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.

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.

Basic overview of method validation in the clinical virology laboratory.

Diagnostic virology laboratories are an essential part of the health system and are often relied upon to provide information to clinicians that will inform clinical decision making. It is therefore imperative that diagnostic results produced in the laboratory are reliable. One way of ensuring quality results is by ensuring that all tests are either validated (for tests developed in-house) or verified (for commercial assays that are FDA-approved or CE-labeled). In the diagnostic virology laboratory, these processes can be complex as both qualitative and quantitative measurements for serological and molecular tests are routinely offered. While there are numerous guidelines governing quality assurance in the virology laboratory, all accrediting agencies would insist on tests being validated or verified prior to implementation without providing explicit guidance to the process. As there is no universal guideline on the optimal way to perform validation/verification experiments, this review will provide a basic overview of method validation/verification, specific for clinical virology laboratories, and includes explanation of statistical analysis and acceptance/rejection criteria.

CD4 count-based failure criteria combined with viral load monitoring may trigger worse switch decisions than viral load monitoring alone.

OBJECTIVE: CD4 count decline often triggers antiretroviral regimen switches in resource-limited settings, even when viral load testing is available. We therefore compared CD4 failure and CD4 trends in patients with viraemia with or without antiretroviral resistance. METHODS: Retrospective cohort study investigating the association of HIV drug resistance with CD4 failure or CD4 trends in patients on first-line antiretroviral regimens during viraemia. Patients with viraemia (HIV RNA >1000 copies/ml) from two HIV treatment programmes in South Africa (n = 350) were included. We investigated the association of M184V and NNRTI resistance with WHO immunological failure criteria and CD4 count trends, using chi-square tests and linear mixed models. RESULTS: Fewer patients with the M184V mutation reached immunologic failure criteria than those without: 51 of 151(34%) vs. 90 of 199 (45%) (P = 0.03). Similarly, 79 of 220 (36%) patients, who had major NNRTI resistance, had immunological failure, whereas 62 of 130 (48%) without (chi-square P = 0.03) did. The CD4 count decline among patients with the M184V mutation was 2.5 cells/mm(3) /year, whereas in those without M184V it was 14 cells/mm(3) /year (P = 0.1), but the difference in CD4 count decline with and without NNRTI resistance was marginal. CONCLUSION: Our data suggest that CD4 count monitoring may lead to inappropriate delayed therapy switches for patients with HIV drug resistance. Conversely, patients with viraemia but no drug resistance are more likely to have a CD4 count decline and thus may be more likely to be switched to a second-line regimen.

Irreproducible positive results on the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qual test are different qualitatively from confirmed positive results.

Criteria that define low positive results on the COBAS® AmpliPrep/COBAS® TaqMan (CAP/CTM) HIV-1 Qual test as inconclusive have been adopted by all academic centres in South Africa that conduct infant HIV PCR, following previous investigations that showed poor specificity of these results. Retesting all inconclusive specimens has considerable cost implications. Therefore, it was attempted to characterise such inconclusive results, by comparing those that prove to be either negative or positive on follow-up testing. This retrospective, laboratory-based study found that 193 of 211 (91.5%) patients with previous inconclusive results (defined as reported positive by CAP/CTM but with cycle threshold [Ct ] values of >32 and/or fluorescence intensity [FI] values of <5) tested negative and only 18 (8.5%) tested positive using independently obtained follow-up samples after a median of 28 days. The only significant independent predictor of a later positive result was a higher FI value (3.326 vs. 0.495, P < 0.0001), whereas Ct values were not predictive independently. Specimens from patients negative on follow-up testing differed qualitatively from specimens that proved to be true positives. As the lower FI values in false-positive compared to true-positive results probably are indicative of a non-specific signal, the incorporation of stringent amplification slope criteria in the assay's test definition file may improve correct classification and thus reduce the need for repeat testing of a large number of inconclusive specimens.

An analysis of the HIV testing cascade of a group of HIV-exposed infants from birth to 18 months in peri-urban Khayelitsha, South Africa.

BACKGROUND: Despite the reduction of HIV mother-to-child transmission, there are concerns regarding transmission rate in the breastfeeding period. We describe the routine uptake of 6 or 10 (6/10) weeks, 9 months and 18 months testing, with and without tracing, in a cohort of infants who received HIV PCR testing at birth (birth PCR) (with and without point of care (POC) testing) in a peri-urban primary health care setting in Khayelitsha, South Africa. METHODS: In this cohort study conducted between November 2014 and February 2018, HIV-positive mothers and their HIV-exposed babies were recruited at birth and all babies were tested with birth PCR. Results of routine 6/10 weeks PCR, 9 months and 18 months testing were followed up by a patient tracer. We compared testing at 6/10 weeks with a subgroup from historical cohort who was not tested with birth PCR. RESULTS: We found that the uptake of 6/10 weeks testing was 77%, compared to 82% with tracing. When including all infants in the cascade and comparing to a historical cohort without birth testing, we found that infants who tested a birth were 22% more likely to have a 6/10 weeks test compared to those not tested at birth. There was no significant difference between the uptake of 6/10 weeks testing after birth PCR POC versus birth PCR testing without POC. Uptake of 9 months and 18 months testing was 39% and 24% respectively. With intense tracing efforts, uptake increased to 45% and 34% respectively. CONCLUSION: Uptake of HIV testing for HIV-exposed uninfected infants in the first 18 months of life shows good completion of the 6/10 weeks PCR but suboptimal uptake of HIV testing at 9 months and 18 months, despite tracing efforts. Birth PCR testing did not negatively affect uptake of the 6/10 weeks HIV test compared to no birth PCR testing.

Epidemiology and outcomes of SARS-CoV-2 infection associated with anti-nucleocapsid seropositivity in Cape Town, South Africa.

Background: In low- and middle-income countries where SARS-CoV-2 testing is limited, seroprevalence studies can characterise the scale and determinants of the pandemic, as well as elucidate protection conferred by prior exposure. Methods: We conducted repeated cross-sectional serosurveys (July 2020 - November 2021) using residual plasma from routine convenient blood samples from patients with non-COVID-19 conditions from Cape Town, South Africa. SARS-CoV-2 anti-nucleocapsid antibodies and linked clinical information were used to investigate: (1) seroprevalence over time and risk factors associated with seropositivity, (2) ecological comparison of seroprevalence between subdistricts, (3) case ascertainment rates, and (4) the relative protection against COVID-19 associated with seropositivity and vaccination statuses, to estimate variant disease severity. Findings: Among the subset sampled, seroprevalence of SARS-CoV-2 in Cape Town increased from 39.2% in July 2020 to 67.8% in November 2021. Poorer communities had both higher seroprevalence and COVID-19 mortality. Only 10% of seropositive individuals had a recorded positive SARS-CoV-2 test. Antibody positivity before the start of the Omicron BA.1 wave (28 November 2021) was strongly protective for severe disease (adjusted odds ratio [aOR] 0.15; 95%CI 0.05-0.46), with additional benefit in those who were also vaccinated (aOR 0.07, 95%CI 0.01-0.35). Interpretation: The high population seroprevalence in Cape Town was attained at the cost of substantial COVID-19 mortality. At the individual level, seropositivity was highly protective against subsequent infections and severe COVID-19. Funding: Wellcome Trust, National Health Laboratory Service, the Division of Intramural Research, NIAID, NIH (ADR) and Western Cape Government Health.

Reduced amplification efficiency of the RNA-dependent-RNA-polymerase target enables tracking of the Delta SARS-CoV-2 variant using routine diagnostic tests.

Routine SARS-CoV-2 surveillance in the Western Cape region of South Africa (January-August 2021) found a reduced RT-PCR amplification efficiency of the RdRp-gene target of the Seegene, Allplex 2019-nCoV diagnostic assay from June 2021 when detecting the Delta variant. We investigated whether the reduced amplification efficiency denoted by an increased RT-PCR cycle threshold value (RΔE) can be used as an indirect measure of SARS-CoV-2 Delta variant prevalence. We found a significant increase in the median RΔE for patient samples tested from June 2021, which coincided with the emergence of the SARS-CoV-2 Delta variant within our sample set. Whole genome sequencing on a subset of patient samples identified a highly conserved G15451A, non-synonymous mutation exclusively within the RdRp gene of Delta variants, which may cause reduced RT-PCR amplification efficiency. While whole genome sequencing plays an important in identifying novel SARS-CoV-2 variants, monitoring RΔE value can serve as a useful surrogate for rapid tracking of Delta variant prevalence.

Acute extrapyramidal dysfunction in two HIV-infected children.

Involvement of the basal ganglia is well documented in children with human immunodeficiency virus (HIV) encephalopathy, often with calcification. High concentrations of HIV protein have been detected in affected basal ganglia, although extrapyramidal dysfunction, in contrast to adults, is infrequently encountered in HIV-infected children. We describe the clinical course, magnetic resonance imaging appearance and outcome of two HIV-infected children who presented with acute debilitating extrapyramidal dysfunction. The cases highlight the importance of immune competence, co-existence of opportunistic infections, HIV testing of all children of HIV-infected mothers and magnetic resonance imaging when assessing the severity and anticipating outcomes of movement disorders in HIV-infected children.

Molecular characterisation and epidemiology of enterovirus-associated aseptic meningitis in the Western and Eastern Cape Provinces, South Africa 2018-2019.

BACKGROUND: Enteroviruses are amongst the most common causes of aseptic meningitis. Between November 2018 and May 2019, an outbreak of enterovirus-associated aseptic meningitis cases was noted in the Western and Eastern Cape Provinces, South Africa. OBJECTIVES: To describe the epidemiology and phylogeography of enterovirus infections during an aseptic meningitis outbreak in the Western and Eastern Cape Provinces of South Africa. METHODS: Cerebrospinal fluid samples from suspected cases were screened using a polymerase chain reaction targeting the 5'UTR. Confirmed enterovirus-associated meningitis samples underwent molecular typing through species-specific VP1/VP2 primers and pan-species VP1 primers. RESULTS: Between November 2018 and May 2019, 3497 suspected cases of aseptic meningitis were documented in the Western and Eastern Cape Provinces. Median age was 8 years (range 0-61), interquartile range (IQR=4-13 years), 405/735 (55%) male. 742/3497 (21%) cases were laboratory - confirmed enterovirus positive by routine diagnostic PCR targeting the 5'UTR. 128/742 (17%) underwent molecular typing by VP1 gene sequencing. Echovirus 4 (E4) was detected in 102/128 (80%) cases. Echovirus 9 was found in 7%, Coxsackievirus A13 in 3%. 10 genotypes contributed to the remaining 10% of cases. Synonymous mutations were found in most cases, with sporadic amino acid changes in 13 (12.7%) cases. CONCLUSION: The aseptic meningitis outbreak was associated with echovirus 4. Stool samples are valuable for molecular typing in CSF confirmed EV-associated aseptic meningitis.

HIV neuropathy in South Africans: frequency, characteristics, and risk factors.

The purpose was to estimate the frequency, characteristics, and risk factors of HIV-associated distal sensory polyneuropathy (DSP) among South Africans who attend an urban community-based clinic. In a cross-sectional study, neuropathy status was determined in 598 HIV-infected adults using validated tools (Brief Peripheral Neuropathy Screen and a modified version of the Total Neuropathy Score) to categorize subjects as DSP versus no DSP. Symptomatic DSP (SDSP) required the presence of at least two neuropathic signs together with symptoms. Clinical, anthropometric, and laboratory evaluations were prospectively performed. CD4 counts, antiretroviral therapy (ART), and questionnaires regarding previous tuberculosis (TB) and alcohol exposure were collected retrospectively. Approximately half (49%) of the study population were diagnosed with DSP, and 30% of the study population were diagnosed with SDSP. In multivariate analyses the odds ratio (OR) (95% confidence interval) of DSP were independently associated with ART use (OR 1.7, 1.0-2.9), age (per 10 year increment) (OR 1.7, 1.4-2.2), and prior TB (OR 2.0, 1.3-3.0). Pain or paresthesias were reported as moderately severe by 70% of those with SDSP. Stavudine use was significantly associated with DSP. DSP is a clinically significant problem in urban HIV-infected Africans. Our findings raise the possibility that the incidence of DSP may be reduced with avoidance of stavudine-containing regimens in older subjects, especially with a history of prior TB infection.

Pandemic influenza A (H1N1) 2009: the experience of the first six months.

After a break of 41 years, 2009 saw the first influenza pandemic of the 21st century caused by a triple-reassortant influenza A (H1N1) virus. The current estimated case fatality rate is lower than that of previous influenza pandemics, but this may change as the pandemic evolves. Illness frequently occurs in previously healthy, young adults with a wide range of clinical presentations. The majority of circulating pandemic viruses remain susceptible to neuraminidase inhibitors, although all strains are intrinsically resistant to the adamantanes. Monovalent vaccines against the pandemic strain are available in both live attenuated and inactivated forms. This review aims to summarise important virological, epidemiological and clinical aspects of the pandemic influenza A (H1N1) virus for physicians and other clinical personnel.

Polymerase Chain Reaction and Goldmann-Witmer Coefficient to Examine the Role of Epstein-Barr Virus in Uveitis.

PURPOSE: To use polymerase chain reaction (PCR) and Goldmann-Witmer Coefficient (GWC) calculation to search for evidence that Epstein-Barr virus (EBV) causes uveitis. METHODS: A prospective cross-sectional study where participants with positive multiplex EBV PCR results were further investigated by: 1) real-time PCR for EBV viral loads (VL) and 2) EBV GWC. RESULTS: Eleven of 106 consecutive uveitis patients (10.4%) had positive multiplex PCR for EBV on aqueous humor sampling and 7/11 (63.6%) were HIV-positive. Only 4/10 (40%) cases had detectable intraocular EBV VLs which were always lower than the blood or plasma VL. EBV GWC was negative in all 10 cases tested. In 9/11 (81.8%) of these cases an alternative, more plausible cause of uveitis was identified. CONCLUSION: We found no evidence of active intraocular replication or antibody production to prove that EBV caused uveitis in these cases. In most cases an alternative treatable cause of uveitis was identified.

Differentiating clearly positive from indeterminate results: A review of irreproducible HIV-1 PCR positive samples from South Africa's Early Infant Diagnosis Program, 2010-2015.

We describe the extent of and variables associated with irreproducible HIV-1 PCR positive results within South Africa's Early Infant Diagnosis (EID) program from 2010 to 2015 and propose criteria for differentiating indeterminate from clearly positive results using the COBAS® AmpliPrep/COBAS® TaqMan HIV-1 Qualitative Test version 2.0 (CAP/CTM Qual v2.0). Fourteen percent of specimens with an instrument-positive result that were repeat-tested yielded a negative result for which cycle threshold (Ct) proved to be the only predictive variable. A Ct <33.0 was found to be the most accurate threshold value for differentiating clearly positive from irreproducible cases, correctly predicting 96.8% of results. Among 70 patients with an irreproducible positive result linked to a follow up HIV-1 PCR test, 67 (95.7%) were negative and 3 (4.3%) were instrument-positive. Criteria differentiating clearly positive from indeterminate results need to be retained within EID services and infants with indeterminate results closely monitored and final HIV status determined.

Rapid decline of HIV-1 DNA and RNA in infants starting very early antiretroviral therapy may pose a diagnostic challenge.

OBJECTIVE: Birth diagnosis of HIV-1 infection offers an ideal opportunity for early antiretroviral therapy (ART) to limit HIV-1 reservoir size and limit disease progression. Although data on cellular HIV-1 DNA decay exist for children commencing treatment from 2 to 3 months of age, data are lacking for starting shortly after birth. DESIGN: We studied infants who initiated ART within 8 days after birth to assess HIV-1 DNA levels longitudinally. METHODS: Children were recruited from public health clinics in Cape Town where birth diagnosis of HIV-1 coupled with early ART initiation occurred. Total cellular HIV-1 DNA levels were determined using a sensitive quantitative PCR targeting a conserved region in integrase. RESULTS: Of 11 infants diagnosed and beginning ART within 8 days of birth with detectable pre-ART HIV-1 DNA, three subsequently had undetectable HIV-1 DNA after 6 days, 3 months and 4 months on treatment, respectively. In seven who had virologic suppression (defined as a continuous downward trend in plasma HIV-1 RNA, and <100 copies/ml after 6 months) total HIV-1 DNA continued to decay over 12 months [mean half-life of 64.8 days (95% confidence interval: 47.9-105.7)]. CONCLUSION: In infants initiated on ART within 8 days of life the combination of maternal ART, and early ART for prophylaxis and treatment contribute to rapid decline of HIV-1 infected cells to low or undetectable levels. However, rapid decline of HIV-1 RNA and DNA may complicate definitive diagnosis when confirmatory testing is delayed.

Interpretation of indeterminate HIV-1 PCR results are influenced by changing vertical transmission prevention regimens.

BACKGROUND: Suppression of HIV by antiretroviral drugs may be one of the reasons that indeterminate HIV-1 PCR results are obtained from testing HIV-exposed infants. This complicates the early identification of infected infants, potentially delaying initiating treatment early. There is uncertainty as to how different vertical HIV transmission prevention regimens (VTP) affect the rate and predictive value of indeterminate PCR results. OBJECTIVES: To investigate rates of indeterminate PCR results, outcomes of subsequent samples and the predictive value of an indeterminate PCR for a later positive result in the setting of intensifying VTP in the Western Cape province of South Africa. STUDY DESIGN: Retrospective laboratory data analysis. Diagnostic PCR data of a public health laboratory from June 2009 to October 2014 was analysed and categorised by South African VTP regimens. First indeterminate HIV-1 PCRs in patients younger than 12 months were linked with follow-up HIV-1 PCRs and/or serological tests. Linked results sets were analysed by PCR amplification characteristics and subsequent patient outcome. RESULTS: Over intensified VTP regimens, the rate of indeterminate and positive PCRs decreased significantly (5.6-3.2% and 2.4-0.4%, respectively; both p<0.001). Most notably, significantly more patients with indeterminate results had positive PCRs on subsequent samples during WHO Option B+ use compared to older regimens (64.1% vs. 14.7%, p<0.001) at a median 28days later. CONCLUSIONS: Indeterminate HIV PCRs, although decreasing in frequency with Option B+, should be regarded with a high index of suspicion for being representative of true HIV-1 infections. Additional virological testing is required to arrive at a definitive diagnosis.

Antiretroviral Treatment Initiated in the First Month of Life.

BACKGROUND: Earlier diagnosis of HIV-infected infants facilitates earlier access to therapy and improved clinical outcomes. The aim of this study was to describe the management of infants who started antiretroviral therapy (ART) in the first month of life. METHODS: A retrospective review was performed on HIV-infected neonates who started ART within the first month of life between January 2013 and March 2015. RESULTS: A total of 997 neonates had 1 HIV polymerase chain reaction test. Of the 997 neonates, 26 (2.6%) tested positive for HIV and 22 initiated therapy in the first month of life. The median age of first HIV polymerase chain reaction test was 7 days. Neonates were started on ART within a median of 7 days of their first HIV test, which equated to a median age of 13.5 [interquartile range (IQR) 7-20] days of life. Median gestational age was 35 weeks (IQR 33-38 weeks), and birth weight was 2170 g (IQR 1773-2480). Nineteen (86.4%) had low birth weight (<2.5 kg) and 16 (72.7%) were premature. Median baseline HIV viral loads were log 4.444 copies/mL (IQR 3.457-5.125), median CD4 counts were 1338 (IQR 803-1928) and CD4% percentages were 36.1% (22.2-45.4). All children initiated zidovudine and lamivudine, 10 with lopinavir/ritonavir and 12 with nevirapine. All children in care are now receiving lopinavir/ritonavir. Of the 22 neonates initiated on treatment, 11 are in care (mean age, 2.1 years), and 2 of these infants had a viral load of <50 copies/ mL when last measured. CONCLUSIONS: Early ART initiation in neonates is feasible. Challenges include safe, palatable regimens and continued close follow-up of mothers and infants.

Recommendations for the management of indeterminate HIV PCR results within South Africa's early infant diagnosis programme.

Indeterminate HIV PCR results represent missed diagnostic opportunities within South Africa's early infant diagnosis programme. These results not only delay diagnosis and appropriate management but are also a source of confusion and apprehension amongst clinicians and caregivers. We describe the extent of indeterminate HIV PCR results within South Africa's early infant diagnosis programme and provide recommendations for the management of these cases, both in terms of laboratory practice and the clinical care of the infants.