COVID-19 serology in nephrology healthcare workers

Background Chronic kidney disease patients show a high mortality in cases of a severe acute respiratory syndrome coronavirus-2 (SARS-CoV‑2) infection. Thus, information on the sero-status of nephrology personnel might be crucial for patient protection; however, limited information exists about the presence of SARS-CoV‑2 antibodies in asymptomatic individuals. Methods We examined the seroprevalence of SARS-CoV‑2 IgG and IgM antibodies among healthcare workers of a tertiary care kidney center during the the first peak phase of the corona virus disease 2019 (COVID-19) crisis in Austria using an orthogonal test strategy and a total of 12 commercial nucleocapsid protein or spike glycoprotein-based assays as well as Western blotting and a neutralization assay. Results At baseline 60 of 235 study participants (25.5%, 95% confidence interval, CI 20.4–31.5%) were judged to be borderline positive or positive for IgM or IgG using a high sensitivity/low specificity threshold in one test system. Follow-up analysis after about 2 weeks revealed IgG positivity in 12 (5.1%, 95% CI: 2.9–8.8%) and IgM positivity in 6 (2.6%, 95% CI: 1.1–5.6) in at least one assay. Of the healthcare workers 2.1% (95% CI: 0.8–5.0%) showed IgG nucleocapsid antibodies in at least 2 assays. By contrast, positive controls with proven COVID-19 showed antibody positivity among almost all test systems. Moreover, serum samples obtained from healthcare workers did not show SARS-CoV‑2 neutralizing capacity, in contrast to positive controls. Conclusion Using a broad spectrum of antibody tests the present study revealed inconsistent results for SARS-CoV‑2 seroprevalence among asymptomatic individuals, while this was not the case among COVID-19 patients. Trial registration number CONEC, ClinicalTrials.gov number NCT04347694 Supplementary Information The online version of this article (10.1007/s00508-021-01848-5) contains supplementary material, which is available to authorized users.


Table of contents
. Details of commercial laboratory tests used for follow-up confirmation of all 60 participants who tested borderline positive or positive for SARS-CoV-2 antibodies at baseline. 17 Table S2. Detailed IgM and IgA antibody test results at baseline and follow-up of all 18 subjects with a positive antibody test at follow-up, and of five Covid-19 patients. 18 Table S3. Detailed IgG antibody and total antibody test results at baseline and at follow-up of 18 subjects with a positive antibody test at follow-up, and of five Covid-19 patients. 19

SARS-CoV-2 neutralization assay
The virus used for the serum neutralization assay was passaged and expanded twice before titration by TCID50 on Vero 76 clone E6 cells (CCLV-RIE929, Friedrich-Loeffler-Institute, Riems, Germany) after its initial isolation from a clinical sample (nasopharyngeal swab, sampled in mid-March 2020 from a 25-year old male patient in Lower Austria; the isolate was submitted for next generation sequencing, full length sequence pending). The neutralization assay was set up in flat-bottom 96-well tissue culture plates. Human sera were heat-treated for 30 min at 56°C and 1 in 10 diluted in serum-free HEPES medium (Cell culture service, AGES, Mödling, Austria) as starting point for the assay. The two-fold serially diluted sera were incubated with an equal volume of 50 μl SARS-CoV-2 at a minimum of 2000 TCID50/ml for 90 min at 37°C. Afterwards, 25,000 Vero 76 clone E6 cells were added to each well to the serum/virus mixture in a volume of 100 µl in Eagle's minimum essential medium supplemented with 10% FBS and incubated for 4 days at 37°C, 5% CO2 in a humidified incubator. The cytopathic effect (CPE) in every well was scored under an inverted optical microscope and the reciprocal of the highest serum dilution that protected more than 50% of cells from CPE was taken as the neutralizing titer.

Detection of SARS-CoV-2
We used the Roche Cobas® SARS-CoV-2 PCR test on the Roche® Cobas 6800 platform (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) for the detection of SARS- CoV-2 RNA in nasopharyngeal or oropharyngeal swab specimens. Tests were done individually or as pool-tests (up to ten samples per analysis).

Definition of SARS-CoV-2 contact category 1 and category 2 individuals
A category 1 person is defined as an individual who has high risk of exposure to SARS-CoV-2 (such as living with a Covid-19 patient, direct contact to a Covid-19 patient), whereas category 2 person refers to an individual who has a low risk of exposure to SARS-CoV-2 (such as being in a room with a Covid-19 positive patient for a certain amount of time within a specific distance).

SARS-CoV-2 antibodies -follow-up negatives
At follow-up, 28 of 60 initially borderline positive or positive participants tested negative with all ten commercial laboratory tests and Western blots for nucleocapsid protein and spike glycoprotein. Of the 28 negatives at follow-up, results at baseline using the Immunodiagnostics ELISAs, were as follows: four tested borderline positive and 20 positive for IgM, two borderline positive for IgG, one combined borderline positive for IgM and IgG, and 1 combined borderline positive for IgM and positive for IgG, Figure 1.

SARS-CoV-2 antibodies -follow-up borderline positives
Fourteen participants showed borderline positive results in one or two of the ten commercial One participant tested positive only by nucleocapsid protein Western blot.

Nucleocapsid protein immunoblotting for presence of anti SARS-Cov-2 IgG antibodies in serum.
One µg of original purified SARS-Cov-2 nucleocapsid protein was loaded onto a 12% IPG/prep-well comb gel (Bio-Rad #456-1041). The resultant PAGE gel was blotted onto nitrocellulose membrane, which was then inserted into the membrane processor (Milliblot-MP; Millipore, Bedford, MA, USA) and 230 µL of 1:100 diluted donor serum was applied into each slot. After incubation of 180 min at RT the entire membrane was removed from the membrane processor and incubated for two washing steps for 10 min each with TPBS. Finally, the membrane was exposed to the HRP-conjugated antibody detection solution for 60 min at RT and developed using chemiluminescence reagent. Upper panel #4, 13, 18, C4 represent #9, 200, 222, C4 at the HEATMAP (Fig 2). Middle panel #5, 16, 21, C4 represent #127, 133, 148, C4 at the HEATMAP (Fig 2). Lower panel #1, 13, C4 represent #65, 67, C4 at the HEATMAP (Fig 2). Number 7 at lower panel depicts patient #127 at the HEATMAP two weeks earlier. Blots, which were processed on different days were clearly separated from others by a white space. The arrow indicates the recombinant nucleocapsid protein as obtained from Prospec-Tany Technogene Ltd. (Ness-Ziona, Israel).

Spike protein immunoblotting for presence of anti SARS-Cov-2 IgG antibodies in serum.
One and a half µg of original purified spike surface glycoprotein (as indicated in Amanat F, Stadlbauer D, et al. Nature Med., 2020) was loaded onto a 12% IPG/prep-well comb gel (Bio-Rad #456-1041). The resultant PAGE gel was blotted onto nitrocellulose membrane, which was then inserted into the membrane processor (Milliblot-MP; Millipore, Bedford, MA, USA) and 230 µL of 1:100 diluted donor serum was applied into each slot. After incubation of 180 min at RT the entire membrane was removed from the membrane processor and incubated for two washing steps for 10 min each with TPBS. Finally, the membrane was exposed to the HRP-conjugated antibody detection solution for 60 min at RT and developed using chemiluminescence reagent. Upper panel #13 (termed C3) represents C3 at the HEATMAP (Fig 2). Lower panel #23 (termed C4) represents C4 at the HEATMAP (Fig 2). The arrow indicates the spike surface glycoprotein as obtained (Amanat F. et al., 2020). Molecular size markers are presented to the left. Blots, which were processed on different days were clearly separated from others by a white space.   ◆ First infections registered in Austria.
Vienna city authorities extended measures taken by the federal government in Austria and issued an escalation plan for adequate medical care (marked striped). Designated hospitals were primarily responsible for medical care of Covid-19 patients while others were intended to be Covid-19 free.
In addition, the Medical University of Vienna took several steps to ensure the readiness of the medical staff and to designate Covid-19 patients as early as possible to specialized centers (marked in grey).