-child transmission (MTCT), little has been done to ensure the same for hepatitis B virus (HBV) transmission. The introduction of HBV immunization at six weeks of age has reduced HBV horizontal transmission in South Africa. However, in order to eliminate HBV MTCT, further interventions are needed. The risk of hepatitis C virus (HCV) MTCT in HIV-infected (HIV+) African women is not yet well described. This study aimed to determine the rate of HBV and HCV vertical transmission in HIV-exposed infants in South Africa. METHODS: Serum samples from infants enrolled in an isoniazid prevention study (P1041) were screened for HBV and HCV serology markers; screening was performed on samples collected at approximately 60 weeks of age of the infants. HBV DNA was quantified in HBsAg positive samples and HBV strains characterized through gene sequencing. All HCV antibody samples with inconclusive results underwent molecular testing. RESULTS: Three of 821 infants were positive for both HBsAg and HBV DNA. All HBV strains belonged to HBV sub-genotype A1. The rtM204I mutation associated with lamivudine resistance was identified in one infant, a second infant harboured the double A1762T/G1764A BCP mutation. Phylogenetic analysis showed clustering between mother and infant viral genomic sequences. Twenty-one of 821 HIV-exposed infants tested had inconclusive HCV antibody results, none were HCV PCR positive. CONCLUSIONS: This study suggests that HBV vertical transmission is likely to be occurring in HIV-exposed infants in South Africa.. A more robust strategy of HBV prevention, including birth dose vaccination, is required to eradicate HBV MTCT. HCV infection was not detected.

Congenital Rubella Syndrome Surveillance in South Africa Using a Sentinel Site Approach: A Cross-sectional Study.

BACKGROUND: Congenital rubella syndrome (CRS) includes disorders associated with intrauterine rubella infection. Incidence of CRS is higher in countries with no rubella-containing vaccines (RCV) in their immunization schedules. In the World Health Organization African region, RCVs are being introduced as part of the 2012-2020 global measles and rubella strategic plan. This study aimed to describe the epidemiology of confirmed CRS in South Africa prior to introduction of RCVs in the immunization schedule. METHODS: This was a descriptive study with 28 sentinel sites reporting laboratory-confirmed CRS cases in all 9 provinces of South Africa. In the retrospective phase (2010 to 2014), CRS cases were retrieved from medical records, and in the prospective phase (2015 to 2017) clinicians at study sites reported CRS cases monthly. RESULTS: There were 42 confirmed CRS cases in the retrospective phase and 53 confirmed CRS cases in the prospective phase. Most frequently reported birth defects were congenital heart disease and cataracts. The median age of mothers of CRS cases was 21 years in the retrospective phase (range: 11 to 38 years) and 22 years in the prospective phase (range: 15 to 38 years). CONCLUSION: Baseline data on laboratory-confirmed CRS will enable planning and monitoring of RCV implementation in the South African Expanded Programme on Immunization program. Ninety-eight percent of mothers of infants with CRS were young women 14-30 years old, indicating a potential immunity gap in this age group for consideration during introduction of RCV.

Pooled PCR testing of dried blood spots for infant HIV diagnosis is cost efficient and accurate.

BACKGROUND: Access to qualitative HIV PCRs for early infant diagnosis (EID) is restricted in resource-limited settings due to cost. We hypothesised that pooling of dried blood spots (DBS), defined as combining multiple patient samples in a single test with subsequent individual testing of positive pools, would be cost saving while retaining clinical accuracy compared to individual patient testing. METHODS: Cost savings: A model was developed to simulate reagent and consumable cost saving of pooled compared to individual sample testing. Daily sample/result data of a public health laboratory in South Africa were used to illustrate outputs from the model. Samples were randomly allocated to pools and the process was repeated 1000 times to measure variation in estimates due to this stochasticity. Clinical accuracy: 1170 patient samples were tested using the Roche CAP/CTM Qual assay in pools of five 50 µl DBS. Negative pools comprised DBS previously tested in single reactions; positive pools included 1 positive sample. RESULTS: Pooling would have saved 64% of laboratory costs in 2015. The model is published as an R-based web tool, into which the user enters sample/positivity estimates and workflow management parameters to obtain cost saving estimates at an optimal pool size. Sensitivity of pooled testing was 98.8% overall; 100% for strongly reactive pools. One pool tested false positive which would not impact clinical specificity as individual patient testing is performed prior to reporting. CONCLUSIONS: Pooled PCR testing for EID remains accurate and dramatically reduces costs in settings with moderate to low prevalence rates and sufficient sample numbers.