Patients with kidney disease on Hemodialysis (HD) are susceptible to Coronavirus Disease (COVID-19) due to multiple risk factors.
AimThis study aims to report the prevalence of antibodies against SARS-CoV-2 among patients on hemodialysis before vaccination in Brazil and to compare with clinical, demographic, and laboratory data.
MethodsBlood samples from 398 Chronic Kidney Disease (CKD) patients treated in three different private institutions in Rio de Janeiro State, Brazil were submitted to the total anti-SARS-CoV-2 testing. Kidney, liver, and hematological markers were also determined. Respiratory samples were tested by real-time PCR for SARS-CoV-2 RNA and positive samples were subjected to high-throughput sequencing on the MinION device.
ResultsOverall, anti-SARS-CoV-2 prevalence was 54.5 % (217/398) and two individuals had SARS-CoV-2 RNA with variant B.1.1. High anti-SARS-CoV-2 seroprevalence was found in male gender and those with hospital admission in the last 3-months before the inclusion in the study. Lower red blood cell count was observed in the anti-SARS-CoV-2 seropositive group. High levels of anti-SARS-CoV-2 were found in those who reported symptoms, had low levels of eosinophils and low hematocrit, and who practiced physical activity.
ConclusionHigh prevalence of anti-SARS-CoV-2 was found in CKD patients before the universal immunization in Brazil suggesting that dialysis patients were highly exposed to SARS-CoV-2.
On March 11, 2020, the World Health Organization (WHO) characterized COVID-19 as a pandemic disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), after 118,000 cases and 4291 deaths reported in 114 countries.1 On September 28, 2020 (date referring to the first collection of this study) over 32.7 million cases of COVID-19 and 991,000 deaths were reported by WHO.2 By the week of November 5 (the second collection of our study), the numbers accumulated to over 49.7 million reported cases and over 1.2 million deaths worldwide.3 By the week of January 19, 2021 (the last collection date of our study) cases have reached over 98.2 million cases and over 2.1 million deaths worldwide.4
It has been observed that Chronic Kidney Disease (CDK) patients are at over 4 fold increased mortality risk compared to healthy individuals.5 CDK leads to marked immunosuppression and has been associated with poor COVID-19 outcomes.6 Risk factors as hypertension, diabetes, obesity, elderly age, cardiovascular disease have also been associated to increase of COVID-19 severity.7,8 According literature, the HD patients presented 10 %‒50 % of asymptomatic COVID-19 infections.6 Renal patients on hemodialysis have a higher risk of transmission. Normally, they attend clinics three times a week, having frequent contact with doctors, nurses and other patients on their dialysis. Even with strict protocols and specific recommendations, many infections are under risk among these individuals.9
During the pandemic, several Variants of Concern (VOCs) have emerged, which are identified by their high transmissibility and their ability to evade innate and acquired immune responses by vaccination.5,10 Between late 2020 and early 2021, four major VOCs emerged worldwide: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta).10,11 The first wave had the predominance of the variant B.1.1.33, being identified in Brazil between April and May 2020. During this wave, 99,760 cases were reported, with 24,174 hospitalizations and 11,270 deaths. The 2nd wave occurred between November 2020 and January 2021, where P.2 (Zeta) was predominant. These isolates were first detected in Rio de Janeiro and then in the other Brazilian states. In this period, 282,339 cases were reported, 27,778 hospitalizations, and 10,621 deaths.11,12
Serological testing is paramount to investigate patient's previous contact with the virus or the development of long-lasting immunity.13 Individuals on hemodialysis have an affected humoral immune response, leading to lower seroconversion and decrease in antibodies compared to healthy individuals.14
It has been observed that individuals undergoing HD constitute a population susceptible to severe COVID-19. Accessing SARS-CoV-2 seroprevalence in a cohort of patients with CKD on HD is important to a better understanding of their natural immunity. The main question of the study was to assess SARS-CoV-2 prevalence in CKD patients and its influence on clinical and laboratorial aspects of kidney damage.
Materials and methodsStudy populationThis cross-sectional study included 398 CKD patients undergoing HD treatment (level 5) in three different private institutions in Rio de Janeiro State (Brazil), from September 2020 to January 2021, during the end of COVID-19 first wave and the beginning of the second wave. These HD units were in the following municipalities in Rio de Janeiro State: (i) Rio de Janeiro City (State capital; n = 171), (ii) Japeri (70 km from the State capital; n = 117), and (iii) Queimados (45 km from the State capital; n = 110).
Inclusion criteria comprised individuals who agreed to sign the Informed Consent Form (ICF) and who were under active dialysis treatment. Exclusion criteria included limitations in understanding ICF terms and insufficient sample volume for posterior tests. Demographic data, clinical characteristics, and risk factors were collected through a questionnaire application. Clinical data and risk factors were not obtained at the Rio de Janeiro unit. Volunteers were argued about their physical activity where it was considered adequate when they reported average weekly volumes of 150–300 min of moderate intensity. Clinical manifestations were also assessed by questionnaire where they should report respiratory symptoms or other clinical manifestations before inclusion the study. The current study was conducted in compliance with the Declaration of Helsinki; it was approved by the National Ethics Committee of Brazil (CONEP) under CAAE number 34049514.7.0000.5248.
Sample collectionParticipants underwent blood and intranasally/oropharyngeal swab collection. Blood was collected by peripheral venipuncture based on using hypodermic needles and sterile 8.5 mL gel vacutainer tubes (SSTTM II Advance, BD Vacutainer®, USA). Serum was stored at −20 °C. Two swabs per patient were collected from nasal (1) and oropharyngeal (1) sites. After the collection procedure was over, swabs were placed in 0.9 % saline (NaCl) solution. The material was aliquoted and stored at −70 °C until testing time.
Serological assaysSerum samples were tested for total anti-SARS-CoV-2 antibodies through Elecsys Anti-SARS-CoV-2 qualitative immunoassay (Roche Diagnostics, Basel, Switzerland). This test uses a recombinant protein that corresponds to SARS-CoV-2 nucleocapsid protein; it was performed in Cobas and in the e801 platform (Roche diagnostics). Reactive samples showed a relative Cut-Off Index (COI) higher than 1.0. According to the manufacturer, test specificity and sensitivity reached 99.81 % and 100 %, respectively, in cases showing more than 14-days of RT-qPCR positivity. Springer et al.15 demonstrated the highest sensitivity for detecting nucleocapsid specific antibodies against SARS CoV-2 compared to other assays and Di Meo et al.16 also used the same assay to measure anti-SARS CoV-2 in hemodialysis patients with good sensitivity.
Molecular assays for SARS CoV-2 RNA detectionSwab samples were submitted to SARS-CoV-2 RNA extraction using QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), as well as to quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) added with a set of probe-associated primers (assay) aimed at SARS-CoV-2 nucleocapsid (N1 and N2) and Envelope (E) genes.17,18 Ribonuclease P/MRP Subunit P30 (RPP30) detection was carried out as endogenous control. The reaction was performed with AgPath-ID™ One-Step RT-PCR (Thermo Fisher Scientific, Waltham, USA) in Rotor Gene Plex-5 equipment (QIAGEN). Negative Template Control (NTC) was added to the extraction procedure; two negative synthetic controls for SARS-CoV-2 (SARS-CoV and MERS-CoV) were included in each RT-qPCR procedure to help monitoring RNA extraction and RT-qPCR quality.
RT-qPCR reaction conditions were initially performed at 45 °C for 15 min (reverse transcription); it was followed by 95 °C for 10 min (initial denaturing), and by 45 cycles of 95 °C for 15 s, and of 55 °C for 45 s. All samples were tested in duplicate. Fluorescence readings were detected at FAM channel. Cycle threshold (Ct) values were automatically provided in each run. Simultaneous Ct values lower than 40, for 2 out of 3 genes, represented positive results.
SARS-CoV-2 lineage genotyping was carried out by high-throughput sequencing via MinION device (Oxford Nanopore Technologies, Oxford, United Kingdom). Initially, SuperScript™ IV First-Strand Synthesis System (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to reverse transcription. Next, PCR amplification of SARS-CoV-2 complete genome with two separate pools of primers19 has been carried out using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Massachusetts, USA). Subsequently, end-repair and dA-tailing was performed with commercial reagent NEBNext Ultra II End Repair/dA tailing module (New England Biolabs). For native barcode ligation, a mixture with end prep products, native barcodes (EXP-NBD104 and EXP-NBD114, Oxford Nanopore Technologies, Oxford, UK) and Blunt/TA Ligase master mix (New England Biolabs) was prepared. Pooled barcoded libraries were purified using ProNex® Size-Selective Purification System (Promega, Madison, WI, USA), quantified by fluorometer Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), and used for adaptor insertion with an NEBNext Quick Ligation Module (New England Biolabs). Reagents from Ligation Sequencing Kit (SQK-LSK109, Oxford Nanopore Technologies) were used to result in an eluted sequencing library. A primed R9.4.1 flow cell (FLO-MIN106D) was used to load the library and was sequenced on a MinION Mk1B device.
Evaluation of the hematological and the biochemical markersBlood samples were sent to central laboratory of Federal Hospital of Servers of the State in Rio de Janeiro, where biochemical tests were performed for evaluation of liver function, with dosage of the following enzymes: Alanine Amino Transferase (ALT), Aspartate Amino Transferase (AST), Gamma Glutamyl Transferase (GGT), Alkaline Phosphatase (ALP), Total Bilirubin (BT) and its fractions [Direct Bilirubin (BD), and Indirect Bilirubin (BI)]. All tests were performed using the dry chemistry analysis methodology through the equipment Clinical Chemistry Analyzer AU680 (Beckman Coulter, California, USA).
Based on the Electrical Impedance method, data from complete blood (hematocrit, hemoglobin, leukocytes, red blood cells, eosinophils, basophils, neutrophils, lymphocytes and monocytes), and platelet count was performed using the Coulter LH 750 Hematology Analyzer (Beckman Coulter).
Kidney function biochemical parameters, such as urea, creatinine, phosphorus, and calcium were measured. Urea was measured by UV Enzymatic methodology, creatinine by Jaffe Colorimetric-Kinetic method, phosphorus by Colorimetric-Phosphomolybdate (PVP) methodology and calcium by O-cresolphthalein method.
Statistical analysisDemographic, clinical, laboratory and epidemiological data were inserted in electronic spreadsheet in Excel® software, version 2019. Relative and absolute frequencies observed for biochemical, hematological, sociodemographic, and behavioral profiles were measured based on using chi-square test for the homogeneity of categorical variables and Student's t-test for continuous variables. Variables showing p-value < 0.05 in the homogeneity Chi-Square test and in Student's t-test during the modeling process were selected for the multivariate model. Analyses were performed in the Statistic Package for Social Science software (SPSS for Windows, version 21.0). In addition, mean antibody levels were compared between groups by taking into consideration the investigated variables as well as their significance after multivariate analysis; Mann-Whitney U test was performed at significance level corresponding to p < 0.05.
ResultsSARS CoV-2 serological and molecular prevalenceIn total, 217 (54.5 %) individuals in the investigated sample (n = 398) had IgG/IgM anti-SARS-CoV-2 detected in serum and 2 individuals (0.8 %) had SARS-CoV-2 RNA detected in swab samples. With respect to molecular results, SARS-CoV-2 N1, N2 and E genes’ Ct values recorded for the two patients detected in the SWAB reached 36.85, 39.17, 42.81, and 23.37, 24.56, 23.96, respectively. One of the aforementioned patients was from the Japeri unit, whereas the other one came from Queimados. Sequencing was only possible in the sample collected from the patient treated at the Japeri HD unit; this patient was the one presenting variant B.1.1.
Anti-SARS-CoV-2 prevalence according demografic characteristics and clinical factors in this study, mean age of the individuals was 52.1 ± 15.2 years and most of them was male 216/398 (54.3 %) and African ethnicity 228/313 (72.9 %). Mean time at hemodialysis treatment was 49.2 months.
Information about the clinical characteristic were obtained in the Japeri and Queimados units. Regarding the clinical factors, 72.4 % of individuals reactive for SARS-CoV-2 did not report any respiratory symptoms before the inclusion in the study. Regarding the physical activity, 106/2017 (48.9 %) did not report the minimum of physical activity recommended by WHO guidelines (average weekly volumes of 150–300 min of moderate intensity).
Hospitalization was more frequent among individuals who anti-SARS-COV-2 reactive compared to non-reactive patients (24 vs. 8) and 25 % of these hospitalized patients (6/24) were known to be due to respiratory infection caused by COVID-19.
Hypertension was the most frequent comorbidity (156/226 %–69.1 %), followed by diabetes (51/226 %–22.5 %). Table 1 shows the bivariate analysis of anti-SARS-CoV-2 prevalence according to sociodemographic factors where male gender (p = 0.025) and lower mean time at hemodialysis treatment (p = 0.000) was associated to high anti-SARS-CoV-2 positivity.
Bivariate analysis of sociodemographic characteristics in relation to seropositivity to anti-SARS-CoV-2.
Feature | Non-reactive (n=178) | Reactive (n=217) | p-value |
---|---|---|---|
Age (years, mean ± SD) | 52.2 ± 14.1 | 53.4 ± 14.7 | 0.759 |
HD treatment (months, mean ± SD) | 74.1 ± 63.6 | 45.8 ± 34.2 | 0.000 |
Government Assistance | 20 (28.6 %) | 31 (33 %) | 0.546 |
Gender□ | |||
Female (n/N,%) | 92/178 (51.7 %) | 87/217 (40 %) | 0.025 |
Male (n/N,%) | 86/178 (48.3 %) | 130/217(60 %) | |
Ethnicitya | |||
White (n/N,%) | 37/179 (20.6 %) | 31/134 (23.1 %) | |
Black (n/N,%) | 133/179 (74.3 %) | 95/134 (70.8 %) | |
Indigenous (n/N,%) | 2/179 (1.1 %) | NA | 0.678 |
Asian (n/N,%) | 7/179 (4 %) | 8/179 (6 %) | |
Years of educationa | |||
8-years (n/N,%) | 93/99 (94 %) | 116/123 (94.3 %) | 1.000 |
More than 8-years (n/N,%) | 6/99 (6 %) | 7/123 (5.7 %) |
N, Total of individuals in this specific analysis; n, Total of patients within N; NA, Does Not Apply, means no result (p-value < 0.053).
At bivariate analysis, hospital admission in the last 3-months before the inclusion in the study was associated with anti-SARS-CoV-2 prevalence (p = 0.017) where most of the reactive individuals reported this hospitalization. The presence of respiratory infection was also frequent among reactive individuals (p = 0.05) although it was not significant. On the other hand, there was no significant difference between the risk factors (hypertension, diabetes, cardiovascular disease, obesity) for seropositivity (Table 2).
Bivariate Analysis of clinical and risk factors in relation to seropositivity to anti-SARS-CoV-2 in patients with CKD, who were treated at Japeri and Queimados units. 2020 and 2021 (n = 226).
Clinical Factors | Non-reactive (n=99) | Reactive (n=127) | p-value |
---|---|---|---|
Physical Activitya | 8 (8.1 %) | 21 (16.5 %) | 0.063 |
Current Smoker | 8 (8.1 %) | 4 (3.1 %) | 0.101 |
Hospital admissionb | 8 (8.1 %) | 24 (18.9 %) | 0.017 |
Respiratory Infection | 3 (3.0 %) | 12 (9.4 %) | 0.054 |
Previous contact with COVID-19 infected individualc | 21 (21.2 %) | 30 (23.6 %) | 0.599 |
Reported symptoms of COVID-19d | 14 (14.2 %) | 27 (21.2 %) | 0.212 |
Comorbidities | |||
Hypertension | 73 (73.7 %) | 83 (65.3 %) | 0.127 |
Diabetes Mellitus | 20 (20.2 %) | 30 (23.6 %) | 0.411 |
Chronic Cardiovascular Disease | 5 (5.0 %) | 6 (4.7 %) | 0.997 |
Obesity | 3 (3.0 %) | 2 (1.5 %) | 0.510 |
Hematological and biochemical markers were investigated in Queimados and Japeri units due to sample volume availability. In this population, 85.3 % of individuals presented low levels of hemoglobin and 58.3 % of them had antibodies against SARS-CoV-2. Regarding anti-SARS-CoV-2 reactive patients, we also found nine individuals with leukopenia and eight with leukocytosis.
Table 3 demonstrates a bivariate analysis of anti-SARS-CoV-2 positivity according to hematological markers. Patients reactive for anti-SARS-CoV-2 had lower mean values of red blood cells compared to the non-reactive group (p = 0.029). Even there was no found significance in white blood cell counts considering the seropositivity investigation, low counts of eosinophils (474.8 vs. 399.5) as well as high counts of monocytes (371.8 vs. 429.3) and basophils (88.2 vs. 98.7) were observed in the group of anti-SARS-CoV-2 seropositive patients (Table 3).
Hematological profile regarding total antibody positivity for SARS-CoV-2 in patients with CKD, who were treated at Japeri and Queimados units. 2020 and 2021 (n = 227).