key: cord-1053128-r4rv5v9h
authors: du Bruyn, E.; Stek, C.; Daroowala, R.; Said-Hartley, Q.; Hsiao, M.; Goliath, R. T.; Abrahams, F.; Jackson, A.; Wasserman, S.; Allwood, B.; Davis, A. G.; Lai, R.; Coussens, A. K.; Wilkinson, K. A.; De Vries, J.; Tiffin, N.; Cerrone, M.; Ntusi, N.; Riou, C.; Wilkinson, R. J.; investigators, HIATUS
title: Communicable and non-communicable co-morbidities and the presentation of COVID-19 in an African setting of high HIV-1 and tuberculosis prevalence
date: 2021-05-11
journal: nan
DOI: 10.1101/2021.05.11.21256479
sha: 9d128ec6caa869d8965b05661f75cb430a218959
doc_id: 1053128
cord_uid: r4rv5v9h

Objectives To describe the presentation and outcome of SARS-CoV2 infection in an African setting of high non-communicable co-morbidity and also HIV-1 and tuberculosis prevalence. Design Case control analysis with cases stratified by HIV-1 and tuberculosis status. Setting A single-centre observational case-control study of adults admitted to a South African hospital with proven SARS-CoV-2 infection or alternative diagnosis. Participants 104 adults with RT-PCR-proven SARS-CoV2 infection of which 55 (52.9%) were male and 31 (29.8%) HIV-1 co-infected. 40 adults (35.7% male, 30.9% HIV-1 co-infected) admitted during the same period with no RT-PCR or serological evidence of SARS-CoV2 infection and assigned alternative diagnoses. Additional in vitro data from prior studies of 72 healthy controls and 118 HIV-1 uninfected and infected persons participants enrolled to a prior study with either immune evidence of tuberculosis sensitization but no symptoms or microbiologically confirmed pulmonary tuberculosis. Results Two or more co-morbidities were present in 57.7% of 104 RT-PCR proven COVID-19 presentations, the commonest being hypertension (48%), type 2 diabetes mellitus (39%), obesity (31%) but also HIV-1 (30%) and active tuberculosis (14%). Amongst patients dually infected by tuberculosis and SARS-CoV-2, clinical features could be dominated by either SARS-CoV-2 or tuberculosis: lymphopenia was exacerbated, and some markers of inflammation (D-dimer and ferritin) elevated in singly SARS-CoV-2 infected patients were even further elevated (p less than 0.05). HIV-1 and SARS-CoV2 co-infection resulted in lower absolute number and proportion of CD4 lymphocytes, with those in the lowest peripheral CD4 percentage strata exhibiting absent or lower antibody responses against SARS-CoV2. Death occurred in 30/104 (29%) of all COVID-19 patients and in 6/15 (40%) of patients with coincident SARS-CoV-2 and tuberculosis. Conclusions In this South African setting, HIV-1 and tuberculosis are common co-morbidities in patients presenting with COVID-19. In environments in which tuberculosis is common, SARS-CoV-2 and tuberculosis may co-exist with clinical presentation being typical of either disease. Clinical suspicion of exacerbation of co-existent tuberculosis accompanying SARS-CoV-2 should be high.

It has been quite widely thought that Africa has been spared the worst effects of the COVID-19

pandemic. There are very few reported case series and no case-control studies comparing COVID-19 patients admitted to hospital to those admitted for other reasons. However several studies have indicated both HIV-1 and tuberculosis co-infection that are endemic in Africa constitute risk factors for poor outcome. In addition Africa is subject to demographic transition and the prevalence of non-communicable co-morbidities such as type 2 diabetes, hypertension and cardiovascular disease is rising rapidly. No study from Africa has described the clinical impact on the presentation of COVID-19 infection.

Two or more co-morbidities were present in over half COVID-19 presentations, including HIV-1 (30%) and active tuberculosis (14%). Patients dually infected by tuberculosis and SARS-CoV-2, presented as either SARS-CoV-2 or tuberculosis. HIV-1 and SARS-CoV2 co-infection resulted in lower absolute number and proportion of CD4 lymphocytes, and those with low CD4 counts had absent or lower antibody responses against SARS-CoV2. Death occurred 29% of all COVID-19 patients and in 40% of patients with coincident SARS-CoV-2 and tuberculosis.

Thus in environments in which tuberculosis is common, SARS-CoV-2 and tuberculosis may coexist with clinical presentation being typical of either disease and clinical suspicion of exacerbation of co-existent tuberculosis accompanying SARS-CoV-2 should be high.

In 2020, COVID-19 caused by SARS-CoV-2 infection replaced HIV-1 infection and tuberculosis as the leading global infectious cause of death: 3.19 million by 1st May 2021 1 . It is Africa that routinely witnesses the bulk of HIV-1 tuberculosis associated mortality, yet with respect to SARS-CoV-2 the continent has been described as a puzzle 2 . Fewer cases and deaths from COVID-19 than predicted have been notified. This has been attributed to limited molecular testing (serological data suggests infection has been more widespread 3 4 ), a much younger population (and thus fewer severe cases and deaths), and the possible influences of pre-existing immunity, genetic factors, and the early implementation of public health measures, especially restriction on already limited intracontinental travel.

In the Republic of South Africa, early importation of SARS-CoV-2 into its Western Cape province in March 2020 led, notwithstanding initial very stringent lockdown measures, to a substantial first wave peaking in July 2020 that exerted extraordinary demand on hospital services 5 6 . Over 1. 5 

Research Council estimates 155,859 excess deaths nationally since 3rd May 2020 (https://www.samrc.ac.za/reports/report-weekly-deaths-south-africa). Multiple mutations giving rise to a novel variant referred to as 501Y.V2 contributed to a worse second wave in this region 7 .

The Western Cape province of South Africa has high HIV-1 prevalence and tuberculosis incidence to which considerable and often co-incident non-communicable disease burden is added with obesity, type 2 diabetes mellitus, hypertension, and vascular co-morbidities being very common 8 9 . Robust provincial health information systems triangulate multiple data sources to enumerate health conditions, with assignment of certainty levels for each enumeration 10 .

These systems were deployed rapidly during the COVID-19 first wave to powerfully identify comorbidities associated with COVID-19 amongst (i) public sector patients, (ii) laboratorydiagnosed COVID-19 cases and (iii) hospitalized COVID-19 cases. Among 3,460,932 patients (16% HIV-1 infected), 22,308 were diagnosed with COVID-19, of whom 625 (28%) died. In addition to widely recognised risk factors (male sex, increasing age, diabetes, hypertension and chronic kidney disease) COVID-19 death was associated with HIV-1 (adjusted hazard ratio [aHR] 2.14), with similar risks irrespective of viral load and immunosuppression, and also current and previous tuberculosis (aHR 2.70 and 1.51 respectively 11 ).

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The copyright holder for this preprint this version posted May 11, 2021. ;  https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint 7 A number of clinical studies have examined interaction between HIV-1 and SARS-CoV-2: mostly cohorts lacking HIV-1-or SARS-CoV-2-uninfected control participants [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] . Definitive conclusions are thus difficult to reach but the presentation and outcome of SARS-CoV-2 in HIV-1 co-infected patients is reported as not differing greatly from that in HIV-1 uninfected persons [22] [23] [24] . A single report associated the use of tenofovir as disoproxil fumarate with better outcome than as alafenamide 18 . Two reports suggest worse outcomes associated with lower CD4 count 17 25 and a further report suggests prolonged SARS-CoV-2 shedding (determined by RT-PCR) and reduced anti-SARS-CoV-2 antibody response in those whose antiretroviral therapy (ART) has been interrupted 26 . Interestingly convalescence from COVID-19 appears associated with a transient increase in HIV-1 viral load despite ART 19 27 . We therefore instigated a series of health facility-based observational studies to investigate interaction and overlap between SARS-CoV-2, HIV-1 and M. tuberculosis infections (HIATUS).

The purpose was to describe the clinical presentation, radiographic appearances, and clinical laboratory features of patients hospitalized with proven or suspected SARS-CoV-2 infection in an African setting not only endemic for HIV-1 and tuberculosis but also subject to demographic transition such that other non-communicable co-morbidities are common 9 .

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This was a single-centre observational case-control study of adult (age > 18 years) patients admitted to Groote Schuur Hospital, Cape Town, South Africa with RT-PCR proven, or suspected, SARS-CoV-2 infection. Between 11 th June and 28 th August 2020, the study team assessed suspected or confirmed SARS-CoV-2 (by nasopharyngeal or oropharyngeal aspirate) participants and expedited written electronic consent for inclusion was provided. Where participants did not have capacity to provide informed consent, e.g. in the case of those admitted in the intensive care unit, informed consent was sought from family members. Informed consent was provided by all participants who regained their capability to provide informed consent after incapacity. Known HIV-1 status or willingness to undergo testing was an inclusion criterion.

During the initial surge of hospital admissions recruitment could not be sequential due to caseload. Emphasis was on known SARS-CoV-2 PCR positive inpatients in a variety of dependency settings ranging through person under investigation (PUI), low dependency known positive, high-flow nasal oxygen and intensive care settings. As caseload decreased emphasis on PUI recruitment increased such that appropriate inpatient controls without laboratory evidence of SARS-CoV-2 infection (other disease controls, ODC) could be recruited. Data were entered directly into an electronic REDCap data entry system, hosted by the University of Cape Town 34 .

The initial aim was to recruit equal numbers of HIV-1-co-infected and -uninfected participants.

Participants presenting as persons under investigation (PUI) for SARS-CoV-2 but persistently negative by RT-PCR (Seegene, Roche or Gene Xpert) for SARS-CoV-2 were subsequently serologically tested. In the absence of either RT-PCR or serological evidence of SARS-CoV-2 infection and in the presence of an alternative diagnosis such participants were designated controls. The WHO ordinal severity score was used to categorize participants on admission according to the following categories: . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 11, 2021. ; https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint 9 7. Hospitalized, on invasive mechanical ventilation with other organ support.

The study was conducted according to the declaration of Helsinki, conformed to South African Good Clinical Practice guidelines, and was approved by the University of Cape Town's Health Sciences Research Ethical Committee (HREC 207/2020).

For some analyses we included data from HIV-1 uninfected and infected persons with either immune evidence of tuberculosis sensitization but no symptoms (latent tuberculosis, LTBI) or microbiologically confirmed pulmonary tuberculosis who had been recruited to prior studies (HREC 050/2015) 35 36 . The details of these persons are presented in Supplemental Table 2 .

Serological testing was performed by Elecsys® (Roche, Basel, Switzerland) Anti-SARS-CoV-2 immunoassay which detects antibodies (including IgG) to SARS-CoV-2. The following routine clinical assays were performed in the laboratories of the National Health Laboratory Service (NHLS): full blood count and automated differential cell count, HIV-1 ELISA, Microbiology including tuberculosis Gene Xpert nucleic acid amplification testing, HIV-1 viral load, peripheral blood CD4 percentage and absolute count, SARS-CoV-2 diagnostic PCR (Seegene, Roche, Gene Xpert), blood electrolytes, C-reactive protein, D-dimer, ferritin and lactate dehydrogenase. The NHLS via its quality assurance division provides proficiency testing to its own and other laboratories schemes and is certified to ISO/IEC 17043:2010 requirements.

The assessment of the percentage of CD4 and CD8 T cells was performed on cryopreserved fixed whole blood. Briefly, samples were thawed, washed and permeabilized with a Transcription Factor perm/wash buffer (eBioscience, San Diego, CA, USA). Cells were then stained with an antibody cocktail containing among others anti-CD3, -CD4 and -CD8 antibodies.

Samples were acquired on a BD LSR-II and analyzed using FlowJo (v9.9.6, FlowJo LCC, Ashland, OR, USA). The percentage of CD4 and CD8 T cells were defined as a % of total lymphocytes identified based on their FSC and SSC characteristics.

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Chest radiographs were reported blind to clinical status by an experienced radiologist (QS-H). The BSTI coding system was employed to report chest radiographs as described 37 and the Brixia severity score was also calculated as described 38 . Posteroanterior chest radiographs were assessed for the total percentage of the lung fields unaffected by any visible pathology. Thus, in the COVID-19 group this score quantified the percentage of normal lung that was not visibly affected by known features of COVID-19 pneumonia on the radiograph. In those with tuberculosis, or in ODC with other respiratory infections this score similarly quantified the percentage of normal lung, not visibly affected by the relevant pathology on the radiograph.

Those with a normal chest radiograph would thus score 100%.

Analyses were conducted in Prism 8 (GraphPad Software, San Diego, CA). The normality of data was assessed by a Shapiro-Wilk test. Descriptive statistics were calculated and presented as percentage or median and IQR. Unpaired normally distributed variables were compared by the student's unpaired t test and non-parametric comparisons by the Kruskal-Wallis test.

Corresponding assessment of non-parametric correlation was by the Spearman method.

• At what stage in the research process were patients/public first involved in the research and how?

It was difficult to engage relevant communities in the design of the research as it was conceived during a period of stringent national lockdown during which unnecessary gatherings and travel were illegal. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 11, 2021. ; https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint As above • How were they involved in the recruitment to and conduct of the study?

Recruitment was shortly after admission to hospital with suspected SARS-CoV2 infection and accompanied by an electronic informed consent procedure with proxy consent (and subsequent reconsent for those incapacitated). For those who did not regain capacity or died the research ethical committee was consulted to determine if such data could be included on a case-by-case basis.

• Were they asked to assess the burden of the intervention and time required to participate in the research?

Yes, a consent procedure as outlined above was undertaken. The research was designed to minimally burden participant and the service during the time of an emergency.

• How were (or will) patients and the public be involved in choosing the methods and agreeing plans for dissemination of the study results to participants and wider relevant communities?

Results have already been shared via academic and lay fora. The lesson that HIV-1 and tuberculosis may co-exist in SARS-CoV2 patients has been widely disseminated and appreciated.

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The copyright holder for this preprint this version posted May 11, 2021. ; https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint Table 1 Figure 1 ), ODC were more likely to exhibit cardiovascular or other respiratory comorbidities (6.7% and 6.7% versus 42.9 and 26.2% , respectively). The majority of participants in both groups had two or more comorbidities (COVID-19 57.7% and ODC 74.4%). 46.2% COVID-19 patients were assessed as grade 5 or above on the WHO ordinal scale for COVID-19. There was greater use of adjunctive corticosteroid therapy (78.8% versus 26.1%, p < 0.0001) and a longer hospital stay (13 versus 7 days, p=0.0008) in COVID-19 patients.

The analysis was neither designed nor powered to determine risk factors for death amongst COVID-19 patients. 30 deaths occurred (29% of the total diagnosed SARS-CoV-2 RT-PCR positive) of which 23 (77%) were males, the median age was 55 years (IQR, 46-66), 25 (83%) were classified as WHO grade 5 or above, and 28 (93%) received corticosteroid therapy (Supplemental Table 2 ). 20% patients who died were HIV-1 co-infected, 20% had tuberculosis and 10% had the triple combination of SARS-CoV-2, tuberculosis and HIV-1. 6/15 (40%) of patients with coincident tuberculosis died of whom equal numbers were HIV-1 infected and uninfected (Supplemental Tables 2 and 3) .

Chest radiographs at enrolment were evaluated by three scoring systems: Brixia 38 , British Society for Thoracic Imaging (BSTI) 37 and the percentage of unaffected lung. There was a close . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2021. ; https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint 13 relationship between Brixia and BSTI radiographic severity scores (Supplemental Figure 2A) ; and an inverse relationship between both the BSTI and Brixia scores and the percentage of unaffected lung (supplemental figures 2B and C). The percentage of unaffected lung was lower in COVID-19 patients than ODC (Median 35 vs 70%, p < 0.0001, Supplemental Figure 2D ).

There was no significant effect of either HIV-1 or tuberculosis co-infection alone on the percentage unaffected lung in COVID-19 patients, but patients with triple infection tended to exhibit a higher percentage of unaffected lung (p = 0.007, Supplemental Figure 2E ). Table 3 and examples of radiographic appearances are provided in Figure 1A and B and Supplemental Figure Figure 1F ). These data therefore indicate that in persons SARS-CoV-2 RT-PCR positive, radiographic appearances were most often typical of COVID-19 with the finding of tuberculosis being incidental, but that in a smaller number of cases appearances were predominantly of tuberculosis, with SARS-CoV-2 being co-prevalent. HIV-1 is known to be associated with non-specific radiographic appearances of tuberculosis 39 and appears in triple combination to associate with atypical appearances also of COVID-19 that were ostensibly less severe. table 4 ). The total white cell count was raised in both ODC and COVID-19 patients by comparison with HC with no differences between the patient groups, and the count increased with increasing COVID-19

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The copyright holder for this preprint this version posted May 11, 2021. ; https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint 14 severity (Figure 2A ). Amongst COVID-19 patients, the total white cell count was highest in HIV-1 uninfected patients with coincident tuberculosis (median: 15.34 x 10 9 /L, IQR: 11.97-17.13). Lymphocyte counts were lower in both ODC and COVID-19 patients (with no differences between these groups) compared to HC. The count trended towards a decrease with increasing COVID-19 severity and was significantly lowest in triply infected patients (p = 0.03, Figure 2B ). Both neutrophil and monocyte counts were raised in both ODC and COVID-19 patients with no differences between these groups. Neutrophil, but not monocyte, count related to increasing COVID-19 severity ( Figure 2C&D ). Those with coincident tuberculosis had higher monocyte counts than those with combined HIV-1 and COVID-19 infections without tuberculosis (p = 0.04, Figure 2D ). By comparison with HC, eosinophil counts were lower in both ODC and COVID-19 patients were lowest in those with COVID-19 infection with a trend observed in relation to disease severity (p = 0.004, Figure 2E ).

We further assessed the nature of lymphopenia by flow cytometric analysis of whole blood.

There was strong correlation between absolute CD4 numbers and the % CD4 determined by flow cytometry (Supplemental Figure 4A ). Amongst COVID-19 patients increasing disease severity correlated with a decrease in CD4 and CD8 positive lymphocytes (Supplemental Figure 4B ). To investigate lymphocyte differences in COVID-19 patients, additional values were obtained from a subset of 118/163 similar ambulant participants enrolled to a prior study of HIV-1 uninfected and infected persons with either immune evidence of tuberculosis sensitization but no symptoms or microbiologically confirmed pulmonary tuberculosis treated at a community health clinic (Supplemental Table 4 

A number of soluble biomarkers have been associated with COVID-19 and its severity 40 . In our study, higher C-reactive protein, D-dimer, lactate dehydrogenase (LDH) and ferritin levels were Figure   3E ). The relationship between (WCC) and neutrophil counts, and between ferritin and LDH was also observed in ODC ( Figure 3F ).

We next determined relationships between disease severity amongst COVID-19 patients, the presence of HIV-1 and/or tuberculosis as co-morbidities, and the level of anti-SARS-CoV-2 nucleocapsid specific antibody levels determined by the Roche Elecsys assay 41 . We have previously reported increased antinucleocapsid antibody levels in COVID-19 patients with more severe disease although no overall association with survival was noted 42 . Although no significant quantitative difference in cut-off index was observed by HIV-1 and or tuberculosis status, 8/15 tuberculosis patients in total and 5/8 with HIV-1 and tuberculosis were below the threshold for positivity ( Figure 4A ). There was a significant trend towards decreased antibody levels in participants with a lower percentage CD4 lymphocytes with just 3/10 antibody responders amongst patients with a CD4 percentage of lymphocytes below 10% ( Figure 4B ).

Amongst HIV-1 co-infected COVID-19 patients, there was positive correlation between the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2021. ; https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint Version 1.1 last edited by RJW 11MAY2021 16 percentage of CD4 T cells and the antibody cut-off index with triply infected patients tending to show low levels of both parameters ( Figure 4C ).

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By means of a case-control analysis, we have set out one of the first accounts of the clinical presentation and laboratory features of COVID-19 in an African setting in which there is high non-communicable, but also communicable (HIV-1 and tuberculosis) co-morbidity. An important conclusion is that the triple occurrence of SARS-CoV-2, HIV-1 and tuberculosis is not uncommon. Clinical features may be dominated by either SARS-CoV-2 or tuberculosis and, although overall radiographic severity may be modulated, lymphopenia is exacerbated, and some markers of inflammation are even more elevated in such patients. Forty percent of patients with coincident COVID-19 and tuberculosis died.

Our study benefitted inclusion of participants (ODC) admitted to the same hospital at the same time as the surge in COVID-19 admissions who were subsequently assigned alternative diagnoses with negative virological and serological tests for SARS-CoV-2. We cannot exclude under the circumstances that such tests were false negative. However, the existence of this control group allowed us to compare blood markers that have been used to characterise and prognosticate SARS-CoV-2 infection. We found that neither raised total white count, neutrophilia or lymphopenia can be inferred as specific for COVID-19 ( Figure 2 ). By contrast, the plasma levels of several markers including the CRP, D-dimer, LDH and especially ferritin were markedly elevated in COVID-19 patients (Figure 3 ). Of these markers however, only LDH showed an increasing trend with COVID-19 disease severity. A potential confounder of results on peripheral blood markers is the greater use of corticosteroid therapy at the time of sampling in COVID-19 patients. The results of the Recovery trial were announced shortly after we began recruitment to our study and were accompanied by an overnight change in clinical guidance to prescribe such therapy to those severely ill 43 . Notwithstanding this potential blunting of values, our results again reflect the marked severe systemic effects of acute SARS-CoV-2 infection? that are now very widely documented. A greater proportion of participants who died also received steroids. This therapy is indicated in severe COVID-19 43 and this association is therefore probably confounded by the fact that more severely ill participants were more likely to receive this treatment.

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in not documenting major differences in presentation and outcome except in those profoundly immunosuppressed. ART use was variable and not equally distributed between COVID-19 cases and ODC with the latter more likely to be ART-naïve: perhaps reflecting late presentations of HIV-1 infection at a time when pressure on health services was acute. This feature also precludes meaningful and well-powered comparisons according to ART status. Overall, HIV-1 infected patients recruited to this study were not more likely to die. There was a predictable decrease in CD4 T cell numbers and percentage in HIV-1 infected participants, not reflected however in CD8 counts. Of potentially greater significance, in terms of longer lasting immunity in convalescence, there was a direct relationship between the peripheral CD4 percentage and acute antibody levels. Whilst this may reflect duration of COVID-19 illness and its severity (both tending to increase antibody levels), it remains a grave concern that both natural and vaccineinduced immune responses to SARS-CoV-2 may be less effective in persons immunosuppressed by HIV-1, irrespective of ART status (https://ir.novavax.com/news-releases/news-releasedetails/novavax-covid-19-vaccine-demonstrates-893-efficacy-uk-phase-3).

At the beginning of the SARS-CoV-2 pandemic there was false optimism that the age structure of African populations may mitigate against severe continental impact. Our study does not uphold this conclusion. A wide range of now well-recognised co-morbidities (most commonly hypertension, type 2 diabetes mellitus and obesity) that associate with COVID-19 severity and death were present in the bulk of our cases and also in relevant other disease controls (ODC).

Overall, 29% COVID-19 patients died. SARS-CoV-2 acutely brings into focus interaction between infectious and non-communicable conditions in Africa and the necessity that multimorbidity research must encompass such interaction much more in the future.

The analysis was not designed or powered to reproduce the previously documented relationships between HIV-1 co-infection and tuberculosis and risk of death from COVID-19 11 . The intent was rather to describe clinical presentation. Against an overall study mortality from COVID-19 of 28.8%, 19.4% of those HIV-1 co-infected, 40% of those with coincident tuberculosis, and 3/8 (37.5%) with coincident HIV-tuberculosis, died. Lymphocyte numbers were lowest, some inflammatory markers were higher and anti-SARS-CoV-2 antibody responses were most . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2021. ; https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint Version 1.1 last edited by RJW 11MAY2021 19 depressed in those exhibiting triple infection. The late detection of unsuspected tuberculosis in several otherwise typical COVID-19 patients is a cause for concern and increased clinical vigilance. We have documented here and elsewhere 42 that tuberculosis may adversely impact both antibody and T cell responses to SARS-CoV-2. Conversely, the immunological milieu set up in the lung by SARS-CoV-2, to which corticosteroid therapy will add, may have contributed to reactivation of subclinical or latent tuberculosis 44 . Thus, in environments in which tuberculosis is common, clinical suspicion of reactivation and also that SARS-CoV-2 may coexist in typical presentations of tuberculosis are the most important clinical lessons from our work.

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JdV and NT on ethical aspects. MSM, GM, NN and SW supervised clinical recruitment. EdB, RD, CS recruited patients with logistic assistance from RTG. AJ designed the clinical research forms and database and supervised data cleaning. BWA conceptualised the percentage unaffected radiographic score. QSH performed the radiographic interpretation and scoring. FA co-ordinated the receipt and assay of laboratory specimens and MH was responsible for processing and reporting of samples in the NHLS analyses. EdB, CS and CR analyzed the data.

All authors critically reviewed and provided input to the manuscript.

All authors have completed the Unified Competing Interest form (available on request from the corresponding author) and declare: no financial relationships with any organisations that might have an interest in the submitted work in the previous three years, no other relationships or activities that could appear to have influenced the submitted work

The corresponding authors (the manuscript's guarantors) affirm that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

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The copyright holder for this preprint this version posted May 11, 2021 is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted May 11, 2021. ; https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint Comparisons were performed using a Kruskal-Wallis test.

F Brixia score in relation to the presence or absence of HIV-1 and/or tuberculosis co-infection.

The extent of changes related COVID-19 was decreased amongst those with coincident HIV-1 associated tuberculosis. Comparisons were performed using a Kruskal-Wallis test with Dunn's correction.

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A The total white cell count was raised in both COVID-19 and ODC patients with no differences between the groups. The count increased with increasing COVID-19 severity. Amongst COVID-19 patients the total white cell count was highest in HIV-1 uninfected patients with coincident tuberculosis.

B The lymphocyte count was lower in both COVID-19 and ODC patients with no differences between these groups. The count tended to decrease with increasing COVID-19 severity.

Amongst COVID-19 patients the lymphocyte count was lowest in triply infected patients with HIV-1 and coincident tuberculosis. All comparisons were performed using a Kruskal-Wallis test with Dunn's correction. D Lactate dehydrogenase was significantly higher in COVID-19 patients than ODC, with levels tending to increase with increasing COVID-19 disease severity. There was however no . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2021. ;  https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint relationship with HIV-1 and/or tuberculosis status. Comparisons between the ODC and COVID-19 groups were performed using a Mann Whitney test and other comparisons were performed using a Kruskal-Wallis test.

E&F Correlation matrix of serum biomarkers in COVID-19 patients (E) and ODC participants (F). Amongst COVID-19 patients there was strong positive correlation between total white cell (WCC) and neutrophil counts, and moderate between ferritin and LDH, and between lymphocyte and monocyte counts. In ODC participants, the relationship between total white cell (WCC) and neutrophil counts, and between ferritin and LDH was also observed. Positive correlations are indicated in blue and negative correlation in red. Spearman R-values are indicated in each square. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 B There was a significant trend towards decreased antibody levels in participants with Lower percentage CD4 lymphocytes. Comparisons were performed using a Kruskal-Wallis test.

C Amongst HIV-1 co-infected COVID-19 participants, there was positive correlation between the percentage of CD4 lymphocytes and the antibody cut-off index with triply infected patients tending to show low levels of both parameters. Correlation was tested by a two-tailed nonparametric Spearman rank test.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2021 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2021. ; https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint There was no significant trend in relation to severity of COVID-19 disease. HIV-1 infected patients with both COVID-19 and tuberculosis had higher levels of all three markers than those with COVID-19 alone. D Lactate dehydrogenase was significantly higher in COVID-19 patients than ODC, with levels tending to increase with increasing COVID-19 disease severity. There was however no relationship with HIV-1 and/or tuberculosis status. Comparisons between the ODC and COVID-19 groups were performed using a Mann Whitney test and other comparisons were performed using a Kruskal-Wallis test. E&F Correlation matrix of serum biomarkers and white cell counts in COVID-19 patients (E) and ODC participants (F). Amongst COVID-19 patients there was strong positive correlation between total white cell (WCC) and neutrophil counts, and moderate between ferritin and LDH, and between lymphocyte and monocyte counts. In ODC participants, the relationship between total white cell (WCC) and neutrophil counts, and between ferritin and LDH was also observed. Positive correlations are indicated in blue and negative correlation in red. Spearman R-values are indicated in each square. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 11, 2021. ; https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint A Although no significant quantitative difference in cut-off index was observed by HIV-1 and or tuberculosis status, 8/15 tuberculosis patients in total and 5/8 with HIV-1 and tuberculosis were below threshold for positivity of 1.0. The >1 cut-off value represents the manufacturer's cut-off. The >0.165 cut-off value represents the optimal cut-off value defined by ROC curve analyses on 197 participants that improved the performance of the test to give a sensitivity of 100% (95%CI: 94.0-100%) (Reference 45). B There was a significant trend towards decreased antibody levels in participants with Lower percentage CD4 lymphocytes. Comparisons were performed using a Kruskal-Wallis test. C Amongst HIV-1 co-infected COVID-19 participants, there was positive correlation between the percentage of CD4 lymphocytes and the antibody cut-off index with triply infected patients tending to show low levels of both parameters. Correlation was tested by a two-tailed non-parametric Spearman rank test.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 11, 2021. ; https://doi.org/10.1101/2021.05.11.21256479 doi: medRxiv preprint

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