SARS-CoV-2 PCR positivity rate and seroprevalence of related antibodies among a sample of patients in Cairo: Pre-wave 2 results of a screening program in a university hospital.

BACKGROUND: Research has revealed that asymptomatic and pre-symptomatic infections are important contributors to the transmission of SARS-CoV-2 in populations. In Egypt, the true prevalence of infections is veiled due to the low number of screening tests. The aim of this study was to determine the SARS-CoV-2 PCR positivity rate as well the seroprevalence of the SARS-CoV-2 antibodies before the ultimate development of a second wave of the epidemic in Cairo, Egypt.
METHODS: Our study was carried out between May 5 and the end of October 2020. It included all patients requiring admission to Ain Shams University hospitals. An interview questionnaire was used to collect demographic and clinical data. Laboratory tests for all participants included RT-PCR and total antibody assay for SARS-CoV-2.
RESULTS: A total of 4,313 subjects were enrolled in our study, with females representing 56% of the sample. Adults and middle-aged individuals represented around 60% of the study sample. The positivity rate of SARS-CoV-2 PCR was 3.84% (95% CI 3.29-4.48), and the SARS-CoV-2 antibody seroprevalence was 29.82% (95% CI: 28.16-31.51). Males showed a higher risk for getting the COVID-19 infection, while middle-age group had significantly higher antibody seroprevalence rates.
CONCLUSION: SARS-CoV-2 infection imposes a high burden on the community as detected by high seroprevalence rates.

Introduction

The new coronavirus, officially named “severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2),” infected around 155 million people worldwide by the beginning of May 2021, with more than 3 million related deaths [1], and the numbers are steeply increasing every day. In Egypt, the first peak of reported cases was recorded by June 2020, with about 65,000 infected cases and 2,789 deaths. Reported cases were stable over the summer season, with around 6,000 cases per month. The infected cases began to surge by November 2020 and reached around 231,803 by the beginning of May 2021, with 13,591 deaths [2]. Egypt is ranked 68th in the world in terms of COVID-19 infections and 8th in terms of COVID-19 deaths. The capital, Cairo, with its high-density population, took the lead among Egyptian governorates in terms of the number of COVID-19 cases, according to data declared by the Egyptian Ministry of Health [3].

COVID-19 surveillance in Egypt mainly depends on the reported PCR detection that is usually carried out in symptomatic cases. According to the Ministry of Health’s announcement in October 2020, the number of daily PCR tests can hardly reach 6,000 when compared to other Arabic countries such as Saudi Arabia and United Arabic Emirates, during the same period [4]. Studies show that about 30%–60% of COVID-19 patients have mild or no symptoms and still can spread the infection [5]. Previous studies have suggested that only a small fraction of asymptomatic persons may eventually develop symptoms [6-8]. These facts add to the struggle of estimating the magnitude of COVID-19 in a community.

Many researchers are attempting to estimate the rates of infection in the community through epidemiological models [9, 10] or structural assumptions [11]. With limited testing availability and a high proportion of mild and asymptomatic infections, there is an under-ascertainment of SARS-CoV-2 infections through passive case reporting [6-8]. In such cases, seroprevalence surveys of SARS-CoV-2 antibodies are important for refining estimates of infection and transmission [12]. Moreover, seroprevalence studies can provide information on risk factors for the disease, such as a patient’s age, location, or underlying health conditions. Furthermore, they could show significant medical data on immune reactions to the virus and provide an understanding of the immune response following infection of SARS-CoV-2 [13].

In hospital settings, the SARS-CoV-2 infection presents a great challenge, where it is highly infectious during the pre-symptomatic period in patients. The nosocomial transmission of COVID-19 to healthcare workers and other patients can have serious impacts on hospital operation, including the suspension of new admissions and the closing of hospital wards. Preadmission screening by PCR is a policy recommended by different agencies, including the Centers for Disease Control and Prevention (CDC) in the US and the Public Health England guidelines, depending on testing capacity and disease prevalence [14].

By the beginning of May 2020, the Ain Shams University (ASU) hospitals in Cairo adopted a universal screening program for all patients requiring admission to the hospitals. The screening process included PCR testing and total antibody assay prior to admission.

With scarce data available on the epidemiology of COVID-19 in Egypt, the aim of the present research was to determine the SARS-CoV-2 PCR positivity rate as well the seroprevalence of SARS-CoV-2 antibodies before the ultimate development of a potential second wave of the epidemic in Cairo, Egypt. The findings are based on the results of a universal screening program for patients in ASU hospitals in Cairo.

Subjects and methods

The current study was carried out between May 5 and the end of October 2020. The protocol of the research was approved by Ain Shams University Faculty of Medicine Research Ethics Committee [FWA00017585], research approval number (FMASU R/2020). Positive cases were reported to the Ministry of Health and Population (MOHP). The guidelines of isolation and treatment protocol of the MOHP were followed. A written informed consent in Arabic was obtained from participants or their guardians in the case of minors (Arabic and English translations of the consent form are attached in the supplementary materials).

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:

An interview questionnaire. The study questionnaire included:

Background characteristics (age, gender, residence, and contact details)

History of contact with a COVID-19 case

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.

History of comorbidities: Self-reported diabetes and hypertension

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.

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.

Specimen collection and handling

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.

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)

Nucleic acid extraction

Samples were mixed well by gentle vortex and a volume of 300ul was used to extract the viral RNA. The viral RNA was automatically extracted by binding to the surface of magnetic beads using the Chemagic 360 automatic extractor (Perkin Elmer, Germany) and the Viasure RNA/DNA extraction kit (CerTest Biotec, Spain). Usually, RNA was processed immediately after extraction, except in cases where the PCR equipment was busy, and we had to wait less than 2 hours to process the samples; therefore, we stored the extracted RNA at −20°C for less than 2 hours in exceptional situations, as per manufacturer recommendations.

Detection of SARS-CoV2 RNA by rRT-PCR

SARS-CoV-2 Real Time PCR Detection Kit (CerTest Biotec, Spain) was used for the detection of SARS-CoV-2 in the respiratory samples. Detection is done in one step real time (RT) format, where the reverse transcription and the subsequent amplification of the specific target sequence take place in the same reaction well. The isolated RNA target is transcribed, generating complementary DNA by reverse transcriptase, which is followed by the amplification of a conserved region of ORF1ab and N genes for SARS-CoV-2 using specific primers and a fluorescent-labeled probe.

The amplification protocol

The thermal cycler was programmed as follows:

Quality control

Samples were processed only once. In each run, a positive and a negative control were included to allow for correct interpretation of the results. Moreover, the presence of an internal positive control (IPC) in each run ensures the correct performance of the amplification mix.

Interpretation of the test results

The sample was considered positive for SARS-COV2 when the obtained Ct value was less than 38 and the IPC showed an amplification signal. On the other hand, a negative sample would have no amplification signal, but the IPC would be positive to exclude the inhibition of the PCR reaction.

The absence of a signal in the positive control or the presence of amplification in the negative control would denote invalid test results.

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

Results

The current research enrolled 4,313 subjects during the study period. A total of 4,008 and 2,951 patients had the PCR test and the antibody assay, respectively. Females constituted 56% of the study sample. Adults and middle-aged individuals represented around 60% of the sample. Most patients (91.3%) did not complain about any related COVID-19 symptoms (Table 1).

Table 1

Characteristics of the study group.

	No. (%)
Total no	4313
Age (years)
<18	928 (21.5)
18–	1356 (31.4)
40–	1214 (28.1)
≥60	815 (18.9)
Gender
Male	1885 (43.7)
Females	2428 (56.3)
Hospital
1. Obstetrics and gynecology	703 (16.3)
2. Oncology	49 (1.1)
3. Surgery	1463 (33.9)
4. Pediatrics	443 (10.3)
5. Internal medicine	1421 (32.9)
6. Cardiothoracic	234 (5.4)
Symptoms
1. No COVID-19 related symptoms	3939 (91.3)
2. Fever	262 (6.1)
3. Cough	165 (3.8)
4. Diarrhea	85 (2.0)
5. Sore throat	106 (2.5)
6. Vascular event	44 (1.0)
Morbidities
1. (N = 3659)	298 (8.1)
2. HTN (N = 3659)	352 (9.6)
No. of PCR done	4008 (92.9)
No. of AB assay done	2951 (68.4)

The unadjusted positivity rate of SARS-CoV-2 PCR during the study period was 154 (3.84%; 95% CI 3.29–4.48), while that of SARS-CoV-2 antibodies in the negative PCR group was 877 (29.96%; 95% CI 28.33%–31.65%) during the same period. With adjustment for test sensitivity and specificity, the positive PCR rate ranged from 3.94% in the high sensitivity scenario (95% CI: 3.34–4.62) to 5.28% (95% CI: 4.47–6.18) in the low sensitivity scenario. The adjusted SARS-CoV-2 antibody seroprevalence was 29.82 (95% CI: 28.16–31.51) (Table 2).

Table 2

Results of SARS-CoV-2 screening by PCR and total antibody.

	No. (unadjusted %, 95% CI)	Adjusted * % (95% CI)
Positive PCR in total group (N = 4008)	154 (3.84, 3.29–4.48)	Scenario1 (sensitivity 71%)
		5.28 (4.47–6.18)
		Scenario 2 (sensitivity 95%)
		3.94 (3.34–4.62)
Positive Antibody among negative PCR group (N = 2927**)	877(29.96, 28.31–31.66)	29.82 (28.16–31.51)
Negative PCR and Negative AB (N = 2927*)	1927(65.84, 64,1–67.53)
Positive PCR and Negative AB (N = 2927*)	55 (1.88, 1.45–2.44)
Positive PCR and positive AB (N = 2927*)	68 (2.32, 1.84–2.94)

* Adjustment for sensitivity and specificity of the test.

**The total number of subjects with both tests (PCR and total AB) determined.

Among the positive antibody group, the level of antibodies did not show any statistical difference between the negative and positive PCR subjects. The median and IQR of SARS-CoV-2 antibodies among the PCR positive group was 26.6 (11.90–68.40) versus 23.70 (6.60–65.60) among the PCR negative group (P value = 0.11) (Fig 1).

Fig 1

Box plot of antibody level in the positively tested antibody group.

Males showed a higher risk for getting the COVID-19 infection as detected by positive PCR (OR adjusted for age was 1.45, 95% CI 1.06–1.98). On analyzing the adult group separately for comorbid conditions, males preserved their risk differential after adjustment (diabetes and hypertension). Age and comorbid conditions did not show any significant relation to PCR positivity rate (Table 3).

Table 3

Epidemiological profile of SARS-C-V-2 PCR positive and antibody seropositive subjects.

	Total no.	PCR +ve No. (%)	Adjusted OR (95% CI)	Total no.	AB +ve* No. (%)	Adjusted OR (95% CI)
All age groups
Age (years)
<18	841	25 (3.0)	1	642	135 (21.0)	1
18–	1268	43(3.4)	1.067(0.66–1.73)	923	361 (39.1)	2.19 (1.73–2.77)
40–	1136	54(4.8)	1.39 (0.87–2.26)	768	273 (35.5)	1.94 (1.52–2.47)
≥60	763	32(4.2)	1.55 (0.933–2.62)	471	108 (22.9)	1.05 (0.79–1.40)
Gender
Male	1732	76 (4.4)	1.45(1.06–1.98)	1258	313 (24.9)	0.63(0.53–0.75)
Female	2276	78 (3.4)	1	1546	564 (36.5)	1
Adult Group (>18 years)
Age (years)
18–	1268	43(3.4)	1	923	361 (39.1)	1
40–	1136	54(4.8)	1.42 (0.89–2.27)	768	273 (35.5)	0.93 (0.75–1.16)
≥60	763	32(4.2)	1.2 (0.73–2.01)	471	108 (22.9)	0.48(0.37–0.77)
Gender
Male	1255	68 (5.4)	1.63 (1.09–2.43)	878	241(27.4)	0.63 (0.52–0.77)
Female	1912	61 (3.2)	1	1284	501 (39)	1
DM
Negative	2470	93 (3.8)	1	1794	597 (33.3)	1
Positive	265	9 (3.4)	1.19(0.56–2.52)	142	44 (31.0)	1.03 (0.7–1.56)
HTN
Negative	2398	95 (4.0)	1	1732	579 (33.4)	1
Positive	337	7(2.1)	0.47 (0.20–1.08)	204	62 (30.4)	0.92 (0.65–1.31)

* The percentage is calculated among the PCR-ve group.

Regarding the seroprevalence of SARS-CoV-2 antibodies, the adult and middle-age group had significantly higher seroprevalence rates when compared to the younger age group of less than 18 years (39% and 35% versus 21%, respectively), while males showed lower seroprevalence rates when compared to females (24.9% versus 36.5%). The effect of gender remained significant in the adult group analysis after adjustment for age and comorbid conditions. The odds ratio of male gender was 0.63 (95% CI: 0.52–0.77) after adjustment for age and comorbid conditions (Table 3).

The older age group (over 60 years) had lower seroprevalence rates when compared to the adult group (from 18 to less than 40) (22.9 versus 39.1, respectively) with an odds ratio of 0.48 (95% CI 0.37–0.77), adjusted for gender and comorbid conditions. Diabetic and hypertensive subjects showed comparable seroprevalence rates to those of non-diseased subjects.

Discussion

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].

Study limitations

This study was carried out for patients attending ASU seeking hospital services, which makes the sample not fully representative of the Cairo population. The laboratory tests were not done for all patients for sampling problems, unavailability of certain kits, or laboratory errors. Some data on hypertension and diabetes were based on participant’s self-reporting.

Conclusion

A total of 4,313 subjects were enrolled in our study. SARS-CoV-2 PCR was 3.84%, and SARS-CoV-2 antibody seroprevalence was 29.82%, which highlights a high burden of infection in the community. Males had higher PCR detection rates in contrast to females, who showed higher seroprevalence rate of anti-SARS-CoV-2 antibodies. The younger age group (less than 18 years) expressed the least PCR positivity rate and the least seroprevalence rate. Expanding testing of persons without symptoms would be valuable in reducing the silent spread of SARS-CoV-2 in healthcare facilities. This study results emphasize the importance of continuing public health prevention steps, such as the use of face masks and social distancing, to keep SARS-CoV-2 from spreading.

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14 Apr 2021

PONE-D-21-06534

SARS-CoV-2 PCR positivity rate and seroprevalence of related antibodies among a sample of patients in Cairo: Pre-wave 2 results of a screening program in a university hospital

PLOS ONE

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Additional Editor Comments:

This is a cross-sectional study of SARS-CoV-2 antibody and RNA status in a university hospital setting in Egypt.

The population size studied is large and had an RNA positivity rate of 3.84%.

The introduction should be updated to appropriately reflect the current number of infections in Egypt.

There are a number of awkward phrases and grammatical / spelling mistakes.  The manuscript should be reviewed carefully by a native English speaker and/or a professional editing service.

The methods should include an estimate of how many unique individuals are seen in this university hospital setting per year.

It is unclear why extracted RNA was kept at -20C.  This almost certainly led to sample degradation prior to RT-PCR testing.

Was real-time PCR performed in duplicate for each sample?

Were RT-PCR negative samples re-tested to confirm the negative finding?

Were symptoms and co-morbid conditions determined by self-report or was fever actually measured?  Were co-morbid conditions determined by patient chart review?

Figure 1 can be simplified by removing the horizontal lines and by give antibodies levels as 0 to 140 (rather than .00 to 140.00).

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[This study was part of regular policy and procedure of Ain Shams University Hospital.]

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10. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://ovarianresearch.biomedcentral.com/articles/10.1186/s13048-020-00734-4

- https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(20)30706-0/fulltext

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Girgis et al, evaluated the SARS-CoV-2 PCR positivity rate and seroprevalence of anti-SARS-CoV-2 among 4313 patients in Cairo between May 5th and end of October 2020 (Pre-wave 2) in a university hospital. The study is important and addressed interesting questions about the seroprevalence of SARS-CoV-2 in Egypt. To improve the quality of the manuscript, the following should be addressed:

1. Do you have anti- SARS-CoV-2 IgM antibodies especially in PCR positive samples?

2. The discrepancy between the PCR positive cases and antibodies negative needs to be addressed and explained since about half of the PCR positive patients are antibodies negative. Is it because of early testing and if so what about the anti- SARS-CoV-2 IgM antibodies, or because of the false positive PCR or the false negative antibodies?

3. Do you have samples to follow some of the PCR positive antibodies negative to evaluate if they will be seroconverted?

4. Cross-reactive antibodies to other Corona viruses should be considered especially for PCR negative antibodies positive samples and may be needed to be discussed or confirmed with other serological kits.

Reviewer #2: The manuscript titled 'SARS-CoV-2 PCR positivity rate and seroprevalence of related antibodies among a sample of patients in Cairo: Pre-wave 2 results of a screening program in a university hospital' by Girgis et al. contains some important information which may be beneficial to the scientific and policy makers. However, the manuscript in its current state needs to be revised to meet a certain standard.

Most of the issues raised such as comments and edits have are in track changes in the attachment. Unfortunately, since the authors did not insert line numbers, it was difficult writing out a comprehensive report.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Mohamed Tarek Mohamed Shata

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-21-06534_reviewer_AAS.pdf

Click here for additional data file.

10 Jun 2021

Editor comments

Please include the following items when submitting your revised manuscript:

*A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers': Done on resubmission.

*A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes: Done on resubmission.

*An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript: done on resubmission.

*The introduction should be updated to appropriately reflect the current number of infections in Egypt: Done.

*There are a number of awkward phrases and grammatical / spelling mistakes. The manuscript should be reviewed carefully by a native English speaker and/or a professional editing service: The manuscript was reviewed by a professional editing service.

*The methods should include an estimate of how many unique individuals are seen in this university hospital setting per year: Referred to page 4 line 14 .This statement was added to the study setting in the methodology "It a campus including seven hospitals (surgery hospital, internal medicine hospital, pediatrics hospital, obstetrics, and gynecology hospital, emergency hospital , and geriatrics hospital), and multiple specific units and centers as clinical toxicology center, psychiatric center, bone marrow transplant unit, digestive endoscopy unit and clinical research unit. It involves more than 3000 inpatient beds, in addition to ICUs at the different hospitals., and serving about one and a half million patients annually".

*It is unclear why extracted RNA was kept at -20C. This almost certainly led to sample degradation prior to RT-PCR testing: Most of the time RNA was processed immediately after extraction except in few times where the PCR instrument is busy and we had to wait for less than 2 hours to process the samples. Therefore, extracts we stored at the -20 in accordance to manufacturer’s recommendations.

*Was real-time PCR performed in duplicate for each sample? In page 8 line 2 we mentioned that samples were processed only once.

*Were RT-PCR negative samples re-tested to confirm the negative finding? In this study we aimed to screen patients admitted to the hospital for medical and/or surgical purposes. Usually, they were symptom-free and hence there was no need to repeat the testing. Yet, only in suspected cases, with CT findings suggestive of SARS-COV2 infection, re-sampling and re-testing was done.

*Were symptoms and co-morbid conditions determined by self-report or was fever actually measured? Were co-morbid conditions determined by patient chart review? Refereed to page 4 line 6.

Regarding patients' symptoms body temperature was measured for all patients as it’s a part of patient general evaluation that done on admission, other symptoms like sore throat and cough were collected based on patient self-reporting

Regarding co-morbid conditions, it was collected also, based on the patients self-reporting and we mentioned this point as one of our study limitations.

*Figure 1 can be simplified by removing the horizontal lines and by give antibodies levels as 0 to 140 (rather than .00 to 140.00): Done.

Journal requirements comments:

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified

(1) whether consent was informed

(2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed).

(3) If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information: Referred to page 10 line 14

Yes, all participant provided documented written consent, the following statement was added at the ethical considerations section.

" A written informed consent in Arabic was obtained from participants or their guardians in the case of minors (Arabic and English translations of the consent form are attached in the supplementary materials"

If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

In our study we made active data collection, we didn't used archived data

*Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).: Refereed to page 10 line 11

The following statement was added to the ethical considerations section " The protocol of the research was approved by the University Ethical Research Committee [ FWA00017585] , the research approval number (FMASU R /2020)"

, please provide the following:

• The name of the colleague or the details of the professional service that edited your manuscript

• A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

• A clean copy of the edited manuscript (uploaded as the new *manuscript* file

Done on resubmission.

*please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. Moreover, please include more details on how the questionnaire was pre-tested, and whether it was validated: Done.

*Please ensure that you refer to Figure 1 in your text as, if accepted, production will need this reference to link the reader to the figure: Done.

*We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 1, 2 and 3 in your text; if accepted, production will need this reference to link the reader to the Table: Done.

*We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://ovarianresearch.biomedcentral.com/articles/10.1186/s13048-020-00734-4

- https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(20)30706-0/fulltext

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed :Done.

Reviewer 1

*Do you have anti- SARS-CoV-2 IgM antibodies especially in PCR positive samples? In SARS-CoV-2 infections, IgM and IgG antibodies can arise nearly simultaneously in serum within 2 to 3 weeks after illness onset. Thus, detection of IgM without IgG is uncommon (CDC, 2020). That is why we used the ROCHE chemiluminescence immunoassay (Elecsys anti-SARS-COV2 total) which is qualitative assay that is used to detect the total antibodies (including IgG) against SARS-COV2.

*The discrepancy between the PCR positive cases and antibodies negative needs to be addressed and explained since about half of the PCR positive patients are antibodies negative. Is it because of early testing and if so what about the anti- SARS-CoV-2 IgM antibodies, or because of the false positive PCR or the false negative antibodies:

The discrepancy between the PCR positive cases and antibodies negative may be explained by:

1) Antibodies usually start to appear 7 to 10 days post-infection and even become detectable during the third week as recommended by the CDC. By that time the patient infectiousness start to decline. The antibodies determine whether the patient has been previously exposed to infection but cannot be used to assess the current infection status.

2) `33% of COVID 19 patients were found to have no detectable antibodies. That is why the CDC recommended not to use the antibody test neither in the diagnosis of COVID-19 cases nor in the determination of immunity.

Refer to CDC Interim Guidelines for COVID-19 Antibody Testing

*Do you have samples to follow some of the PCR positive antibodies negative to evaluate if they will be seroconverted : This was beyond the scope of the study.

*Cross-reactive antibodies to other Corona viruses should be considered especially for PCR negative antibodies positive samples and may be needed to be discussed or confirmed with other serological kits The analytical specificity of the Elecsys anti-SARS COV2 assay is 99.5% as determined by the manufacturer (Elecsys anti-SARS COV2 assay package insert). A total of 792 potentially cross-reacting samples were tested and no-cross reactivity has been reported with the common cold panel (number of tested samples = 40) nor the coronavirus panel (number of tested samples = 40).

Reviewer 2

*Introduction COV-2 over the entire population is likely much higher, and accordingly the mortality rate should be fundamentally lower.

What are these numbers? Agencies sited?

The total number of screening tests done in Egypt has been substantially lower than that done in other countries

*The number at writing the manuscript should be included

Numbers of daily PCR test that conducted daily at the time of writing the manuscript was added " Page 2 line 14"

as duration antibodies in patients after infection

*this statement is incomplete The statement was corrected

Gynecology and Obstetrics, Oncology, Pediatrics, Psychiatry, Internal Medicine, surgical, cardiothoracic and Geriatrics

Are these different hospitals or departments within the same hospital Yes, these are different hospitals.

Ain Shams university hospitals include 7 hospitals (surgery hospital, internal medicine hospital, pediatrics hospital, obstetrics, and gynecology hospital, emergency hospital , and geriatrics hospital), and multiple specific units and centers as clinical toxicology center, psychiatric center, bone marrow transplant unit, digestive endoscopy unit and clinical research unit. It involves more than 3000 inpatient beds, in addition to ICUs at the different hospitals.

Methodology:

*Nucleic acid extraction: Viral RNA was extracted using the Viasure RNA/DNA extraction kit (CerTest Biotec, Spain) as follows: � Each sample was mixed well by gentle vortexing and a volume of 300ul was transferred into a deep-well plate. � A total volume of 314ul lysing solution containing 300ul of lysis buffer, 10ul proteinase K solution and 4ul of carrier RNA was added into each sample well. � Sample plate was then loaded onto the Chemagic 360 automatic extractor (Perkin Elmer, Germany). Viral RNA was then automatically extracted by binding to the surface of magnetic beads. Impurities such as salts, metabolites, and soluble macromolecular cellular components were efficiently removed by a series of quick washing steps. � Unless immediately processed, extracted RNA was kept at -20�C.

Comment : follow manufacturers protocol Done

Ethical considerations:

The protocol of the research was approved by the University Ethical Research Committee

Comment: Insert the approval number Done

Results: The current research enrolled 4313 subjects during the study period. A total of 4008 and 2951 patients had the PCR test and the antibody assay done, respectively. Females constituted 56% of the study sample. Adults and middle age represented around 60% of the sample. The vast majority of patients (91.3%) did not complain of any related COVID-19 symptoms.

*Comment : table or figure should be stated: Done.

Males showed higher risk for getting the COVID-19 infection as detected by positive PCR (OR adjusted for age was 1.45, 95% CI 1.06 8-1.98). On analyzing the adult group separately for comorbid conditions, males preserved their risk differential after adjustment for comorbid conditions (Diabetes and hypertension). Age and comorbid conditions did not show any significant relation to PCR positivity rate

*Comments: no tables or figures :Done.

Submitted filename: Response to reviwers.docx

Click here for additional data file.

30 Jun 2021

SARS-CoV-2 PCR positivity rate and seroprevalence of related antibodies among a sample of patients in Cairo: Pre-wave 2 results of a screening program in a university hospital

PONE-D-21-06534R1

Dear Dr. Mossad,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jason T. Blackard, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

None

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors responded adequately to the comments raised by the reviewers. They addressed the main concerns

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Mohamed Tarek M Shata

Reviewer #2: No

5 Jul 2021

PONE-D-21-06534R1

SARS-CoV-2 PCR positivity rate and seroprevalence of related antibodies among a sample of patients in Cairo: Pre-wave 2 results of a screening program in a university hospital

Dear Dr. Mossad:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr. Jason T. Blackard

Academic Editor

PLOS ONE

Cycles	Step	Time	Temperature
1	Reverse Transcription	15 min	45°C
1	Initial denaturation	2 min	95°C
45	Denaturation	10 sec	95°C
	Annealing/Extension (Data collection)	50 sec	60°C