The novel coronavirus disease 2019 (COVID-19), a contagious acute respiratory syndrome, has spread worldwide from China, causing more than 200,000 deaths, as of May 2020. Despite high number of people living with human immunodeficiency virus (HIV)-1 globally (about 37 million) and higher severity impact for certain viral infections in this category, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)/HIV-1 coinfection has been described in few cases.[3–6] Furthermore, black people are overrepresented among reported COVID-19 cases and deaths, especially in some areas of US.
Chronic immune activation and persistent interferon (IFN)-I response are well-known driver of HIV-1 disease progression.[8,9] Notably, SARS-CoV-2 mono-infection has been also associated to alteration in IFNα/β and T cell immune activation,[10,11] suggesting that HIV-1 and SARS-CoV-2 might both promote deleterious immunological and clinical consequences.
This study reports a severe case of SARS-CoV-2 in a black female patient co-infected by HIV-1 under protease inhibitors (PI) regimen, who was treated with hydroxychloroquine. To address the role of IFN-I response and T cell activation in SARS-CoV-2/HIV-1 coinfection, we have analyzed IFN-I transcript levels and frequencies of CD4 and CD8 T cells expressing CD38 and/or human leukocyte antigen-DR [HLA-DR] in the SARS-CoV-2/HIV-1-coinfected subject, compared to those of HIV-1-monoinfected patients and healthy blood donors.
A black Ethiopian 52-year-old woman with SARS-CoV-2/HIV-1 coinfection was admitted to Department of Infectious Diseases at Policlinico Umberto I, Hospital of Sapienza University of Rome (Italy) during the epidemic wave in Italy. Nasopharyngeal swabs were collected within 48 hours of hospital admission and before hospital discharge (Fig. 1, Panel A). Blood samples were also collected from SARS-CoV-2-positive patient (Fig. 1, Panel A), age-matched virologically suppressed HIV-1-infected women (n = 10) and healthy individuals (n = 5). The study was approved by the institutional review board (Ethics Committee of Umberto I General Hospital, Rome, approval number/ID Prot. 109/20209). All study participants gave written informed consent.
2.2 RT-qPCR detection of SARS-CoV-2 RNA
Viral RNA was extracted from nasopharyngeal swabs using Versant SP 1.0 Kit (Siemens Healthcare Diagnostics). Ten μl of extracted RNA was reverse-transcribed and simultaneously amplified using a Real time RT PCR system (RealStar SARS-CoV2 RT PCR, Altona Diagnostics), targeting E and S viral genes.
2.3 Flow cytometry assay
Phenotypes and activation markers were evaluated by Miltenyi Biotec flow cytometer-MACSQuant Analyzer on lysed and washed whole-blood samples by the following antihuman monoclonal antibodies: CD3-PerCP, CD4-APC-Vio770, CD8-FITC, CD38-APC and HLA-DR-PE (Miltenyi Biotec, Bergisch Gladbach, Germany). Gating analysis and data were analyzed using MACSQuantify software 2.5 (Miltenyi Biotec).
2.4 Real-time polymerase chain reaction assay
Total RNA was extracted using Total RNA Purification kit (Norgen, Thorold, Canada) and reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). Relative quantification of IFNα, IFNβ and housekeeping gene β-glucuronidase target mRNA expression levels were carried out with LightCycler 480 instrument (Roche, Basel, Switzerland), as previously described.
2.5 Statistical analysis
Data on IFNs gene expression level and activated T cells frequencies were expressed as median relative difference (MRD), as previously reported. In particular, MRD was defined as MRD = Δ/X2=(X1–X2)/X2 where X1 is the level of immunological measurements, expressed as median, in the SARS-CoV-2/HIV-1-coinfected patient, while X2 is measure of the same variables referred to age-matched HIV-1-positive female patients or healthy women. For values greater than reference, MRD should be a positive number (Δ > 0) and for values that are smaller, MRD should be negative (Δ < 0). MRD with 1 sigma is uncertain on the meaning for each parameter.
3 Case report
A 52-year-old woman with SARS-CoV-2/HIV-1 coinfection under darunavir/cobicistat regimen was analyzed. SARS-CoV-2 diagnosis, symptoms and therapy timeline is shown in Figure 1, Panel A. For the past 23 years, her HIV-1 status has been immunologically and virologically stable with no reported opportunistic diseases. Laboratory test results at admission time revealed a HIV-1 viral load below level of detection (<37 HIV-1 RNA copies/ml) in line with results of past months and a CD4 T cell count of 242 cells/μl, CD8 T cell count of 336 cells/μl, with a CD4/CD8 T cells ratio of 0.72. Besides her HIV-1 infection diagnosed in 1997, she had a 15 pack-year smoking history, sobriety from alcohol and no intravenous drug abuse habits. At hospital admission, the patient was febrile (37.5°C), with a blood pressure of 120/70 mm Hg, a pulse of 120 beats/min, a peripheral blood saturation of 96%, a respiratory rate of 24 breaths/min and Glasgow Coma Scale of 15. Furthermore, the patient had a normal total leucocyte count (7210 cells/μl) with an absolute lymphocytopenia (610 cells/μl), elevated PCR (15.67 mg/dl), interleukin-6 (50.96 pg/ml) and normal PCT value (0.09 ng/ml). Arterial blood gas analysis showed a pH of 7.48, a value of 35 mm Hg for CO2 arterial pressure and 70 mm Hg for O2 arterial pressure, a P/F ratio of 333 mm Hg, based on a FiO2 of 21%. M. pneumoniae IgM and C. pneumoniae IgM on blood test resulted negative, as peripheral blood smear, TB-GOLD QuantiFERON and urinary antigen of Legionella. Thus, she took a nasopharyngeal swab, resulted positive for PCR amplification of SARS-CoV-2 E and S viral genes. She, then, performed a thorax high-resolution computed tomography scan that detected multiple peripheral ground-glass areas bilaterally and rare crazy paving signs, highly suspicious of COVID-19 pneumonia (Fig. 1, Panel B1-B2).
Hence, she underwent high flow nasal cannula with a FiO2 of 50% and 60 l/min, associated with a 7-days therapy based on hydroxychloroquine 200 mg bid and enoxaparin 4000 UI bid, maintaining darunavir/cobicistat. At the end of treatment, on day 7, patient reported resolution of fever, amelioration of her fatigue and normalization of respiratory rate (14 breaths/min), requiring less intensive oxygen support. Then, she interrupted hydroxychloroquine. Contemporarily, she took an arterial blood gas analysis that showed improved oxygen arterial pressure (98 mm Hg) with a FiO2 of 30%. Besides, her PCR declined (2.1 mg/dl) and lymphocytes increased (800 cells/μl). On day 14, patient interrupted oxygen support with normalization of arterial blood gases pressures in ambient air and performed 2 consecutive nasopharyngeal swabs 24 hours apart resulted negative. Lymphocyte count was repeated with detection of lymphocytopenia (810 cells/μl), CD4 T cell count 528 cells/μl, CD8 T cell count 533 cells/μl and a CD4/CD8 T cell ratio of 0.99.
3.2 Type I IFN and T cell activation
Because of the key role of chronic immune activation and persistent IFN-I response in driving HIV-1 disease,[8,9] we evaluated IFNα and IFNβ gene expression and T cell activation levels in patient with SARS-CoV-2/HIV-1 coinfection. MRDs of IFNα and IFNβ mRNAs and T cell activation levels were calculated referring to age-matched HIV-1-positive female patients and healthy women. We found that MRDs for IFNα and IFNβ and T cells were on average approximatively equal to 0 when refereed to HIV-1-positive female patients [MRDs IFNα/β: median −0.2545 (range: −0.5/0.1); T cells: median −0.11(range: −0.8/1.3) (Fig. 2, Panel A, B)]. By contrast, MRDs values of IFNs and T cells immune activation were always ≥6 when referred to healthy individuals [MRDs IFNα/β: median 28.45 (range: 15/41.9); T cells: median 10 (range 6/22) (Fig. 2, Panel A, B)].
Little is known about clinical and immunological outcomes of HIV-1-patients infected with SARS-CoV-2. Our case report is consistent with previous researches that described progression of SARS-CoV-2 viral pneumonia in the second week of illness. Furthermore, as reported in a study by Thevarajan et al, peripheral CD4 and CD8 T cell count increases during host immune response, despite CD4/CD8 T cell ratio was dissimilar to the one found in the same case reported. At the present, COVID-19 in people living with HIV-1 has been described in few cases in China,[3,4] in a case series of 5 patients from Spain, with just 2 patients on PI based regimen and in a retrospective Italian study, with an 11% of patients receiving PI.
PI drugs, especially lopinavir/ritonavir have been considered, together with hydroxychloroquine, as first-line drug in COVID-19 therapeutic regimens. Darunavir/cobicistat and darunavir/ritonavir have also been included as alternative regimen to lopinavir/ritonavir by the Italian society in infectious and tropical diseases (SIMIT), especially in case of intolerable side effects.
However, a recent clinical trial showed that lopinavir/ritonavir was ineffective as a monotherapy against SARS-CoV-2 pneumonia.
Since in vitro activity against SARS-CoV and Middle East respiratory syndrome CoV (MERS-CoV) was reported for PI drugs in combination with IFNβ,[20,21] it is reasonable to suppose existence of a synergistic PI and IFNβ effect against SARS-CoV-2, as also suggested by stronger efficacy of combined IFNβ, lopinavir-ritonavir and ribavirin treatment compared to lopinavir/ritonavir alone in patients with COVID-19.
Thus, the aim of this report is suggesting further investigation on efficacy of treatment in patients with COVID-19, especially in potential prevention and earlier stages of disease.
In addition, we presented a severe case of COVID-19 in a black person who recovered without serious complications. It is reported in literature how non-Hispanic black people, especially in the US, seem to have higher mortality rate, compared to white persons, even though more studies would be needed in this field. Blacks are disproportionately affected by poverty, mass incarceration, infant mortality, limited health care access, and health-related conditions including heart disease, diabetes, stroke, kidney disease, respiratory illness, and HIV-1. All these facts and the tendency of black African people to live in close and numerous communities seem to increase the exposure risk of this population.
Interestingly, our results also indicate, for the first time to our knowledge, that SARS-CoV-2/HIV-1-coinfected woman had levels of IFNα/β and activated CD4 and CD8 T cells higher than those recorded in healthy donors, but comparable to those found in HIV-1-monoinfected women. Levels of HLA-DR CD38 CD4 and HLA-DR CD38 CD8 T cells have been reported to be increased in SARS-CoV-2-infected patients together with common inflammation cytokines. Moreover, it has found that SARS-CoV-2 triggers lower levels of IFN-I/III in ex vivo human lung tissue explants from cancer patients. It has also showed that low IFN-I-III levels are produced during SARS-CoV-2 infection despite a consistent chemokine signature in cell lines, primary cell cultures, ferrets and COVID-19 patients.[27,28] Then, our findings suggest that SARS-CoV-2 infection did not adversely affect IFNα/β and activated T cells levels in HIV-1-infected patients.
In summary, we found that SARS-CoV-2 infection can cause severe disease in HIV-1-positive patient under PI regimen. The outcome of COVID-19 was reversed by a 7-days hydroxychloroquine therapy, although, to date, there have been no robust clinical trials that have shown efficacy of these agents for this illness. Notably, active SARS-CoV-2 infection was not associated with increased levels of IFNα and IFNβ gene expression and T cell immune activation compared to HIV-1-monoinfected women. Although SARS-CoV-2/HIV-1-coinfected patient had no significant alterations in levels of immune dysregulation compared to HIV-1-positive patients, caution must be exercised because analysis was performed in only one HIV-1-coinfected patient. Studies with larger patient series, currently under way, will be needed to explore this topic.
Consent for publication: Informed written consent was obtained from the patient for publication of this case report.
CP, GPI, and CB wrote the paper, carried out the experiment and performed statistical analysis. LS, FF, and CB collected the samples and participated in carrying out the experiments. GR, MR, GS, GD, and GC provided patient's samples and participated in the design and revision of the manuscript. GA and CMM participated in the design and revision of the manuscript. GD and CS conceived the study, analyzed the data, wrote the paper and supervised the work. All authors reviewed the work and approved the final manuscript.
. World Health Organization Coronavirus disease 2019 (COVID-19) Situation Report. Published online, April 2020.
. González Álvarez DA, López Cortés LF, Cordero E. Impact of HIV on the severity of influenza. Expert Rev Respir Med 2016;10:46372.
. Wang M, Luo L, Bu H, et al. One case of coronavirus disease 2019 (COVID-19) in a patient co-infected by HIV with a low CD4+ T-cell count. Int J Infect Dis 2020;96:14850.
. Zhu F, Cao Y, Xu S, et al. Co-infection of SARS-CoV-2 and HIV in a patient in Wuhan city, China. J Med Virol 2020;92:52930.
. Blanco JL, Ambrosioni J, Garcia F, et al. COVID-19 in patients with HIV: clinical case series. Lancet HIV 2020;7:e3146.
. Härter G, Spinner CD, Roider J, et al. COVID-19 in people living with human immunodeficiency virus: a case series of 33 patients. Infection 2020;16.
. Ferdinand KC, Nasser SA. African American COVID-19 mortality: a sentinel event. J Am Coll Cardiol 2020;75:27468.
. Paiardini M, Müller-Trutwin M. HIV-associated chronic immune activation. Immunol Rev 2013;254:78101.
. Scagnolari C, Antonelli G. Type I interferon and HIV: Subtle balance between antiviral activity, immunopathogenesis and the microbiome. Cytokine Growth Factor Rev 2018;40:1931.
. Zhuo Z, Ren L, Zhang L, et al. Heightened innate immune responses in the respiratory tract of COVID-19 patients. Cell Host Microbe 2020;27:883.e290.e2.
. Wen W, Su W, Tang H, et al. Immune cell profiling of COVID-19 patients in the recovery stage by single-cell sequencing. Cell Discov 2020;6:31.
. Santinelli L, Statzu M, Pierangeli A, et al. Increased expression of IL-32 correlates with IFN-(, Th1 and Tc1 in virologically suppressed HIV-1-infected patients. Cytokine 2019;120:27381.
. Statzu M, Santinelli L, Viscido A, et al. Increased SAMHD1 transcript expression correlates with interferon-related genes in HIV-1-infected patients. Med Microbiol Immunol 2019;208:67991.
. Santinelli L, Ceccarelli G, Borrazzo C, et al. Sex-related differences in markers of immune activation in virologically suppressed HIV-infected patients. Biol Sex Differ 2020;11:23.
. Gervasoni C, Meraviglia P, Riva A, et al. Clinical features and outcomes of HIV patients with coronavirus disease 2019. Clin Infect Dis 2020;ciaa579.
. Huang C, Wang Y, Li X, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020;395:497506.
. Thevarajan I, Nguyen THO, Koutsakos M, et al. Breadth of concomitant immune responses prior to patient recovery: a case report of non-severe COVID-19. Nat Med 2020;26:4535.
. Società italiana di Malattie infettive e tropicali, Vademecum per la cura delle persone con malattia da COVID-19 Edizione 2.0, 13 marzo 2020.
. Cao B, Wang Y, Wen D, et al. A trial of lopinavir-ritonavir in adults hospitalized with severe Covid-19. N Engl J Med 2020;382:178799.
. Chan JFW, Chan KH, Kao RY, et al. Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus. J Infect 2013;67:60616.
. Chu CM, Cheng VC, Hung IF, et al. Role of lopinavir/ritonavir in the treatment of SARS: initial virological and clinical findings. Thorax 2004;59:2526.
. Hung IF, Lung KC, Tso EY, et al. Triple combination of interferon beta-1b, lopinavir–ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial. Lancet 2020;395:1695704.
. Laurencin CT, McClinton A. The COVID-19 pandemic: a call to action to identify and address racial and ethnic disparities. J Racial Ethn Health Disparities 2020;7:398402.
. Milam AJ, Furr-Holden D, Edwards-Johnson J, et al. Are clinicians contributing to excess African American COVID-19 deaths? Unbeknownst to them, they may be. Health Equity 2020;4:13941.
. Qin C, Zhou L, Hu Z, et al. Dysregulation of immune response in patients with COVID-19 in Wuhan. China Clin Infect Dis 2020;71:7628.
. Chu H, Chan JF, Wang Y, et al. Comparative replication and immune activation profiles of SARS-CoV-2 and SARS-CoV in human lungs: an ex vivo study with implications for the pathogenesis of COVID-19. Clin Infect Dis 2020;ciaa410.
. Blanco-Melo D, Nilsson-Payant BE, Liu WC, et al. Imbalanced host response to SARS-CoV-2 drives development of COVID-19. Cell 2020;181:1036.e945.e9.
. Antonelli G, Turriziani O, Pierangeli A, et al. Type I interferons can be detected in respiratory swabs from SARS-Cov-2 infected patients. J Clin Virol 2020;128:104450.
. Geleris J, Sun Y, Platt J, et al. Observational study of hydroxychloroquine in hospitalized patients with Covid-19. N Engl J Med 2020;382:24118.