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 Table of Contents  
Year : 2020  |  Volume : 22  |  Issue : 3  |  Page : 93-97

Antibody response among nucleic acid amplification test confirmed COVID-19-positive patients: A cross-sectional study

1 Classified Specialist (Microbiology), MH Namkum, Ranchi, Jharkhand, India
2 Commandant, MH Namkum, Ranchi, Jharkhand, India
3 Officer Commanding 48 FHO, Bathinda, Punjab, India
4 Graded Specialist (Medicine), MH Namkum, Ranchi, Jharkhand, India

Date of Submission31-Jul-2020
Date of Decision01-Aug-2020
Date of Acceptance30-Sep-2020
Date of Web Publication26-Oct-2020

Correspondence Address:
Lt Col (Dr.) Rachana Warrier
Graded Specialist (Medicine), MH Namkum, Ranchi - 834 010, Jharkhand
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmms.jmms_107_20

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Introduction: Coronavirus disease 2019 (COVID-19) pandemic has caused immense loss of lives, enormous devastation, and has affected lives globally. Timely diagnosis is necessary to contain its spread. While it is important to test symptomatic individuals and high-risk contacts through reliable and confirmatory test (nucleic acid amplification test [NAAT]) to find out the positive cases of COVID-19, it is also vital to check their immunity status through the serological tests to find out the varied immunological response in the affected population. Materials and Methods: All NAAT confirmed COVID-19 patients were included in the study. With a repeat respiratory sample for NAAT, simultaneous serum sample was taken for the detection of IgM and IgG antibodies. Data were compiled and analyzed statistically. Results: During the first repeat NAAT, 71.43% had mounted an antibody response, whereas 28.57% had no response. Irrespective of the antibody status, 68.57% tested positive for SARS-CoV-2 virus in NAAT. Although the number dwindled on second repeat NAAT, it raised important questions regarding humoral response and its clinical and diagnostic applicability. Conclusion: Not all, COVID-19 patients mount a humoral response due to yet unexplained factors. Furthermore, exclusive dependence on serology assays will miss the diagnosis in these patients. More research is needed for description and applicability of humoral response in COVID-19 patients.

Keywords: COVID-19, antibody response, nucleic acid amplification test, SARS-CoV-2, serology assay

How to cite this article:
Bhalla GS, Shukla R, Bandyopadhyay K, Warrier R. Antibody response among nucleic acid amplification test confirmed COVID-19-positive patients: A cross-sectional study. J Mar Med Soc 2020;22, Suppl S1:93-7

How to cite this URL:
Bhalla GS, Shukla R, Bandyopadhyay K, Warrier R. Antibody response among nucleic acid amplification test confirmed COVID-19-positive patients: A cross-sectional study. J Mar Med Soc [serial online] 2020 [cited 2022 Jan 24];22, Suppl S1:93-7. Available from: https://www.marinemedicalsociety.in/text.asp?2020/22/3/93/299130

  Introduction Top

Coronavirus disease 2019 (COVID-19) outbreak caused by the novel coronavirus was declared a pandemic by the WHO on March 12, 2020.[1],[2] In our nation, as on August 27, 2020, we have a total of 2,114,140 confirmed cases with 1,447,332 recovered patients.[3] The pandemic has caused immense loss of lives, enormous devastation, and has affected lives globally.

Timely diagnosis of cases, besides other preventive measures, is one of the most important links in the chain of reducing the spread of COVID-19. It is of paramount importance, especially in hospitalized patients who can spread the disease to health-care workers and other contacts, resulting in the loss of precious man-hours, panic, and even shutting down of hospitals. Asymptomatic individuals harboring the virus are a serious threat to the community and can spread it unknowingly.

Currently, the Indian Council of Medical Research (ICMR) has approved nucleic acid amplification tests (NAAT) (real-time reverse transcription polymerase chain reaction [RT-PCR], cartridge-based NAAT [CB-NAAT], and Truenat™), and rapid antigen detection for diagnostic purpose. Nucleic acid testing modalities are confirmatory but are expensive and require expertise. With RT-PCR, more than 90 samples can be processed at one time; however, it has a long turn-around-time and requires dedicated infra-structure and trained technicians. Thus, it is justifiable in hospitals with a large number of patients. CB-NAAT and Truenat™ have limited sample testing capacity; however, turn-around-time is very less, even single samples can be tested, and the results are available within 1 h. CB-NAAT is a one-step confirmatory test, whereas Truenat™ is a two-step test, screening followed by confirmation. These testing methods do not require huge infrastructure and are ideal for zonal hospitals. Antigen detection is an easy to do point-of-care test, but a negative result needs to be reconfirmed by the nucleic acid detection method. All the above modalities can only be used for testing respiratory samples. As on date, ICMR has not approved serological methods for the clinical diagnosis of COVID-19.[4]

While it is important to test symptomatic individuals and high-risk contacts through reliable and confirmatory test (NAAT) to find out the positive cases of COVID-19, it is also vital to check their immunity status through serological tests to find out the varied immunological response in the affected population. Besides the diagnostic aspect, understanding of humoral response in COVID-19 patients will help clear the speculations about the success of plasma therapy and vaccination. Hence, this study was conducted to understand the antibody response in patients of COVID-19 and infer diagnostic and clinical implications of antibody response in them.

  Materials and Methods Top

The present study was conducted as a cross-sectional, descriptive study. The study was conducted for duration of 3 months (June 2020 to August 2020). All individuals who tested positive for COVID-19 by NAAT during the period of study (both symptomatic and asymptomatic) were included in the study. The exclusion criteria were individuals who tested negative on initial NAAT. Nasopharyngeal and oropharyngeal swab samples from all individuals who reported to the center (either with symptoms suggestive of the disease such as sore-throat, cough, fever or breathlessness, or as high-risk contacts of a confirmed case of COVID-19), were subjected to NAAT. Out of the 387 individuals who were tested during the period of this study, 105 initial CB-NAAT confirmed COVID-19 patients were included in the study [Figure 1]. These 105 cases were subjected to a repeat NAAT (as per the organizational policy). Simultaneously, the serum samples of these 105 patients were collected by venipuncture using the standard aseptic technique in serum separator vacutainers for serology assay for IgM and IgG antibodies (14 days after initial NAAT positive). All individuals who tested positive on first repeat NAAT (72 cases) were subjected to a second repeat NAAT (as per the organizational policy). Testing kits used for CB-NAAT were GeneXpert ® Xpress SARS-CoV-2 (Cepheid, Sunnyvale, CA, USA) [Figure 2], and those used for serology testing were SD Biosensor Standard Q ® COVID-19 IgM/IgG Duo (ICMR approved lot number) [Figure 3]. Written informed consent was obtained from all patients.
Figure 1: Flowchart of study protocol

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Figure 2: GeneXpert: Equipment, cartridge, and sample result

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Figure 3: SD Biosensor Standard Q® COVID-19 IgM/IgG Duo Kit with positive IgM and IgG response

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Descriptive analysis by Microsoft Excel (2016), Redmond, Washington, U.S and Epi-Info™ ver (by Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, U.S) was carried out. The appropriate measures of descriptive and inferential statistics were used for the statistical analysis. The present study was approved by the Institutional Ethics Committee.

  Results Top

The present study included a total of 105 participants whose mean age was found to be 31.64 ± 7.79 years (minimum age being 21 years and maximum age being 46 years). The study participants were all healthy participants with no known co-morbidities, and they had a minimum of Std XII educational level. They were all laboratory confirmed COVID-19 cases (either being a symptomatic individual or being a high-risk contact of a suspect/confirmed case in the station), who had reported to the hospital.

[Table 1] shows the distribution of various categories of individuals based on their antibody response with first repeat NAAT results. A total of 75 (71.43%) participants showed development of antibodies (IgM/IgG/both), whereas the remaining 30 (28.57%) did not show any antibody response at all. It was found that among the 72 (68.57%) participants who were found positive on first repeat testing, 12 (11.43%) did not show any antibody response at all and remaining 60 (57.14%) showed antibody response. Among the 33 (31.43%) participants who tested negative on first repeat NAAT, 15 (14.29%) showed antibody response, whereas 18 (17.14%) had no humoral response. A significant association was found (P < 0.001) between NAAT positivity on first repeat testing and the antibody response among the participants.
Table 1: Association between first repeat nucleic acid amplification test positivity and antibody response among the participants

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[Figure 4] shows the distribution of study participants based on their antibody response (either IgM only or IgG only or both) on first repeat NAAT. Among the 60 participants who had tested positive on first repeat NAAT, 12 (20%) showed IgM only response, 12 (20%) showed IgG only response, and 36 (60%) showed both IgM and IgG response. Among the 15 participants who were negative on first repeat testing with NAAT, three (20%) showed IgM only response, six (40%) showed IgG only response, and six (40%) showed both IgM and IgG response.
Figure 4: Distribution of study participants based on antibody response

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Seventy-two participants who tested positive on first repeat NAAT were subjected to a second repeat NAAT and were followed up. Among these 72 participants, three remained positive on second repeat NAAT as well, whereas the remaining 69 individuals were tested negative.

  Discussion Top

Dynamics of immune response and protection against infections caused by many viral infections are well understood and have been successfully modeled.[5] The same is yet to be understood for SARS-CoV-2 infection.

As we can see from the results, 60 patients (57.14%) mounted a humoral response but virus was also detected in their respiratory samples. Possible explanations for the same being that humoral response may take a long time (>14 days) to clear infection from the body, as evidenced by 69 patients who tested negative on second repeat NAAT. A significant association was found (P < 0.001) between NAAT positivity on repeat testing and the antibody response among the participants, suggesting that humoral response is triggered after prolonged infection with SARS-CoV-2. Whether the patients remain infective during this stage is yet to be determined as nucleic acid detection techniques available are qualitative and not quantitative and performing viral culture assays are beyond the scope of this institute.

Even on the 14th day, 12 patients (11.43%) did not show any antibody response; however, their repeat NAAT was positive. Possible explanation for the same is that these patients are nonresponders and do not form any detectable antibodies in response to infection. Since they are NAAT positive, they may still be infectious. It may also be possible that viremia is not achieved, virus remains localized in certain patients and no antibody response is triggered; or, they may form incomplete or nonneutralizing antibodies which are not detected by commercially available kits. It may also be hypothesized that multiple strains of virus are in circulation, and serology assay kits may not detect antibodies against all strains. It could also be there that the virus has entered a latent/nonpropagative stage where its nucleic acid is detected, but no antibodies are formed. Thus, it implies that the exclusive use of serology assays for diagnostic purpose will miss this set of population. Although a large number of serology assays are available from various manufacturers, their clinical use is questionable owing to unacceptable sensitivity and/or specificity.[6],[7],[8]

Among the first repeat NAAT negative patients, 15 (14.29%) showed antibody response. This is an ideal scenario where the virus is neutralized by the antibodies and gradually cleared from the body rendering the patient noninfectious. Correlation between antibody response and severity of the disease has not yet been defined. Moreover, no data are available on the number of circulating strains and whether antibodies against one strain would be helpful in the prevention against reinfection with same or another strain. The duration for which the protection lasts is another caveat which needs careful consideration.[9]

Finally, 18 patients (17.14%) tested negative for virus nucleic acid despite the absence of circulating antibodies. With 30 patients mounting no humoral response and more than half of them (18 participants) testing negative by NAAT, suggest other mechanisms of viral clearance like CD8+-mediated killing of infected cells, role of interferons, and cell-mediated immunity in protection against COVID-19. Genetic make-up of an individual determines the inflammatory response; hence, its role, though questionable/not studied, may determine the susceptibility/immunity to the development of disease following SARS-CoV-2 infection.

No data is available till date regarding humoral response in public postvaccination; however, if approximately 29% are nonresponders, the clinical protection offered by vaccination may be sub-optimal.

Amorim Filho et al. studied the antibody titers to SARS-CoV-2 in 2857 blood donors from Rio de Janeiro. Overall, antibody prevalence was 4%; remaining population being either nonexposed to virus or nonresponders. All antibody positive samples were subjected to molecular assay which was negative.[10] It may imply that there is no viremia; however, virus may still be present in the respiratory tract.

Similarly, Stringhini, et al. studied the seroprevalence of anti SARS-CoV-2 IgG antibodies in Geneva and found that most of the study population were IgG negative.[11]

A seroprevalence study conducted in Spain on 66805 study participants revealed that the prevalence of IgG antibodies against coronavirus is around 5%. They concluded that the majority of the Spanish population is seronegative to SARS-CoV-2 infection, and at least a third of infections determined by serology were asymptomatic.[12] However, no correlation was done with PCR; hence, nonresponders were not defined. They might have been infectious and spreading the disease.

Wu et al. conducted a study on 537 patients in Taiwan which showed that in the first 2-week period, RT-PCR was the most sensitive method of detecting the virus. During the convalescent phase of the disease antibodies were detected in serum. Neutralization test, enzyme-linked immunosorbent assay, immunofluorescence test, and immunochromatographic test were evaluated and compared for antibody responses to SARS-CoV-2 infection, in which the neutralization test was held as a reference method. In this study, the antibody positive rate for probable COVID-19 patients was 54.2%.[13] Similar antibody response was observed in the present study.

Padoan et al. studied the IgA antibody response to SARS-CoV-2 infection. They concluded that the IgA antibody levels were persistently high and peaked at 20–22 days of infection.[14] This method was not included in the present study. Theoretically, it is logical to believe that IgA, being a secretory antibody on epithelial surfaces offers effective neutralization of virus particles and more initial protection than the circulating antibodies (IgM and IgG).

  Conclusion Top

Although the present study was conducted with a small sample population, it raises important questions regarding the absence of humoral response in approximately 29% of SARS-CoV-2 infected patients. Clinical and diagnostic use of serology assays too need exhaustive research. Thus, it also probes the early availability of an effective vaccine and role of convalescent plasma for the treatment. With further research, more light will be shed on pathophysiology of the disease, and hence, its management; thereby shifting the pandemic response back to normalcy.

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Conflicts of interest

There are no conflicts of interest.

  References Top

World Health Organization. Coronavirus Disease (COVID-19) – Events as They Happen. Geneva: World Health Organization; 2020. Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/events-as-they-happen. [Last acessed on 2020 Aug 27].  Back to cited text no. 1
Odor PM, Neun M, Bampoe S, Clark S, Heaton D, Hoogenboom EM, et al. Anaesthesia and COVID-19: Infection control. Br J Anaesth 2020;125:16-24.  Back to cited text no. 2
Coronavirus Outbreak in India. Available from: https://www.covid19india.org/. [Last accessed on 2020 Aug 27].  Back to cited text no. 3
Indian Council of Medical Research – Information forTesting Laboratories. Available from: https://www.icmr.gov.in/ctestlab.html. [Last accessed on 2020 Jul 29].  Back to cited text no. 4
Hens N, Habteab Ghebretinsae A, Hardt K, Van Damme P, Van Herck K. Model based estimates of long-term persistence of inactivated hepatitis A vaccine-induced antibodies in adults. Vaccine 2014;32:1507-13.  Back to cited text no. 5
Lassaunière R, Frische A, Harboe ZB, Nielsen AC, Fomsgaard A, Krogfelt KA, et al. Evaluation of nine commercial SARS-CoV-2 immunoassays. medRxiv 2020.04.09.20056325; doi: https://doi.org/10.1101/2020.04.09.20056325.  Back to cited text no. 6
Whitman JD, Hiatt J, Mowery CT, Shy BR, Yu R, Yamamoto TN, et al. Test performance evaluation of SARS-CoV-2 serological assays. medRxiv [Preprint]. 2020 May 17:2020.04.25.20074856. doi: 10.1101/2020.04.25.20074856. Update in: Nat Biotechnol. 2020 Aug 27;: PMID: 32511497; PMCID: PMC7273265.  Back to cited text no. 7
Adams ER, Anand R, Andersson MI, Auckland K, Baillie JK, Barnes E, et al. Evaluation of antibody testing for SARS-Cov-2 using ELISA and lateral flow immunoassays. medRxiv [Preprint] 2020. [www.medrxiv.org/content/10.1101/2020.04.15.20066407v1.full.pdf]  Back to cited text no. 8
Krammer F, Simon V. Serology assays to manage COVID-19. Science 2020;368:1060-1.  Back to cited text no. 9
Amorim Filho L, Szwarcwald CL, Mateos SOG, Leon ACMP, Medronho RA, Veloso VG, et al. Seroprevalence of anti-SARS-CoV-2 among blood donors in Rio de Janeiro, Brazil. Rev Saude Publica. 2020;54:69. doi: 10.11606/s1518-8787.2020054002643. Epub 2020 Jul 6. PMID: 32638883; PMCID: PMC7334006.  Back to cited text no. 10
Stringhini S, Wisniak A, Piumatti G, Azman AS, Lauer SA, Baysson H, et al. Seroprevalence of anti-SARS-CoV-2 IgG antibodies in Geneva, Switzerland (SEROCoV-POP): A population-based study. Lancet 2020;396:313-9.  Back to cited text no. 11
Pollán M, Pérez-Gómez B, Pastor-Barriuso R, Oteo J, Hernán MA, Pérez-Olmeda M, et al. Prevalence of SARS-CoV-2 in Spain (ENE-COVID): A nationwide, population-based seroepidemiological study. Lancet 2020;396:535-44.  Back to cited text no. 12
Wu HS, Chiu SC, Tseng TC, Lin SF, Lin JH, Hsu YH, et al. Serologic and molecular biologic methods for SARS-associated coronavirus infection, Taiwan. Emerg Infect Dis 2004;10:304-10.  Back to cited text no. 13
Padoan A, Sciacovelli L, Basso D, Negrini D, Zuin S, Cosma C, et al. IgA-Ab response to spike glycoprotein of SARS-CoV-2 in patients with COVID-19: A longitudinal study. Clin Chim Acta 2020;507:164-6.  Back to cited text no. 14


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1]


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