Study setting

This study took place in ASU hospitals. It included seven hospitals (surgery hospital, internal medicine hospital, pediatrics hospital, obstetrics and gynecology hospital, emergency hospital, and geriatrics hospital). The hospitals have a capacity of more than 3,000 inpatient beds and serve about 1.5 million patients annually.

Study population

All patients needing admission to ASU hospitals were eligible for the study.

The hospital screening program

By the beginning of the epidemic in Egypt, ASU hospitals established a symptom-based screening clinic for all patients seeking hospital services. SARS-CoV-2 PCR and total antibody assay were done for all patients requiring hospitalization.

Study methods

Every enrolled patient was subjected to:

1. An interview questionnaire. The study questionnaire included:
   1. Background characteristics (age, gender, residence, and contact details)
   2. History of contact with a COVID-19 case
   3. Clinical data: Temperature was measured on admission as part of the preadmission screening. Patients were asked about other symptoms, e.g., cough and sore throat.
   4. History of comorbidities: Self-reported diabetes and hypertension
2. Laboratory Tests: Reverse Transcription Polymerase Chain Reaction (RT-PCR) and total antibody assay for SARS-CoV-2. Tests were done only once before admission. PCR was repeated for initially negative tests only if the patient developed suspected symptoms.

Specimen collection and handling

1. 1. Following the recommendations of the US CDC, combined oropharyngeal and nasopharyngeal swabs were collected from studied patients using sterile swabs with synthetic tips (dacron/nylon) and plastic, flexible shafts.

The swabs were rubbed against the posterior pharyngeal wall and tonsillar pillars, and then, the same swab was inserted into the patient’s nostril while tilting the patient’s head 70 degrees, and it passed slowly parallel to the palate until resistance was encountered. The swab was left in place for a few seconds to allow for secretion absorption and then was slowly removed while being twisted. Finally, the swab was immersed into a sterile tube containing 2 mL of viral transport media and was immediately transported to the laboratory at a temperature of 4±1°C.

1. 2. Serum samples: A sample of 3 ml whole blood was collected from each patient by peripheral venipuncture on a clot activator and gel separator vacutainer tube. The tubes were immediately centrifuged, and the separated serum was used to measure the SARS-COV2 total antibodies using the Elecsys® Anti-SARS-CoV-2 immunoassay (ROCHE).

I-Detection of SARS-COV2 RNA by reverse transcription real time polymerase chain reaction (rRT-PCR)

The amplification protocol.

The thermal cycler was programmed as follows:

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https://doi.org/10.1371/journal.pone.0254581.t001

II-Detection of SARS-COV2 total antibodies

The Elecsys® Anti-SARS-CoV-2 is an immunoassay for the invitro qualitative detection of antibodies (including IgG) to SARS-CoV-2 in human serum and plasma. The assay uses a recombinant protein representing the nucleocapsid (N) antigen in a double-antigen sandwich assay format, which favors detection of high affinity antibodies against SARS-CoV-2. The test is intended as an aid in the determination of the immune reaction to SARS-CoV-2.

A volume of 20 μL of the patient serum was incubated with a mix of biotinylated and ruthenylated nucleocapsid (N) antigen. Double-antigen sandwich immune complexes are formed in the presence of corresponding antibodies. After the addition of streptavidin-coated microparticles, the double-antigen complexes bind to the solid phase via the interaction of biotin and streptavidin. The reagent mixture is transferred to the measuring cell, where the microparticles are magnetically captured on the surface of the electrode. Unbound substances are subsequently removed. Electrochemiluminescence is then induced by applying a voltage and is measured using a photomultiplier. The signal yield increases with the antibody titer.

A cutoff index of <1.0 is considered non-reactive, whereas a cutoff index of ≥1.0 is considered reactive.

Statistical analysis

Data were validated, cleaned, and entered into a spreadsheet. Qualitative data were presented in frequency and related percentages. The level of antibodies was presented by median and interquartile range with the Mann–Whitney U test used for comparison. Unadjusted frequency of positive screening among the total was calculated with a 95% confidence interval. Given that SARS-CoV-2 PCR sensitivity was reported to be between 71%–95% [15], the PCR positivity was adjusted for test sensitivity for both scenarios with a specificity of 99.9%. The antibody seroprevalence was adjusted for kit sensitivity and specificity. According to the manufacturer’s package insert, Elecsys®.Anti-SARS-CoV-2 exhibits a high overall clinical specificity of 99.81% with no cross-reactivity to the common cold coronaviruses and a sensitivity of 100%. We used the Clopper–Pearson exact method to calculate 95% confidence intervals.

Comparison between groups was done using a Chi-square test with a P value of 0.05 as the level of significance. The odds ratio was calculated for the estimation of risk with a 95% confidence interval. Logistic regression was used for adjustment of the confounding factors.

SPSS program version 15 was used for the analysis. Epitools Epidemiological Calculators. Ausvet. was used for adjustment for tests’ sensitivity and specificity. Available at: http://epitools.ausvet.com.au

PCR detection rate

The PCR detection rate in the study group was 3.84%. Most estimations of the disease incidence in various countries are based on the vigorous surveillance system [16]. In Egypt, the reported cases are consensually believed to be highly underreported [17]. This study adds insight on the number of active cases in Cairo, one of the highest density population areas. Although the frequency of infected cases in the community varies geographically as well temporally, the findings of this research revealed relatively higher rates when compared to other published figures. Reported prevalence rates in Italy of SARS-CoV-19 by PCR were 2.6% at the start of the lockdown, with a comparable rate (2.5%) in Sweden. The PCR detection rate was reported as less than 1% in Iceland [18]. Given the limitation of this hospital-based study and possible preferential testing, these findings still support wide community transmission in Cairo before the second wave of the epidemic.

Seroprevalence of anti-SARS-CoV-2 antibodies

Epidemiological data of SARS-CoV-2 are mostly restricted to laboratory-confirmed cases for symptomatic patients. Conversely, the SARS-CoV-2 infection can present as an asymptomatic or mild disease in major sections of the population that do not seek medical advice. Therefore, the actual burden of SARS-CoV-2 can be minimized. Improved serological detection of specific SARS-CoV-2 antibodies could help calculate the true numbers of infections and enhance the understanding of related epidemiology [19–21].

Among the 2,927 subjects tested for both PCR and SARS-CoV-2 antibodies, 877 subjects (almost 30%) tested positive for antibodies with negative PCR (95% CI 28.33–31.65), denoting a past infection of SARS-COV-2 in previous months.

The literature shows that SARS-CoV-2 seroprevalence varies markedly, as expected, among geographic regions, which is sensibly elucidated by the variation in the community transmission of the infection. The results of the current study revealed a seroprevalence rate of 30%. The published data in the US show seroprevalence that ranges from less than 1% to 23% [22]. In Europe, reported seroprevalence rates have varied among different countries, with about 3.4% in Demark, 5% in Spain, and up to 23% in some areas of Italy [23–25]. An earlier study reported a seroprevalence of 17% in Iran [26].

Once more, the seroprevalence results underscore the high transmission of the infection in the community.

The timing of the study may be related to the observed high seroprevalence rates. The present study measured the seroprevalence at the end of wave 1 of the epidemic and may really reflect the cumulative infection rate in the community in contrast to many studies that measured it at the beginning or in the middle of the first wave.

This implies that the infection may be much more commonly spread than is indicated by the number of confirmed cases. Other seroprevalence studies have been directed in various territories of the world, demonstrating that for each reported case, the genuine number of diseases in the population is higher [27–30]. The discrepancy between the PCR positive cases and negative antibodies is expected as antibodies usually take 7–10 days and can even become detectable by the third week. By that time, the patient’s infectiousness begins to decline, and PCR may turn negative. The antibodies determine whether the patient has previously been exposed to infection but cannot be used to assess their current infection status. Adding to this, about 33% of COVID-19 patients were found to have no detectable antibodies.

Factors associated with infection and seroprevalence

The present study showed that males had higher PCR detection rates in contrast to females who showed higher seroprevalence rate of anti-SARS-CoV-2 antibodies. These differential findings are not supported consistently in previous research. Early epidemiological studies in China, India, and Iran indicated that SARS-CoV2 infected fewer females [31–37]. Females may be less vulnerable to SARS-CoV-2 infection and/or less likely to show signs of COVID-19, according to these findings. However, with the rapid spread of SARS-CoV-2 across the world and the rise in epidemiological research, more recent studies have found no substantial differences in COVID-19 incidence between men and women [38]. On the contrary, female patients have better outcomes than male patients, according to several reports [39–41].

A point to mention is that this study was carried out around the peak of the first wave and the following 3 months, compared to other studies that were carried out earlier during the first wave of the epidemic. Clearance of antibodies is a point to be further investigated if it has a longer duration in females.

Although the mechanisms underlying sex-specific COVID-19 outcomes are unclear, a complex interaction of physiological, behavioral, ecological, and sociocultural influences is likely to be involved. There have been reports of sex differences in immune responses to infectious diseases, as well as the role of sex steroids in immunity [39, 42]. Estrogen can protect against COVID-19, according to some researchers [42–44].

Hypertension and diabetes failed to show any relation with either infection or seroprevalence. The relation of diabetes to infection and seroprevalence is controversial. There is wide acceptance that diabetes increases the severity of and mortality from COVID-19 [45, 46]. On the other hand, little published research has highlighted the risk of infection of SARS-CoV-2 among diabetics [47, 48]. Although there are some hints of increased susceptibility to infection among diabetics, the findings are inconsistent, with some research pointing to a similar prevalence of diabetes in COVID-19 patients to that in the overall population, suggesting no relation of diabetes to susceptibility of the infection [49, 50]. Hypertension is another non-communicable disease that has been linked to the severity and fatality of COVID-19, but its relation to the infection risk is lagging [51]. One limitation of this study is that it depended on self-reporting of hypertension and diabetes.

The younger age group (less than 18 years) expressed the least PCR positivity rate and the least seroprevalence rate (3% and 21%, respectively). This observed difference between the different age groups was not statistically significant in the positivity rate but was in the seroprevalence analysis. These findings of seroprevalence rates are in line with another research [25, 27, 29, 52].

The peak of the first wave in Egypt was in June 2020, which corresponded with the highest PCR and positive antibody detection in the study sample. The seroprevalence rate showed a decline in subsequent months, which is aligned with other studies. Röltgen et al. showed that outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to 5 months post-infection [53]. Findings from some research propose a weaker immune response to SARS-CoV-2 infection in asymptomatic individuals and state that the antibody level begins to decrease within 2–3 months after infection [54, 55]. Wang et al. also concluded that the antibody level was highest during the 31–40 days since onset and then decreased slightly [56].