1 Repeat Testing Outcomes of Inconclusive SARS-CoV-2 RT- PCR results in inpatients at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) Student Name: Nombuso Precious Nxele BSc (Hons), MBChB, Dip HIV Man, DTM&H Student Number: 2503547 Masters in Medicine (MMed) Virology Research Dissertation 23 August 2024 Supervisors: Supervisor: Dr Zinhle Makatini BSc (Hons), MSc, MBChB, FCPath Viro (SA), DTM&H, PhD Co-supervisor: Dr Lerato Sikhosana MBhCB, MPH, FCPath Viro (SA) Co-supervisor: Hloni Mthiyane BTEC, MSc Co-supervisor: Dr Mokopi Moloi BSc (Hons), MSc, MBChB, FCPath Viro (SA), DTM&H Copyright © Dr Nombuso Nxele All rights reserved. 2 Declaration I declare that the dissertation hereby submitted to the University of Witwatersrand, Faculty of Health Sciences for the Degree in Masters of Medicine (Virology), has not previously been submitted by me for a degree at this or any other university; that it is my work in design and in execution, and that all material contained herein has been duly acknowledged. 10 March 2024 3 Dedication I dedicate this work with gratitude to my late parents Mr JR and Mrs TA Nxele for instilling a culture of positive attitude and hard work in me. A special thank you to my children Phathisiwe and Khethokuhle Nxele, my number one fans for the sacrifices they made in allowing me to ensure that this work is a success. My deepest gratitude to my mentor and supervisor Dr Zinhle Makatini for her invaluable support and guidance. The experience I gained working with her is priceless. A final thank you to family and friends for the constant support and prayers throughout this journey. 4 Abstract Background With an analytical sensitivity of > 99% in samples harbouring viral loads between 500 – 5000 copies/ml, SARS-CoV-2 RT-PCR is regarded as the gold standard test method for the diagnosis of COVID-19. The reality of inconclusive results occurring, presents a laboratory diagnostic challenge that has an impact on patient management as well as infection and control preventative measures. Methods Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) Virology laboratory performed a total of 42790 SARS-CoV-2 RT PCR tests using the Allplex™ 2019-nCoV (Allplex; Seegene, Seoul, Korea) molecular assay on nasopharyngeal swabs collected from inpatients during the period of June 2020 to December 2020. Inpatient samples with initial inconclusive result outcomes, were subjected to repeat testing of the residual sample within 24 – 48 hours. Results Of the overall samples tested, 709 (1.7%) yielded inconclusive results. A finding of single gene target detection was observed in 98.4% (n=698) of inpatient samples, with the N gene predominance at 73.6% (n=514). Repeat testing provided negative and positive results in 47% (n=335) and 16% (n=112) respectively, with 28% (n=198) remaining persistently inconclusive. Inconclusive samples which subsequently tested positive detected 2 (56.3%) and 3 (47.3%) gene targets. Inpatients samples testing repeat inconclusive, whilst displaying a single gene target distribution, 66% detected the same gene target at initial and repeat testing (N-N, (n=131); RdRP-RdRP, n=19); with the initial gene target failing to detect the same gene target in 34%, displaying N- RdRP, n=12; N-E, n=6, RdRP-N, n=6; E-RdRP, n=3 and E-N, n=3. Conclusion Our relatively low prevalence rate of 1.7% is consistent with other studies. Although clinical history was not furnished, the finding of N gene predominance is explained by inpatients tested at either initial presentation or late stage of COVID-19 infection. Retesting of samples that are initially inconclusive, should be advocated for it allows for establishment of a definitive SARS-CoV-2 outcome. 5 CONTENTS Title Page ..........................................................................................................1 Declaration ....................................................................................................... 2 Dedication ........................................................................................................ 3 Abstract ........................................................................................................... 4 Table of Content…………………………………………………………………… 5-6 1. Literature Review ..................................................................................... 10 1.1 SARS-CoV-2 Epidemiology................................................................................ 10 1.1.1 SARS-CoV-2 Variant .............................................................................. 11 1.1.2 SARS-CoV-2 Transmission………………………………………………… 12 1.2 SARS-CoV-2 Virology .......................................................................................... 12 1.2.1 Classification .............................................................................................. 12 1.2.2 Structure .................................................................................................... 12 1.2.2.1 SARS-CoV-2 Genome .......................................................................... 13 1.3 Clinical Manifestations of SARS-CoV-2 ............................................................... 14 1.4 SARS-CoV-2 Laboratory Diagnosis ...................................................................... 14 1.4.1 Molecular Assay Test Principles ............................................................... 14-15 1.4.1.1 Molecular Assay Gene Targets ............................................................... 15 1.5 SARS-CoV-2 Ct Values ....................................................................................... 16 1.6 Result Interpretation ............................................................................................. 18 1.6.1 SARS-CoV-2 Positive Outcomes ............................................................. 18 1.6.2 Inconclusive SARS-CoV-2 RT-PCR Outcomes ......................................... 19 1.7 Study Rationale.................................................................................................... 20 1.8 Study Objectives and Aim ..................................................................................... 21 1.8.1Study Aim .................................................................................................. 21 1.8.2 Primary Objectives ..................................................................................... 21 1.8.2 Secondary Objectives……………………………………………………………21 2. Materials and Methods ................................................................................ 22 2.1 Study Design ........................................................................................................ 22 2.2 Study Setting and Population ............................................................................... 22 6 2.3 Eligibility Criteria ........................................................................................................... 23 2.3.1 Inclusion Criteria ................................................................................................. 23 2.3.2 Exclusion Criteria ................................................................................................ 23 2.4 Laboratory Methods ...................................................................................................... 23 2.5 Study Definitions ........................................................................................................... 25 2.6 Data Collection and Management ................................................................................. 25 2.7 Statistical Analysis ........................................................................................................ 25 2.7.1 Cost Analysis ...................................................................................................... 25 2.8 Ethical Considerations ........................................................................................... 26 3. Results ................................................................................................................ 27 3.1 Initial Testing Outcomes ................................................................................................ 27 3.1.1 Rates of SARS-CoV-2 Positivity.......................................................................... 28 3.2 Baseline Characteristics of Initial Inconclusive Inpatients…………………………….… 28 3.3 Repeat Testing Outcomes ............................................................................................ 30 3.3.1 Negative Results on Repeat Testing ................................................................... 30 3.3.2 Positive Results on Repeat Testing .................................................................... 31 3.3.3 Repeatedly Inconclusive Results ........................................................................ 31 3.4 Overall Gene Target Distribution ................................................................................... 31 3.5 SARS-CoV-2 Ct Values ................................................................................................ 32 4. Discussion ....................................................................................................... 36-42 5. Study Limitations .................................................................................................. 42 6.Conclusion ........................................................................................................ 42-43 7. References ...................................................................................................... 44-52 8.Appendices……………………………………………………………………………53-57 7 List of Figures Figure 1.1 Estimated cumulative excess deaths per 100, 000 people during COVID-19 South Africa…………………………………………………………………………….10 Figure 1.2 COVID-19 cases, hospital admissions and deaths in South Africa from NICD, MRC……………………………………………………………………………...11 Figure 1.3 Figure 1.4 Figure 1.5 Schematic presentation of the structure and genome organization of SARS-CoV- 2.…………………………………………………………………………………13 SARS-CoV-2 RT-PCR amplification cycle………………………………………17 Estimated variation over time in diagnostic tests for detection of SARS-CoV-2 relative to symptom onset……………………………………………………….18 Figure 3.1 Flow diagram for the selection of CMJAH Inconclusive inpatient samples…………………………………………………………………………..27 Figure 3.2 Correlation between the rate of SARS-CoV-2 to inconclusivity and positivity in samples analysed by RT-PCR…………………………………………………...28 8 List of Tables Table 3.1 Demographic and baseline characteristics of inpatients with initially inconclusive outcomes……………………………………………………………………………29 Table 3.2 Characteristics of CMJAH Inpatients Repeat Testing Outcomes ……………………30 Table 3.3 Gene Target distribution from initial to Repeat Testing Outcomes……. ……………32 Table 3.4 SARS-CoV-2 median Ct Values and gene detection in positive and inconclusive inpatients nasopharyngeal swabs………………………………………………….33 Table 3.5 Univariate and Multivariate Analysis of factors Associated with positive and negative SARS-CoV-2 Outcomes on Repeat Testing………………………………………….34 9 Abbreviations ACE-2 CDW CMJAH COVID-19 Angiotensin- converting enzyme- 2 Central Data Warehouse Charlotte Maxeke Johannesburg Academic Hospital Coronavirus Disease-19 Ct DATCOV HCoV ICTV Cycle threshold Daily hospital surveillance Human coronavirus International Committee on Taxonomy of Viruses ICU MERS-COV MRC NAAT NHLS NICD NPS PHEIC PUI Intensive care unit Middle East Respiratory Syndrome Coronavirus Medical research council Nucleic acid amplification test National health laboratory service National institute of communicable diseases Nasopharyngeal swab Public Health Emergency of International Concern Person under investigation RT-PCR Real time polymerase chain reaction SARS-COV SARS-COV-2 VTM Severe acute respiratory syndrome coronavirus Severe acute respiratory syndrome coronavirus- 2 Viral transport medium WHREC WHO WITS Health and research executive committee World Health Organisation 10 1. Literature Review 1.1 Epidemiology Coronavirus disease (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a respiratory pathogen reported on the 31st of December 2019 in Wuhan, Hubei, China (World Health Organisation [WHO], 2020). The then novel pathogen was isolated from a cluster of patients presenting with a respiratory illness later identified as COVID-19. The World Health Organisation (WHO) identified COVID-19 as a public health emergency of international concern (PHEIC) and accorded COVID-19 a global pandemic status in January 2020 (WHO, 2020). The devastation of the pandemic is reflected in the high mortality rates with 6.98 million COVID-19 related deaths globally and 102 595 deaths reported in South Africa alone by the end of 2023 (WHO, 2023) (Figure 1.1). 11 Figure 1.1 Estimated cumulative excess deaths per 100,000 during COVID-19, South Africa 1.1.1 Variants Genetic variability of SARS-CoV-2 has led to the formulation of an ongoing surveillance strategy for early detection of potential variants which would otherwise have a negative public health implication. The current WHO variant classification recognises variants of high consequence, variants of concern and variants being monitored (WHO, 2023). The variants have an impact on SARS-CoV-2’ s transmissibility and severity of clinical symptoms (Cui et al., 2019). In addition, the impact also extends to diagnostic assays which may result in false negative results, should a mutation occur in the part of the gene target for the detection of SARS-CoV-2. During the course of the pandemic, there have been surges and subsequent declines in new cases of COVID-19 and these have been termed “waves”. These waves on the whole are caused by the emerging variants of concern (Lin et al., 2022). South Africa has experienced five waves driven sequentially by the ancestral strain with an Asp614Gly mutation, Beta variant 12 (B.1.351), Delta variant and Omicron BA4 and BA5 subvariants, representing the fourth and fifth wave respectively. Figure 1.2 COVID- 19 cases, hospital admissions and deaths in South Africa (FT analysis of data from NICD, MRC) Of interest, from figure 1.2, is the much reduced number of Covid-19 related deaths from the fourth and fifth waves caused by BA4 and BA5 respectively. 1.1.2 Transmission Infection with SARS-CoV-2 is mainly through contact with respiratory droplets and SARS-CoV-2 infected surfaces. The incubation period varies between 5 to 7 days depending on the circulating variant (Lauer et al., 2020). Although the virus replicates in the upper respiratory tract, viral replication has been reported to occur in the gastrointestinal tract. In addition, the presence of viral RNA has been reported in the peripheral blood (Salzberger et al., 2021). 1.2 Virology 1.2.1 Classification The International Committee on Taxonomy of Viruses (ICTV) classifies the Severe acute respiratory syndrome-2 (SARS-CoV-2) under the order Nidovirales within the 13 Coronaviridae family. The Coronaviridae family is further sub-divided into two subfamilies - Orthocoronavirinae and Torovirinae, with the Betacoronavirus genus encompassing Human coronavirus (HCoV) HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome human coronavirus (SARS-CoV), Middle Eastern respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2. Phylogenetic analysis identified subgenus Sarbecovirus as the ancestral origin of SARS-CoV-2 and frequently displays recombination in its evolutionary history (Rastogi et al., 2020). 1.2.2 Virus Structure SARS-CoV-2 is 65 -125 nm in diameter and the 29 to 30 kb single stranded RNA positive sense non-segmented genome is contained inside a capsid (Harcourt et al., 2020; Zhou et al., 2020; Wu et al., 2020). The capsid consist of the nucleocapsid N protein as a ribonucleic complex and the capsid is further surrounded by a membrane that contains three proteins – membrane protein (M) and envelope protein (E) involved in the virus budding process and the spike protein (S). The spike protein binds to angiotensin converting enzyme (ACE-2) receptor, allowing for membrane fusion and ultimate entry of the virus into the cell (Li et al., 2016; Cai et al., 2020). 1.2.2.1 Genome The SARS CoV-2 genome spans from an untranslated 5’end to a polyadenylated 3’end (Wu et al., 2020), with the 5’end of the genome comprising of two open reading frames (ORF) designated ORF1a and ORF 1b, allowing for the encoding of two polypeptides which are subsequently cleaved to produce 16 non-structural proteins, NSp1-16 (Mousavizadeh et al., 2020). These non-structural proteins play a role in the viral life cycle (Gordon et al., 2020). The 3′-end encodes structural proteins S, N, M and E. The nucleocapsid binds viral genomic RNA in addition to its role in replication and transcription and the viral envelope is in turn composed of the S, M and E proteins (Paules et al., 2020). Figure 1.3 a and b Schematic presentation of the structure and genome organization of the SARS- CoV-2 (Adapted from Rahimi et al, 2021) 14 1.3 Clinical manifestation Disease manifestation is dependent on the site where the infection has been established (Mehta et al., 2021). Clinical manifestation of SARS-CoV-2 infection is driven by the presence of ACE-2 receptors in organs such as the heart, intestine, kidney as well as the mucosal lining of the respiratory tract and the vascular endothelial cells. COVID-19 infection is categorised into asymptomatic, mild, moderate to severe form of disease. Symptoms range from a mild febrile illness, dry cough, sore throat to shortness of breath, productive cough with signs of hypoxia. In severe cases, patients present with severe pneumonia, respiratory failure, cardiac failure, multiple organ failure ultimately leading to death (Ganesh et al., 2021). 15 1.4 Laboratory diagnosis Nucleic acid amplification tests (NAAT) and serological based assays which amplify viral RNA and viral protein or antibody from respiratory samples, are now widely available. Real time polymerase chain reaction (RT-PCR) is established as “gold standard” method in SARS-CoV-2 diagnostics (Ishige et al., 2024). However, the low sensitivity of RT-PCR assays available at the beginning of the pandemic, were reported to have resulted in a high number of false-negative results (Yang et al., 2020). In that regard, a false negative RT-PCR result could be possibly obtained a few days before symptom onset and at the tail end of SARS-CoV-2 infection - typically from 20 days after symptom onset due to a low SARS-CoV-2 viral load and a viral shedding below analytical RT-PCR sensitivity threshold (Lippi et al., 2020). It is clear however, that the accuracy of tests for SARS-CoV-2 detection in a given sample, will depend on the on the reliability of the SARS-CoV-2 RT-PCR detection methods in use. 1.4.1 Molecular Assay Test Principles The principle of SARS-CoV-2 RT-PCR is based on the in-vitro amplification of at least two molecular targets (Carter et al., 2020). Commercial SARS CoV-2 assays rely on variable primer probe sets with different gene targets (Carter et al., 2020). The primer probe set selected, will ultimately affect the sensitivity of the assay (Vogels et al., 2020, Dhama et al., 2020). In addition, mutations that may be present on primer probe binding site, could potentially reduce the ability of the assay to detect SARS CoV-2 (Vogels et al., 2020). To mitigate against this, at minimum two different gene targets – with one from the conserved and the other from specific regions of the genome, are recommended to avoid the potential genetic drift and the cross reaction with other endemic coronaviruses and to reduce the chance of spontaneous mutations occurring in both selected gene targets (Tang et al., 2020). 1.4.1.1 Molecular Assay Gene targets The challenges of decreased sensitivities of various SARS-CoV-2 assays, highlights the importance of strategic selection of gene targets for the detection of SARS-CoV-2. Other than the emergence of mutation in the target area of primer/probe binding, the reasons for reduced RT-PCR sensitivity could be due to the platform itself, the kit and including the methodology in use. Mutations in primer or probe binding sites would not 16 however be easy to evidence as a reason for a decrease in PCR efficacy (Vogel et al., 2020). The strategy of concomitant detection of two or more gene targets will allow for optimisation of the performance of the SARS-CoV-2 assay. A number of SARS-CoV-2 molecular targets have been selected for use in various commercial RT-PCR assays. These gene targets, amongst others are nucleocapsid, envelope and the glycoprotein spike protein (Tang et al., 2020; Chan et al., 2020; Konrad et al., 2020; Tombuloglu et al., 2021). Species specific accessory gene targets include RNA dependent RNA polymerase (RdRP), open reading frame 1a (ORF1a) and ORF1b genes (Corman et al., 2020; Lu et al., 2020; Colton et al., 2020). Nucleocapsid gene target and the ORF1a/b, are selected due to their highly conserved nature as well as their unique sequences that are specific for SARS-CoV-2 respectively. E gene on the other hand, serves as a pan-Sarbecovirus marker (Roche et al., 2020). The three WHO recommended SARS-CoV-2 gene targets are RdRP, E and N genes (Corman et al., 2020) with E gene targeted for first line screening and with RdRP and N gene serving as confirmatory targets. The widely utilised Allplex 2019-nCoV (Allplex; Seegene, Seoul, Korea) and TaqPath COVID-19 Combo (ThermoFisher) commercial assays in South Africa are based on the detection of E, RdRP and N gene as well as ORF1ab, N and S respectively. The group specific E gene target is similar and detectable in all viruses from Sarbecovirus subgenus species. RNA dependent RNA polymerase has been shown to have the highest diagnostic sensitivity (Corman et al., 2020; Böger et al., 2021) despite having more than 300 mutations and raises the concern that the mutations could potentially interfere with SARS-CoV-2 detection (Yashvardhini et al., 2021). The N protein and the corresponding mRNA are the most abundant in the replication cycle of coronaviruses (Hiscox et al.,1995; Das et al., 2006). Prolonged SARS-CoV-2 positivity has been widely reported in the literature (D’ardes et al., 2020; Cento et al., 2020; Mallet et al., 2020) as well as positivity before the onset of the clinical symptoms. It has been reported that the N gene was the first and the last to be expressed in the infected cells (Lim et al., 2021; Kim et al., 2021). The basis for species specificity of the RdRP, N and S gene targets is in the fact that these gene targets display low homology when compared to other related coronaviruses (Habibzadeh et al., 2021). 17 1.5 Ct values With a maximum of 40 thermal cycles for a typical RT-PCR assay, a cycle threshold (Ct) of a specimen denotes the number of cycles required for the fluorescent signal to cross the threshold (Choudhuri et al., 2020; Engelmann et al., 2021). Viral RNA, measured in Ct value, is detectable as early as the first day of being symptomatic, peaking within 7 days of symptom onset in the majority of COVID-19 infected individuals (Fox-Lewis et al., 2022) (Fig 1.4). Figure 1.4 SARS-CoV-2 RT-PCR amplification cycle (Adapted from Public Health England, 2020) (Jayakody et al., 2021) In routine diagnostic practice, RT-PCR Ct values are used as represent the viral load in a given sample. The lower the Ct value the higher the quantity of viral RNA in the sample as an approximate proxy for viral load (Walker et al., 2021). Interpretation of the patient’s SARS-CoV-2 results, is based on the amplification Ct values (Choudhuri et al., 2020; Engelmann et al., 2021). Following review of the performance of quality and internal controls of the assay, together with the gene targets amplified, the test results are then reported as either positive, negative, inconclusive 18 or invalid (Engelmann et al., 2021). It is important to note that Ct values cannot be directly compared between assays of different types due to variation in the sensitivity chemistry of reagents, gene targets, cycle parameters, analytical interpretive methods, sample preparation and extraction techniques (Rhoads et al., 2022). 1.6 Results Interpretation 1.6.1 SARS-CoV-2 Positive Outcome Generally, and according to most assay manufacturers, a positive sample is one with a Ct value less than 40. Comparison between severely ill patients and those with mild infections reflect differences in viral loads – with the severely ill patients displaying lower Ct values and therefore higher viral loads compared to those with mild COVID19 infections. In addition, when retesting is conducted, PCR positivity in the severely ill group is likely to persist beyond 3 weeks after illness – when a negative result is likely to be an outcome in mildly infected patients on retesting (Yang et al., 2020). A positive PCR in severely ill patients may not necessarily reflect infectivity but rather, possible detection on non-viable viral RNA (Wölfel et al., 2020). Figure 1.5 Estimated Variation Over Time in Diagnostic Tests for Detection of SARS-CoV-2 Infection Relative to Symptom Onset (Adapted from Sethuraman et al., 2020) 19 The clinical significance of positive results with high Ct values are challenging to interpret in the absence of clinical history and context. 1.6.2 Inconclusive SARS-CoV-2 RT-PCR Outcomes Whereas clear-cut positive or negative results are easy to interpret, 3-5% of all samples fall in the grey indeterminate zone, making interpretation a challenge (Bhoyar et al., 2021; Pajudas et al., 2020). Although it is already established that Ct cut offs are in place to ensure specific result outcomes, some SARS-CoV-2 assays have had indeterminate cut-off values introduced. When there is amplification signal of only one of the target genes and the other targets in the assay are not detected, manufacture’s recommendation is that the resulting outcomes be called out as inconclusive and the sample merits repeat testing. The virological causes for these inconclusive outcomes are varied and when E gene target alone is detected, the results signify betacoronavirus infection which is not due to SARS-CoV-2. Different analytical sensitivities of individual viral gene targets, moreso at low viral loads, also have a role to play. In addition, mutations occurring in the target genes, impact the performance of RT-PCR primers or probes (Bhattacharya et al., 2021; Sung et al., 2020; Kim et al., 2020; Peñarrubia et al., 2020). Other preanalytical and analytical reasons include sample collection and sample quality related issues as well as technical issues related primarily to challenges in RNA extraction (Bhattacharya et al., 2020). It is clear therefore that, retesting of inconclusive RT-PCR results is important as it allows for results confirmation that will ultimately influence decision-making regarding patient management strategy. Inconclusive cases might indicate the initial or late stage of COVID-19 infection which require a different patient management strategy, including isolation and testing approach (Falasca et al., 2020). The Allplex 2019-nCoV (Allplex; Seegene, Seoul, Korea) and TaqPath COVID-19 Combo (ThermoFisher) commercial assays in use at the CMJAH Virology Laboratory are based on the detection of E, RdRP and N gene as well as ORF1ab, N and S respectively. In our laboratory, a cut off Ct value of ≤ 38 and > 38 is clinically reported as SARS-CoV-2 PCR positive and negative respectively for both platforms. In addition, 20 both TaqPath COVID-19 Combo Kit and Allplex 2019-nCoV assays, inconclusive outcomes are reported when only one or two of these targets is detected. It has already been established that inconclusive results in symptomatic patients cause diagnostic, clinical, and infection control uncertainty. These inconclusive samples will most likely require multiple consecutive tests to convert to a negative or positive outcome. Although there are reports of up to 5% SARS-CoV-2- RT- PCR results being inconclusive (Bhattacharya et al., 2020), there is limited information on the extent of results released as inconclusive by diagnostic laboratories, together with the number of repeats and the final outcomes of inconclusive results in the South African public sector setting, where a significant proportion of the SARS-CoV-2 testing is conducted. There is therefore an urgent need to have reliable diagnostic test for SARS-CoV-2 that will provide a clear within turn- around time initial laboratory SARS-Cov-2 testing outcome. Our goal was to determine the extent of inconclusive results from SARS-CoV-2 testing at CMJAH Virology Laboratory and to discern true and false SARS-CoV-2 outcomes from repeat testing of initially inconclusive results from CMJAH inpatients. 1.7 Study Rationale Although SARS-CoV-2 RT-PCR is considered the gold standard, interpretation of result outcomes remains a challenge, often calling on the laboratory to retest multiple times. Negative results do not always rule out SARS-CoV-2 infection and are often used in conjunction with other clinical features to determine patient management., The concern of false negatives may be due to sampling error and inappropriate timing of sampling. False negative results may lead to spread of the epidemic in the community. Similarly, the false positive results may lead to unnecessary treatment and mental trauma to the patients. There is therefore an urgent need to have a reliable diagnostic test for SARS- CoV-2 infection. Inconclusive results in symptomatic patients can cause clinical, diagnostic and infection control uncertainty. Inconclusive results require multiple consecutive tests to convert to a negative or positive outcome. Although up there are reports of up to 5% COVID RT PCR being inconclusive, there is limited information on the extent of results released as inconclusive by diagnostic 21 laboratories, in the South African public sector setting, where most of the testing is conducted. Our goal is to determine the extent of inconclusive results from SARS-CoV-2 testing at CMJAH Virology Laboratory and to determine the final outcomes of inconclusive results from inpatients. 1.8 Study Objectives and Aims 1.8.1 Study Aim This study aims to characterise repeat testing outcomes of inconclusive SARS-CoV-2 RT-PCR results from analytical processing with the Allplex 2019 –nCoV assays in inpatients at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH). 1.8.1.1 Primary objectives a) To describe the proportion of CMJAH inpatients with initial inconclusive SARS- CoV-2 RT-PCR results; b) To describe the gene target distribution of inpatient’s inconclusive testing outcomes. 1.8.1.2 Secondary objectives a) To describe gene target patterns in inpatient samples following repeat testing; b) To determine factors associated with SARS-CoV-2 RT-PCR repeat testing in CMJAH inpatients; 22 MATERIALS AND METHODS 2.1 Study Design The study is a cross-sectional design of SARS-CoV-2 infected inpatients with inconclusive testing outcomes admitted at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) and whose SARS-CoV-2 RT PCR was conducted at the National Health Laboratory Service (NHLS) Virology Academic Laboratory. 2.2. Study Setting and Population The study was conducted at CMJAH, a 1088-bed Johannesburg based hospital with intensive and high-care facilities offering tertiary, secondary and highly specialised services. Diagnostic laboratory work-up is supported by the NHLS. The Virology laboratory is equipped with a number of SARS-CoV-2 platforms as well as Pathologists on site. The CMJAH Virology Laboratory at the time, was the main centre for SARS-COV-2 testing in Gauteng, receiving samples from inpatients, health care workers (HCWs) and in addition community screening samples. Hospital policy then required that all individuals admitted at the hospital including those with non-COVID-19 related pathologies, to be screened for SARS-CoV-2. As such, the patient population in our study includes symptomatic and non-symptomatic inpatients. CMJAH Area 161, served as main intake and holding area for all incoming patients. Patients would then be allocated to wards once SARS-CoV-2 RT PCR results were released. The following wards were re-purposed as COVID-19 adult inpatient wards – 376, 377, 577, with 587 and 567 serving as Intensive Care Unit (ICU) wards. COVID-19 symptomatic children were admitted to wards 276, 286 and with 357 and 567 serving as paediatric intensive care units. CMJAH staff were managed in Ward 356. In addition, ward 194 was designated for pregnant inpatients with suspected COVID-19. Collected SARS-CoV-2 23 nasopharyngeal swabs (NPS) were sent to the NHLS Virology laboratory for analytical processing. The NHLS CMJAH Virology laboratory conducts SARS-CoV-2 RT-PCR through initially use of Allplex 2019-nCoV (Allplex; Seegene, Seoul, Korea) and later with the addition of TaqPath COVID-19 Combo (ThermoFisher). All inconclusive results from SARS- CoV-2 tested samples received from admitted patients from the period of April 2020 to March 2021 and that were subsequently repeat- tested, were requested from the NHLS data repository – Central Data Warehouse (CDW). 2.3. Eligibility Criteria 2.3.1. Inclusion criteria • All SARS-CoV-2 test results from the inpatient population that were processed on the Allplex 2019-nCoV (Allplex; Seegene, Seoul, Korea) platforms and that initially had inconclusive results and subsequently re-tested on Allplex 2019nCoV; • Inpatient samples tested between 1st April 2020 to 31 March 2021 for SARSCoV-2 with subsequent repeat testing; • Inpatient samples of all ages; • Inconclusive samples repeat-tested within 24-48 hours. 2.3.2 Exclusion criteria • All inconclusive samples from community screens; • All inconclusive samples that were not subsequently re-tested; • Initially inconclusive results repeated on newly recollected samples; • Inconclusive samples repeated > 48 hours from initial testing; All results with missing patient laboratory and clinical data. 2.4 Laboratory Methods The NHLS Virology Laboratory accepted nasopharyngeal swab samples transported in viral transport medium (VTM) or phosphate buffered saline (PBS) from people under investigation (PUIs) for SARS-COV-2 diagnostic testing. The samples were extracted 24 in the Seegene Nimbus or the Magnipure extraction platforms, followed by real time amplification on the thermocycler. For amplification, the Allplex RT-PCR assay which amplifies 3 specific targets has the E gene serving as a screening test (pan-sarbecovirus target), and the RdRP and N genes which are SARS-CoV-2 species specific, serving as a confirmatory test. For amplification, the protocol followed as per manufacturer instructions, involves a master mix preparation of 5µl one-step buffer, 5µl water, 5µl 2019 nCoV MOM and real- time 2µl enzyme as well as 8 µl of quality controls. A total reaction mixture of 25µl was aliquoted into the amplification plate. In addition, PCR inhibitors were monitored by the inclusion of an internal control and the reaction mixture amplified in the Seegene Allplex platform. Results from the Allplex thermocycler were analysed and uploaded by the software for pathologist decision and resulting. Worksheets with run information for all the specimens with inconclusive results were checked for validity of specific runs. In addition, the amplification curves were visually checked for atypical amplification curves above the threshold that signal inconclusive outcomes. However, checking if inconclusive specimens were not adjacent to another highly positive specimen (defined as Ct <20) was not conducted – hence cross contamination could be ruled out. According to the laboratory standard operating procedure, inconclusive samples were repeat-tested. For the retesting was conducted by re-extracting nucleic acids from the residual samples. We selected only samples that were initially tested on Allplex SARSCoV-2 assay (Seegene Inc.; Seoul, Korea) and rested on the same platform. According to the standard operating procedure (SOP), retesting would be conducted within 2448 hours of an inconclusive result outcome. 2.5 Study Definitions Our laboratory defined criteria for result interpretation are as follows: SARS-CoV-2 RT PCR is reported as positive when all 3 gene targets are detected. Results are invalid when the internal control has not amplified. Inconclusive results on the other hand are called out when only 1 or 2 gene targets are detected and the Ct values is less than 38. 25 2.6. Data Collection and Management Variables from SARS-CoV-2 test results, that met the inclusion criteria were requested from CDW. These included episode number, date of birth, gender, ward, dates of initial and repeat testing and gene targets detected – all captured on Trakcare laboratory Information System. For the following clinical variables, approval was sought and attained from DATCOV sentinel surveillance system: i) Demographic, ii) anthropometric measures and iii) comorbidities - in particular HIV and CD4 T cell count. SARS-CoV-2 RT-PCR results from April to May 2020 were not available. The study period therefore included data from June to December 2020, allowing for only a 7 month’s data analysis. In addition, data from January 2021 to March 2021 was missing and therefore not extracted. 2.7 Statistical Analysis Descriptive statistics were determined for continuous measures using the median and interquartile ranges (IQR), whereas categorical data was presented as frequencies and percentages. Where appropriate, Chi-square, paired t-test, Pearson’s correlation coefficient and the Wilcoxon signed-rank test were used to evaluate statistical differences. Univariate and multivariate logistic regression analysis was used to examine the relationship of independent variables with the primary outcomes. All variables were included in the multivariate analysis model with logistic regression using odds ratio. Variables with p values < 0.05 were be considered statistically significant. All data was analysed using Microsoft Excel and Graph Pad Software (Graph Pad Inc., USA). 2.7.1. Cost Analysis The study aimed to reflect major and minor laboratory costs associated with repeat testing of inconclusive SARS-CoV-2 samples during the pandemic. Costing of consumables such as sampling swabs, viral transport media and test kit reagents were going to be considered and calculated per test. Microsoft excel spreadsheet as well as the Stata statistical package was going to be used to capture both the parameters of test activity as well as the breakdown of test kit contribution. A bottom up method to 26 estimate cost per result was planned for as well as soliciting quotations will be solicited from the manufacturer as well as from the National Health Laboratory Service (NHLS). However, due to academic time constraints the cost sub-analysis of this project will be conducted separately from the dissertation and published. 2.8. Ethical Considerations The study protocol was submitted to the University of Witwatersrand Health Research Ethics Committee and approval granted (Appendix i). In addition, approval was also sought from the hospital management of CMJAH as well as from the NHLS CDW and DATCOV (Appendices ii, iii, iv) to allow for access to patient SARS-CoV-2 laboratory results and clinical information respectively as well as information on comorbidities. 27 3. RESULTS 3.1 Initial Testing Outcomes In our laboratory, during the period from June to December 2020, 42 790 nasopharyngeal swabs (NPS) were collected from inpatients at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) and tested for SARS-CoV-2 using the Allplex SARS-CoV-2 assay. Of 42 790 samples analysed,1605 (3.7%) were invalid and subsequently excluded from the study and of the remaining overall 41 185, 37 224 (90.4%) and 3252 (7.9%) samples tested negative and positive for SARS-CoV-2 respectively. Seven hundred and nine SARS-CoV-2 inconclusive samples where either E, RdRP or N was isolated regardless of the Ct value, were identified. Fig. 3.1 Flow diagram for the selection of CMJAH inconclusive inpatient samples Total number of samples tested for SARS - CoV - 2 n = 42 790 Final cohort under study n=41 185 Positive n = 37224 Negative n = 3252 Inconclusive n = 709 Invalid n = 1605 28 3.1.1 Rates of SARS-CoV-2 Positivity Our study period coincided with the first pandemic wave characterised by the circulating WT D614G variant, with the peak in SARS-CoV-2 positivity occurring in July 2020. From stratifying both positive and inconclusive samples by month covering the study period, we observed that as the number of samples testing SARS-CoV-2 positive in our laboratory increased, the number of inconclusives also displayed a corresponding increase (Fig 3.2). Figure 3.2 Correlation between the rate of SARS-CoV-2 inconclusivity and positivity in samples analysed by RT-PCR 3.2 Baseline characteristics of Initial Inconclusive Cohort Of the 709 inpatients with inconclusive results, the median age (interquartile range [IQR]) was 34 years [25.5-49.5 years; p<0.810], while 445 (63 %) of the patients were female. The cohort was predominately adult inpatients (n=643; 91%), with only 66 29 (9.3%) children. Although the clinical background on tested inpatients was not provided for, requests for testing from laboratory requisition forms were determined to be predominantly (n=378; 53.3%) from the medical wards (Table 1). Table 3.1 Demographic and baseline characteristics of inpatients with initially inconclusive outcomes. Characteristic Overall (N=709) N (%) Age, median (IQR) 34 (25.5, 49.5) Adults 643.0 90.7 Children (<14yrs) 66.0 9.3 Gender, n (%) Female 445.0 62.7 Male 264.0 37.2 Sub-specialities Medical 327.0 52.3 O and G 143.0 22.8 Surgical 130.0 20.8 Oncology 20.0 3.2 Transplant 5.0 0.8 Paediatrics Medical 51.0 60.7 Oncology 12.0 14.3 Surgical 7.0 8.3 Transplant 0.0 0.0 Others 14.0 16.7 Of the 709 inconclusive results, the majority (n=698; 98.4%) of Allplex based gene targets detected, were single genes – N, RdRP or E. The remaining 11 had 2 genes detected in the following combinations – RdRP+/N+ (n=7; 0.4%), E+/N+ (n=3; 1%) and E+/RdRP+ (n=1; 0.1%). The N gene target was the more frequently detected, with 73.6% (514/698), 13.3% (93/698) and 13.0% (91/698) being N, RdRP and E gene respectively. The median Ct values of N, RdRP and E were 38, 37 and 36 respectively. The Ct range of N (minimum = 29.0, maximum = 39.0) was much narrower than that of E (minimum 30 = 26.0, maximum = 39.0) and RdRP (minimum =24,0 maximum 39,0). The CMJAH Virology defined Ct cut off for positive detection of 38 was applied to filter out all gene targets with Ct values > 38. The revised frequency of detection was therefore 484 (69.3%), 81(11.6%) and 86 (12.3%) of N, RdRP and E respectively – with the N gene consistently the most frequently detected. 3.3 Repeat Testing Outcomes The Laboratory’s SOP stipulate that SARS-CoV-2 specimens yielding an initial inconclusive outcome, should be repeat-tested within 24 hours. Our finding was that the time interval between all original specimen samples testing inconclusive and repeat testing being conducted, was 12 to 48 hours. From the inconclusive cohort of 709, the time interval was 1.2 ± 1.2, 1 ± 1.3 and 1.1 ± 1.4 (p <0.000) for samples that yielded a positive, a negative and a persistent inconclusive SARS-CoV-2 outcome on repeat testing (Table 3.2). 3.3.1 Negative Results on Repeat-Testing For this analysis, of the 709 initially inconclusive samples, 47.2% (n=335) on repeat testing were negative, thereby pointing to the likelihood that the initial test was most likely false-positive. Table 3.2 Characteristics of CMJAH Inpatient Repeat Testing Outcomes Variable Total (n=709) Positive (n=112; 16%) Negative (n=335; 47.2%) Inconclusive (n=198; 28%) Invalid (n=64; 9.0%) p-value Age, median (IQR) 34 (25.5, 49.5) 34 (27.3, 47.8) 34 (25, 47) 35.5 (26, 51.3) 37 (27, 50.0) 0.582 Adults 643 (90.7) 104 (92.9) 301 (89.9) 181 (91.4) 57 (89.1) 0.747 Children (<14yrs) 66 (9.3) 8 (7.1) 34 (10.1) 17 (8.6) 7 (10.9) Gender, n (%) Female 445 (62.8) 80 (71.4) 201 (60.0) 125 (63.1) 39 (60.9) 0.187 Male 264 (37.2) 32 (28.6) 134 (40.0) 73 (36.9) 25 (39.1) Hospital ward Medical 327 (46.1) 47 (41.9) 154 (46.0) 97 (48.98) 29 (8.9) 0.693 O and G 143 (20.2) 30 (26.8.) 63 (18.8.) 37 (26) 13 (9.1) 0.295 Surgical 130 (18.3) 19 (17.0) 68 (20.3) 33 (16.7) 10 (15.6) 0.640 31 Oncology 20 (2.8) 4 (3.6) 8 (2.4) 7 (3.5) 1 (1.6) 0.754 Transplant 5 (0.7) 0 (0) 1 (0.3) 2 (1) 2 (3.1) 0.066 Paediatrics Medical 51 (7.2) 6 (5.3) 25 (7.4) 14 (7.1) 6 (9.4) 0.786 Oncology 12 (1.7) 2 (1.8) 6 (1.8) 2 (1) 2 (3.1) 0.713 Surgical 7 (1) 2 (1.8) 4 (1.2) 1 (0.5) 0 (0) 0.575 Transplant 0 (0) 0 (0) 0 (0) 0 (0) 1 (2) - Others 14 (2) 2 (1.8) 6 (1.8) 5 (2.5) 1 (1.6) 0.931 Ct-Values 36.9 ± 2 36.1 ± 2.4 36.9 ± 1.9 36.6 ± 2.1 0.000 Time interval (days) 1.2 ± 1.2 1 ± 1.3 1.1 ± 1.4 1.9 ± 2.1 0.000 3.3.2 Positive Results on Repeat Testing Only 112 out of 709 (16%) inpatients with an initial inconclusive test result, tested positive in the follow-up test on the same sample. The time interval from an initial inconclusive to positive follow up results was 1.2 ± 1.2 days. A change in Ct value from an initial inconclusive to the final positive outcome was observed – with the Ct-value decreasing from an initial inconclusive median Ct [IQR] result of 38 [37–38] to a positive median Ct [IQR] result of 34 [32–36]. 3.3.3 Repeatedly Inconclusive Results Of significance were 198 (27.9%) inpatient samples that were repeatedly inconclusive. As per protocol of the CMJAH Virology laboratory, inpatients with repeat inconclusive results lead to a request for a new nasopharyngeal swab on the inpatients concerned. 3.4 Overall Gene Target Distribution With 698 (98.4%) initial inconclusive outcomes being single gene targets, the most frequently detected single target was the N gene at 73.6% in CMJAH inpatients. From repeat testing, where the initial outcome was repeatedly inconclusive, the predominance of N (66%) persisted (Table 3.3). Where repeat testing yielded a positive outcome (n=112), the presence of two and three gene targets was observed in 52.6% and 47.3% respectively. The two gene target combination of RdRP+/N+ and N+/E+ predominated at a frequency of 27 (24.1%) and 26 (23.2%) respectively. 32 The remaining 53 (47.3%) had all 3 gene targets detected, with 34 (67%) with a Ct value <36. Table 3.3 Gene target distribution from initial to repeat testing outcomes Targeted gene Initial inconclusive outcome (n=709) N% Repeat testing Positive Outcome (n=112) N% Repeat Testing Inconclusive Outcome (n=198; N%) N% Single Gene Targets RdRP gene only (%) 93 13.3 0.0 0.0 47.0 23.7 N gene only (%) 514 73.6 0.0 0.0 132.0 66.7 E gene only (%) 91 13.0 0.0 8.0 18.0 9.1 2Gene Targets E gene and N gene (%) 3.0 0.4 26 23.2 0.0 0.0 RdRP gene and N gene (%) 7.0 1.0 27 24.1 1.0 0.5 E gene and RdRP 1.0 0.1 6.0 5.3 0.0 0.0 3Gene Targets E, RdRP, N 0.0 0.0 53.0 47.3 0.0 0.0 3.5 SARS-CoV-2 Ct values The Ct values of 36.9 ± 2.4 for initially inconclusive, 36.1 ± 2.4 positive on repeat testing and 36.9 ± 1.9 for repeated inconclusive were significantly different between the three groups (p>0.000). The overall average Ct value of initial inconclusive (36.9+/-2) results reflect high Ct values that are close to the laboratory defined cut off Ct value. The median Ct values in repeat-tested positive cases with the E+/N+ pattern was 37, RdRP+/N+ with a median Ct of 35 and E+/RdRP+ with a median Ct 36 as shown in Table 3.4. 33 Table 3.4 SARS-CoV-2 median Ct values and genes detected in positive and inconclusive swabs Positive Inconclusive (N = 112) (N = 198) Number of genes detected N% Median CT* N% Median CT* (IQR) [range] (IQR) [range] 1 0.0 (0.0%) - 198.0 (100) 37 (36-38) [25- 39.78] 2 59 (52.6%) 36.5 (35-38) [20 - 39] 0.0 - 3 53 (47.3%) 34 (32–36) [15– 39] 0.0 - Genes detected N only 0.0 - 133.0 38 (37-38) [25- 39.78] E only 0.0 - 18.0 36 (35-36) [33- 38] RdRP only 0.0 - 47.0 36.87 (35-37) [29- 39] E and N 26 37 (35-38) [20- 39] 0.0 - RdRP and N 27 35 (30-39) [30- 39] 0.0 - E and RdRP 6 36 (35-36) [30- 36] 0.0 - 34 Table 3.5 Univariate and multivariate analyses of factors associated with positive and negative SARS-Cov-2 outcomes following repeat testing of an initial inconclusive result in inpatients at CMJAH: June to December 2020 Variable Positive Negative Univariate Multivariate n % n % p OR 95% CI OR 95% CI Lower Upper Lower Upper Adults 104 92.9 301 89.9 0.454 1.468 0.659 3.274 5.583 .258 120.906 Children (<14yrs) 8 7.1 34 10.1 0.454 0.681 0.305 1.518 Gender, n (%) Female 80 71.4 201 60 .032 1.667 1.047 2.652 1.576 .879 2.827 Male 32 28.6 134 40 .032 0.600 0.377 0.955 Testing interval ≤ 24 hours 25 22.3 99 29.6 0.146 .685 .414 1.133 .680 .389 1.190 Hospital ward Medical 47 42 154 46 0.511 0.850 0.551 1.310 1.361 .155 11.942 O and G 30 26.8 63 18.8 0.081 1.580 0.958 2.604 2.499 .280 22.313 Surgical 19 17 68 20.3 0.492 0.802 0.458 1.405 1.373 .149 12.684 Oncology 4 3.6 8 2.4 0.506 1.514 0.447 5.127 2.449 .201 29.901 Transplant 0 0 1 0.3 1.000 - - - Paediatrics Medical 6 5.4 25 7.5 0.525 0.702 0.280 1.758 4.941 .122 200.017 Oncology 2 1.8 6 1.8 1.000 0.997 0.198 5.012 9.281 .228 378.241 Surgical 2 1.8 4 1.2 0.643 1.505 0.272 8.327 11.954 .187 763.919 Others 2 1.8 6 1.8 1.000 0.997 0.198 5.012 E-1 gene 13 11.6 52 15.5 0.355 0.715 0.373 1.368 14.498 1.644 127.878 RdRp-1 gene 24 21.4 41 12.2 0.020 1.956 1.120 3.414 52.380 5.840 469.798 N-1 gene 83 74.1 243 72.5 0.807 1.084 0.666 1.762 47.171 5.263 422.767 Ct-Values (Mean ± SD) 36.1 ± 2.4 37.3 ± 1.7 0.000 .716 .632 .811 Age, median (IQR) 34 (27.3, 47.8) 34 (25, 47) 0.689 1.004 .987 1.021 35 Factors found to be associated with SARS-CoV-2 positivity from repeat tested samples were likely to be adults 92.9% (OR: 1.46, 95%CI:0.65-3.27), being female 71.4% (OR: 1.66, 95%CI: 1.047- 2.65) and for adults from O&G and oncology wards 26.8% (OR: 1.580 95%CI:0.958-2.604; 3.6% (OR:1.514 95%CI:0.447-5.177) respectively. In paediatric patients, admission to the surgical ward, was associated with SARS-CoV-2 positivity on repeat testing - 1.2% (OR:1.505, 95%CI:0.272-38.327). 36 4. DISCUSSION There is no test that gives a 100% accurate result and as such tests need to be evaluated to determine their sensitivity and specificity ideally by comparison with a gold standard. Since the onset of the pandemic, the SARS-CoV-2 real- time RT- PCR not only has been regarded as the gold standard but continues to be widely used for detection of infection. Throughout the pandemic to date, SARS-CoV-2 RT-PCR negative and positive outcomes presented little or no challenge in the clinical environment. Inconclusive results however create a diagnostic uncertainty and the challenge of inconclusive results continues to be reported. In this present study we therefore aimed to determine the extent of true or false positive findings from initially inconclusive results from SARS-CoV-2 testing at a large academic hospital in Johannesburg South Africa. Inconclusive SARS-CoV-2 RT-PCR results reduce diagnostic confidence in terms of establishing a definitive COVID-19 diagnosis, direction in terms of clinical management as well as infection control preventative measures that can be undertaken (Chan et al., 2020). In addition, inconclusive results released by the diagnostic laboratory, are not always easy for the clinicians to discern (Yang et al., 2021). It is for these reasons that repeat testing following a finding of an inconclusive result is advocated for as this diagnostic laboratory practice allows for a definite conclusion of the test result in most cases (Boeckmans et al., 2021; Munne et al., 2021). The standard practice following an inconclusive result outcome in our laboratory, is to retest the same nasopharyngeal residual sample that yielded an initial SARS-CoV-2 inconclusive result within the laboratory defined interval of 24 - 48hrs. In our study, the time to repeat testing was within the stipulated time limit and does not represent variation factor in the final outcomes. Charlotte Maxeke Johannesburg Academic Hospital during our study period, admitted patients categorised as having moderate to severe COVID-19 symptomatic infection. In addition, the screening algorithm in place at CMJAH mandated SARS-CoV-2 testing of all hospital attendees including asymptomatic individuals coming in as 37 preadmissions for planned procedures. We were not able to however, confirm a history of COVID-19 related symptoms nor its absence in inpatients admitted for elective procedures, clinical follow-up, obstetrics and gynaecology, those presenting at casualty and health care workers. However, as there were COVID-19 and non- COVID19 designated wards, in this study wards are used as a proxy for stratifying inpatients into symptomatic and asymptomatic categories for COVID-19. A relatively low rate of 4 – 5% SARS-CoV-2 inconclusive results has been generally reported (Bhoyer et al., 2021; Pujudas et al., 2020). In our study, inconclusive results conducted on the Allplex 2019-nCoV (Allplex; Seegene, Seoul, Korea) platform were detected in 709 (1.7%) of the overall 41185 inpatient samples tested for SARS-CoV-2 during the first COVID-19 wave driven by the ancestral strain. The 1.7% prevalence rate of SARS-CoV-2 inconclusivity coincides with the previous findings of 0.3% to 2.9 % from studies conducted in the United States, Belgium, Bulgaria, Ireland, Indonesia and Korea (Dolan et al., 2024; Boeckmans et al., 2021; Stoykova et al., 2022; Yang et al., 2021; Ardianto et al., 2021; Lim et al., 2021). Jeon et al reported much higher inconclusive rates of 81.7% in their study reflecting the dynamic factors influencing the rate of inconclusive results in different study settings (Jeon et al., 2023). Of note is that studies supporting our inconclusive findings were also conducted during the first pandemic wave, however the fact that different testing platforms with different gene targets and different patient sample sub-populations were used, makes a comparison of these SARS-CoV-2 results a challenge. In addition, we observed a correlation between the frequency of SARS-CoV-2 positives detected monthly and the number of corresponding inconclusive results by month. However, the higher the total number of samples tested, the higher the inconclusive and positive detection rates - wherein, as the number of the inpatient samples testing positive increased, the number of inconclusive inpatient samples also increased. In our study, the majority of initially inconclusive results were characterised by a high frequency of single gene target positivity (n=698; 98.4%). Overall, the N gene was solely amplified in 74% (n=514) of samples across the study inpatient subpopulation, with no preponderance for gender and age. The predominance of the isolated N gene in inconclusive results is supported by studies reporting frequency of N gene detection at 65% and 100% respectively (Ardianto et al., 2022; Stoykova et al., 2022). The finding 38 of a high frequency of the isolated N gene target compared to the E and RdRP gene targets, can be attributed to the stability of the N gene, a gene that is conserved, possessing less nucleotide variation compared to the E gene and RdRP gene (Grifoni et al., 2020). The N protein and its corresponding mRNAs dominate the replication cycle of coronaviruses and the partial amplification of viral RNA remnants by SARS- CoV-2 RT PCR, together forms the basis of the rationale that inconclusive single gene N target outcomes in inpatient samples represents initial or late stage of COVID-19 infection (Das et al., 2006; Falasca et al. 2020; Kim et al., 2020; Lim et al., 2021). Of the 514 inpatient samples with inconclusive single N gene positivity, 190 (36.9%) were inpatients from the designated medical COVID-19 wards and thus assumed to be COVID-19 symptomatic. The remaining 324 (63.0%) inpatients were SARS-CoV-2 screening cases assumed to be asymptomatic. There are also reports of a high proportion of inconclusive SARS-CoV-2 RT PCR results being false positive, especially amongst asymptomatic patients. In a setting of low prevalence, even a test with high specificity could generate a substantial percentage of false positive results (Yang et al., 2020; CDC 2020). In our study therefore, the hospital policy of screening all patients with low pre-test probability for COVID-19 infection- including asymptomatic preadmission and presurgical attendees, we assume inevitably led to false positive results. Since clinical decisions including timing of elective surgery may be based in part on COVID-19 screening test results, false-positive results can delay care for patients and have unintended consequences, including delayed surgery or delays in diagnosis, as has been reported in the literature (Schizas et al., 2020; Katz et al., 2020). It is therefore important for clinicians to be able to understand the meaning of inconclusive tests. There are a number of factors that may cause inconclusive results ranging from inadequate nasopharyngeal sampling, cold chain transport temperature deviations, poor quality of viral transport medium (VTM) and extraction failure (Bhattacharya et al., 2020). In addition, false positivity could be found in the non-specific binding during the late phases of the PCR reaction –an assumption that is supported by the relatively high Ct values obtained (Bhattacharya et al., et al, 2020). Re-testing in case of inconclusive results has been widely advocated for since repeat testing allows for a definite conclusion of the test result in most cases (Boeckmans et al., 2021). The retesting of the 709 inconclusive inpatient samples in our study cleared 39 112 samples (16%) as positive, 335 samples (47.5%) as negative and 198 samples (28%) as inconclusive. The probability of a negative outcome from an initial inconclusive test, has also been recognised in a number of studies (Gubbay et al., 2021; Lim et al., 2021). Our study demonstrated that 47.2% of the SARS-CoV-2 inconclusive RT-PCR results were subsequent negatives on retesting – this indicating that the index test was most likely false positive, although it is still possible that SARS-CoV-2 RT PCR partially amplified small amounts of remnant RNA in the initial testing (Boeckmans et al., 2021; Munne et al., 2021). The majority of these inpatient samples were from the medical, obstetrics and surgical wards at 36.7%, 21.5% and 20% respectively. Our finding of subsequent negative results is consistent with reports of 31.5%, 42.1%, 46%, 52% from studies conducted in South Korea and the United States respectively (So et al., 2020; Jeon et al., 2023; Green et al., 2020; Yang et al., 2021). Higher false positive of 86% and 86.9% results were reported in studies in the Netherlands and Belgium (Rondaan et al., 2023; Boeckmans et al., 2021). In addition, a negative result when following retesting of an inpatient inconclusive sample implies that there might have been an error in the previous inconclusive result. These results suggest that contamination from adjacent wells with positive samples or previously analysed positive samples, cannot be ruled out. Human errors from mishandling of samples or reagent instability are factors that should also be considered (Hong et al., 2020; Sung et al., 2020; Kim et al., 2020). Given a high analytical sensitivity of SARS-CoV-2 RT PCR of over 99%, repeat-tested inpatients that yield a positive outcome can be considered true positives. The finding of 16% subsequent SARS-CoV-2 RT PCR positive inpatient samples from repeat testing in our study, can thus be regarded as true positive (Zhou et al., 2020; Pfefferle et al., 2020; Smithgall et al., 2020; Cordes et al., 2020; Zhen et al., 2020). The majority of these inpatients (n=71; 63.4%) were from non-COVID-19 designed wards and therefore assumed to be COVID-19 asymptomatic. Of the 112 subsequently positive inpatients, 61 (54.5%) and 51 (45.5%) were positive in only 2 or 3 gene targets. providing assurance in overall results. Whilst Ct values are not directly comparable between studies, according to the Public Health England study, only samples with Ct value <37 were positive on repeat testing of the same nasopharyngeal swab (Ladhani et al., 2020; Riley et al., 2020). Of 112 https://elifesciences.org/articles/64683#bib16 https://elifesciences.org/articles/64683#bib16 40 CMJAH inpatients with a repeat-testing positive outcome, 34 (67%) had all 3 target genes detected in addition to having a Ct value < 36 – thus likely reflecting true positivity of these samples. The remaining 59 inpatients had 2 gene target detected with a combination RdRP+/N+ (n=27), N+/E+ (n=26) and E+/RdRP+ (n=6). Our results are supported by findings from Ardianto et al showing that when two genes (N+/E+) were detected, the probability of positive result on retest was significantly higher (80%) (p- value = 0.001) than when only one gene was detected at 45.16% (Ardianto et al., 2022). One hundred and ninety-eight (28%) of samples in our initially inconclusive inpatient cohort, returned inconclusive outcomes following repeat testing. Individuals with repeated inconclusive results, without a confirmed history of COVID-19 as is the case with our cohort - are often reported to be patients in the incubation period or at an early stage of infection (Sung et al., 2020). Although two thirds (n=131; 66%) of repeated initially inconclusive samples displayed identical gene target amplification at retesting, 34% (n=67) were persistently inconclusive and had a different single gene target detected. The majority (66%) of all inconclusive results we obtained, amplified the N gene at initial and repeat testing (N+N+). This is an expectation as the N gene is reported to be the first and last to be expressed in the infected cells (Lim et al., 2021; Kim et al., 2020). The other initial- repeat testing inconclusive result combinations were N+ - RdRP+ (n=12; 25%), RdRP+ - RdRP+ (n=19; 9.6 %), N+ - E+ (n=6; 3%), RdRP+ - N+ (n=6, 3%) E+ - RdRP+ (n=3; 1.5%) and E+ - N+ (n=3; 1.5%). These changes in gene target detection from initial to repeat testing may indicate that the same genes targets may have been present in the sample or that some gene targets are lower than the analytical sensitivity of the assay used (Drew et al., 2020; Lim et al., 2021). In addition, the different analytical sensitivity of the RT-PCR assay for the individual genes could also play a role, with each primer probe set having a specific target and even single-point mutations that potentially could lead to amplification failure of the corresponding gene (Artesi et al., 2020; Miller et al., 2021). Gene target combination of initial inconclusive E gene target changing to a different gene target on repeat testing, could be a reflection of the non-specificity problems of the gene as well as E gene’s vulnerability to sample contamination (Benrahma et al., 2020; Lim et al., 2021; Bezier et al., 2020). Overall, our data indicated 41 that retesting inconclusive samples was beneficial, particularly when two genes were detected, but was ineffective when only E gene was detected. In laboratory medicine, it is widely recognised that restricting diagnostic test results to either a positive or negative outcome, whilst desirable for diagnostic decision making, is not a reality in a clinical setting as there often is a subset of results that are relatively uninformative, ultimately categorised as inconclusive or indeterminate (Feinstein et al., 1990). Our study supports the finding that when the SARS-CoV-2 positive rate is high, there likely will be a corresponding increase in the inconclusive rate ultimately increasing the amount of resource input through warranting repeat testing until a definitive diagnosis is made. To reduce the consumption of resources, an inconclusive test should be interpreted flexibly with the patient’s characteristics or even the epidemiological setting at the time of repeat testing (Stoykova et al., 2022). Therefore, there is a high probability of an initially inconclusive result yielding a positive outcome on repeat testing when the patient is local and the positivity rate at the time is high. Three hundred and thirty-five (47.2%) of our initially inconclusive samples tested negative following repeat testing – making subsequent negative outcomes the most frequent finding on repeat testing. The initial inconclusive signal in those that are newly infected with COVID-19 or those that are asymptomatic but with past COVID-19 infection, necessitate retesting – allowing for a definitive COVID-19 diagnosis. With association between Ct value and SARS-CoV-2 viral load, inconclusive results from COVID-19 patients may indicate that the viral load decreased during recovery from infection and as such inconclusive results could derive from a natural process during follow-up of patients with positive SARS-CoV-2 RT-PCR. Furthermore, the SARS-CoV-2 diagnostic RT-PCR assays detect viral RNA even when the virus has lost its infectivity (Atkins et al., 2020; Dighe et al., 2020) therefore, inconclusive results associated with a lower viral load than positive results may indicate no infectivity. In addition, there is a school of thought that mutations of SARS-CoV-2 target genes have a bearing of inconclusive results (Penarrubia et al., 2020). However, the concern is challenged by the finding of alternating target gene targets upon request i.e. initial detection of N gene and replaced by RdRP detection on retesting without the presence of N. It is for this reason that primer-probe mutations are unlikely to have caused inconclusive results in our study and others (Penarrubia et al., 2020; Kim et al., 2020). 42 RT-PCR, serological testing including the epidemiological setting – all need to be considered in the formulation of a differential diagnosis, allowing for rapid release of accurate results and the subsequent isolation of infected patients thus limiting the spread of COVID-19 (CDC, 2020, Lou et al., 2020., Yong et al., 2020). When a negative SARS-CoV-2 RT-PCR result is obtained from symptomatic patients, a retest is advocated for and a respiratory multiplex PCR that includes viral/bacterial/fungal pathogens should be considered (Kim et al., 2020; Zhu et al., 2020). 5. Study Limitations Our study analysed data of SARS-CoV-2 RT-PCR inconclusive results obtained only from inpatients admitted at CMJAH between June to December 2020 period as data from April to May 2020 was missing. Our study period therefore only allows for analysis of the first pandemic wave and cannot be generalizable to the period of other COVID19 pandemic waves and subsequent variants. Clinical data to confirm presence or absence of COVID- 19 symptoms and history of previous SARS-CoV-2 infection in inpatients whose samples were analysed was not available. From repeatedly inconclusive samples, retesting for SARS-CoV-2 RT-PCR on new samples, was not routinely conducted timeously. 6. Conclusion Although SARS-CoV-2 RT- PCR inconclusive studies have been conducted in other parts of the world, our study addressed the gap of inconclusive findings in the South African public sector health context. Despite the high sensitivity and specificity of SARS-CoV-2 RT-PCR, the occurrence of inconclusive results in the molecular laboratory is inevitable. 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Elife, 10, p. e64683. https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas#individual-molecular https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas#individual-molecular https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas#individual-molecular https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas#individual-molecular https://w