Increased incidence of abnormal TREC/ KREC levels is revealed in virtually healthy individuals over the post-pandemic period

The COVID-19 pandemic has posed an unprecedented challenge to global public health. Poor clinical outcomes in COVID-19 were associated with a pathogenic triad of interrelated mechanisms: immune dysregulation, coagulopathy, and secondary immunodeficiency, which collectively establish a selfperpetuating cycle of progressive multiorgan dysfunction. The failure of immune defense stems, in part, from T and B cell depletion due to immune resource exhaustion during acute infection. The extent of immune system impairment may be assessed by quantifying biomarkers of lymphocyte function: T cell receptor excision circles (TREC) and κ-deleting recombination excision circles (KREC) for T and B cells, respectively. The aim of our study was to evaluate the prevalence of abnormal TREC/KREC levels in peripheral blood among apparently healthy individuals during the post-pandemic period. We analyzed whole peripheral blood samples from 4,433 apparently healthy adults (≥ 18 years), residents of St. Petersburg and Leningrad Region. The samples were collected during two population studies: June 15-21, 2020 (n = 717) and September 4-29, 2023 (n = 3,716). TREC/KREC quantification was performed by means of real-time PCR with “TREC/KREC-AmpPS” commercial kit (St. Petersburg Pasteur Institute, St. Petersburg, Russia) following manufacturer’s protocol. The prevalence of reduced TREC/KREC levels increased significantly from pre-pandemic (14.2% and 5.7%, respectively) to post-pandemic periods (50.1% and 21.2%, respectively), thus representing a 3.6-fold increase. The mentioned differences in pathological biomarker levels were observed across all age groups. Characteristic immunological disturbances manifested as either isolated T cell deficiencies or combined T/B cell dysfunction, with no isolated B cell defects detected, thus suggesting the pivotal role of T cell impairment in observed immunopathological changes. Post-pandemic findings included decreased minimal population TREC values in older age groups and a paradoxical 50.5% increase in lower threshold KREC levels. Our findings demonstrate a substantially increased prevalence of abnormal TREC/KREC values, which seems to reflect long-term immune alterations following COVID-19. These changes underscore the critical need for the ongoing immunological surveillance in the post-pandemic era. Future research should focus on temporal dynamics of these disturbances and develop preventive strategies to mitigate long-term COVID-19 sequelae.

1. Arsentieva N.A., Liubimova N.E., Batsunov O.K., Korobova Z.R., Stanevich O.V., Lebedeva A.A., Vorobyov E.A., Vorobyova S.V., Kulikov A.N., Lioznov D.A., Sharapova M.A., Pevtcov D.E., Totolian Areg A. Plasma Cytokines in Patients with COVID-19 During Acute Phase of the Disease and Following Complete Recovery. Meditsinskaya Immunologiya = Medical Immunology (Russia), 2021, Vol. 23, no. 2, pp. 311-326. (In Russ.) doi: 10.15789/1563-0625-PCI-2312.

2. GOST R 53022.3-2008. Clinical laboratory technologies. Requirements for the quality of clinical laboratory studies. Part 3. Rules for assessing the clinical information content of laboratory tests. Moscow: Standartinform, 2009. 19 p.

3. Evgina S.A., Saveliev L.I. Current Theory and Practice of Reference Interval. Laboratornaya sluzhba = Laboratory Service, 2019, Vol. 8, no. 2, pp. 36-44. (In Russ.)

4. Zurochka A.V., Dobrynina M.A., Safronova E.A., Zurochka V.A., Zuikova A.A., Sarapultsev G.P., Zabkov O.I., Mosunov A.A., Verkhovskaya M.D., Ducardt V.V., Fomina L.O., Kostolomova E.G., Ostankova Y.V., Kudryavtsev I.V., Totolian A.A. Alterations in T Cell Immunity Over 6–12 Months Post-COVID-19 Infection in Convalescent Individuals: A Screening Study. Infektsiya i immunitet = Russian Journal of Infection and Immunity, 2024, Vol. 14, no. 4, pp. 756-768. (In Russ.) doi: 10.15789/2220-7619-AIT-17646.

5. Immunology According to Yarilin: Textbook. Ed. Nedospasov S.A., Kuprash D.V. 2nd ed., revised and enlarged. Moscow: GEOTAR-Media, 2021. 808 p.

6. Kudryavtsev I.V., Golovkin A.S., Totolian A.A. T helper cell subsets and related target cells in acute COVID-19. Infektsiya i immunitet = Russian Journal of Infection and Immunity, 2022, Vol. 12, no. 3, pp. 409-426. (In Russ.) doi: 10.15789/2220-7619-THC-1882.

7. Ostankova Yu.V., Saitgalina M.A., Arsentieva N.A., and Totolian A.A. Evaluation of TREC/KREC Levels in HIV-Infected Individuals. VICh-infektsiya i immunosupressii = HIV Infection and Immunosuppressive Disorders, 2024, Vol. 16, no. 2, pp. 51-59. (In Russ.)

8. Popova A.Yu., Gorbunova A.Yu., Ostankova Yu.V., Egorova S.A., Reingardt D.E., Ivanova A.R., Schemelev A.N., Drozd I.V., Zhimbaeva O.B., Danilova E.M., Milichkina A.M., Ezhlova E.B., Melnikova A.A., Bashketova N.S., Buts L.V., Totolian Areg A. Herd Immunity to Hepatitis A Virus in the Saint Petersburg and Leningrad Region. Meditsinskaya Immunologiya = Medical Immunology (Russia), 2025, Vol. 27, no. 3, pp. 625-642. (In Russ.) doi: 10.15789/1563-0625-HIT-3224.

9. Popova A.Yu., Egorova S.A., Smirnov V.S., Ezhlova E.B., Milichkina A.M., Melnikova A.A., Bashketova N.S., Istorik O.A., Buts L.V., Ramsay E.S., Drozd I.V., Zhimbaeva O.B., Drobyshevskaya V.G., Danilova E.M., Ivanov V.A., and Totolian A.A. Herd Immunity to Vaccine Preventable Infections in Saint Petersburg and the Leningrad Region: Serological Status of Measles, Mumps, and Rubella. Infektsiya i immunitet = Russian Journal of Infection and Immunity, 2024, Vol. 14, no. 6, pp. 1187-1208. (In Russ.) doi: 10.15789/2220-7619-HIT-17797.

10. Popova A.Yu., Ezhlova E.B., Mel’nikova A.A., Bashketova N.S., Fridman R.K., Lyalina L.V., Smirnov V.S., Chkhindzheriya I.G., Grechaninova T.A., Agapov K.A., Arsent’eva N.A., Bazhenova N.A., Batsunov O.K., Danilova E.M., Zueva E.V., Komkova D.V., Kuznetsova R.N., Lyubimova N.E., Markova A.N., Khamitova I.V., Lomonosova V.I., Vetrov V.V., Milichkina A.M., Dedkov V.G., and Totolyan A.A. Herd Immunity to SARS-CoV-2 Among the Population in Saint-Petersburg During the COVID-19 Epidemic. Problemy osobo opasnykh infektsiy = Problems of Particularly Dangerous Infections, 2020, no. 3, pp. 124-130. (In Russ.)

11. Saitgalina M.A., Liubimova N.E., Ostankova Yu.V., Kuznetzova R.N., and Totolian A.A. Determination of Reference Values for TREC and KREC in Circulating Blood of Persons Over 18 Years. Meditsinskaya Immunologiya = Medical Immunology (Russia), 2022, Vol. 24, no. 6, pp. 1227-1236. (In Russ.) doi: 10.15789/1563-0625-DOR-2587.

12. Saitgalina M.A., Ostankova Yu.V., Arsentieva N.A., Korobova Z.R., Lyubimova N.E., Kashchenko V.A., Kulikov A.N., Pevtsov D.E., Stanevich O.V., Chernykh E.I., Totolian Areg A. Levels of TREC and KREC Molecules Significance Determining in Peripheral Blood for Predicting the Outcome of COVID-19 Disease in the Acute Period. Rossiiskii immunologicheskii zhurnal = Russian Journal of Immunology, 2023, Vol. 26, no. 4, pp. 611-618. (In Russ.) doi: 10.46235/1028-7221-14714-LOT.

13. Saitgalina M.A., Ostankova Y.V., Arsentieva N.A., Korobova Z.R., Liubimova N.E., Kashchenko V.A., Kulikov A.N., Pevtsov D.E., Stanevich O.V., Chernykh E.I., Totolian A.A. Assessment of trec and krec levels in COVID-19 patients with varying disease severity. Infektsiya i immunitet = Russian Journal of Infection and Immunity, 2023, Vol. 13, no. 5, pp. 873-884. (In Russ.) doi: 10.15789/2220-7619-AOT-16937.

14. Saitgalina M.A., Ostankova Y.V., Liubimova N.E., Semenov A.V., Kuznetsova R.N., Totolian A.A. Modified quantitative approach for assessing peripheral blood TREC and KREC levels in immunodeficient patients. Infektsiya i immunitet = Russian Journal of Infection and Immunity, 2022, Vol. 12, no. 5, pp. 981-996. (In Russ.) doi: 10.15789/2220-7619-MMF-2039.

15. Al-Aly Z., Xie Y., Bowe B. High-dimensional characterization of post-acute sequelae of COVID-19. Nature, 2021, Vol. 594, no. 7862, pp. 259-264.

16. Alijotas-Reig J., Anunciacion-Llunell A., Esteve-Valverde E., Morales-Pérez S., Rivero-Santana S., Trapé J., González-García L., Ruiz D., Marques-Soares J., Miro-Mur F. Pituitary-Adrenal Axis and Peripheral Immune Cell Profile in Long COVID. Biomedicines, 2024, Vol. 12, no. 3, 581. doi: 10.3390/biomedicines12030581.

17. Bergwerk M., Gonen T., Lustig Y., Amit S., Lipsitch M., Cohen C., Mandelboim M., Levin E.G., Rubin C., Indenbaum V., Tal I., Zavitan M., Zuckerman N., Bar-Chaim A., Kreiss Y., Regev-Yochay G. Covid-19 Breakthrough Infections in Vaccinated Health Care Workers. N. Engl. J. Med., 2021, Vol. 385, no. 16, pp. 1474-1484.

18. Bowe B., Xie Y., Al-Aly Z. Postacute sequelae of COVID-19 at 2 years. Nat. Med., 2023, Vol. 29, no. 9, pp. 2347-2357.

19. Brown J.J., Datta V., Browning M.J., Swift P.G.F. Graves’ disease in DiGeorge syndrome: patient report with a review of endocrine autoimmunity associated with 22q11.2 deletion. J. Pediatr. Endocrinol. Metab., 2004, Vol. 17, pp. 1575-1579.

20. Dasouki M., Jabr A., AlDakheel G., Elbadaoui F., Alazami A.M., Al-Saud B., Arnaout R., Aldhekri H., Alotaibi I., Al-Mousa H., Hawwari A. TREC and KREC profiling as a representative of thymus and bone marrow output in patients with various inborn errors of immunity. Clin. Exp. Immunol., 2020, Vol. 202, no. 1, pp. 60-71.

21. Davis H.E., McCorkell L., Vogel J.M., Topol E.J. Long COVID: major findings, mechanisms and recommendations. Nat. Rev.Microbiol., 2023, Vol. 21, no. 3, pp. 133-146.

22. De Ioris M.A., Scarselli A., Bracaglia C., Perrotta D., Bernardi S., Santilli V., Ceglie G., Fabozzi F., Agrati C., Prencipe G., Alaggio R., Mastronuzzi A., De Vito R. Common bone marrow signature in COVID-19-associated multisystem inflammatory syndrome in children: A first-wave small case series experience. Pediat. Blood Cancer, 2022, Vol. 69, no. 11, e29919. doi: 10.1002/pbc.29919.

23. Files J.K., Boppana S., Perez M.D., Sarkar S., Lowman K.E., Qin K., Sterrett S., Carlin E., Bansal A., Sabbaj S., Long D.M., Kutsch O., Kobie J., Goepfert P.A., Erdmann N. Sustained Cellular Immune Dysregulation in Individuals Recovering From SARS-CoV-2 Infection. J. Clin. Invest., 2021, Vol. 131, no. 1, 140491. doi: 10.1172/JCI140491

24. García-Abellán J., Padilla S., Fernández-González M., García J.A., Agulló V., Andreo M., Ruiz S., Galiana A., Gutiérrez F., Masiá M. Antibody Response to SARS-CoV-2 Is Associated With Long-Term Clinical Outcome in Patients With COVID-19: A Longitudinal Study. J. Clin. Immunol., 2021, Vol. 41, no. 7, pp. 1490-1501.

25. Gonçalves R., Couto J., Ferreirinha P., Costa J.M., Silvério D., Silva M.L., Fernandes A.I., Madureira P., Alves N.L., Lamas S., Saraiva M. SARS-CoV-2 variants induce distinct disease and impact in the bone marrow and thymus of -mice. iScience, 2023, Vol. 26, no. 2, 105972. doi: 10.1016/j.isci.2023.105972.

26. Gong F., Dai Y., Zheng T., Cheng L., Zhao D., Wang H., Liu M., Pei H., Jin T., Yu D., Zhou P. Peripheral CD4+ T Cell Subsets and Antibody Response in COVID-19 Convalescent Individuals. J. Clin. Invest., 2020, Vol. 130, no. 12, pp. 6588-6599.

27. Hu B., Huang S., Yin L. The cytokine storm and COVID-19. J. Med. Virol., 2021, Vol. 93, no. 1, pp. 250-256.

28. Ioannou M., Zacharouli K., Doukas S.G., Diamantidis M.D., Tsangari V., Karakousis K., Koukoulis G.K., Vageli D.P. Hemophagocytic lymphohistiocytosis diagnosed by bone marrow trephine biopsy in living postCOVID-19 patients: case report and mini-review. J. Mol. Histol., 2022, Vol. 53, no. 4, pp. 753-762.

29. Khadzhieva M.B., Kalinina E.V., Larin S.S., Sviridova D.A., Gracheva A.S., Chursinova J.V., Stepanov V.A., Redkin I.V., Avdeikina L.S., Rumyantsev A.G., Kuzovlev A.N., Salnikova L.E. TREC/KREC Levels in Young COVID-19 Patients. Diagnostics, 2021, Vol. 11, no. 8, 1486. doi: 10.3390/diagnostics11081486.

30. Korobova Z.R., Arsentieva N.A., Butenko A.A., Kudryavtsev I.V., Rubinstein A.A., Turenko A.S., Ostankova Y.V., Boeva E.V., Knizhnikova A.A., Norka A.O., Rassokhin V.V., Belyakov N.A., Totolian A.A. T Cell Dynamics in COVID-19, Long COVID and Successful Recovery. Int. J. Mol. Sci., 2025, Vol. 26, no. 15, 7258. doi: 10.3390/ijms26157258.

31. Korobova Z.R., Zueva E.V., Arsentieva N.A., Batsunov O.K., Liubimova N.E., Khamitova I.V., Kuznetsova R.N., Rubinstein A.A., Savin T.V., Stanevich O.V., Kulikov A.N., Pevtsov D.E., Totolian A.A. Changes in Anti-SARS-CoV-2 IgG Subclasses over Time and in Association with Disease Severity. Viruses, 2022, Vol. 14, no. 5, 941. doi: 10.3390/v14050941.

32. Kwiecień I., Rutkowska E., Kłos K., Więsik-Szewczyk E., Jahnz-Różyk K., Rzepecki P., Chciałowski A. Maturation of T and B Lymphocytes in the Assessment of the Immune Status in COVID-19 Patients. Cells, 2020, Vol. 9, no. 12, 2615. doi: 10.3390/cells9122615.

33. Kwok J.S.Y., Cheung S.K.F., Ho J.C.Y., Tang I.W.H., Chu P.W.K., Leung E.Y.S., Lee P.P.W., Cheuk D.K.L., Lee V., Ip P., Lau Y.L. Establishing Simultaneous T Cell Receptor Excision Circles (TREC) and K-Deleting Recombination Excision Circles (KREC) Quantification Assays and Laboratory Reference Intervals in Healthy Individuals of Different Age Groups in Hong Kong. Front. Immunol., 2020, Vol. 11, 1411. doi: 10.3389/fimmu.2020.01411.

34. Li M., Guo W., Dong Y., Wang X., Dai D., Liu X., Wu Y., Li M., Zhang W., Zhou H., Zhang Z., Lin L., Kang Z., Yu T., Tian C., Qin R., Gui Y., Jiang F,. Fan H., Heissmeyer V., Sarapultsev A., Wang L., Luo S., Hu D. Elevated Exhaustion Levels of NK and CD8+ T Cells as Indicators for Progression and Prognosis of COVID-19 Disease. Front. Immunol., 2020, Vol. 11, 580237. doi: 10.3389/fimmu.2020.580237.

35. Malkova A., Kudryavtsev I., Starshinova A., Kudlay D., Zinchenko Y., Glushkova A., Yablonskiy P., Shoenfeld Y. Post COVID-19 Syndrome in Patients With Asymptomatic/Mild Form. Pathogens, 2021, Vol. 10, no. 11, 1408. doi: 10.3390/pathogens10111408.

36. Mehta P., McAuley D.F., Brown M., Sanchez E., Tattersall R.S., Manson J.J.; HLH Across Specialty Collaboration, UK. COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet, 2020, Vol. 395, no. 10229, pp. 1033-1034.

37. Middelkamp V., Kekäläinen E. Measuring thymic output across the human lifespan: a critical challenge in laboratory medicine. GeroScience, 2025. doi: 10.1007/s11357-025-01555-3.

38. Ochani R., Asad A., Yasmin F., Shaikh S., Khalid H., Batra S., Sohail M.R., Mahmood S.F., Ochani R., Hussham Arshad M., Kumar A., Surani S. COVID-19 pandemic: from origins to outcomes. A comprehensive review of viral pathogenesis, clinical manifestations, diagnostic evaluation, and management. Infez. Med., 2021, Vol. 29, no. 1, pp. 20-36.

39. Quinti I., Soresina A., Spadaro G., Martino S., Donnanno S., Agostini C. Long-term follow-up and outcome of a large cohort of patients with common variable immunodeficiency. J. Clin. Immunol., 2007, Vol. 27, no. 3, pp. 308-316.

40. Rossetti C.L., Cazarin J., Hecht F., Beltrão F.E.L., Ferreira A.C.F., Fortunato R.S., Ramos H.E., de Carvalho D.P. COVID-19 and thyroid function: what do we know so far? Front. Endocrinol., 2022, Vol. 13, 1041676. doi: 10.3389/fendo.2022.1041676.

41. Savchenko A.A., Tikhonova E., Kudryavtsev I., Kudlay D., Korsunsky I., Beleniuk V., Borisov A. TREC/KREC Levels and T and B Lymphocyte Subpopulations in COVID-19 Patients at Different Stages of the Disease. Viruses, 2022, Vol. 14, no. 3, 646. doi: 10.3390/v14030646.

42. Sekine T., Perez-Potti A., Rivera-Ballesteros O., Strålin K., Gorin J.-B., Olsson A., Llewellyn-Lacey S., Kamal H., Bogdanovic G., Muschiol S., Wullimann D.J., Kammann T., Emgård J., Parrot T., Folkesson E.; Karolinska COVID-19 Study Group; Rooyackers O., Eriksson L.I., Henter J.-I., Sönnerborg A., Allander T., Albert J., Nielsen M., Klingström J., Gredmark-Russ S., Björkström N.K., Sandberg J.K., Price D.A., Ljunggren H.-Gu., Aleman S., Buggert M. Robust T Cell Immunity in Convalescent Individuals With Asymptomatic or Mild COVID-19. Cell, 2020, Vol. 183, no. 1, pp. 158-168.e14.

43. Shakerian L., Pourpak Z., Shamlou S., Domsgen E., Kazemnejad A., Dalili H., Nourizadeh M. Determining Laboratory Reference Values of TREC and KREC in Different Age Groups of Iranian Healthy Individuals. Iran. J. Allergy Asthma Immunol., 2019, Vol. 18, no. 2, pp. 143-152.

44. Sottini A., Serana F., Bertoli D., Chiarini M., Valotti M., Vaglio Tessitore M., Imberti L. Simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) by real-time PCR. J. Vis. Exp., 2014, Vol. 94, 52184. doi: 10.3791/52184.

45. Sudre C.H., Murray B., Varsavsky T., Graham M.S., Penfold R.S., Bowyer R.C., Pujol J.C., Klaser K., Antonelli M., Canas L.S., Molteni E., Modat M., Cardoso M.J., May A., Ganesh S., Davies R., Nguyen L.H., Drew D.A., Astley C.M., Joshi A.D., Merino J., Tsereteli N., Fall T., Gomez M.F., Duncan E.L., Menni C., Williams F.M.K., Franks P.W., Chan A.T., Wolf J., Ourselin S., Spector T., Steves C.J. Attributes and Predictors of Long COVID. Nat. Med., 2021, Vol. 27, no. 4, pp. 626-631.

46. Thoo L., Gumowski P.I., Kammermann K., Nussli S., Grabscheid B., Hausmann O., Axius U., Pichler W.J., Yerly D. Highly Specific and Reliable In Vitro Diagnostic Analysis of Memory T and B Lymphocytes in a Swiss Cohort of COVID-19 Patients. Swiss Med. Wkly, 2021, Vol. 151, w30005. doi: 10.4414/smw.2021.w30005

47. Wiech M., Chroscicki P., Swatler J., Stepnik D., De Biasi S., Hampel M., Brewinska-Olchowik M., Maliszewska A., Sklinda K., Durlik M., Wierzba W., Cossarizza A., Piwocka K. Remodeling of T Cell Dynamics During Long COVID Is Dependent on Severity of SARS-CoV-2 Infection. Front. Immunol., 2022, Vol. 13, 886431. doi: 10.3389/fimmu.2022.886431.

48. Woodruff M.C., Ramonell R.P., Haddad N.S., Anam F.A., Rudolph M.E., Walker T.A., Truong A.D., Dixit A.N., Han J.E., Cabrera-Mora M., Runnstrom M.C., Bugrovsky R., Hom J., Connolly E.C., Albizua I., Javia V., Cashman K.S., Nguyen D.C., Kyu S., Singh Saini A., Piazza M., Tipton C.M., Khosroshahi A., Gibson G., Martin G.S., Maier C.L., Esper A., Jenks S.A., Lee F.E., Sanz I. Dysregulated Naive B Cells and De Novo Autoreactivity in Severe COVID-19. Nature, 2022, Vol. 611, no. 7934, pp. 139-147.

49. World Health Organization. Timeline: WHO’s COVID-19 response. Available at: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/interactive-timeline.

50. Wu Q., Thakolwiboon S., Ali A.S., Wang Q., Dwyer D., Fox D.A., Mao-Draayer Y. MOG-specific T cell response amplified in para- and post-SAR-CoV-2 infection in myelin oligodendrocyte glycoprotein antibodyassociated disease. J. Neuroimmunol., 2023, Vol. 382, 578143. doi: 10.1016/j.jneuroim.2023.578143.

51. Xu C.S., Yang W.X. ACE2 in male genitourinary and endocrine systems: does COVID-19 really affect these systems? Histol. Histopathol., 2023, Vol. 38, no. 3, pp. 261-272.