Immunosuppressive therapy reduction and early post-infection graft function in kidney transplant recipients with COVID-19

Abstract

Background: Kidney transplant (KT) recipients with COVID-19 are at high risk of poor outcomes due to the high burden of comorbidities and immunosuppression. The effects of immunosuppressive therapy (IST) reduction are unclear in patients with COVID-19.
Methods: A retrospective study on 45 KT recipients followed at the University Hospital of Modena (Italy) who tested positive for COVID-19 by RT-PCR analysis.
Results: The median age was 56.1 years (interquartile range,[IQR] 47.3-61.1), with a predominance of males (64.4%). Kidney transplantation vintage was 10.1 (2.7-16) years, and 55.6 % of patients were on triple IST before COVID-19. Early immunosuppression minimization occurred in 27 (60%) patients (reduced-dose IST group) and included antimetabolite (88.8%) and calcineurin inhibitor withdrawal (22.2%). After SARS-CoV-2 infection, 88.9% of patients became symptomatic and 42.2% required hospitalization. One patient experienced irreversible graft failure. There were no differences in serum creatinine level and proteinuria in non-hospitalized patients before and post-COVID-19, whereas hospitalized patients experienced better kidney function after hospital discharge (P=0.019). Overall mortality was 17.8%. without differences between full- and reduced-dose IST. Risk factors for death were age (odds ratio [OR]: 1.19; 95%CI: 1.01-1.39), and duration of kidney transplant (OR: 1.17; 95%CI: 1.01-1.35). One KT recipient developed IgA glomerulonephritis and two ones experienced symptomatic COVID-19 after primary infection and SARS-CoV-2 mRNA vaccine, respectively.
Conclusions: Despite the reduction of immunosuppression, COVID-19 affected the survival of KT recipients. Age of patients and time elapsed from kidney transplantation were independent predictors of death . Early kidney function was favorable in most survivors after COVID-19.

Keywords: COVID-19, kidney transplant, immunosuppressive therapy, graft function, proteinuria, mortality, transplant, SARS-COV-2, reinfection

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Introduction

Since SARS CoV-2 infection was first identified in December 2019, the pandemic spread quickly around the world, with a disruptive impact on social and economic life. This virus yielded several new challenges to our healthcare systems that had to cope with an increased rate of morbidity and mortality among the most vulnerable populations [1]. Kidney transplant (KT) recipients are a subset of the population at high risk of severe COVID-19 due to the high burden of comorbidities and the cumulative side effects of immunosuppressive therapy (IST) [2]. Data collected so far show that transplant recipients are extremely susceptible to the SARS-CoV-2 infection, much more than the general population [3, 4]. The causes are multiple, but principally revolve around the use of long-term IST.

Despite the great emphasis on early IST reduction to face the potentially lethal consequences of COVID-19, no confirming data supports its beneficial effect in terms of survival or clinical manifestations. Additional uncertainty arises from the recent literature reporting that a tempered immune response is thought to prevent COVID‐19–induced systemic inflammatory syndrome. To date, data regarding early graft outcomes after COVID-19 are scarce [5]. It is worth noting that graft survival may be threatened by non-reversible episodes of kidney injury [6, 7]. Lastly, a concerning issue may be the hyporesponsiveness to anti-SARS-CoV-2 vaccination [8, 9]. Numerous studies have confirmed that KT recipients have a blunted immune response to mRNA vaccines [10]. Only 48% of patients were able to develop a protective serologic response to SARS-CoV-2 [11]. Caillard et al [12] reported that about one-third of kidney transplant patients had severe manifestations, including a fatal outcome, despite COVID-19 vaccination. This group of patients is therefore expected to remain vulnerable to the severe complications of COVID-19 until new strategies will be implemented to reduce the susceptibility of these subjects.

Considering all the uncertainties in the management of KT recipients and the high risk of severe COVID-19 manifestations within this cohort of patients, we report our experience in managing KT recipients with COVID-19. In particular, we focus on the impact of early IST reduction, and early graft function after the resolution of the infection.

 

Material and methods

Kidney transplant outpatient clinic

This kidney transplant outpatient clinic follows more than 500 KT recipients, including combined liver and pancreas-kidney transplantation. Outpatient service was delivered by a senior nephrologist with experience in kidney transplantation, one fellow and three nurses. A 24-h, 7/7 days per week service was available for KT recipients in case of kidney-related pathologic processes (anuria, fluid overload) or infections. This service was also offered to the subjects transplanted in our Center but living far away from it.

During COVID-19 all the patients were instructed to call the clinic in case of COVID-19 symptoms. Despite the reduction of non-essential healthcare services, our outpatient clinic continued to deliver care to KT recipients, adopting all the containment measures (triage at entry, masking, social distancing and hands hygiene) to prevent COVID-19 diffusion. A telephonic triage was performed for all patients before reaching the hospital to intercept paucisymptomatic patients.

Patients with symptoms were invited to perform nasal swabs using RT-PCR and were visited in a dedicated room to assess vital parameters and clinical conditions. According to the severity of the symptoms, patients were sent home or to the emergency room. To reduce the workload of the emergency room, patients were managed as outpatients unless they developed severe symptoms that required hospital admission. The monitoring of noncritical patients was mostly performed via phone calls and emails.

According to our internal protocol and taking into account the opinions of European experts [13, 14], immunosuppression was modulated as follow:

  • for asymptomatic or mild COVID-19 patients (i.e., mild upper respiratory and/or gastrointestinal symptoms, temperature <38°C without dyspnea) in triple therapy (calcineurin-inhibitors [CNI] + mycophenolate acid [MPA]/azathioprine [AZA] + steroids), MPA or AZA was withdrawn, and a dual therapy (CNI + steroid) was continued. If the patients were on dual therapy (CNI + mammalian target of rapamycin inhibitor [mTOR-i] or CNI + MPA), MPA/mTOR was withdrawn and replaced with a low dose of steroids (i.e., methylprednisolone 4 or 8 mg once-daily).
  • for moderate (signs and symptoms of lower respiratory disease or saturation of oxygen [SpO2] ≥94% on room air at sea level) and severe COVID-19 (SpO2 <94% on room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen [PaO2/FiO2] <300 mm Hg, respiratory frequency >30 breaths per minute, or lung infiltrates >50%) all immunosuppressors, but steroids, were stopped. The prescription of anti-inflammatory and immunomodulant steroid therapy for symptomatic COVID-19 patients (dexamethasone at a dose of 6 mg once daily for up to 10 days) was not part of the anti-rejection therapy and was administered by COVID-19 experts.

COVID-19 population

The study population was comprised of kidney transplant recipients with COVID-19 with a complete follow-up, including death or discharge from hospital.

We retrospectively reviewed the electronic charts of all KT recipients with COVID-19 from March 7, 2020, to June 25, 2021. During this period we performed 144 nasopharyngeal swabs. The diagnosis of COVID-19 was performed through reverse transcriptase-polymerase chain reaction (RT-PCR) assay on a nasopharyngeal swab. We excluded patients aged <18 years. Kidney function was estimated by glomerular fraction rate (eGFR) using the CKD-EPI equation. Occasionally, some data were missing for patients admitted to a hospital located far from our Center.

This study has been authorized by the local Ethical Committee of Emilia Romagna (n. 839/2020). The study protocol complies with the guidelines for human studies and includes evidence that the research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki.

Statistical analysis

Baseline characteristics were described using median (interquartile range [IQR]) or frequencies, as appropriate. The chi-square or Fisher’s test, and student’s t-test were used to compare categorical and continuous variables between groups, respectively. Univariate and multivariate logistic regressions were performed to test the association between mortality and baseline patient characteristics. Variables that were significant on univariate analysis (P=<0.05) were entered into the multivariate model to identify independent predictors. Results were expressed as odds ratios (OR) and 95% confidence intervals (CI). Univariate and multivariate logistic regression analysis determined risk factors for death. A P value of <0.05 was considered statistically significant. All statistical analyses were performed using SPSS® statistical software.

 

Results

Characteristics of COVID-19 population

From the beginning of the COVID-19 pandemic in Italy, 45 KT recipients followed in our center contracted COVID-19. The demographic and clinical characteristics of these patients are detailed in Table I. This group of patients included two (4.4%) combined liver-kidney and one (2.2%) heart-kidney transplant recipient. Seven (15.5%) patients were hospitalized in another structure because they lived far from our Center.

Variable All patients
(n.=45)
Reduced-dose IST
(n.=27)
Full-dose IST
(n.=18)
p-value
Age, year 56.1 (47.3-61.1) 55.9 (47.6-61.2) 56.1 (44.4-62) 0.85
Range 19.2-83.5 19.2-79.8 28.1-83.5
Males, n. (%) 29 (64.4) 18 (66.7) 110 (61.1) 0.75
Race/ethnicity 0.61
White, n. (%) 41 (91.1) 26 (92.6) 16 (88.9
Black, n. (%) 4 (8.9) 2 (7.4) 2 (11.1)
Transplant vintage, year 10.1 (2.7-16.01) 7.8 (2.4-15.2) 11.1 (4.7-21.1) 0.29
sCr pre-COVID-19, mg/dl 1.45 (1.18-1.84) 1.44 (1.18-1.81) 1.28 (1.14-1.82) 0.68
eGFR pre-COVID-19, ml/min 48.4 (36-64) 47.7 (35-64) 49.5 (38.6-67.9) 0.83
24-h proteinuria, mg/dl 87.4 (0.52-188.5) 72 (0.25-183) 145.5 (6.2-205) 0.69
Immunosuppressive therapy, n. (%)
CNI 39 (86.7) 24 (88.9) 15 (83.3) 0.67
mTOR-i 8 (17.8) 4 (14.8) 4 (22.2) 0.69
MPA 31 (68.9) 24 (88.9) 7 (38.9) 0.01
Steroid 36 (80) 23 (85.2) 13 (72.2) 0.44
IS regimen 0.001
Triple therapy 25 (55.6) 21 (77) 4 (22.2)
Double therapy 19 (42.2) 6 (22.2) 13 (72.2)
Monotherapy 1 (2.2) 0 (0) 1 (5.6)
Reduction IS therapy, n. (%) 27 (60) 27 (100) 0 (0) N/A
MPA withdrawal 24 (53.3) 24 (88.9) 0 (0) N/A
CNI or mTOR-i withdrawal 6 (13.3) 6 (22.2) 0 (0) N/A
Increase steroid 9 (5,4) 8 (29.6) 1 (5.6) 0.064
Comorbidities, n. (%)
HIV, HCV or HBV 6 (13.3) 3 (11.1) 3 (16.7) 0.65
Diabetes 5 (11.1) 4 (14.8) 1 (5.6) 0.63
Neoplasia 10 (22.2) 7 (25.9) 3 (16.7) 0.71
Graft rejection 4 (8.9) 1 (3.7) 3 (16.7) 0.13
CVD 12 (26.7) 7 (25.9) 4 (22.2) 77
Autoimmune disease 4 (8.9) 1 (3.7) 3 (16.7) 0.13
Previous severe infection 13 (28.9) 8 (29.6) 5 (27.7) 1
Symptomatic COVID-19, n. (%) 40 (88.9) 27 (100) 13 (72.2) 0.45
Hospitalization, n. (%) 19 (42.2) 14 (51.9) 5 (27.8) 0.13
Graft failure, n. (%) 1 (2.2) 1 (3.7) 0 (0) 1
ICU admission, n. (%) 9 (20) 4 (14.8) 5 (27.8) 0.28
Mortality, n (%) 8 (17.8) 4 (14.8) 4 (22.2) 0.69
Post-COVID-19 follow-up, day 70.5 (51-109) 76  (50.5-116.5) 69 (66-76) 0.57
Notes: eGFR denotes estimated glomerular filtration rate; CNI, calcineurin inhibitor; CVD, cardiovascular disease; HCV, hepatitis C; HBV, hepatitis B; IST, immunosuppressive therapy; MPA, mycophenolate acid; mTOR-I, mammalian target of rapamycin inhibitor; sCr, serum creatinine.
Table I:Demographics and clinical characteristics of KT recipients

The age of patients ranged from 19.2 to 83.5 years and the median was 56.1 (IQR, 47.3-61.1) years. COVID-19 was more prevalent in males than in females (64.4% vs 35.6%) and occurred after a median of 10.1 (2.7-16.01) years from transplantation.

Before the COVID-19 infection, serum creatine (sCr) was 1.45 (IQR 1.1-1.8) mg/dl corresponding to a median eGFR of 48.4 (IQR 36-64) ml/min. At the time of the COVID-19 diagnosis, more than half of the patients were in triple standard IST. Forty patients (88.9%) developed symptoms of COVID-19 and 19 of them (42.2%) required hospitalization. One patient returned to dialysis following acute kidney injury. Overall, nine patients (20%) were admitted to ICU for severe manifestations of COVID-91 and eight (17.8%) died.

Reduced- vs full-dose IST group

The entire population was subdivided into two groups: reduced-dose (n.=27; 60%) and full-dose IST (n.=18; 40%). There were no significative statistical differences in terms of demographic and clinical characteristics between the two groups. Statistical analysis detected significant differences in the prescription of IST. Patients who underwent reduction of immunosuppression (reduced-dose IST) were treated with a higher dose of IST before COVID-19; indeed, the rate of prescribed triple-drug IST was higher in this group than in full-dose IST patients (77% vs. 22.2%; P=<0.001).

In the reduced-dose IST group, MPA (88.8%) and CNI or mTOR-i (22.2%) were the most frequent discontinued agents. Conversely, the dose of steroids was increased in a third of patients and, in all of them, the administration of steroids changed from alternate days (methylprednisolone 2/0 or 4/0) to a daily regimen.

Hospitalization, ICU admission and death rate in patients who underwent IST reduction were 51.8%, 14.8% and 14.8%, respectively. However, despite IST reduction, hospitalization (P=0.13), ICU admission (P=0.28) and death (P=0.69) rates were not different from those of the full-dose IST group.

Outcomes of KT recipients with COVID-19

Univariate and multivariate logistic regression was performed to detect predictors of mortality (Table II). Multivariate analysis found that age (OR=1.19 [95%CI 1.01-1.39]; P=0.034) and years spent on immunosuppressive therapy (OR=1.17 [95%CI 1.01-1.35]; P=0.040) were associated with mortality in this group of patients.

Univariate Multivariate
Variable OR CI (95%) p-value OR CI (95%) p-value
Sex
Male 4.40 0.78 24.81 0.09  
Age (1-yr increase) 1.11 1.02 1.22 0.016 1.19 1.01 1.39 0.034
KT vintage (1-yr increase) 1.10 1.00 1.21 0.053 1.17 1.01 1.35 0.040
Steroid-based IST 1.93 0.21 18.08 0.56
Reduction IST 1.33 0.26 6.869 0.74
Increase of steroid 0.52 0.06 4.85 0.56
Triple IST 0.51 0.10 2.620 0.42
Double IST 1.96 0.38 10.026 0.42
GFR 0.99 0.95 1.026 0.57
GFR< 45ml/min 1.47 0.32 6.80 0.62
GFR 45-59 ml/min 0.68 0.15 3.16 0.62
sCr 1,33 0,26 6.87 0.73
Graft rejection 1.52 0.14 16.91 0.73
Autoimmune disease 0.00 0.00 0.99
HIV/HCV/HBV 2.58 0.38 17.43 0.33
Previous sever infection 0,73 0.13 4.19 0.72
Diabetes 1.11 0.11 11.49 0.93
Neoplasm 1.12 0.19 6.70 0.89
Cardiovascular disease 1.73 0.34 8.76 0.50
Notes: eGFR denotes estimated glomerular filtration rate; HCV, hepatitis C; HBV, hepatitis B; IST, immunosuppressive therapy; MPA, mycophenolate acid; mTOR-I, mammalian target of rapamycin inhibitor; sCr, serum creatinine.
Table II: Univariate and multivariate predictors of mortality through logistic regression analysis

Among the survivors (82.2%), one patient with a CKD stage 4 (GFR=20 ml/min) before SARS-CoV-2 infection developed irreversible graft failure requiring HD. One patient (2.7%) manifested de-novo proteinuria (4100 mg/die) after the resolution of COVID-19 and graft biopsy revealed IgA glomerulonephritis (the lack of data on the cause of CKD did not allow us to classify these histological findings as either de-novo or recurrent IgA glomerulonephritis). Lastly, one patient experienced symptomatic COVID-19 reinfection after the primary infection and another one following the SARS-CoV-2 mRNA vaccine. Early post-COVID-19 follow-up of 25 out of the 37 survivors showed that pre- and post-COVID variations of sCr, eGFR and 24-hour proteinuria were not statistically significant in outpatients after the resolution of COVID-19. A significantly lower sCr level (P=0.019) and eGFR (P=0.028) were measured after hospital discharge in hospitalized patients. No differences were noted in the level of daily proteinuria (Table III). The early follow-up of KT recipients after COVID-19 resolution did not show any new episodes of graft rejection.

Non-hospitalized patients Hospitalized patients
Pre-COVID-19 Post-COVID-19 p-value Pre-COVID-19 Post-COVID-19 p-value
sCr, mg/dl 1.31 (1.2-1.76) 1.33 (1.08- 1.7) 0.85 1.49 (1.1-1.8) 1.21 (0.9-2.1) 0.019
eGFR, ml/min 48.8 (40.5-62.1) 56.7 (41.5-67) 0.25 46.7 (36-64) 56.7 (41.5-67) 0.028
24-h proteinuria, mg/die 102 (6.2-205) 89.4 (37.2-246.4) 0.08 13(2.5-183) 44.7 (10.8-1141) 0.29
Notes: eGFR, estimated glomerular filtration rate; sCr, serum creatinine.
Table III: Early graft function post-COVID-19 in hospitalized and non-hospitalized KT recipients

 

Discussion

Numerous reports have alerted the scientific community regarding the unfavorable outcome of COVID-19 in patients with a reduced immune response [1, 15]. The results of this study confirmed that COVID-19 poses KT recipients at high risk of severe consequences.

In our cohort of KT recipients, COVID-19 carried with it a higher rate of symptoms, hospitalization and mortality compared to the general population [16, 17]. We found that in this cohort (45 KT recipients with COVID-19, median age 56.1), 40% of patients developed severe symptoms requiring hospitalization. Overall mortality was 17.8%, higher than the mortality reported in the general population, which ranges between 0.1-19.2% around the world and accounts for about 2.02% globally [18].

In an attempt to reconstitute the immune system against SAR-CoV-2 infection, we minimized the burden of IST in these patients. All KT recipients who communicated their COVID-19 positivity to our center, were advised to discontinue the antimetabolite agents (i.e., MFA or AZA) (88.9%) and CNI or m-TOR-i (22.2%). In the hospitalized patients, IST was further reduced or suspended, according to the clinical conditions of the patient. Nevertheless, hospitalization and death rates in the reduced-dose IST group were not dissimilar from the full-dose IST group.

At first glance, these results show that the reduction of immunosuppression did not confer any advantage in terms of patient survival. However, some considerations should be considered before drawing firm conclusions. Most patients who underwent IST reduction carried a significantly higher burden of IST compared to KT recipients whose therapy was left unmodified. The higher prevalence of triple-drug immunosuppressive regimen in patients who underwent IST minimization (77% vs. 22.2%; P=<0.001) has probably increased the vulnerability to COVID-19. Conversely, patients with a full-dose IST spent more time (11.2 vs 7.8 years) on kidney transplantation compared to the reduced-dose IST group. Lastly, we believe that the slight increase of steroid therapy (from alternate days to a daily administration) in the reduced-dose IST group (P=0.064) was too small to mitigate the inflammatory response driven by COVID-19.

Although the reduction of IST did not lead to a favorable outcome, it is worth mentioning that the overall mortality in our cohort was tendentially lower than that reported in other studies, where this approached up to 32.5% [1926]. Our results are in line with the population-based data on 1013 KT recipients affected by COVID-19 collected by the French and Spanish national registries, which reported a 28-day mortality of 20% [27]. In Italy, Bossini et al. [24] reported a higher overall mortality rate (28%) during the first wave of COVID-19 in the city of Brescia. Similarly to our therapeutic strategy, they discontinued immunosuppression in all hospitalized patients and introduced or increased the dose of steroids. The causes underlying these different mortality rates are unknown. The different timing of enrollment made the two cohorts not perfectly comparable. All patients in the Brescia cohort were enrolled during the first wave of COVID-19 in Europe, in an overwhelmed and unprepared hospital setting, within a timespan characterized by a high rate of experimental regimens and relative side effects [28, 29]. Lastly, a lower median age (56.1 vs. 60 years) in our cohort of patients probably contributed to the better prognosis.

Multivariate analysis showed that the predictors of death were age and time elapsed on IST, in line with previous studies. Age is widely associated with COVID-19 severity and death in KT recipients [30, 31] as well as in the general population [32]. The Centers for Disease Control (CDC) claims that 8 out 10 COVID-19 deaths in the U.S. occurred in adults over 65 and that the risk of hospitalization and death increases enormously with age [33].

The effect of immunosuppression is still controversial in KT recipients [34]. Immunosuppression is known to dysregulate innate and adaptive immunity, exposing the patients to severe infections. On the other hand, severe COVID-19 infection has been associated with a dysregulated inflammatory response (IL-6, IL-1, and chemokines) leading to ARDS and sepsis. The new insights support a promising role of immunosuppressants (i.e., tocilizumab, steroid) in tempering the immune response of patients with severe manifestations of COVID-19 [35].

Lastly, we report a short-term good graft function in patients who survived COVID-19. These data indicate a stable early graft function (sCr and 24-hour proteinuria) in outpatients who were not hospitalized. Conversely, hospitalized KT recipients had a statistically significant improvement in renal function. As stated also by Dacina et al. [5], we speculate that lower sCr after SARS-CoV-2 is due to the minimization or withdrawn of CNI, a ‘drug holiday’ apparently without dire consequences in terms of graft rejection.

Finally, the limitations of the study should be enumerated. It is a retrospective study, with a small sample size and a short follow-up after COVID-19. The small number of patients and the short observation period may have reduced the probability to observe an underlying difference between these two groups. Long-term follow-up is required to verify if the early improvement of kidney function after COVID-19 is maintained in the survivors. Furthermore, we cannot exclude that, in some cases, the reduction of IST occurred with a short delay after the diagnosis of COVID-19; however, all patients with symptoms underwent nasopharyngeal swabs as fast as possible in an ambulatory setting.

 

Conclusion

In our cohort of patients, the reduction of immunosuppression did not decrease the risk of severe COVID-19 or death. COVID-19 was associated with hospitalization (42%), graft failure (2.2%), IgA glomerulonephritis (2.2%) and death (17.8%). Age and time elapsed from kidney transplantation were independent predictors of death in our patients. Short-term follow-up after COVID-19 showed an excellent graft function in most survivors. Primary infection or vaccination did not exclude the risk of SARS-CoV-2 infection in KT recipients.

 

Authorship credit

Conception: Gaetano Alfano and Francesca Damiano

Collection of data: Camilla Ferri, Francesco Giaroni, Andrea Melluso, Martina Montani, Niccolò Morisi, Lorenzo Tei, Jessica Plessi

Analysis and interpretation of data: Gaetano Alfano, Francesco Giaroni, Francesca Damiano

Drafting the article: Gaetano Alfano, Francesco Fontana, Silvia Giovanella, Giulia Ligabue, Giacomo Mori

Intellectual Contribution: Francesco Fontana Gianni Cappelli, Giovanni Guaraldi

Revising the article: Gianni Cappelli, Giovanni Guaraldi

Approval of the version to be published: all authors

 

Acknowledgments

Special thanks are due to Marco Ballestri, Elisabetta Ascione, Roberto Pulizzi and Francesca Facchini, skilled and experienced nephrologists involved in the “Kidney Transplant Program”, and to Laura Bonaretti and all nurses of the “Kidney Transplantation Outpatient Clinic” at the University Hospital of Modena for their precious support in managing KT recipients.

 

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  25. Cravedi P, Mothi SS, Azzi Y, et al. COVID-19 and kidney transplantation: Results from the TANGO International Transplant Consortium. Am J Transplant 2020; 20:3140-3148. https://doi.org/10.1111/ajt.16185
  26. Nair V, Jandovitz N, Hirsch JS, et al. COVID‐19 in kidney transplant recipients. Am J Transplant 2020. https://doi.org/10.1111/ajt.15967
  27. Jager KJ, Kramer A, Chesnaye NC, et al. Results from the ERA-EDTA Registry indicate a high mortality due to COVID-19 in dialysis patients and kidney transplant recipients across Europe. Kidney Int 2020; 98:1540-1548. https://doi.org/10.1016/j.kint.2020.09.006
  28. Gérard A, Romani S, Fresse A, et al. “Off-label” use of hydroxychloroquine, azithromycin, lopinavir-ritonavir and chloroquine in COVID-19: A survey of cardiac adverse drug reactions by the French Network of Pharmacovigilance Centers. Therapies 2020; 75:371-379. https://doi.org/10.1016/j.therap.2020.05.002
  29. Izcovich A, Siemieniuk RA, Bartoszko JJ, et al. Adverse effects of remdesivir, hydroxychloroquine, and lopinavir/ritonavir when used for COVID-19: systematic review and meta-analysis of randomized trials. Infectious Diseases (except HIV/AIDS) 2020. https://doi.org/10.1101/2020.11.16.20232876
  30. Coll E, Fernández-Ruiz M, Sánchez-Álvarez JE, et al. COVID-19 in transplant recipients: The Spanish experience. Am J Transplant 2021; 21:1825-1837. https://doi.org/10.1111/ajt.16369
  31. Oto OA, Ozturk S, Turgutalp K, et al. Predicting the outcome of COVID-19 infection in kidney transplant recipients. BMC Nephrol 2021; 22:100. https://doi.org/10.1186/s12882-021-02299-w
  32. Levin AT, Hanage WP, Owusu-Boaitey N, et al. Assessing the age specificity of infection fatality rates for COVID-19: systematic review, meta-analysis, and public policy implications. Eur J Epidemiol 2020; 35:1123-1138. https://doi.org/10.1007/s10654-020-00698-1
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AKI in pazienti ospedalizzati con COVID-19: l’esperienza di un singolo centro

Ci spiace, ma questo articolo è disponibile soltanto in Inglese Americano. Per ragioni di convenienza del visitatore, il contenuto è mostrato sotto nella lingua alternativa. Puoi cliccare sul link per cambiare la lingua attiva.

Dear Editor,

since December 2019, the COVID-19 pandemic is straining hospitals and nephrology services worldwide. Although this disease manifests mostly with pneumonia, acute kidney injury (AKI) is recognized as a common complication in patients with severe manifestations of COVID-19. The pathogenesis of COVID-19 is still unclear but recent evidence supports a multifactorial etiology [1]. Generally, kidney involvement following SARS-CoV-2 infection is proportionate to the gravity of the infection and is commonly diagnosed in hospitalized patients with lung involvement [2]. As in another clinical scenarios, kidney injury is independently associated with morbidity and mortality in patients with SARS-CoV-2 infection [3,4].

The distribution of AKI in patients with COVID-19 is extremely variable across countries [5]. The first reports from China described a low prevalence of AKI in hospitalized patients [6] but subsequent evidence, coming from the USA and Europe, suggested a higher kidney involvement, especially in the intensive care setting [7] and among vulnerable patients [8]. Few studies have estimated the rate of AKI in hospitalized patients admitted to non-intensive care units in Italy. It ranges between 13.7-22.6% [911] and is similar to the prevalence detected in other European countries (4.5-22%) [1214]. In order to broaden the knowledge of this phenomenon, we report the data on the prevalence and clinical characteristics of AKI in COVID-19 patients.

We evaluated a cohort of 792 COVID-19 patients hospitalized at the University Hospital of Modena, Italy, between February 25 and December 14, 2020 for severe symptoms of COVID-19. The diagnosis of COVID-19 was performed through reverse transcriptase-polymerase chain reaction (RT-PCR). We excluded patients aged <18 years (n=2), patients on dialysis (n=5), and patients without serum creatinine on admission (n=19). The diagnosis of AKI was defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria [15], without considering the urine output criteria. Baseline serum creatinine (sCr) coincided with sCr at admission. All the enrolled patients were discharged or died at the end of the follow-up.

According to the Istituto Superiore di Sanità (ISS), the coronavirus pandemic in Italy can be subdivided in three waves during 2020: first wave (February-May), transitional period (June-August) and second wave (September- December) [16]. As a result, the study population was subdivided into three groups: wave-1 (n=389), transitional period (n=57) and wave-2 (n=346).

Data are expressed as mean ± standard deviation or a percentage (%). Statistical differences were tested using Student’s t-test or Chi-square as appropriate. Cox regression analysis evaluated the influence of AKI on the hazard of death. The study was approved by the regional ethical committee of Emilia Romagna (n. 0013376/20).

In a cohort of 792 hospitalized patients, 122  cases (15.4%) of AKI were diagnosed. Patients with AKI were older (77.4 vs 64.3 years; P=<0.001) and had a higher baseline sCr (1.37 vs 0.96 mg/dl; P=0.004) than non-AKI patients (Table I). As expected, patients with AKI showed increased levels of inflammatory markers (CRP; P=0.001), tissue damage (LDH; P=0.01) and hypoxia (PO2/Fi02;P=<0.001). We detected a higher burden of morbidity and comorbidity compared to non-AKI patients, as indicated by a higher SOFA (P=<0.001) and Charlson score (P=<0.001), respectively. In particular, AKI patients had a high rate of non-invasive ventilation (NIV; P=0.001), high flow nasal oxygen (HFNO; P=<0.001), mechanical ventilation (P=0.001) and, consequently, ICU admission (P=0.01). Given the burden of multiorgan dysfunction, AKI patients experienced a prolonged hospital stay (22.4 vs 13.2 days; P=0.008).

AKI stage 1 was the most frequent event (n=82; 67.2%) followed by AKI stage 2 (n=15; 12.2%) and AKI stage 3 (n=25; 20.4%). In this latter group, renal replacement therapy was necessary for 11 patients (44%).

The overall mortality rate was 19.1% and it increased up to 61.5% in patients with an acute worsening of kidney function (Figure 1). AKI was an independent risk factor for death after adjustment for age, sex, PO2/FiO2, baseline creatinine, BMI, LDH, CRP, diabetes and cardiovascular disease (HR, 3.39; CI95% 1.032-11.1; P=0.04). Of the survivors with AKI, 40.4% did not recover kidney function at discharge.

Variable All patients (n=792) No AKI (n=670) AKI patients (n=122) p-value
Age 66.3±16.1 64.3±16.18 77.4±10.92 0.012
Males (%) 511 (64.5) 425 (63.4) 86 (70.5) 0.15
White blood cells (cell/mm3) 8587±7170 7657.1±5696.6 9737.7±7380.5 0.053
Hemoglobin (gr/dl) 12.7±1.8 12.6±1.8 13.1±1.6 0.084
Platelets (103/mm3) 253.7±116.3 261.8±111.2 207.8±133.5 0.85
CRP (mg/dl) 8.9±8.2 8.3±7.89 12.3±9.6 0.001
LDH (U/L) 648.9±991.9 592±283.4 950±238.9 0.01
Baseline sCr (mg/dl) 1±0.71 0.96±0.25 1.37±0.08 0.004
sCr peak (mg/dl) 1.2±1.1 1.01±0.65 2.72±1.76 <0.001
sCr at discharge (mg/dl) 1±0.89 0.84±0.44 2.23±1.57 <0.001
MAP 90.3±13.2 88.8±12.2 95.5±14.2 0.12
PO2/FO2 250.6±105.2 261.11±101.3 184.87±106 <0.001
SOFA score 2±2 1.7±1.6 3.5±2.7 <0.001
Charston score 3.4±2.9 3 ±2.8 5.1±3.1 <0.001
Comorbidities§ (%)
COPD (%) 32 (14.5) 22 (12.1) 10 (26.3) 0.04
Diabetes (%) 75 (31.9) 61 (31.1) 14 (35.9) 0.576
Hypertension (%) 182 (65.9) 150 (64.7) 32 (72.6) 0.386
CVD (%) 50 (22.5) 30 (16.5) 20 (50) <0.001
CKD (%) 35 (15.8) 24 (13.1) 11 (28.9) 0.025
BMI>30 (%) 113 (32.2) 99 (33.6) 14 (25) 0.275
ACE inibitors (%) 102 (12.9) 90 (13.4) 12 (9.8) 0.307
FANS (%) 21 (2.7) 17 (2.5) 4 (3.3) 0.550
Nephrotoxic antibiotic (%) 20 (2.5) 15 (2.2) 5 (4.1) 0.216
Use of chonic diuretic therapy (pre-AKI) (%) 259 (32.7) 182 (27.2) 77 (63.1) 0.001
Antiviral (%) 253 (31.9) 215 (32.1) 38 (31.1) 0.91
IV hydratation with cystalloids pre-AKI (%) 233 (29.4) 189 (28.2) 44 (36.1) 0.085
Steroid (%) 287 (38.5) 232 (36.7) 55 (48.7) 0.021
Immunotherapy  (%) 326 (43.4) 275 (43.4) 326 (43.7) 0.758
O2 therapy (%) 537 (67.8) 454 (67.8) 83 (68) 1
HFNO (%) 101 (18.5) 71 (15.4) 30 (35.7) <0.001
NIV (%) 172 (31.3) 128 (27.8) 44 (49.4) 0.001
Mechanical ventilation (%) 91 (12.2) 59 (9.3) 32 (28.3) 0.001
ICU admission (%) 153 (20.5) 111 (7.5) 42 (37.2) 0.001
BMI 28.5±5.3 28.5±5.1 28.1±6.2 0.109
Time elapsed from admission to AKI (day) 11.8±9.34 NA 11.8±9.34 0.063
Hospitalization (day) 14.7±13.7 13.28±11.3 22.45±21.38 0.008
Death (%) 151 (19.1) 76 (11.3) 75 (61.5) <0.001
Legend: CRP, C-reactive protein; LDH, lactate dehydrogenase; MAP, mean arterial pressure; sCr, serum creatinine; SOFA, Sequential Organ Failure Assessment; COPD, chronic obstructive pulmonary disease; CVD, cardiovascular disease; CKD, chronic kidney disease; BMI, body mass index; HFNO, high-flow nasal oxygen; NIV, noninvasive ventilation; AKI, acute kidney injury; ICU, intensive care unit.
Table I: Demographics and clinical manifestation of COVID-19 patients
Figure 1: Kaplan Mayer curves showing survival of AKI and non-AKI patients with COVID-19
Figure 1: Kaplan Mayer curves showing survival of AKI and non-AKI patients with COVID-19

From an epidemiological point of view, the prevalence of AKI remained similar during the first (15.9%) and the second wave (14.7%) (P=0.89) (see Figure 2). The rate of ICU admission (P=0.42) was similar in these two groups but during the second wave AKI patients were more frequently treated with steroids (P=0.007), HFNO (P=0.001) and required less mechanical ventilation (P=0.019) compared to patients admitted during the first wave. Nevertheless, the mortality of AKI patients did not change between the first (59.7%) and the second (70.6%) wave of COVID-19 (P=0.243).

Figure 2: AKI prevalence during the first wave, the trasitional period and the second wave
Figure 2: AKI prevalence during the first wave, the trasitional period and the second wave

The findings of this study provide new information on the epidemiology of AKI in COVID-19. We found that the overall rate of AKI in unvaccinated hospitalized patients with COVID-19 accounted for 15.4% and that the prevalence of AKI remained relatively steady (about 15%) during the three phases of the COVID-19 pandemic that hit Italy and Europe during 2020. These data are in line with the results of a recent metanalysis, reporting a comparable pooled incidence (15.4%) among 25,566 patients, enrolled in 39 studies [17]. However, the distribution of AKI is not homogeneous among the published studies, where prevalence ranged from 0.5%-60%. Multiple factors may have affected this epidemiological variability including the surge capacity of the healthcare system, how the care was delivered (publicly or privately) and the method of patients selection (e.g. criteria for hospital admission).

In our study, subjects with AKI showed different demographic and clinical characteristics compared to non-AKI patients. Kidney injury was indeed experienced by elder patients affected by a more severe disease than non-AKI patients. COVID-19 patients with kidney involvement had a higher rate of morbidity (lung involvement, ICU admission, length of stay) and a 3.4-fold increase in mortality than non-AKI patients. No clear differences were detected in terms of AKI prevalence between the first and second wave, despite some therapeutic improvements (steroids, remdesivir, immunomodulant) were made in the management of these patients.

Since AKI is an independent risk factor  in COVID-19, many efforts should be made to identify and correct predisposing factors for kidney injury. From a practical point of view, the prevention measures that we put in place were not different from those we follow for AKI from other causes in critically ill patients [15,16]. These were based mainly on surveillance of kidney function, maintenance of normovolemia and avoidance of nephrotoxic agents.

In conclusion, our study confirms that AKI is a common event (15.4%) in COVID-19 and its prevalence was stable through 2020. AKI was more common in older patients who experienced a severe COVID-19. The outcome of patients with AKI was poor, as more than half died at the end of the follow-up and 40% of survivors had not recovered kidney function at hospital discharge. The heterogeneity of COVID-19-associated AKI in terms of incidence and etiology presents many challenges to its prevention and management. Further studies are required to investigate the effects of new virulent SARS-CoV-2 variants on the development of AKI, the impact of vaccination in the prevention of kidney involvement and the long term consequences of AKI.

 

Bibliography

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Efficacia dei test sierologici per COVID-19 in emodializzati asintomatici: esperienza di un centro dialisi italiano

Abstract

È qui riportata la breve esperienza di un piccolo Centro di Nefrologia situato nell’Ospedale di Massa Marittima, individuato, dalla direzione aziendale, come struttura no-COVID.

Si descrive l’approccio per la prevenzione della diffusione dell’infezione da COVID-19 tra i pazienti emodializzati ed il personale del reparto, le metodiche attuate per l’individuazione dei soggetti COVID-19 positivi con focus sull’uso dei test sierologici rapidi e dei tamponi rinofaringei (RT-PCR) in soggetti asintomatici.

I risultati dei primi test sierologici per le IgM e le IgG eseguiti su 34 pazienti, negativi ai tamponi rinofaringei, mostravano positività nel 41,18% dei casi. Essi sono classificabili come falsi positivi sia per i referti dei tamponi, che ripetutamente hanno dato risultati negativi, sia per la negatività dell’anamnesi e della clinica, sia per gli esiti della seconda determinazione, avvenuta dopo 18 giorni, in cui tutti i soggetti sono risultati negativi.

L’interpretazione dei test sierologici è complessa e deve essere inserita in una più ampia valutazione del soggetto che comprenda un’accurata anamnesi (storia epidemiologica, comorbidità), la presenza/assenza di segni e sintomi, i risultati di test di conferma con riferimento ad un periodo di osservazione ben preciso, per evitare di classificare come immunologicamente protetti, i soggetti falsi positivi.

Parole chiave: COVID-19, SARS CoV-2, test sierologici, soggetti asintomatici, falsi positivi

Introduzione

L’infezione realizzata dal nuovo coronavirus SARS-CoV-2 presenta forme variabili di malattia, dal contagio tra soggetti asintomatici o paucisintomatici [1] a forme simil-influenzali più o meno importanti, fino a gravi quadri con distress respiratorio necessitanti cure intensive in reparti di rianimazione [2].

Il primo focolaio di COVID-19 è stato segnalato a Wuhan, in Cina, il 31 dicembre 2019. Il primo focolaio italiano è esploso nelle settimane successive al ricovero di un caso COVID-19 positivo all’Ospedale di Codogno (Lodi) in data 21 febbraio 2020. Il 25 febbraio 2020 è stato segnalato il primo caso di infezione da SARS-CoV-2 in Toscana, a Firenze (un sessantenne rientrato in Italia da Singapore). Il 05 marzo 2020 è stato accertato il primo caso nella provincia di Grosseto. In data 08 marzo il Governo italiano ha dichiarato lo stato di isolamento dell’intera Lombardia e di ulteriori 14 province del Nord Italia. Il 09 marzo il Governo ha poi esteso le restrizioni a tutta l’Italia. L’11 marzo l’Organizzazione Mondiale della Sanità (OMS) ha dichiarato “Pandemia” il focolaio internazionale di infezione da nuovo coronavirus SARS-CoV-2.

L’esperienza riportata è relativa al Centro Dialisi situato in uno dei cinque ospedali della provincia di Grosseto, la provincia più estesa (4503 Km2) e meno popolata (223.652 abitanti) della Toscana.

Con la diffusione dell’epidemia l’Azienda ha predisposto percorsi separati per pazienti COVID-19 positivi ed individuato Ospedali COVID e Ospedali no-COVID (come il nostro). All’ingresso, oltre alla tenda pre-triage per l’accesso al Pronto Soccorso, è stata approntata anche una postazione di accoglienza per tutte le persone che entrano in Ospedale (utenti e personale). Chi entra è sottoposto ad una breve intervista, alla disinfezione delle mani ed alla misurazione della temperatura corporea. Anche i nostri pazienti transitano davanti alla postazione dell’accoglienza e subiscono un primo controllo che viene poi ripetuto, in reparto, dall’infermiere che ha in carico il paziente.

Al Centro Dialisi abbiamo progressivamente attuato le procedure per mantenere il distanziamento dei pazienti nelle aree comuni, implementato l’uso dei dispositivi di protezione individuali (DPI) e potenziato la sanificazione dei locali. Dal 10 marzo abbiamo dotato i pazienti di mascherine chirurgiche da indossare durante il viaggio da casa all’Ospedale e viceversa, durante il tragitto intraospedaliero e durante tutto il trattamento dialitico. Il personale medico ed infermieristico è stato dotato di mascherina chirurgica, da indossare per tutto il turno di lavoro, e di camici monouso da indossare sopra la divisa per tutta la durata del turno di lavoro, oltre ai DPI forniti regolarmente nel periodo precedente la pandemia [3].

Il lavoro (osservazionale, retrospettivo, monocentrico) ha lo scopo di valutare la diffusione del SARS CoV-2 tra la popolazione dializzata trattata nel Centro Dialisi di Massa Marittima mediante l’uso di RT-PCR e test sierologici.

 

Pazienti e metodi

Nell’ottica di mantenere l’Ospedale no-COVID sono stati pianificati tamponi naso-faringei per i pazienti dializzati a partire dal 27 marzo al 17 aprile, ripetuti ogni 7-10 giorni. Contestualmente, abbiamo eseguito anche una rilevazione di COVID-19 IgG e IgM con test rapido (immunochromatographic assay) in data 2 e 3 aprile, ripetuto dopo 18 giorni, in data 20 e 21 aprile.

Abbiamo limitato l’osservazione ai pazienti presi in carico dal Centro Dialisi antecedentemente al 15 marzo ed ivi trattati fino al 30 aprile 2020, ritenendo il periodo di osservazione di 45 giorni sufficiente a rilevare un eventuale contagio da SARS-CoV-2. Analogamente, abbiamo considerato soltanto il personale sanitario che effettivamente ha prestato servizio nel reparto dal 15 marzo al 30 aprile 2020. In questo modo abbiamo ottenuto un insieme di persone che si sono incontrate, nello stesso ambiente, con cadenza regolare, nell’arco dei 45 giorni di sorveglianza. Motivo di esclusione era il mancato consenso alla partecipazione allo studio.

Nessuno dei pazienti presentava il criterio epidemiologico che caratterizza i casi sospetti o i contatti stretti e nessuno aveva o aveva avuto, nelle settimane precedenti, sintomatologia riferibile all’infezione da SARS-CoV-2. Anche medici ed infermieri, tutti asintomatici e in assenza di criterio epidemiologico per caso sospetto o contatto stretto, sono stati sottoposti al test sierologico per COVID-19 IgG/IgM in data 09 aprile 2020.

Abbiamo utilizzato il test sierologico rapido come test diagnostico di screening su un campione di soggetti a basso rischio, confrontandone i risultati con i tamponi rinofaringei, ad oggi considerati metodica Gold Standard.

Il test utilizzato dal nostro laboratorio, ossia “COVID-19 IgG/IgM Rapid Test Cassette (sangue intero/siero/plasma)”, realizzato da Zhejiang Orient Gene Biotech Co. Ltd., esplicita, nella scheda tecnica, che le proprie performance, confrontate con i referti di RT- PCR, sono le seguenti: sensibilità del test per le IgM = 87,9% con specificità del 100%, sensibilità del test per le IgG = 97,2% con specificità del 100%.

L’osservazione dei 2 gruppi di soggetti, pazienti ed operatori, limitato ad un determinato periodo di tempo, ha permesso di contestualizzare i dati e stimare la prevalenza di COVID-19. Abbiamo scelto di procedere soltanto con l’elaborazione dei dati relativi ai pazienti poiché soltanto i pazienti sono stati sottoposti al tampone rinofaringeo.

Le prestazioni del test sierologico sono state valutate mediante il calcolo di: percentuale, specificità osservata, prevalenza apparente, valore predittivo positivo (VPP), valore predittivo negativo (VPN), intervalli di confidenza al 95% (IC95%) sia del VPP che del VPN.

 

Risultati

La popolazione dializzata in osservazione era composta da 34 persone di razza caucasica, 25M/9F, età media 72 anni, range 28-93 anni, con comorbidità multiple nel 67,65% dei casi (Figura 1).

I tamponi dei pazienti sono risultati negativi nel 100% dei casi, mentre i referti delle Immunoglobuline (1a rilevazione) hanno dato esiti variabili classificabili in 4 gruppi Quadro A, B, C, D secondo la positività/negatività delle IgM e IgG (Figura 2).

 

Figura 1: Rappresentazione delle comorbidità dei pazienti. Classificazione dei pazienti in 3 gruppi secondo il numero di comorbidità presenti (riquadro in alto)

 

Figura 2: Risultati del primo test rapido di ricerca degli anticorpi suddivisi, secondo le possibili risposte delle 2 linee di lettura del test rapido, in Quadro A, B, C, D

 

Lo staff sanitario, non sottoposto a tampone, costituito da 10 operatori tutti caucasici e di sesso femminile, età media 53,8 anni, range 46-59 anni, presentava al test sierologico soltanto il Quadro A (IgM-, IgG-).

Una prima interpretazione dei risultati, avulsi dalla clinica e dai referti dei tamponi, mostrava il 41,18% dei pazienti positivo al test sierologico (Quadro B, C, D). Nel totale dei soggetti indagati (pazienti + operatori) abbiamo riscontrato una positività del 31,82%, una percentuale molto elevata rispetto all’atteso, data la bassa prevalenza del COVID-19 calcolata sugli operatori sanitari dell’Ospedale di Massa Marittima pari allo 0.01 (2 RT-PCR positivi su 64 RT-PCR effettuati, dopo screening con test sierologico, sui 190 operatori sanitari dell’Ospedale).

Il risultato dei tamponi rinofaringei e la mancanza di sintomatologia da COVID-19 conferivano tranquillità. I successivi 2 controlli dei tamponi eseguiti sui pazienti, a distanza di 7-10 giorni (totale 3 tamponi), confermavano la negatività al COVID-19 di tutti i soggetti.

Non abbiamo messo in atto ulteriori precauzioni rispetto a quanto adottato prima della disponibilità dei referti delle Immunoglobuline, considerando come adeguati i provvedimenti già attivati.

A distanza di 18 giorni dalla 1a rilevazione abbiamo ripetuto il test sierologico a tutti i pazienti ottenendo nella totalità dei casi il Quadro A (IgM-, IgG-). I referti dei soggetti positivi alla 1a valutazione non avevano subito modifiche compatibili con una progressione della malattia e ciò avvalorava l’ipotesi che le positività del primo test potessero essere classificate come falsi positivi.

Prendendo in esame separatamente i risultati dei test sierologici per le IgM e le IgG COVID-19 (il test sierologico valuta le 2 classi di Immunoglobuline con 2 linee di lettura separate), abbiamo osservato i seguenti risultati nei nostri pazienti non contagiati da SARS-CoV-2 (criterio epidemiologico, clinico e diagnostico).

Per le IgM la situazione è esplicitata in Tabella I, utilizzando i dati ivi riportati è possibile calcolare la Specificità osservata IgM (Spo IgM) = 21/34 = 0,618 e la Prevalenza apparente IgM (Preva IgM) = 13/34 = 0,382, che risulta molto elevata rispetto alla prevalenza reale di diffusione del COVID-19, stimata come assimilabile alla prevalenza del campione degli operatori sanitari dell’Ospedale di Massa Marittima pari a 1%.

Il calcolo del Valore Predittivo Positivo IgM (VPP IgM) e del Valore Predittivo Negativo IgM (VPN IgM) sia atteso che osservato, mediante applicazione del Teorema di Bayes [4], ha fornito maggiori informazioni circa la probabilità che un soggetto positivo al test fosse realmente ammalato e che un soggetto negativo fosse realmente sano, formule e calcoli sono riportati in Tabella II. Dato il piccolo campione di popolazione studiato, abbiamo proceduto alla stima dei valori predittivi mediante il calcolo degli intervalli di confidenza al 95% (IC 95%).

Tabella I : Tabella a doppia entrata riassuntiva dei referti relativi alla ricerca di IgM e IgG

 

Tabella II: Test rapido per le IgM: applicazione del Teorema di Bayes per il calcolo dei valori predittivi positivi e negativi, calcolo degli intervalli di confidenza relativi

I soggetti positivi al test rapido per le IgM avevano la probabilità del 2,27% di essere ammalati con un IC 95% ± 0,08; una bassa probabilità, quindi, con intervallo fiduciale esteso dallo 0 al 10,27%, valori estremamente distanti dal VPP atteso (100%).

I soggetti negativi al test avevano la probabilità del 99,8% di essere sani con un IC 95% ± 0,019; una elevata probabilità con intervallo fiduciale molto piccolo dal 97,9% al 100%. In questo caso, il test ha fornito risultati sovrapponibili al VPN atteso (99,88%).

Sempre in Tabella I sono riportati i dati per le IgG con i quali sono stati calcolati la Specificità osservata IgG (Spo IgG) = 27/34 = 0,794 e la Prevalenza apparente (IgG Preva IgG) = 7/34 = 0,206.

Anche in questo caso abbiamo calcolato il Valore Predittivo Positivo IgG (VPP IgG), il Valore Predittivo Negativo IgG (VPN IgG) ed i rispettivi IC 95% (Tabella III).

 

Tabella III: Test rapido per le IgG: applicazione del Teorema di Bayes per il calcolo dei valori predittivi positivi e negativi, calcolo degli intervalli di confidenza relativi

 

I soggetti positivi al test rapido per le IgG avevano la probabilità del 4,5% di essere ammalati con un IC 95% ±0,15, ossia una bassa probabilità con intervallo fiduciale esteso dallo 0 al 19,95%.

I soggetti negativi al test per le IgG avevano la probabilità del 99,96% di essere sani con un IC 95% ± 0,00755, ossia una elevata probabilità con un intervallo fiduciale molto piccolo dal 99,21% al 100%. Il VPN osservato per le IgG mostrava valori sovrapponibili all’atteso (99,97%), mentre il VPP differiva notevolmente dai valori attesi (100%).

Le performance ottenute dai test sierologici utilizzati nel nostro reparto hanno fornito risultati congrui con quanto atteso in popolazioni a bassa prevalenza di malattia. I VPP delle IgM e delle IgG ed i rispettivi IC 95% informano sulla bassa fiducia che dobbiamo riporre nel considerare ammalati o guariti i soggetti positivi al test. I VPN delle IgM e delle IgG con i rispettivi IC 95% forniscono una minima incertezza relativa alla probabilità che i soggetti negativi al test siano realmente sani.

 

Discussione

Secondo la settima edizione delle Linee Guida “Diagnosis and Treatment Guidelines for COVID-19” (03 marzo 2020), elaborate dal Comitato Nazionale per la Salute della Repubblica Popolare Cinese, per la conferma dei casi sospetti è necessario combinare almeno 3 criteri: anamnesi per eventuali contatti, manifestazioni cliniche (segni e sintomi) e referti di indagini diagnostiche [5].

Le metodiche attualmente disponibili per la diagnosi dell’infezione da SARS CoV-2 sono state approntate e sperimentate prevalentemente su soggetti sintomatici con quadro TC positivo, quindi su soggetti con evidenza clinica di patologia. Ad oggi è consigliato valutare i dati dei test sierologici integrandoli con i risultati dei tamponi [6,7], considerati tutt’ora il test disponibile più attendibile. L’integrazione è necessaria poiché test sierologici e tamponi forniscono informazioni differenti, la sierologia rileva la presenza degli anticorpi e fornisce informazioni sulla risposta dell’ospite all’infezione, mentre i tamponi individuano gli acidi nucleici virali [8]. L’interpretazione dei test sierologici è piuttosto complessa, richiede conoscenza di limiti e punti di forza della metodica oltre ai necessari approfondimenti successivi [7]. La sierologia da sola non può confermare o escludere la diagnosi o dare informazioni sullo stato dell’infezione [7].

Anche le indagini microbiologiche soffrono di limitazioni: in letteratura sono segnalati casi di falsi negativi e falsi positivi tra i referti dei tamponi. I falsi negativi potrebbero essere dovuti ad errori di tecnica nell’esecuzione del tampone, alla bassa carica virale presente nel momento del prelievo sia in fase acuta che in convalescenza [7]. Risultati falsi positivi nei RT-PCR possono verificarsi per errori tecnici e/o contaminazione dei reagenti [8]. Un tampone è positivo quando rileva RNA virale e ciò non implica necessariamente la presenza del virus [8].

D’altronde, la risposta immunitaria che il virus SAR-CoV-2 innesca nel corpo umano è stata studiata per un periodo di tempo troppo limitato. Una prolungata clearance virale è stata segnalata in una proporzione di pazienti che potrebbe essere sottostimata [9]. Non sappiamo ancora se il nuovo Coronavirus possa rimanere presente nell’organismo anche dopo aver generato una risposta anticorpale, dando luogo ad una infezione cronica asintomatica.

I test sierologici rapidi per SARS-CoV-2 sono stati costruiti sul modello dei test immunocromatografici di routine; questi test, ormai collaudati, presentano falsi positivi nei soggetti con elevata risposta anticorpale, Fattore Reumatoide (FR) elevato, paraproteinemie, malattie autoimmuni [10,11]. Nel caso specifico del SARS-CoV-2, i falsi positivi possono risultare anche da reazioni crociate con altri Coronavirus per infezioni pregresse non legate al COVID-19 [7,12].

Il test utilizzato dal nostro laboratorio COVID-19, IgG/IgM Rapid Test Cassette, si dichiara, nella scheda tecnica, come un test qualitativo che fornisce risultati preliminari che devono essere confermati mediante esami realizzati con metodiche alternative e dati clinici. IgG ed IgM anti SARS-CoV-2 possono rilevarsi nel sangue a partire dalla settimana precedente la comparsa dei sintomi fino a varie settimane dopo l’esposizione e il loro titolo incrementa rapidamente [12,8]. Alcuni pazienti, nelle prime fasi di malattia, possono presentare positività alle immunoglobuline e negatività all’RNA test con tampone naso-faringeo, per cui la ricerca delle immunoglobuline può aiutare nell’individuare i pazienti esposti, così come nel monitoraggio della malattia accertata [6,12]. Si tratta di un test qualitativo, e non quantitativo, a risposta binaria che rileva anticorpi, ossia entità dosabili; perciò, la risposta positiva è legata alla rilevazione di un quantitativo di Ig adeguato ad essere letto dal test. Per titoli inferiori rispetto alla capacità di lettura del test e in caso di assenza degli anticorpi (a seconda delle fasi della malattia), il test fornisce risultati negativi. Un test rapido negativo non esclude un contatto e/o un contagio con il Nuovo Coronavirus poiché il soggetto potrebbe trovarsi nella condizione di essere stato esposto al SARS-CoV-2 e di non aver ancora sviluppato anticorpi.

Nella scheda tecnica del test da noi utilizzato si avverte che, per un risultato corretto, è necessario che il campione di sangue non sia emolizzato, che i campioni siano conservati a temperatura adeguata e che tutte le fasi del test siano rispettate. La scheda tecnica esplicita anche che, prima dell’immissione in commercio, il test è stato valutato su 113 campioni di sangue ottenuti da pazienti con sintomi respiratori e diagnosi clinica, inclusi i referti di TC torace e RT-PCR, di cui 14 erano COVID-19 negativi.

La situazione di sperimentazione e convalida del test rapido, usato nella nostra esperienza, risulta molto diversa da quella presente nel Centro Dialisi di Massa Marittima, reparto a bassa prevalenza di contagio da COVID-19, in cui tutti i soggetti sottoposti al test erano asintomatici e non vi era consapevolezza di pregressi contatti sospetti.

L’elevata percentuale dei pazienti positivi alla 1a rilevazione del test rapido può chiarirsi ricordando che i valori predittivi di un test di screening variano al variare della prevalenza della malattia ricercata [13]. Nel campione sottoposto al test, minore è la prevalenza reale, minore risulta il valore predittivo positivo del test (incremento dei falsi positivi) e maggiore risulta il valore predittivo negativo (riduzione dei falsi negativi). Gli esami utilizzati come test di screening devono possedere elevata sensibilità per poter individuare anche basse positività, “lo scopo dell’esame è infatti quello di evitare la mancata individuazione di soggetti positivi” [14]. Per riuscire a trovare tutti i soggetti positivi, risulta accettabile un numero anche elevato di falsi positivi, da sottoporre ad ulteriori test diagnostici. Questo è vero per i test di screening in genere, a maggior ragione in caso di una malattia infettiva emergente in corso di pandemia. Il rischio sociale di ottenere falsi negativi sarebbe intollerabile per la rapida diffusione dei contagi che comporterebbe.

L’entità dei falsi positivi dipende anche dal punto di cut-off scelto per la lettura dei test. “Il punto di cut-off è la concentrazione in cui la ripetizione del test sullo stesso campione si rivela positiva nel 50% dei casi e negativa nell’altro 50%. Il leggero incremento o decremento della concentrazione sposta di molto la percentuale positiva o negativa dei risultati ottenuti. Questo dimostra come a concentrazioni vicine al limite del cut-off l’imprecisione dei metodi sia elevata” [14].

La stima del valore predittivo positivo e negativo dipende anche dalla numerosità del campione di popolazione osservato; a parità di prevalenza, un piccolo numero di soggetti valutati comporta un piccolo numero di test (positivi o negativi) al denominatore e quindi un più ampio intervallo fiduciario.

Nel nostro reparto, il 41,18% del campione è risultato mis-classificato dal test sierologico; si tratta di un campione di pazienti con età media elevata e comorbidità multiple. I fattori che possono aver favorito risultati falsi positivi sono: la bassa prevalenza di malattia, concentrazioni anticorpali vicine al cut-off del test, errori nella conservazione dei campioni e/o nella metodologia impiegata. La piccola numerosità campionaria ha influenzato l’incertezza della stima dei valori predittivi.

I vantaggi della metodica immunocromatografica, quali la facilità di esecuzione, la rapidità nell’ottenere i risultati, i bassi costi, ne permetteranno una diffusione capillare sul territorio.

I test sierologici saranno importanti per comprendere l’epidemiologia dell’emergente infezione da SARS CoV-2 nei soggetti asintomatici [6]; a questo scopo, saranno utilizzati in maniera sempre più estesa, nei prossimi mesi, e saranno usati in campioni di popolazione aventi prevalenza differente, variabile nel tempo e di scarsa numerosità. Date le peculiarità dei test sierologici, l’evidenza di una positività alle IgM e/o IgG per SARS-CoV-2 non può, da sola, far ritenere i soggetti “protetti” dalla presenza di anticorpi specifici. Si rischierebbe anzi di esporre i falsi positivi a facili contagi, per omissione di misure di protezione, isolamento e contenimento, fino all’esclusione dalla vaccinazione.

 

Conclusioni

Abbiamo accertato che nel mese di aprile 2020 il Centro Dialisi dell’Ospedale di Massa Marittima, Ospedale no-COVID, non era interessato dal contagio da SARS CoV-2.

L’esito dei test sierologici effettuati sugli operatori ha dato risultati negativi.

L’esito dei test sierologici effettuati sui pazienti ha dato, alla prima rilevazione, il 41,18% dei risultati positivi. Essi sono classificabili come falsi positivi sia per i referti dei tamponi, che ripetutamente hanno dato risultati negativi, sia per la negatività dell’anamnesi e della clinica, sia per gli esiti della seconda determinazione, avvenuta dopo 18 giorni, in cui tutti i soggetti sono risultati negativi.

I risultati della nostra esperienza confermano la necessità di sottoporre ad ulteriori indagini i soggetti positivi ai test sierologici. Suggeriscono, inoltre, prudenza nel classificare i casi risultati positivi al test come protetti, per evitare di sovrastimare la copertura anticorpale della popolazione.

 

 

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