key: cord-0683612-wujpw8dw
authors: Meiring, Susan; Tempia, Stefano; Bhiman, Jinal N; Buys, Amelia; Kleynhans, Jackie; Makhasi, Mvuyo; McMorrow, Meredith; Moyes, Jocelyn; Quan, Vanessa; Walaza, Sibongile; du Plessis, Mignon; Wolter, Nicole; von Gottberg, Anne; Cohen, Cheryl
title: Prolonged shedding of SARS-CoV-2 at high viral loads amongst hospitalised immunocompromised persons living with HIV, South Africa
date: 2022-02-02
journal: Clin Infect Dis
DOI: 10.1093/cid/ciac077
sha: c0974d41eddf1182c3f641d69d47aa22689a8fc7
doc_id: 683612
cord_uid: wujpw8dw

BACKGROUND: We assessed SARS-CoV-2 RNA shedding duration and magnitude amongst persons living with HIV (PLHIV). METHODS: From May through December 2020, we conducted a prospective cohort study at 20 hospitals in South Africa. Adults hospitalised with symptomatic COVID-19 were enrolled and followed every two days with nasopharyngeal/oropharyngeal (NP/OP) swabs until documentation of cessation of SARS-CoV-2 shedding (two consecutive negative NP/OP swabs). Real-time reverse transcription-polymerase chain reaction testing for SARS-CoV-2 was performed and Cycle-threshold (C(t)) values <30 were considered a proxy for high SARS-CoV-2 viral load. Factors associated with prolonged shedding were assessed using accelerated time-failure Weibull regression models. RESULTS: Of 2,175 COVID-19 patients screened, 300 were enrolled and 257 individuals (155 HIV-uninfected and 102 PLHIV) had >1 swabbing visit (median 5 visits (range2-21)). Median time to cessation of shedding was 13 days (inter-quartile range (IQR)6-25) and did not differ significantly by HIV-infection. DISCUSSION: Amongst a subset of 94 patients (41 PLHIV and 53 HIV-uninfected) with initial respiratory sample C(t)-value <30, median time of shedding at high SARS-CoV-2 viral load was 8 days (IQR4-17). This was significantly longer in PLHIV with CD4 count<200cells/µl, compared to HIV-uninfected persons (median 27 days (IQR8-43) versus 7 days (IQR 4-13); aHR 0.14, 95%CI 0.07-0.28, p<0.001), with similar results in unsuppressed-HIV versus HIV-uninfected persons. CONCLUSION: Although SARS-CoV-2 shedding duration did not differ significantly by HIV-infection, amongst a subset with high initial SARS-CoV-2 viral loads, immunocompromised PLHIV shed SARS-CoV-2 at high viral loads for longer than HIV-uninfected persons. Better HIV control may potentially decrease transmission time of SARS-CoV-2.

Immunocompromised persons are thought to shed SARS-CoV-2 for a longer duration, increasing time for viral transmission and potentially driving within host viral evolution.[1-3] South Africa has 7,500,000 persons living with HIV (PLHIV) and there are no systematically collected data on duration of SARS-CoV-2 shedding in PLHIV. We hypothesised that PLHIV may shed SARS-CoV-2 for a longer period of time and at a higher viral load than HIV-uninfected individuals.

SARS-CoV-2 shedding from the upper respiratory tract extends for a mean of 17 days (15.5-18.6) . [4] Unlike SARS-CoV-1, viral shedding of SARS-CoV-2 from the upper respiratory tract peaks on or before symptom onset, allowing some viral transmission to occur before symptom onset in infected individuals. [4] [5] [6] [7] [8] Quantifying SARS-CoV-2 viral load (Log 10 RNA copies/mL) is calculated by converting qualitative rRT-PCR cycle threshold values using calibration curves based on quantified E-gene in vitro RNA transcripts. [9] However, studies on SARS-CoV-2 infectiousness indicate that successful virus isolation is most likely from specimens with a real-time RT-PCR cycle threshold (C t ) value<34(~7 Log 10 copies/mL), however not all these specimens may be positive for viable virus. [9, 10] Using this threshold, infectiousness declines significantly 8 days after becoming symptomatic, even though SARS-CoV-2 viral RNA persistence has been shown to occur for months in some individuals. [9, 11, 12] Various factors have been associated with increased shedding duration, including increased age, male sex, severity of illness and use of corticosteroids. [13, 14] There are several published case reports indicating prolonged SARS-CoV-2 transmission in immunocompromised persons, mostly with cancers or autoimmune conditions, and more recently in an HIV-infected immunocompromised person. [15] [16] [17] [18] We aimed to evaluate the overall duration of SARS-CoV-2 shedding in upper respiratory tract specimens and duration of high viral load shedding in a cohort of PLHIV and HIV-uninfected persons A c c e p t e d M a n u s c r i p t hospitalised with COVID-19 in South Africa. We also estimated SARS-CoV-2 shedding duration in stool and blood specimens, and described serologic responses to SARS-CoV-2 infection.

We conducted a prospective cohort study from May 1 through December 31, 2020 (spanning the first wave and beginning of the second wave of the COVID-19 pandemic in South Africa). Persons hospitalised for symptomatic COVID-19 at one of 20 hospitals situated in 8 of the 9 South African provinces were invited to participate in the study if they met the following inclusion criteria: aged 18 years and older, laboratory-confirmed diagnosis of SARS-CoV-2 in the previous 5 days, resided within a 50km radius of the hospital, and had laboratory-confirmation of their HIV status.

Using standardised case report forms, demographic and clinical details were collected at enrolment, daily whilst in hospital, and at discharge from hospital/cessation of shedding/death of the participant. Included variables are detailed in supplementary text.

At enrolment and every second day thereafter, until cessation of shedding or death, a combined nasopharyngeal/oropharyngeal (NP/OP) swab was collected by trained nursing staff, using two flocked nylon plastic shaft swabs (one for the nasopharynx and one for the oropharynx) which were then inserted together into Universal Transport Medium and transported on ice to the laboratory.

Sensitivity of the combined NP/OP swab has been shown to be 97% for detection of SARS-CoV-2 in the upper airways. [19, 20] A rectal swab or stool was collected from patients at the same time intervals and transported to the laboratory in sealed containers. Results available in supplementary text. Patients were followed up at home if they were still SARS-CoV-2 positive on NP/OP swab on discharge from hospital. M a n u s c r i p t Whole blood and serum specimens were taken on enrolment and at days 7, 14 and 21 post symptom-onset for SARS-CoV-2 testing and serology. All specimens were transported to the National Institute for Communicable Diseases (NICD) in Johannesburg for processing and testing.

Real-time reverse transcription polymerase chain reaction (rRT-PCR) for the qualitative detection of nucleic acid from SARS-CoV-2 was performed on NP/OP, stool/rectal swabs and blood specimens using the Allplex™ nCoV 2019 kit (Seegene, Seoul, South Korea). Specimens were considered positive for SARS-CoV-2 nucleic acids if the C t was <40 for ≥1 of 3 gene targets. A nucleocapsid gene (N gene) C t -value <30 on NP/OP specimens was used as a proxy for a high viral load based on published data showing a high correlation between low C t -values (using various gene targets), high viral load and increased odds of shedding cultivable virus. [10, 12, 21, 22] Antibody detection against trimeric ectodomain HexaPro spike protein was performed on serum specimens as described previously. [23, 24] Absorbance at 450 nm was measured and specimens with Optical Density (OD)>0.4 were considered positive for anti-spike protein antibodies.

SARS-CoV-2 sequencing was performed on the first NP/OP specimens on enrolment from ten randomly selected participants of 29, who demonstrated high viral load shedding for >14 days. Clade and lineage assignments were made using the online Nextclade (https://clades.nextstrain.org/) and Pangolin (https://pangolin.cog-uk.io/) applications, which also enable identification of known variants of concern as well as novel mutations. See supplementary methods for more details.

Shedding was defined as presence of SARS-CoV-2 nucleic acid in a specimen as detected by a positive SARS-CoV-2 rRT-PCR result. Participants were deemed to have stopped shedding SARS-CoV-2 from the respiratory tract once two consecutive NP/OP swabs, taken at least two days apart, tested negative for all 3 gene targets on rRT-PCR. Time to cessation of shedding was taken from date of symptom onset to the date of the last rRT-PCR SARS-CoV-2 positive NP/OP swab prior to the two A c c e p t e d M a n u s c r i p t consecutive negative swabs. Participants were deemed to be shedding SARS-CoV-2 virus at high viral loads if their NP/OP swab N gene C t -value was <30. Persistence of high viral load was measured in the subgroup of individuals whose first study NP/OP swab had an N gene C t -value <30 and was measured from date of symptom onset to the last date where NP/OP swab N gene C t -value was <30.

PLHIV were deemed to be significantly immunocompromised if their CD4 T-lymphocyte count was <200 cells/µl, and not HIV virally suppressed if their HIV viral load measured >400 copies/ml in the 3 months before hospital admission. [25, 26] Severity of COVID-19 was categorised using respiratory rate at time of admission according to the WHO clinical classification of COVID-19 and a quick Sequential (Sepsis-related) Organ Failure Assessment (qSOFA) score. [27, 28] 

Data were captured on a real-time data capture (REDCAP) database and transferred onto a password Patients with missing data were dropped from the models. (Table 1) . Of PLHIV, 84% were currently receiving antiretroviral therapy, median CD4 T-cell count was 221 cells/µl (IQR48-443) (with 53% having CD4 T-cell count>200cells/µl), and 59% were HIV virally suppressed (HIV viral load<400 copies/ml).

Patients were admitted for a median of 9 days (IQR6-13) in hospital. Eight participants had unknown outcome. Overall, 10% (24/250) of participants died, 8% (19) during their hospital admission and a A c c e p t e d M a n u s c r i p t further 2% (5) within 2months post-discharge. This did not differ significantly by HIV-status. Of 214 participants with known parameters to assess disease severity, most (64%, 137/214) had moderate disease severity, with 18% (39/214) having mild and 18% (38/214) having severe COVID-19. HIVuninfected persons and PLHIV had similar clinical presentation. The most common presenting symptoms were cough (79%, 192/243), dyspnoea (66%, 160/243), chest pain (63%, 153/243), malaise (52%, 127/243), and fever (45%, 111/243). Oxygen therapy was required for 53% (125/235), non-invasive ventilation for 7% (15/227), and invasive ventilation for 5% (11/230). Ten percent (23/235) were admitted to an intensive care unit. Glucocorticoids were given to 34% (84/250) of individuals whilst in hospital (Table 1) .

Of the 257 participants with at least 2 swabbing visits: 186 were followed up until cessation of SARS- Table 2) .

Of a subset of 94 participants with N gene C t -value<30 at enrolment (41 PLHIV and 53 HIVuninfected persons), the median duration of SARS-CoV-2 shedding at N gene C t -value<30 from symptom onset was 8 days (IQR4-17). On multivariable analysis, when adjusting for age and glucocorticoid use, PLHIV with a CD4 cell count<200 cells/µl shed at high SARS-CoV-2 viral loads for A c c e p t e d M a n u s c r i p t longer (median 27days, IQR8-43, aHR 0.14, 95%CI 0.07-0.28, p<0.001), whereas PLHIV with CD4 count>200 cells/μl shed at high SARS-CoV-2 viral loads for a similar time period (median 7days, IQR4-10, aHR 1.14, 95%CI 0.56-2.31, p=0.71), compared to HIV-uninfected persons (median 7days, . Similarly, PLHIV with HIV viral loads>400 copies/ml were more likely to shed SARS-CoV-2 at high viral loads for longer (26days, IQR10-41, unadjusted HR 0.34, 95%CI 0.18-0.64, p<0.001) than PLHIV with HIV viral suppression (6days, IQR4-8, unadjusted HR 1.77, 95%CI 0.88-3.56, p=0.107) and those who were HIV-uninfected (7days, IQR4-13) ( Table 3 ; Figure 3 ; Supplementary Table 3) .

The proportion of participants with positive anti-spike protein antibodies increased from 80% 

Eight different SARS-CoV-2 phylogenetic lineages were detected from enrolment NP/OP swabs from the ten randomly selected participants demonstrating high SARS-CoV-2 viral load shedding for >2 weeks. These included two participants with B.1, two B.1.140, and one each of B.1.1.57, B.1.1.448 lineages accounting for 59% of all sequences (n=1705). [36] In this study the multiple SARS-CoV-2 phylogenetic lineages detected reflect the lineage diversity during the earlier waves in South Africa, indicating that persistent shedding was not dominated by a specific lineage or variant. All lineages detected in these individuals had been identified in national genomic surveillance efforts, with 7 of the 8 being lineages first detected in South Africa. [36] Going forward we aim to investigate within host viral evolution by sequencing sequential specimens of the participants with prolonged high viral load shedding, many of whom were immunocompromised PLHIV.

Obese patients in our study showed a 45% increase in median time of SARS-CoV-2 shedding compared to their non-obese counterparts. However, obesity was not associated with prolonged duration of high viral load SARS-CoV-2 shedding. Recent studies indicate that obesity may be associated with prolonged SARS-CoV-2 shedding, in a similar manner to prolonged shedding seen in obese adults with influenza A virus. [37, 38] See supplementary material for further discussion.

Most participants developed anti-spike protein antibodies by day 14, with fewer immunocompromised PLHIV developing antibodies compared to HIV-uninfected persons. Overall A c c e p t e d M a n u s c r i p t lower median anti-spike protein antibody titres were detected in PLHIV. Our study showed 14 individuals (4 HIV-uninfected persons and 10 PLHIV -8 of whom had low CD4 count) who did not develop antibodies within the initial 21 days' follow-up period, half of which showed prolonged shedding (including 5 PLHIV with CD4 count<200 cells/µl). Other studies have found similar findings in immunocompromised individuals with prolonged viral shedding having negative seroconversion. [15, 16] The cohort of participants from 20 public hospitals is likely representative of the hospitalised COVID-19 South African adult population, however the analysis did not account for within-hospital correlations due to case mix, treatment strategies or inter-hospital differences in quality of care. [39] Thrice weekly collection of NP/OP swabs to detect ongoing SARS-CoV-2 viral shedding may result in underestimated viral shedding due to interval censoring of the data. A major limitation of our study is the use of qualitative C t -values as a proxy for viral load. Although all specimens were tested in the same laboratory, using standardised technique and assays, quantitative RT-PCR was not performed using calibration curves to determine viral load. In addition, we were unable to perform viral culture to confirm shedding of infectious virus at proxy C t -values<30. Our findings may not be generalizable to outpatients with mild COVID-19, as hospitalised COVID-19 patients may represent more severe disease and therefore shed for a longer duration. Non-enrolment of participants due to unavailability of HIV test data (37% (702/1875) of non-enrolments) may have introduced bias due to potential differences in persons willing to undergo HIV testing versus those who are unwilling.

Previous South African studies have shown similar unknown HIV results, as opt-out policies on HIV testing are not routinely followed. [39] Although sensitivity analyses in our study did not show much effect when excluding patients who died (data not shown), it should be noted that in-hospital mortality in our study was low (8%) compared to 23% in Jassat, et al., as patients too ill to consent would have been excluded. [39] Follow-up of participants' post-hospital discharge was challenging due to stigma of COVID-19 in the communities, safety concerns and travel limitations. Fifty-eight A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 

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