Diagnostic significance of SARS-CoV-2 IgM positive/IgG negative antibody profile in symptomatic patients with suspected COVID-19 testing negative by RT-PCR.

Dear Editor,

We were very interested to read the article by Pan and colleagues, and related letter by Cancella de Abreu supporting the diagnostic utility of SARS-CoV-2 Lateral Flow Immunochromatographic assays (LFIC) for acute COVID-19 in patients testing negative by RT-PCR at initial screening, provided that serological testing is performed at least one week after symptoms onset. We noted that COVID-19 diagnosis was made on detection of SARS-CoV-2 IgM in the absence of IgG reactivity in a non-negligible fraction of patients in both series (around 50%). , Our experience detailed below casts some doubt on the reliability of an isolated SARS-CoV-2 IgM positive result as determined by LFIC for COVID-19 diagnosis. The current study was approved by the Research Ethics Committee of University Clinic Hospital (HCU), INCLIVA, Valencia (2020-03).

Between March 23 and June 30, 2020 a total of 1,032 patients attended at the emergency department of HCU with clinical suspicion of COVID-19 were tested as per protocol using the ALLTEST 2019-nCoV IgG/IgM Rapid Test Cassette (Hangzhou ALLTEST Biotech Co., Ltd. Hangzhou, China), a LFIC device which detects IgG and IgM against a recombinant SARS-CoV-2 N protein. The test was carried out and interpreted as recommended in the package insert, whereby appearance of the test band was recorded as a positive result, irrespective of intensity. According to the manufacturer, the specificity of the SARS-CoV-2 IgM assay is 97.0% (IC95%:86.3%-99.5%).

The SARS-CoV-2 IgM+/IgG- antibody profile was found in sera from 18 patients (1.7%), which were collected at a median of 7 days (range 5-12 days) after the onset of symptoms (1.7%). Three patients tested positive by RT-PCR in upper respiratory tract (URT) specimens using commercially available RT-PCR assays. , Accordingly, SARS-CoV-2 IgM detected in these 3 patients were deemed to be true positives. The remaining 15 patients (9 females; median age, 67 years; range, 37 to 93 years) tested negative by RT-PCR in URT samples once or several times (Table 1 ). Follow-up sera from these patients were collected at a median of 11 days (range, 2 to 30 days) after initial serological testing. SARS-CoV-2 IgG seroconversion, which is generally documented by 3-4 weeks after SARS-CoV-2 infection,6, 7, 8 could not be demonstrated in any cases. Following thorough review of medical charts and based upon clinical, imaging and laboratory findings, repeat negative RT-PCR testing and lack of IgG seroconversion, we came to the conclusion that patients 1,3,4,5,7,9,10,11 and 15 most likely had no COVID-19, whereas patients 2,6,8,12,13 and 14 experienced either possible or probable COVID-19 (Table 1). Of note, IgM reactivity persisted in 4 out of 6 follow-up sera from patients categorized as likely having COVID-19 (66.6%), and in 3 out of 9 from patients without it (33.3%).

Table 1

Characteristics of patients with clinical suspicion of COVID-19 testing IgG-/IgM+ by lateral flow immunochromatography at initial serological screening and negative by RT-PCR in upper respiratory tract specimens.

Patient	Hospital admission/days of stay	Negative RT-PCR results in URT	Initial serological testing (days after symptoms onset)	LFIC IgM result in initial sera after urea treatmenta	LFIC result in follow-up sera	LFIC result of follow-up sera after urea dissociation	Final diagnosisb
1	No	2	2	-	IgG- /IgM-	NA	Upper respiratory tract infection/No COVID-19
2	No	1	7	+	IgG- /IgM+	+	Possible COVID-19
3	Yes/14	2	4	+	IgG- /IgM+	+	Pulmonary abscess secondary to pneumococcal pneumonia
4	Yes/13	3	7	-	IgG- /IgM+	-	Community acquired pneumonia/No COVID-19
5	No	3	10	-	IgG- /IgM-	NA	Urinary tract infection due to Escherichia coli
6	Yes/7	5	6	+	IgG- /IgM+	+	Probable COVID
7	Yes/10	2	10	-	IgG- /IgM-	NA	Pneumococcal pneumonia
8	Yes/4	3	7	+	IgG- /IgM+	+	Possible COVID
9	Yes/86	4	1	+	IgG- /IgM+	-	Cardiac insufficiency
10	Yes/12	4	1	-	IgG- /IgM-	NA	Hepatic encephalopathy/ ascitic decompensation
11	Yes/5	2	15	-	IgG- /IgM-	NA	Pneumococcal pneumonia
12	Yes/9	5	9	+	IgG- /IgM-	NA	Probable COVID-19
13	Yes/7	2	14	-	IgG- /IgM-	NA	Probable COVID-19
14	Yes/10	2	3	-	IgG- /IgM+	-	Probable COVID-19
15	No	3	16	-	IgG- /IgM-	NA	Fever of unknown origin

LFIC, lateral flow immunochromatographic assay; NA, not applicable; URT, upper respiratory tract.

A volume of 10µL of serum was diluted into 1 mL of sample buffer before depositing (100 µL) into the appropriate location of the cassette (Test T-hole). When the fluid was about to reach the absorbent pad, 100 µL of sample buffer containing 6M urea was added to the T hole on the card. Serum specimens were run in parallel in the absence of urea treatment. Each reading was carried out independently by two observers after 20 min incubation.

According to the European Centre for Disease Prevention and Control (ECDC) https://www.ecdc.europa.eu/en/covid-19/surveillance/case-definition, possible COVID-19 case: any person meeting the clinical criteria; Probable case: any person meeting the clinical criteria with an epidemiological link or person meeting the diagnostic imaging criteria.

Urea dissociation antibody test performed on LFIC matrices has proven helpful in reducing false-positive IgM results in suspected COVID-19 patients resulting from presence of rheumatoid factor (RF) or other undetermined causes. , SARS-CoV-2 IgM reactivity in initial sera disappeared in 2 out of the 6 patients deemed to have COVID-19 (33.3%) following urea (6M) treatment and in 7 out of 9 patients in whom COVID-19 was judged to be unlikely (78%). Regarding follow-up sera, SARS-CoV-2 IgM were eluted from 1 out 4 sera (25%) from patients with COVID-19 and from 2 out of 3 sera (66.6%) from patients without COVID-19.

We acknowledge that a major limitation of the current study is the potential misclassification of patients as either having COVID-19 or not, which may have occurred due to either a lack of RT-PCR testing of lower tract respiratory specimens, that may yield positive results in the absence of URT SARS-CoV-2 shedding, or overly prompt follow-up serological testing in patients displaying delayed SARS-CoV-2 IgG seroconversion. Another drawback of the study is not testing for potential causes of SARS-CoV-IgM false positive reactivity, in particular for presence of RF.

In summary, our data indicate that interpreting the SARS-CoV-2 IgM+/IgG- antibody profile in symptomatic patients with suspected COVID-19, either requiring hospitalization or not, who test negative by RT-PCR is by no means straightforward. In our series, up to 50% of patients displaying that antibody pattern might not have had COVID-19. The prevalence of confirmed COVID-19 cases among patients attended at the emergency department of HCU within the study period was 10-15%. For a SARS-CoV-2 antibody test with a specificity of 97%, such as that of the LFIC used herein, its positive predictive value for COVID-19 diagnosis is expected to be 70-80%, a much higher figure than the one reported herein. Our data also support the assumption that persistent SARS-CoV-2 IgM reactivity in initial or follow-up sera following urea treatment, and repeat positive IgM testing in sequential sera, may both help to reliably establish COVID-19 diagnosis.

Funding

This research did not receive any specific grant from public or private funds.

Declaration of Competing Interest

The authors declare no conflicts of interest.