Original Article Delayed SARS-COV-2 clearance in infected persons in Ghana Mildred A. Adusei-Poku1, James O. Aboagye2, Joseph H. K. Bonney2, John K. Odoom2, Evangeline Obodai2, Helena Lamptey3, Ivy A. Asante2, Seth Agyemang4, Joseph Oliver-Commey5, George B. Kyei2,6, William K. Ampofo2 and Evelyn Y. Bonney2 Ghana Med J 2023; 57(2): 97-101 doi: http://dx.doi.org/10.4314/gmj.v57i2.3 1 Department of Medical Microbiology, University of Ghana Medical School. College of Health Sciences, Uni- versity of Ghana, Accra, Ghana. 2Virology Department. Noguchi Memorial Institute for Medical Research, College of Health Sciences, Univer- sity of Ghana. Off Akilagpa Sawyerr Road, Legon, Accra, Ghana. 3Immunology Department. Noguchi Memorial Institute for Medical Research, College of Health Sciences, Uni- versity of Ghana. Off Akilagpa Sawyerr Road, Legon, Accra, Ghana. 4Korle Bu Teaching Hospital, Korle Bu, Accra, Ghana 5LEKMA Hospital, Agbleza Manet Road, Teshie, Accra, Ghana 6Department of Medicine, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO, USA. Corresponding author: Evelyn Y. Bonney E-mail: ebonney@noguchi.ug.edu.gh Conflict of interest: None declared SUMMARY Objective: This study aimed to determine the duration of SARS-CoV-2 clearance in persons in Ghana. The research question was whether the duration of virus clearance in Ghana matched the 14 days recommended by the World Health Organization (WHO); this had direct implications for transmission, which was key in managing the COVID-19 pan- demic. Design: This was a retrospective analytical study. Setting: All facilities that submitted clinical specimens to Noguchi Memorial Institute for Medical Research (NMIMR) for SARS-CoV-2 diagnosis between March to June 2020 were included in the study. Interventions: Samples from 480 persons who tested positive for SARS-CoV-2 by RT-PCR from March to June 2020 at NMIMR and submitted at least two follow-up samples were retrospectively analysed. Individuals with two consec- utive negative RT-PCR retesting results were considered to have cleared SARS-CoV-2. Results: The median time from the initial positive test to virus clearance was 20 days (IQR: 5-56 days). This was six days longer than the WHO-recommended 14 days, after which infected persons could be de-isolated. Sputum and nasopharyngeal swabs proved more sensitive for detecting viral RNA as the infection progressed. At a significance level of 0.05, age and sex did not seem to influence the time to SARS-CoV-2 clearance. Conclusions: The median time to SARS-CoV-2 clearance in this study was 20 days, suggesting that SARS-CoV-2 infected persons in Ghana take longer to clear the virus. This finding calls for further investigations into whether patients who remain PCR positive continue to be infectious and inform isolation practices in Ghana. Keywords: Virus clearance, SARS-CoV-2, RT-PCR, isolation, sputum Funding: The study was supported by the Ministry of Health/ Ghana Health Service through the provision of labora- tory supplies, the US Naval Medical Research Unit #3, the World Health Organization, the Jack Ma Foundation and the Virology Department of Noguchi Memorial Institute for Medical Research, University of Ghana. Research projects within Noguchi Memorial Institute for Medical Research contributed reagents and laboratory consumables. However, the authors alone are responsible for the contents of this manuscript. INTRODUCTION Coronavirus belongs to the family Coronaviridae, sub- pneumonia.2 Coronaviruses are commonly found in ani- family Coronavirinae.1 This family of viruses may cause mals, with very few species such as a severe acute respir- symptoms such as myalgia, fever, breathing difficulty, atory virus (SARS-CoV) and the Middle East respiratory and clinical syndromes such as the common cold and syndrome-related coronavirus (MERS-CoV) causing dis- ease in humans.3 97 www.ghanamedj.org Volume 56 Number 2 June 2022 Copyright © The Author(s). This is an Open Access article under the CC BY license. Original Article The World Health Organization (WHO) named the dis- test without an exit RT-PCR test.14 Per this recommenda- ease, caused by the novel coronavirus that affected the tion, symptomatic patients could also be discharged 14 lower respiratory tract of patients with pneumonia in Wu- days after the first positive test plus at least three days han, China, in December 2019, COVID-19.4 Severe without symptoms.14 acute respiratory syndrome coronavirus 2 (SARS-CoV- 2), the causative agent for COVID-19 is now responsible Although Ghana revised its discharge from isolation pol- for over 220 million infections and more than 4.7 million icy based on the updated WHO recommendations,14 it is deaths worldwide as at September 20, 2021.5, 6 In Ghana worth noting that the data on which the new WHO rec- there are over 125,000 confirmed cases of COVID-19 in- ommendation was based came mainly from Asia, Europe fections with 1,121 deaths.7 Most COVID-19-infected and the USA, with none from Africa.12, 13 Since SARS- people are either asymptomatic or present with mild to CoV-2 infection in Africa seems to have a different tra- moderate respiratory illness and recover without special jectory in most patients, with fewer deaths, it is important treatment. However, older people and those with under- to determine the duration for viral clearance in African lying medical problems such as cardiovascular disease, patients to help guide policies on the continent. Here, we diabetes, chronic respiratory disease, hypertension and present the results of virus clearance data for 480 people cancer are more likely to develop serious illnesses.8 At tested in Ghana during the first WHO criteria for de-iso- the time of this data, there were no specific vaccines or lation who were infected with SARS-CoV-2 and had at treatments for COVID-19. However, many ongoing clin- least two more RT-PCR tests after the initial positive test ical trials were evaluating potential treatments.9 results. In January 2020, the WHO published its maiden guide- METHODS lines for the clinical management of COVID-19, which Sample collection and laboratory testing included recommendations on when patients were no The Virology Department of the University of Ghana’s longer considered infectious and could be released from Noguchi Memorial Institute for Medical Research isolation.10 Here, patients were required to have been (NMIMR) was the major testing centre for SARS-CoV- clinically recovered and have two negative RT-PCR test 2 in Ghana. The samples reported here were submitted results taken sequentially in a 24-hour window. This rec- between March 12 and June 14, 2020, as part of Ghana’s ommendation was based largely on experience and pre- emergency response to the SARS-CoV-2 pan- vious knowledge of SARS and MERS.10 demic. Samples were accompanied by the Ghana Health Service case forms from which available demographic Ghana adopted and implemented the WHO’s initial rec- information was extracted. Sputum or oropharyngeal or ommendation where patients were required to have two nasopharyngeal swabs were collected from returning consecutive negative RT-PCR tests within a 24-hour travellers, patients suspected of COVID-19, or close con- window before they could be discharged from isolation.10 tacts of confirmed cases. The samples were submitted to Later, some reports indicated that patients with mild NMIMR and taken through ribonucleic acid (RNA) ex- COVID-19 infection shed the virus for approximately 7- traction and real-time reverse transcription polymerase 12 days; this duration may be longer in severe cases chain reaction (RT-PCR). The nucleocapsid (N), the though significant variations have been reported in both open reading frame 1ab (ORF 1ab) and/or the envelope instances.11 A study by Wölfel et al. indicated that the (E) genes of SARS-CoV-2 were amplified using gene- virus could be isolated in culture from patient specimens specific primers as designed by the manufacturer (Da An (swabs or sputum) taken during the first week of symp- Gene, MiRXES, and TIBMOLBIOL). The RT-PCR re- toms but could not be isolated from specimens taken after sults were interpreted as instructed by the kit manufactur- the eighth day following symptoms though they were ers. Cycle threshold (Ct) values below 40 were consid- RT-PCR positive.12 Therefore, the virus might not be in- ered positive for these kits. Internal controls were in- fectious after the eighth day following symptoms onset. cluded as quality control for sample collection, RNA ex- He et al. corroborated this finding in their research, indi- traction and the PCR processes. In the database, 480 peo- cating that viral shedding may begin 5 to 6 days before ple with at least two test results after their initial positive the appearance of the first symptoms, and viral loads sig- test were analysed for this study. Out of these, those with nificantly decline after the onset of symptoms.13 These two consecutive Negative retest results (419) were ana- findings thus suggest that a positive RT-PCR result does lysed for their various duration to virus clearance. Pa- not necessarily equate to infectiousness. Based on these tients who tested positive were quarantined and required and the fact that many countries, including Ghana, had to submit subsequent samples for retesting after 14 days logistical challenges, the WHO revised the criteria for re- and at 7-day intervals (Days 14, 21, 28, etc.) until a neg- leasing patients from isolation such that people in isola- ative test for SARS-CoV-2 was obtained. tion could be de-isolated 14 days after the initial positive 98 www.ghanamedj.org Volume 56 Number 2 June 2022 Copyright © The Author(s). This is an Open Access article under the CC BY license. Original Article By the World Health Organization’s protocol at the time RESULTS of this study, persons in quarantine needed two consecu- Demographics and testing Outcomes tive PCR negative results within 24 hours to be de-iso- Ghana reported its first COVID-19 case on March 12 lated. Clearance of SARS-CoV-2 in infected individuals 2020. As of June 14, 2020, our laboratory had confirmed was defined as having two consecutive negative PCR re- 7,025 COVID-19 cases with follow-up samples from sults after the initial positive result. These two consecu- 1,073 patients. Four hundred and eighty (45%) out of the tive Negative results were obtained at varying patient 1,073 had submitted at least two follow-up samples after sampling times. testing positive. Statistical analysis Of the 480 individuals, 419 had two consecutive negative Categorical variables were described as frequency and results (Table 1), while 61 still tested positive. Of the 419 percentage, while continuous variables were expressed as patients, 54% (228) were 40 years or younger, and 55.6% the median and interquartile range (IQR). Statistical anal- were male (Table 1). Six patients out of 419 (1.4%) tested yses were performed with SPSS version 22. The signifi- negative after submitting a new sample within a week (7 cance level was set at 0.05. days), while 25% became RT-PCR negative two weeks after their first test (8-14 days). However, over 70% (308/419) tested negative after 14 days (Table 1). The median days to test negative for the patients in this study was 20 days (IQR, 5 – 56). Nearly 90% of the patients who became RT-PCR negative submitted only two sam- ples following their first test. Table 1 Characteristics of SARS-CoV-2 infected patients with two consecutive negative results Number of Testing negative Median days OR (CI) p Value Variable patients n(%) within 14 days (%) (IQR) Gender Female 186 (44.4) 51 (27.4) 20 (5 - 56) 1.1 (0.7 - 1.7) 0.7007 Male 233 (55.6) 60 (25.8) 19 (6 - 50) 1 Age (years) < 18 21 (5.0) 4 (19.0) 19 (11 - 47) 1 - 18 - 40 207 (49.4) 53 (25.6) 20 (6 - 56) 1.5 (0.5 - 4.5) 0.5107 > 40 118 (28.2) 33 (28.0) 20 (5 - 48) 1.7 (0.5 - 5.3) 0.3979 Missing 73 (17.4) 21 (28.8) 19 (7 - 44) 1.7 (0.5 - 5.7) 0.3781 Symptoms Asymptomatic 367 (87.6) 103 (28.1) 19 (5 - 47) 2.1 (1.0 - 4.7) 0.0572 Symptomatic 52 (12.4) 8 (15.4) 21 (6 - 56) 1 - Number of follow-ups 2 samples 378 (90.2) 109 (28.8) 19 (5 - 48) 5.7 (1.3 - 24.2) 0.0191 3 samples 30 (7.2) 2 (6.7) 27 (13 - 50) 1 - 4 samples 11 (2.6) 0 (0.0) 31 (21 - 56) - - Days to testing negative ≤ 7 days 6 (1.4) - - - - 8 - 14 days 105 (25.1) - - - - 15 - 21 days 139 (33.2) - - - - > 21 days 169 (40.3) - - - - Total 419 (100.0) 111 (26.5) 20 (5 - 56) - - Factors associated with infected patients testing neg- Table 2 Repeat test results from oropharyngeal, naso- ative pharyngeal and sputum specimens collected after 21 To determine the risk associated with days to recovery, days from 17 patients who had previously tested posi- we assessed gender, age, presentation with symptoms, tive for SARS-CoV-2 by RT-PCR and the type of sample as a factor. There was no signifi- Patient Specimen type and Results cant association between gender and age in testing nega- Oropharyngeal Nasopharyngeal Sputum Patient 1 Negative Negative Positive tive within 14 days after being confirmed with SARS- Patient 2 Negative Negative Positive CoV-2 infection (Table 1). Though not statistically sig- Patient 3 Negative Negative Negative nificant, symptomatic patients required longer to test Patient 4 Negative Negative Positive Negative than asymptomatic patients (Table1). To inves- Patient 5 Negative Negative Negative tigate what type of sample contributed to detecting the Patient 6 Negative Negative Negative Patient 7 Negative Negative Positive virus, oropharyngeal, nasopharyngeal, and sputum sam- Patient 8 Negative Negative Negative ples were collected from seventeen patients who still Patient 9 Negative Negative Negative tested positive 21 days after the initial positive test. As Patient 10 Negative Negative Negative shown in Table 2, eight patients were still SARS-CoV-2 Patient 11 Negative Negative Negative positive. Patient 12 Negative Positive Positive 99 www.ghanamedj.org Volume 56 Number 2 June 2022 Copyright © The Author(s). This is an Open Access article under the CC BY license. Original Article Patient 13 Negative Positive Positive negative results and reduced the stigma of prolonged iso- Patient 14 Negative Negative Negative lation.17-19 However, moving infected persons from isola- Patient 15 Negative Negative Positive tion without confirming virus clearance could have con- Patient 16 Negative Positive Negative Patient 17 Negative Negative Negative tributed to the community spread of SARS-CoV-2 in Ghana. Of these, 7 were sputum samples, and three were naso- pharyngeal samples. All the oropharyngeal samples This study was limited by the inability to accurately esti- tested negative. Two patients had their sputum and naso- mate the day of infection. Thus, the initial positive test pharyngeal samples testing positive for SARS-CoV-2 af- date was used as a reference for analyses. This may affect ter 21 days (Table 2). the estimated duration for virus clearance. The study is further limited by our inability to culture the virus from Three specimen types, oropharyngeal, nasopharyngeal patients to determine infectivity before the RT-PCR neg- and sputum, were collected from the patients 21 days af- ative test; thus, results are based on viral RNA detection ter testing positive for SARS-CoV-2. Nasopharyngeal by RT-PCR, which cannot distinguish between infectious and sputum samples tested positive, while all oropha- and non-infectious viruses. Also, based on the findings ryngeal samples tested Negative. from the 17 patients with the three sample types, it would have been better to use only sputum samples to study vi- DISCUSSION ral persistence or clearance in the 480 patients since using This study estimated the time to SARS-CoV-2 clearance other sample types could mask the findings. However, after the first RT-PCR positive result. Here, data from the data is one of the few African studies documenting 480 patients confirmed as SARS CoV-2 positive and sub- the time to SARS-CoV-2 RNA clearance in infected pa- mitted at least two follow-up samples were assessed. A tients who submitted follow-up samples after their initial total of 419, representing 87%, had two consecutive neg- positive test. ative test results. Sixty-one (13%) patients had not tested negative at the time of this study. CONCLUSION This study showed that the median duration of SARS- Literature has demonstrated that the viral shedding of in- CoV-2 clearance from infected persons in Ghana, as fectious pathogens is significantly associated with the in- measured by two consecutive negative RT-PCR results, fectivity and transmissibility of the pathogens. Here, the was 20 days (IQR: 5-56 days), irrespective of age and median days to test PCR negative was 20 days (IQR 5 - sex. This duration is six days longer than the 14 days ac- 56) (Table 1). While some individuals cleared the virus cepted by WHO as the time for virus clearance, and upon in five days, it took others as long as 56 days to achieve which the updated guidelines for isolation were based. virus clearance. Thus, it may be important to consider in- The findings suggest that SARS-CoV-2-infected persons dividual differences in managing SARS-CoV-2 infec- in Ghana take longer to clear the virus. The finding calls tions. The finding of a median of 20 days for virus clear- for further investigations into whether patients who re- ance is at variance with studies that guided the updated main PCR positive continue to be infectious, for how WHO guidelines for isolation of infected SARS-CoV-2 long and at what virus levels to inform isolation practices patients,12, 13 but concordant with that of Zhou et al., 2020 in Ghana. Sputum and nasopharyngeal swabs proved who reported a similar median range in Wuhan patients.15 more sensitive than oropharyngeal swabs to detect viral Several factors including the age of the patients and type RNA as the infection progressed. of respiratory sample collected could have contributed to the duration for testing positive. Though carried out as an REFERENCES exploratory study, it was observed that sputum and naso- 1. 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