Antiviral and Monoclonal Antibody Combination Therapy in Haematological Patients in the Omicron Era 

Serena Vita1, Emanuela Giombini1, Patrizia De Marco1, Martina Rueca1, Cesare Ernesto Maria Gruber1, Alessia Beccacece1, Laura Scorzolini1, Valentina Mazzotta1, Carmen Pinnetti1, Priscilla Caputi1, Daniele Focosi2, Enrico Girardi1, Andrea Antinori1, Fabrizio Maggi1, Alessandra D’Abramo1# and Emanuele Nicastri1 and Spallanzani COVID-19 case investigation team*.

1 National Institute for Infectious Diseases “Lazzaro Spallanzani” IRCCS- Rome Italy.
2 North-Western Tuscany Blood Bank, Pisa University Hospital, Pisa, Italy.

Correspondence to: Alessandra D’Abramo. National Institute for Infectious Diseases “Lazzaro Spallanzani” IRCCS via Portuense 292, 00149 Rome Italy. E-mail:

Published: May 01, 2024
Received: February 19, 2024
Accepted: April 18, 2024
Mediterr J Hematol Infect Dis 2024, 16(1): e2024043 DOI 10.4084/MJHID.2024.043

This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

To the editor

Immunocompromised (IC) patients are at higher risk for persistent and/or severe SARS-CoV-2 infection caused by different viral variants, with a high case-fatality ratio.[1,2] The first persistent SARS-CoV-2 infection (5 months) was reported in 2020 in an IC patient with a long persistence of SARS-CoV-2,[3] immediately followed by further reports.[2,4] Indeed, the impairment of the immune system changes the natural history of COVID-19. However, no consensus exists on clinical management of IC COVID-19 patients.[5] Several reports emphasize the clinical relevance of a combination therapy between small-molecule antivirals (AV) and anti-spike monoclonal antibodies (MoAbs) both in early and prolonged COVID-19 clinical management.[6,7] In 2022, tixagevimab/cilgavimab (T/C) MoAb fixed combination was introduced as early therapy for outpatient with COVID-19.[8] We describe here a single-center case series of 22 IC COVID-19 in patients with hematological disorders (HD) treated with a combined therapy based on tixagevimab/cilgavimab (T/C) plus small-molecule antivirals (AV), between April 1, 2022, and November 30, 2022.
The viral genomic evolution was assessed by sequencing the whole SARS-CoV-2 genome in a subgroup of patients (pts). Pts were consecutively admitted for COVID-19 to the Lazzaro Spallanzani National Institute for Infectious Diseases, Rome, Italy (INMI). Demographic characteristics, medical history, clinical presentation, treatment, adverse drug reactions, and clinical outcome (survival/death) during follow-up were collected from patient clinical records. Real-time reverse transcription polymerase chain reaction (RT-PCR) on nasopharyngeal swab (NPS) samples was performed according to the laboratory workflow using Alinity m SARS-CoV-2 Assay (Abbott, Chicago, Illinois, United States) targeting RdRp and N genes. When possible, molecular characterization of the SARS-CoV-2 virus was performed using whole genome sequencing (WGS) at diagnosis and during follow-up.[9] Whole Genome sequencing (WGS) was carried out on an Ion Torrent Gene Studio S5 platform using Ion AmpliSeq SARS-CoV-2 in-sight research assay following the manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA, USA). The whole genome reconstruction was performed using ESCA software.[10] All the mutations were identified with respect to the reference suggested by NCBI Wuhan-Hu-1 (NC_045512.2). A phylogenetic tree was built using 16 Italian SARS-CoV-2 sequences that were selected among those available on the GISAID platform with a collection date closer to that of the INMI patients and clustered using cd-hit with 99% identity.[11] The transition model (TIM+I+F+G) was identified as the best-fitting nucleotide substitution model, and a phylogenetic tree was constructed with 5,000 bootstrap replications using the IQ-Tree program.[12]
Table 1 shows the characteristics of the study population. All patients were fully vaccinated against COVID-19 with at least 3 doses, 11 (50%) of them males, with a median age of 78 years old (IQR 69-83) (Table 1).

Table 1
Table 1. Clinical features of study population

Twenty patients were under active chemotherapy. They were admitted with a median of 11 days (IQR 1-33) after the first NPS positive for SARS-CoV-2. The study population had a median total lymphocyte count of 910/µl (IQR 520-1547), and 15 out of 22 (68%) had hypogammaglobulinemia. All patients had pneumonia, but only 14 of them required respiratory support. Seven patients had severe COVID-19 (WHO COVID-19 ordinary scale 5), and 15 patients had moderate/mild COVID-19 (6 patients with a score of 4 and 9 patients with a score of 3). Steroid therapy (oral or intravenous 6 mg dexamethasone daily) was started in 14 patients with respiratory failure. At the admission, NPS for SARS-CoV-2 was positive with a median cycle threshold (Ct) of 20 (IQR 16-24). All patients were treated with a first combination regimen of MoAbs (T/C in 17 cases, sotrovimab in 3 cases, and casirivimab/imdevimab in 2 cases) plus a 5-day course of intravenous remdesivir (200 mg on day one followed by 100 mg on day 2-5). Eleven out of 22 (50%) patients with an NPS<35 Ct required a second course of antivirals (remdesivir in 2 cases and oral nirmatrelvir/ritonavir in 9 cases, 300mg/100 mg twice daily for 5 days) associated with T/C in the five subjects initially treated with different MoAbs. Two patients who, after 2 courses of antivirals and T/C, still had an NPS<35 Ct received at least 2 doses of COVID-19 convalescent plasma (CCP) with > 1:160 SARS-CoV-2 neutralizing antibody titer. Four patients died (all with positive NPS PCR at the last available time point, i.e., at days 103, 115, 43, and 41, respectively, since the first positive NPS) (see Table 2). In particular:
•    Patient #3 died of gastrointestinal severe graft-versus-host diseases (GvHD) at month 2 after hematopoietic stem cell transplantation for acute myeloid leukemia.
•    Patient #9 died of recurrent Clostridioides difficile infection during a relapse of NHL.
•    Patient #19 died from a relapse of NHL.
•    Patient #22 died of respiratory failure and pneumonia sustained by Aspergillus spp. and Stenotrophomonas maltophilia.
In the remaining 18 patients, the SARS-COV-2 NPS PCR was negative at a median of 59 (IQR 47-93) days since the first evidence of SARS-CoV-2 infection (Table 2) and 47 days (IQR 28-51) after starting the treatment. The median duration of hospital stay was 32 days (IQR 24-41).

Table 2
Table 2. Virological, therapeutic features and clinical outcome of study population.

Spike-gene sequencing was possible in 18 out of 22 patients, and identified a BA.2* VoC in 9, a BA.4/5* VoC in 7, a BA.1.1* in 1, and a BQ.1.1* VoC in 1.
The whole SARS-CoV-2 genome was sequenced in 4 out of 22 BA.2 patients (Patient#1, #3, #4, and #7). A deeper analysis was conducted on the Spike glycoprotein. No recurrent amino acid mutations in the 21 sequenced patients were found. In baseline sequences, no mutations that were not lineage-related were found in patients #3 and #4 (Table 3), while V445A mutation in patient #1 and E340Q, R683W, and G798S mutations in patient #7 were found. Patients #1 and #4 exhibited 3 and 1 additional Spike mutations at the available second timepoint (T1), compared to the baseline sequences. In particular, T1 patient #1 sequence showed a deletion in position S: A243-L244. Finally, the phylogenetic tree showed that whole genome sequences collected at baseline clustered with a significative bootstrap with sequences collected after days 22 and 80 for patients #1 and #4, respectively, while the baseline sequence of patient #3 was interspersed between other BA.2 sequences currently circulating in Italy (Figure 1).

Table 3 Table 3. Spike mutations of sequenced samples.

Figure 1 Figure 1. Phylogenetic  analysis on whole genome sequences

In the context of SARS-CoV-2 infection, IC patients face heightened vulnerability. Although they have been underrepresented in previous randomized clinical trials, they are likely overrepresented among currently hospitalized patients with severe and/or persistent symptoms associated with SARS-CoV-2 infection.[5,13] Nevertheless, there is no evidence-based approach for managing these patients. Several recent studies support the use of MoAb and AV combination therapy in IC inpatients and outpatients or, for inpatients, prolonged antiviral therapy.[7,14-18] At admission, the cohort had a median of 11 days since the first SARS-CoV-2 positive NPS, with a median Ct value of 20, suggesting a persistently high viral replication. Notably, Ct-values, a measure of viral burden, between 17 and 32 represent an amount of virus that is likely to be replicative competent.[19] Seventy-one percent of patients had a BA.2* VoC that retains in-vitro susceptibility to cilgavimab; T/C has reduced efficacy against BA.5* VoC, although it was unclear at that time of use. All patients were considered at high risk of clinical progression and underwent a full course of remdesevir and MoAb combined therapy with an off-label 600 mg tixagevimab/cilgavimab prescription with no reported adverse event. Half of them achieved viral clearance after the first course of treatment, whereas the remaining 11 patients necessitated a second AV and MoAb combined course.
Additionally, two patients only partially responder (NPS<35 Ct) after two full combined antiviral regimens, received CCP, a major therapeutic option as a source of exogenous specific antibodies against SARS-CoV-2 Spike glycoprotein: one patient died, and one recovered. We considered the 35 Ct cut-off value during therapy as a surrogate marker of successful viral response. Lower Ct values are commonly related to active viral replication and potential contagiousness.[19,20]
All COVID-19 survival patients had a negative SARS-CoV-2 NPS PCR after combined therapy, with a median time of 52 days since the first positive NPS and of 38 days since hospitalization. The observed case fatality rate in our cohort was 18%, which falls within the previously reported range of 13.8% to 39%.[21] The four deceased patients tested positive for NPS PCR at the time of death: in three patients, the death was due to recurrence of the underlying HD, and in one case, to complication of stem cell transplant.
The literature poorly describes IC patients treated by T/C, and this MoAb has provided new therapeutic opportunities apart from the already two registered indications.[8] Lahouati describes the treatment of a cohort of 223 IC patients, although patients with HD represented 25%, and among them, 12% were treated with T/C, corresponding to 7 pts.[22]
In our cohort, all patients were fully vaccinated against SARS-CoV-2. Indeed, COVID-19 vaccination among IC persons has been found to be highly protective against COVID–19–associated hospitalization, leading to fewer hospitalized patients and deaths.[23] All surviving patients were able to resume treatment for their underlying disease a few weeks after SARS-CoV-2 viral clearance. Although the molecular analysis was performed only in four patients, it showed that affected viruses did not contain any recurrent mutation present in all samples. This suggests that in the 4 sequenced patients, there was no specific mutation pattern that could be associated with the reported long shedding or clinical severity. Although the analysis of a second-time point was possible in only two patients, the follow-up mutation profile of patients #1 and #4 was consistent with the observations of Leung.[2] Patient #4 had a lower number of new mutations than patient #1, considering that the interval period between the two sampling was 80 and 22 days, respectively (Table 3). The V445A variant of SARS-CoV-2 Spike was found in patient #1 at both time points. This mutation is located within the ACE2 receptor-binding domain (RBD; aa 438-506) and causes full resistance to imdevimab and bebtelovimab[24] and partial resistance to but did not induce immune evasion to casirivimab.[25] In the second sampling of patient #4, the additional S: K444N mutations within the RBD were reported, which reduces neutralization by bebtelovimab[26] and imdevimab. A S: E340Q baseline mutation was reported in patient #9, which causes resistance to sotrovimab.[27]
Our case series showed that in IC patients, the use of AV combined with passive immunotherapy (MoAbs or CCP) is safe and can be effective. Indeed, AV blocks viral replication, while MoAbs or CCP directed to the Spike protein can neutralize the ability of the virus to bind and fuse with the target host cell, reduce cytokine storm intensity in COVID-19 patients, and alleviate symptoms.[28] Finally, combined antiviral therapy can reduce or completely limit the emergence of drug-resistant mutations during prolonged sequential antiviral monotherapy and is superior to monotherapy in terms of viral clearance.[6,7,14,15,29]
The study acknowledges limitations inherent to its retrospective, single-center design and restricted sample size. Additionally, the small cohort hinders the ability to analyze the impact of specific variables like hematological disorder types or disease severity. Furthermore, whole genome sequencing data, offering a more comprehensive analysis of viral strains, was only available for a subset of patients.
Despite being a small case series, this study offers valuable insights into a critical gap: the underrepresentation of immunocompromised patients with HD in COVID-19 clinical trials. The findings suggest a potential link between active HD and higher mortality in IC COVID patients, even with mild symptoms. This underscores the importance of treating all IC COVID patients with HD and the need for further research on standardized combination therapies for this population. 

Author Contributions

Conceptualization, SV, EmG, AD'A; Data curation, PD, AB, PC; Funding acquisition, EN, CEMG; Investigation, GM, LS, VM, CP; Experiments, EmG, MR, and CEMG; Supervision, EN, DF and FM; Validation, EN, EG, FV; Writing-original draft, SV, EG, AD'A and EN; Writing-review and editing, AA, LS, MR, CEMG, FM, DF and EN. All authors contributed to the article and approved the submitted version. All authors have read and agreed to the published version of the manuscript.


This work was supported by Line1 Ricerca Corrente “Studio dei patogeni ad alto impatto sociale: emergent, da importazione, multiresistenti, negletti” funded by Italian Ministry of Health, and 5 per Mille- Progetto 5M-2020-23682104.

Institutional Review Board Statement

Since the retrospective nature of our data, ethical approval was not required.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The data presented in this study are available by request to the corresponding author.


Spallanzani COVID-19 Case Investigation Team: Tommaso Ascoli Bartoli, Nazario Bevilacqua, Angela Corpolongo, Ambrogio Curtolo, Francesca Faraglia, Maria Letizia Giancola, Gaetano Maffongelli, Claudia Palazzolo, Andrea Mariano, Silvia Rosati, Maria Virginia Tomassi.  


  1. Singson JRC, Kirley PD, Pham H, Rothrock G, Armistead I, Meek Jet al. Factors Associated with Severe Outcomes Among Immunocompromised Adults Hospitalized for COVID-19 - COVID-NET, 10 States, March 2020-February 2022. MMWR Morb Mortal Wkly Rep. 2022 July 8;71(27):878-884. PMid:35797216 PMCid:PMC9290380
  2. Leung WF, Chorlton S, Tyson J, Al-Rawahi GN, Jassem AN, Prystajecky N, et al. COVID-19 in an immunocompromised host: persistent shedding of viable SARS-CoV-2 and emergence of multiple mutations: a case report. Int J Infect Dis. 2022 Jan;114:178-182. PMid:34757008 PMCid:PMC8553657
  3. Choi B, Choudhary MC, Regan J, Sparks JA, Padera RF, Qiu X, et al. Persistence and Evolution of SARS-CoV-2 in an Immunocompromised Host. N Engl J Med. 2020 December 3;383(23):2291-2293. PMid:33176080 PMCid:PMC7673303
  4. D'Abramo A, Vita S, Maffongelli G, Beccacece A, Agrati C, Cimini E, et al. Clinical Management of Patients With B-Cell Depletion Agents to Treat or Prevent Prolonged and Severe SARS-COV-2 Infection: Defining a Treatment Pathway. Front Immunol. 2022 May 27;13:911339. PMid:35711444 PMCid:PMC9196078
  5. D'Abramo A, Vita S, Nicastri E. Correction: The unmet need for COVID-19 treatment in immunocompromised patients. BMC Infect Dis. 2023 January 31;23(1):61. Erratum for: BMC Infect Dis. 2022 December 12;22(1):930. PMid:36721126 PMCid:PMC9888749
  6. Orth HM, Flasshove, C, Berger M, Hattenhuauer T, Biederbick KD, Mispelbaum R et al. Early combination therapy of COVID-19 in high-risk patients. Infection. 2023; PMCid:PMC10955030
  7. D'Abramo A, Vita S, Beccacece A, et al. B-cell-depleted patients with persistent SARS-CoV-2 infection: combination therapy or monotherapy? A real-world experience. Front Med (Lausanne). 2024 February 29;11:1344267. PMid:38487021 PMCid:PMC10937561
  8. Vita S, Rosati S, Ascoli Bartoli T, Beccacece A, D'Abramo A, Mariano A, et al. Monoclonal Antibodies for Pre- and Postexposure Prophylaxis of COVID-19: Review of the Literature. Pathogens. 2022 Aug 5;11(8):882. PMid:36015003 PMCid:PMC9412407
  9. Berno G, Fabeni L, Matusali G, Gruber CEM, Rueca M, Giombini E, et al. SARS-CoV-2 Variants Identification: Overview of Molecular Existing Methods. Pathogens. 2022 Sep 17;11(9):1058. PMid:36145490 PMCid:PMC9504725
  10. Rueca M, Giombini E, Messina F, Bartolini B, Di Caro A, Capobianchi MR, et al. The Easy-to-Use SARS-CoV-2 Assem-bler for Genome Sequencing: Development Study. JMIR Bioinform Biotech. 2022 Mar 14;3(1):e31536. PMid:35309411 PMCid:PMC8924907
  11. Fu L, NiuB, Zhu Z, Wu S, & Li W. CD-HIT: accelerated for clustering the next-generation sequencing data. In Bioinformatics. 2012; (Vol. 28, Issue 23, pp. 3150-3152). Oxford University Press (OUP). PMid:23060610 PMCid:PMC3516142
  12. Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015 Jan;32(1):268-74. PMid:25371430 PMCid:PMC4271533
  13. Trøseid M, Hentzien M, Ader F, Cardoso SW, Arribas JR, Molina JM, et al.. Immunocompromised patients have been neglected in COVID-19 trials: a call for action. Clin Microbiol Infect. 2022; 28:1182-3. PMid:35623577 PMCid:PMC9130310
  14. Hirai J, Mori N, Sakanashi D, Ohashi W, Shibata Y, Asai N, et al. Real-World Experience of the Comparative Effectiveness and Safety of Combination Therapy with Remdesivir and Monoclonal Antibodies versus Remdesivir Alone for Patients with Mild-to-Moderate COVID-19 and Immunosuppression: A Retrospective Single-Center Study in Aichi, Japan [Internet]. Viruses. 2023; MDPI AG; 2023. p. 1952. PMid:37766358 PMCid:PMC10538070
  15. Calderón-Parra J, Gutiérrez-Villanueva A, Ronda-Roca G, Jimenez MLM, de la Torre H, Ródenas-Baquero M, et al. Efficacy and safety of antiviral plus anti-spike monoclonal antibody combination therapy vs. monotherapy for high-risk immunocompromised patients with mild-to-moderate SARS-CoV2 infection during the Omicron era: A prospective cohort study. Int J Antimicrob Agents. 2024 Mar;63(3):107095. PMid:38244814
  16. Mikulska M, Sepulcri C, Dentone C, Magne F, Balletto E, Baldi F, et al. Triple Combination Therapy With 2 Antivirals and Monoclonal Antibodies for Persistent or Relapsed Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Immunocompromised Patients [Internet]. Clinical Infectious Diseases. 2023; p. 280-6. PMid:36976301
  17. Brosh-Nissimov T, Ma'aravi N, Leshin-Carmel D, Edel Y, Ben Barouch S, Segman Y, et al. Combination treatment of persistent COVID-19 in immunocompromised patients with remdesivir, nirmaltrevir/ritonavir and tixegavimab/cilgavimab. J Microbiol Immunol Infect. 2024;57(1):189-194. PMid:37805361
  18. Vita S, D'Abramo A, Coppola A, Farroni C, Iori A P, Faraglia F,et al. Combined antiviral therapy as effective and feasible option in allogenic hematopoietic stem cell transplantation during SARS-COV-2 infection: a case report. Frontiers in Oncology. 2024;14.22 PMid:38414746 PMCid:PMC10896944
  19. Wölfel R, Corman VM, Guggemos W, Seilmaier M, Zange S, Müller MA, et al. Virological assessment of hospitalized patients with COVID-2019. Nature, 2020 May;581(7809):465-469. Epub 2020 April 1. Erratum in: Nature. 2020 ;588(7839):E35. PMid:32235945
  20. Singanayagam A, Patel M, Charlett A, Bernal JL, Saliba V, Ellis J et al. Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020. Euro Surveill. 2020;25. PMid:32794447 PMCid:PMC7427302
  21. Pagano L, Salmanton-García J, Marchesi F, Busca A, Corradini P, Hoenigl M, et al. COVID-19 infection in adult patients with hematological malignancies: a European Hematology Association Survey (EPICOVIDEHA). J Hematol Oncol. 2021;14(1):168. PMid:34649563 PMCid:PMC8515781
  22. Lahouati M, Cazanave C, Labadie A, Gohier P, Guirlé L, Desclaux A, et al; Bordeaux COVID-19 Treatment Group. Outcomes of targeted treatment in immunocompromised patients with asymptomatic or mild COVID-19: a retrospective study. Sci Rep. 2023 Sep 16;13(1):15357. PMid:37717101 PMCid:PMC10505186
  23. Farroni C, Aiello A, Picchianti-Diamanti A, Laganà B, Petruccioli E, Agrati C, et al. Booster dose of SARS-CoV-2 messenger RNA vaccines strengthens the specific immune response of patients with rheumatoid arthritis: A prospective multicenter longitudinal study. Int J Infect Dis. 2022;125:195-208. PMid:36328289 PMCid:PMC9622025
  24. Focosi D, Maggi F, Franchini M, McConnell S, Casadevall A. Analysis of Immune Escape Variants from Anti-body-Based Therapeutics against COVID-19: A Systematic Review. Int J Mol Sci. 2021 Dec 21;23(1):29. PMid:35008446 PMCid:PMC8744556
  25. Cox M, Peacock TP, Harvey WT, Hughes J, Wright DW; COVID-19 Genomics UK (COG-UK) Consortium et al. SARS-CoV-2 variant evasion of monoclonal antibodies based on in vitro studies. Nat Rev Microbiol. 2023;21(2):112-124. PMid:36307535 PMCid:PMC9616429
  26. last accessed February 9, 2024
  27. Andrés C, González-Sánchez A, Jiménez M, Márquez-Algaba E, Piñana M, Fernández-Naval C, et al. Emergence of Del-ta and Omicron variants carrying resistance-associated mutations in immunocompromised patients undergoing sotrovimab treatment with long-term viral excretion. Clin Microbiol Infect. 2022; S1198-743X(22)00458-X.
  28. Taylor PC, Adams AC, Hufford MM, de la Torre I, Winthrop K, Gottlieb RL. Neutralizing monoclonal antibodies for treatment of COVID-19. Nat Rev Immunol. 2021;21(6):382-393. PMid:33875867 PMCid:PMC8054133
  29. De Forni D, Poddesu B, Cugia G, Chafouleas J, Lisziewicz J, Lori F. Synergistic drug combinations designed to fully suppress SARS-CoV-2 in the lung of COVID-19 patients. PLoS One. 2022 Nov 10;17(11):e0276751. PMid:36355808 PMCid:PMC9648746