Keywords
antigen test - antibody test - point-of-care test
Introduction
Coronavirus disease-2019 (COVID-19) has infected more than 176 million people worldwide
resulting in approximately 3.8 million deaths.[1] With the rapid surge of patients/contacts overwhelming existing laboratory capacities,
there is a need for point-of-care rapid diagnostic tests (RDT) for early diagnosis
of COVID-19. It would allow early treatment and isolation of cases thus reducing the
spread of COVID-19, especially in resource-constrained settings.
Laboratory diagnosis and management of COVID19 have been helpful in combating the
spread of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). At present,
the gold standard for COVID-19 diagnosis is reverse-transcription-quantitative PCR
(RT-qPCR) that uses nasopharyngeal swabs, throat swabs, or saliva samples.[2] RT-qPCR kits that do not require viral RNA extraction and high-throughput RT-qPCR
systems have also been developed. Although such tests are widely utilized in tertiary
care centers and large well-equipped hospitals, they are rarely available in the local
clinics that are more approachable for the patients who are under suspicion.
Studies done on rapid antigen tests have shown sensitivity of 61.70% and specificity
of 98.26% for the diagnosis of COVID-19.[3] A cross-sectional, single-blinded study by Gupta et al showed overall sensitivity
and specificity 81.8 and 99.6%, respectively. The sensitivity of RDT was higher (85.9%)
in participants with a duration of illness up to 5 days.[4]
To aid in the proper management and timely diagnosis of COVID-19, validated and accurate
laboratory testing is crucial. This will help the clinicians and infection control
practitioners to combat infection at the health-care level and detect clinical cases
timely. Therefore, it would further help in appropriate treatment, prompt isolation,
and consequently deceleration of the cases.
Methodology
This is prospective cohort study conducted at the Department of Microbiology at Jai
Prakash Narayan Apex Trauma Center, All India Institute of Medical Sciences, New Delhi.
In this study, a total of 110 subjects were included. Samples were collected from
the hospital inpatients who were laboratory confirmed cases of COVID-19.
RT-PCR for COVID-19 was done for these patients in last 48 to 72 hours to ensure that
the desired number of true positives was being enrolled.
We used STANDARD Q COVID-19 antigen and immunoglobulin M/immunoglobulin G (IgM/IgG)
combo kit (SD Biosensor, Inc., Gurugram, Haryana, India) that is a rapid immunochromatography
test designed for the qualitative detection of specific antigens in human nasopharynx
and IgM/IgG in humoral fluid ([Fig. 1]).
Fig. 1 Schematic diagram of immunoglobulin M/immunoglobulin G (IgM/IgG) severe acute respiratory
syndrome-coronavirus-2 antibody tests (https://doi.org/10.1002/jmv.25727). COVID-19, coronavirus disease 2019.
For antigen-based tests, nasopharyngeal swab was collected (as per manufacturer's
instructions). The samples were immediately placed in the buffer (provided with the
test kit). Test was performed as per manufacturer's instructions. For antibody-based
test, blood/plasma/serum was collected as per manufacturer's instructions. All necessary
reagents are provided with the kit and no equipment is required. The rapid antigen
tests were performed as per the test kit instructions.
The detection time for antibodies is 10 to 15 minutes and the specimen types are whole
blood (20 µL) and serum/plasma (10 µL) suitable for detection. The antigen test, with
nasopharyngeal sample, gives results in 30 minutes. The reading for the rapid antigen
test should be taken as per time mentioned in the manufacturer's instructions. Pictures
of the rapid test strips results were taken for the documentation.
Statistical Analysis
Stata 14.0 Statistical Software (Stata Corp LLC, Texas, United States) was used for
data analysis. Diagnostic characteristics such as sensitivity and specificity of the
test with RT-PCR as reference were calculated. The RDT was also evaluated considering
days since infection. For each of the summary measures, frequency with percentage
and 95% confidence interval (CI) was also computed.
Result
This is a prospective cohort study in which we have evaluated 110 number of patients
for the study. All patients were laboratory confirmed cases of COVID-19. Out of 110
patients, only 88 patients met the inclusion criteria. The gender distribution of
the present population was male 62 (70.4%) and females 26 (29.6%), respectively. The
median duration of illness at the time of testing among symptomatic patients was 1
day (range: 1–10). The most common symptoms among the participants were fever (71.5%),
cough (25.4%), and fatigue/malaise (12.8%).
The study found that 26.7% (95% CI: 17.2–36.3) of total patients tested positive for
antigen test ([Fig. 2A]). The sensitivity for IgM was found to be 48.6% (95% CI: 37.2–60.1), whereas 51.3%
(95% CI: 39.8–62.81) of patients show presence of IgG antibody ([Table 1A]–[D]). The sensitivity of combined antibody test (IgM and IgG both) was 52% ([Fig. 2B]). The sensitivity of combined testing, that is, both antigen and antibody test,
was found to be 72.7%. There were 14.7% patients who were neither positive to antigen
nor to antibody.
Fig. 2 Illustrations of (A) severe acute respiratory syndrome-coronavirus-2 antigen and (B) immunoglobulin M/immunoglobulin G antibody test.
Table 1
(A) Sensitivity and 95% confidence interval of SD antigen–antibody combo kit, (B)
comparison between antigen and IgM antibody, (C) comparison between antigen and IgG
antibody, (D) comparison between IgM and IgG antibody
|
(A) SD antigen–antibody combo kit
|
Frequency
|
Sensitivity (%)
|
95% Confidence interval
|
|
|
Antigen kit
|
23
|
26.74
|
17.2
|
36.29
|
|
Antibody kit (IgM)
|
37
|
48.68
|
37.19
|
60.18
|
|
Antibody kit (IgG)
|
39
|
51.32
|
39.82
|
62.81
|
|
(B) Ab kit (IgM)
|
|
Antigen kit
|
Negative
|
Positive
|
Total
|
|
Negative
|
26
|
28
|
54
|
|
Positive
|
12
|
9
|
21
|
|
Total
|
38
|
37
|
75
|
|
(C) Antibody kit (IgG)
|
|
Antigen kit
|
Negative
|
Positive
|
Total
|
|
Negative
|
18
|
36
|
54
|
|
Positive
|
18
|
3
|
21
|
|
Total
|
36
|
39
|
75
|
|
(D) Antibody kit (IgG)
|
|
Antibody kit (IgM)
|
Negative
|
Positive
|
Total
|
|
Negative
|
24
|
15
|
39
|
|
Positive
|
13
|
24
|
37
|
|
Total
|
37
|
39
|
76
|
Abbreviations: IgG, immunoglobulin G; IgM, immunoglobulin M.
Discussion
RT-PCR is the gold standard test for SARS-CoV-2, but it is time-consuming and requires
specialized laboratory and trained laboratory personnel. At the same time, RT-PCR
is also very expensive. Due to higher contingency of SARS-CoV-2 and increasing number
of patients, antigen and antibody tests may be utilized. Various tests detecting SARS-CoV-2
antigens and antibody have recently been developed and commercially available.[5]
[6] Many studies have been done to evaluate the COVID-19 tests including antibody and
antigen test.[7]
[8]
[9] In the current study, median time from onset of illness at the time of testing was
1 day (interquartile range [IQR]: 1–10 days), which is shorter than that in previous
studies (6 days) (IQR: 3–13 days).[10] IgM antibody was found in 48.6%, IgG antibody in 51.3%, and the sensitivity of the
combined antibody test (IgM and IgG both) was calculated to be 52% from the specimens.
However, in a study from Imai et al, IgM and IgG antibodies were detected in 43.2%
and 14.4% and combined antibody test (IgM and IgG both) was 43.2%, respectively.[10] The clinical effectiveness of serological tests for COVID-19 remains questionable
due to interval between onset of symptoms and appearance of IgM and IgG antibodies
in serum. This discrepancy seemingly reflects differences in timing of sampling because
the health centers vary across countries. In this study, the antigen testing showed
less sensitivity than antibody testing. In fact, the sensitivity to individual antibody
test was more than the sensitivity of antigen test. The sensitivity of IgM and IgG
individually is approximately twice that of antigen test. Present study shows sensitivity
of combined testing is approximately three times than antigen testing alone, whereas
this value is double when compared with only antibody testing. SARS-CoV-2 sensitivity
increases when combined methods are used.
The reason for lower sensitivity of antigen tests is the dependance upon viral load.
Also, RT-PCR may remain positive for longer duration, because it is a nucleic acid
detection-based test. However, antigen will clear from the nasopharynx earlier. Higher
viral load has correlated with higher sensitivity but it shows high specificity (99.3–100%).[11]
[12]
The appearance of IgG antibodies correlates with clearance of antigen in various infectious
diseases; surveilling symptomatic individuals or individuals with mild symptoms who
are likely to be unaware of their disease status would be greatly helped by combining
antigen to the test. However, the overall sensitivity of the kit is not very promising
for diagnostic utility for COVID-19 and there is much scope of improvement.[13]
A major limitation of this study was small sample size. In addition, cycle threshold
values were not followed up in the subjects.
Conclusion
To enhance the laboratory diagnostics in the most challenging time of COVID-19 pandemic
supporting evidence-based medicine, government guidelines, health-care policies should
be formulated and priortized. The efforts of microbiologist–clinician team should
focus on implementing the most reliable diagnostic tools; however, because COVID-19
is a new disease, there are not enough data as of yet that would enable the determination
of standards for the interpretation of serological point of care test. Serological
tests should meet the standards to be put to use in patients having symptomatic and
asymptomatic disease.
