Diagnostic Performance of Pooled RT-PCR Testing for SARS-CoV-2 Detection

ABSTRACTBackgroundWith the high number of COVID-19 cases, a need to optimize testing strategy must be regarded to obtain timely diagnosis for early containment measures. With this, several studies have employed pooled RT-PCR testing for SARS-CoV-2 as this could potentially conserve laboratory resources while has the capacity to test several individuals. However, this was recommended to firstly validate the method as different laboratory reagents and equipment vary with its diagnostic performance.ObjectiveThe aim of this study was to determine the diagnostic performance of pooled SARS-CoV-2 nasopharyngeal/oropharyngeal swabbed samples using RT-PCR technique.MethodsA records review of two-staged pooled RT-PCR testing data from August 10, 26, 30 and September 5, 2020 was utilized from Northern Mindanao Medical Center COVID-19 Satellite Laboratory (formerly CHDNM TB Regional Center). For the first stage, using known samples, a total of 30 pools were made for each of the pooling size, 5- and 10-pooled, on both pooling phase, pre- and post-RNA extraction. One positive individual was used to represent each of the Cycle threshold values given (<24, 25-28, 29-32, 33-36, and 37-40) while the rest of the samples were negative. For the second stage, 54 pools of five from 270 random unknown samples were used to validate the results. Target gene performance of N gene and RdRp was also determined.Key ResultsResults show that 5-pooled sample has higher sensitivity (SN), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) of 100% (95% confidence interval (CI) 88.97-100), 66.95% (95% CI, 60.75-72.6), 28.18% (95% CI, 20.62-37.22), and 100% (95% CI, 97.66-100) compared to 10-pooled sample that has 87.1% (95% CI, 71.15-94.87), 56.9% (50.57-63.02), 20.77% (95% CI, 14.68-28.53) and 97.14% (95% CI, 92.88-98.88). Further, these Ct values were only from the N gene, emphasizing its higher diagnostic performance as well to detect SARS-CoV-2 compared to RdRp as only a few samples were detected, thus, no analysis was made.ConclusionThis study found out that 5-pooled sample has better diagnostic performance compared to 10-pooled samples. Specifically, all positive individual samples were detected in 5-pooled samples in pre-RNA extraction phase which these results are evident and consistent on both known and unknown samples. N gene was found out to detect more SARS-CoV-2 samples compared to RdRp.

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Development and Evaluation of AccuPower® COVID-19 Multiplex Real-Time RT-PCR Kit and AccuPower® SARS-CoV-2 Multiplex Real-Time RT-PCR Kit for SARS-CoV-2 Detection in Sputum, NPS/OPS, Saliva and Pooled Samples

10.1101/2021.11.21.21264927 ◽

2021 ◽

Author(s):

In Bum Suh ◽

Jaegyun Lim ◽

Hyo Seon Kim ◽

Guil Rhim ◽

Heebum Kim ◽

...

Keyword(s):

Real Time ◽

Limit Of Detection ◽

Clinical Performance ◽

Rna Extraction ◽

Kappa Coefficient ◽

N Gene ◽

Clinical Samples ◽

Rt Pcr ◽

Current Standard ◽

Pooled Samples

Rapid and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the successful control of the current global COVID-19 pandemic. The real-time reverse transcription polymerase chain reaction (Real-time RT-PCR) is the most widely used detection technique. This research describes the development of two novel multiplex real-time RT-PCR kits, AccuPower ® COVID-19 Multiplex Real-Time RT-PCR Kit (NCVM) specifically designed for use with the ExiStation ™48 system (comprised of ExiPrep ™48 Dx and Exicycler ™96 by BIONEER, Korea) for sample RNA extraction and PCR detection, and AccuPower ® SARS-CoV-2 Multiplex Real-Time RT-PCR Kit (SCVM) designed to be compatible with manufacturers` on-market PCR instruments. The limit of detection (LoD) of SCVM was 2 copies/µ L and the LoD of the NCVM was 120 copies/mL for both the gene and the SARS-CoV-2 gene (N gene and RdRp gene). The AccuPower ® kits demonstrated high precision with no cross reactivity to other respiratory-related microorganisms. The clinical performance of AccuPower ® kits was evaluated using the following clinical samples: sputum and nasopharyngeal/oropharyngeal swab (NPS/OPS) samples. Overall agreement of the AccuPower ® kits with a Food and Drug Administration (FDA) approved emergency use authorized commercial kit (STANDARD ™ M nCoV Real-Time Detection kit, SD BIOSENSOR, Korea) was above 95% (Cohen`s kappa coefficient ≥ 0.95), with a sensitivity of over 95%. The NPS/OPS specimen pooling experiment was conducted to verify the usability of AccuPower ® kits on pooled samples and the results showed greater than 90% agreement with individual NPS/OPS samples. The clinical performance of AccuPower ® kits with saliva samples was also compared with NPS/OPS samples and demonstrated over 95% agreement (Cohen`s kappa coefficient > 0.95). This study shows the BIONEER NCVM and SCVM assays are comparable with the current standard confirmation assay and are suitable for effective clinical management and control of SARS-CoV-2.

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Methods for Molecular Surveillance of Influenza Used in Macedonia

PRILOZI ◽

10.2478/prilozi-2014-0003 ◽

2014 ◽

Vol 35 (2) ◽

pp. 25-30

Author(s):

Golubinka Bosevska ◽

Elizabeta Janceska ◽

Gordana Kuzmanovska ◽

Vladimir Mikik ◽

Nikola Panovski

Keyword(s):

Influenza Virus ◽

Predictive Value ◽

Influenza A ◽

Rna Extraction ◽

Influenza Virus Infection ◽

Laboratory Diagnosis ◽

Influenza Viruses ◽

Rt Pcr ◽

Two Samples ◽

Nose And Throat

AbstractThe aim: To present and compare different Nucleic Acid Testing assays used for laboratory diagnosis of influenza virus infection in our country.Materials and methods: Respiratory samples used were nose and throat swabs. The RNA extraction was performed with a QIAamp viral RNA kit. During the season 2009–2010 the first 25 samples were tested with: conventional gel-based RT-PCR and CDC rtRT-PCR using published specific matrix and HA gene primers and probes for influenza virus typing and subtyping.Results: Of 25 samples tested with conventional RT-PCR7(28%) were positive for influenza A, but negative for A/H1seasonal and A/H3. Retested with rtRT-PCR 9(36%) were positive for influenza A, 8(32%) were positive for A/H1pdm and 1(4%) was A/H3. Two samples positive with rtRT-PCR for influenza A were negative with RT-PCR. The sensitivity of the RT-PCR in comparison with rtRT-PCR is 100% and the specificity is 88.89%. Positive predictive value for RT-PCR is 77.78%, and negative predictive value is 100%. RT-PCR is a four-step and rtRT-PCR a one-step procedure. The turn-around time of RT-PCR is 6 hours and for rtRT-PCR it is 2 hours.Discussion and conclusion: For surveillance purposes nose and throat swabs are the more easy and practical to collect. It was proved that RT-PCR is too laborious, multi-step and time-consuming. The sensitivity of both assays is equal. The specificity of rtRT-PCR is higher. NAT assays for detection of influenza viruses have become an integral component of the surveillance programme in our country. They provide a fast, accurate and sensitive detection of influenza.

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Pooling of samples to optimize SARS-CoV-2 diagnosis by RT-qPCR: comparative analysis of two protocols.

10.1101/2020.08.24.20181008 ◽

2020 ◽

Author(s):

Fabiana Volpato ◽

Daiana Lima-Morales ◽

Priscila Lamb Wink ◽

Julia Willig ◽

Fernanda de-Paris ◽

...

Keyword(s):

Large Scale ◽

Diagnostic Yield ◽

Rna Extraction ◽

Clinical Samples ◽

The Novel ◽

Positive Individual ◽

Sample Pooling ◽

Novel Coronavirus ◽

The Individual ◽

Pooled Samples

RT-qPCR for SARS-CoV-2 is the main diagnostic test used to identify the novel coronavirus. Several countries have used large scale SARS-CoV-2 RT-qPCR testing as one of the important strategies for combating the pandemic. In order to process the massive needs for coronavirus testing, the usual throughput of routine clinical laboratories has reached and often surpassed its limits and new approaches to cope with this challenge must be developed. This study has aimed to evaluate the use pool of samples as a strategy to optimize the diagnostic of SARS-CoV-2 by RT-qPCR in a general population. A total of 220 naso/orofaryngeal swab samples were collected and tested using two different protocols of sample pooling. In the first protocol (Protocol A); 10 clinical samples were pooled before RNA extraction. The second protocol (Protocol B) consisted of pooling the already extracted RNAs from 10 individual samples. Results from Protocol A were identical (100% agreement) with the individual results. However, for results from Protocol B, reduced agreement (91%) was observed in relation to results obtained by individual testing. Inconsistencies observed were related to RT-qPCR results with higher Cycle Thresholds (Ct > 32.73). Furthermore, in pools containing more than one positive individual, the Ct of the pool was equivalent to the lowest Ct among the individual results. These results provide additional evidence in favor of the clinical use of pooled samples for SARS-CoV-2 diagnosis by RT-qPCR and suggest that pooling of samples before RNA extraction is preferrable in terms of diagnostic yield.

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Assessment of Successful qRT-PCR of SARS-CoV-2 Assay in Pool Screening Using Isopropyl Alcohol Purification Step in RNA Extraction

BioMed Research International ◽

10.1155/2021/6653950 ◽

2021 ◽

Vol 2021 ◽

pp. 1-6

Author(s):

Mayank Gangwar ◽

Alka Shukla ◽

Virendra Kumar Patel ◽

Pradyot Prakash ◽

Gopal Nath

Keyword(s):

Isopropyl Alcohol ◽

Rna Extraction ◽

Group Testing ◽

Research Work ◽

Rt Pcr ◽

Purification Step ◽

E Gene ◽

Qrt Pcr ◽

Pcr Targeting ◽

Pooled Samples

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 μL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 μL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng / uL , which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.

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Comparative analysis of the diagnostic performance of five commercial COVID-19 qRT PCR kits used in India

Scientific Reports ◽

10.1038/s41598-021-00852-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

J. Singh ◽

A. K. Yadav ◽

A. Pakhare ◽

P. Kulkarni ◽

L. Lokhande ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Diagnostic Performance ◽

Diagnostic Testing ◽

Rna Extraction ◽

Clinical Samples ◽

Diagnostic Process ◽

Control Group ◽

Rt Pcr ◽

Short Period ◽

Commercial Kits

AbstractTo meet the unprecedented requirement of diagnostic testing for SARS-CoV-2, a large number of diagnostic kits were authorized by concerned authorities for diagnostic use within a short period of time during the initial phases of the ongoing pandemic. We undertook this study to evaluate the inter-test agreement and other key operational features of 5 such commercial kits that have been extensively used in India for routine diagnostic testing for COVID-19. The five commercial kits were evaluated, using a panel of positive and negative respiratory samples, considering the kit provided by National Institute of Virology, Indian Council of Medical Research (2019-nCoV Kit) as the reference. The positive panel comprised of individuals who fulfilled the 3 criteria of being clinically symptomatic, having history of contact with diagnosed cases and testing positive in the reference kit. The negative panel included both healthy and disease controls, the latter being drawn from individuals diagnosed with other respiratory viral infections. The same protocol of sample collection, same RNA extraction kit and same RT-PCR instrument were used for all the kits. Clinical samples were collected from a panel of 92 cases and 60 control patients, who fulfilled our inclusion criteria. The control group included equal number of healthy individuals and patients infected with other respiratory viruses (n = 30, in each group). We observed varying sensitivity and specificity among the evaluated kits, with LabGun COVID-19 RT-PCR kit showing the highest sensitivity and specificity (94% and 100% respectively), followed by TaqPath COVID-19 Combo and Allplex 2019-nCoV assays. The extent of inter-test agreement was not associated with viral loads of the samples. Poor correlation was observed between Ct values of the same genes amplified using different kits. Our findings reveal the presence of wide heterogeneity and sub-optimal inter-test agreement in the diagnostic performance of the evaluated kits and hint at the need of adopting stringent standards for fulfilling the quality assurance requirements of the COVID-19 diagnostic process.

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Asymptomatic screening of SARS-CoV-2 (COVID-19) virus RNA using reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)

10.21203/rs.3.rs-847286/v1 ◽

2021 ◽

Author(s):

Rebecca Allsopp ◽

Caroline Cowley ◽

Ruth Barber ◽

Carolyn Jones ◽

Christopher Holmes ◽

...

Keyword(s):

Diagnostic Performance ◽

Screening Program ◽

False Negative ◽

Rna Extraction ◽

Lamp Assay ◽

Molecular Diagnostic ◽

Acid Extraction ◽

Rt Pcr ◽

Clinical Patient ◽

The University

Abstract This study demonstrates the diagnostic performance of SARS-CoV-2 RT-LAMP assays, comparing the performance of genomic versus sub-genomic sequence target with subsequent application in an asymptomatic screening population. An RT-LAMP workflow was developed using synthetic positive control RNA and the diagnostic sensitivity and specificity was then determined using clinical patient samples processed through the diagnostic RT-PCR service within the University Hospitals of Leicester NHS Trust. 92 RT-PCR clinically positive and 88 RT-PCR clinically negative swab samples along with 78 clinically positive and 63 clinically negative saliva samples were equally detected at 100% DSe and 100% DSp for all samples reporting a Ct < 20. DSe for all samples reporting a Ct < 30 reduced slightly to around 95% (100% DSp) for both the single genomic (large open reading frame; orf1a) and dual sub-genomic (nucleocapsid plus envelope) targeting RT-LAMP assays. Lastly, the diagnostic performance of a saliva direct workflow was only about 50% that of the saliva RNA extraction workflow. Subsequently, a swab based RNA -RT-LAMP assay was implemented to ISO 15189:2012 standards supporting an advisory COVID-19 screening program for staff and students at the University of Leicester between October and December 2020. Within a 24-hour period, total nucleic acid extraction was followed by genomic target RT-LAMP plus an internal total RNA control to mitigate the possibility of false negative reporting. SARS-CoV-2 RT-LAMP positive samples were confirmed by an RT-PCR test in an NHS diagnostic laboratory and results were included within national statistics. Nine confirmed positive samples were detected in 1680 symptom free individuals (equivalent to 540 cases per 100,000) thus demonstrating the utility of RT-LAMP molecular diagnostic tool for the detection of SARS-CoV-2 in an asymptomatic population.

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Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y, and del156-157/R158G) in settled solids using digital RT-PCR v2

10.17504/protocols.io.b2mmqc46 ◽

2021 ◽

Author(s):

Bridgette Hughes ◽

Bradley J. White ◽

Marlene K. Wolfe ◽

Krista Wigginton ◽

Alexandria B Boehm

Keyword(s):

Quantitative Analysis ◽

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Population Level ◽

Digital Pcr ◽

N Gene ◽

Rt Pcr ◽

Pcr Inhibitor ◽

Wastewater Samples

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one for HV69-70, one for E484K/N501Y, and one for del156-157/R158G) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

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Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y,del156-157/R158G, and Del143-145) in settled solids using digital RT-PCR v2

10.17504/protocols.io.b2qmqdu6 ◽

2021 ◽

Author(s):

Bridgette Hughes ◽

Bradley J. White ◽

Marlene K. Wolfe ◽

Krista Wigginton ◽

Alexandria B Boehm

Keyword(s):

Quantitative Analysis ◽

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Population Level ◽

Digital Pcr ◽

N Gene ◽

Rt Pcr ◽

Pcr Inhibitor ◽

Wastewater Samples

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one each for HV69-70, E484K/N501Y, del156-157/R158G, and Del143-145) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

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Clinical Relevance of Human Mammaglobin mRNA in Pleural Effusion from Patients Undergoing Thoracoscopy: A Pilot Study

The International Journal of Biological Markers ◽

10.5301/jbm.2012.9305 ◽

2012 ◽

Vol 27 (2) ◽

pp. 99-104 ◽

Cited By ~ 1

Author(s):

Pier Aldo Canessa ◽

Carmen Manta ◽

Paola Ferro ◽

Maria Cristiana Franceschini ◽

Massimiliano Sivori ◽

...

Keyword(s):

Relative Risk ◽

Predictive Value ◽

Clinical Relevance ◽

Diagnostic Performance ◽

False Negative ◽

Malignant Diseases ◽

Performance Parameters ◽

Rt Pcr ◽

Risk Of Cancer

Background human mammaglobin (hMAM) expression has been reported in pleural effusions (PE). The aim of this study was to assess the clinical relevance of hMAM mRNA in PE from patients who underwent thoracoscopy. Material and methods A total of 288 patients with PE were studied, 155 of which were diagnosed with malignant and 133 with non-malignant diseases by thoracoscopy. Cells from PE were analyzed by nested hMAM RT-PCR. Statistical analyses were performed to evaluate the diagnostic performance parameters (DPP), the association between hMAM expression and benign or malignant status and the relative risk of cancer for patients with negative thoracoscopy showing hMAM positivity. Results hMAM mRNA was found in 68/288 (23.6%) PE samples of which 51 were from the 155 patients diagnosed with malignant diseases and 17 were from the 133 patients diagnosed with non-malignant diseases. A significant correlation between hMAM expression and malignancy was found (OR=3.04) and the DPP were as follows: sensitivity=32.9%, specificity=87.2%, accuracy=58.0%, positive predictive value=75.0% and negative predictive value=52.7%. Among the patients with negative thoracoscopy (n=133), 5/17 (29.4%) hMAM-positive patients had or developed a tumor during the 18-month follow up period, as compared to 10/116 (8.6%) hMAM-negative patients (relative risk of 4.6 for developing a malignancy). Conclusion These findings suggest a possible application of hMAM RT-PCR detection in PE as to identify a false-negative thoracoscopy in non-specific pleuritis.

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Prevalence of Sars-Cov-2 Infection in Health Workers (HWs) and Diagnostic Test Performance: The Experience of a Teaching Hospital in Central Italy

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17124417 ◽

2020 ◽

Vol 17 (12) ◽

pp. 4417 ◽

Cited By ~ 14

Author(s):

Edith Lahner ◽

Emanuele Dilaghi ◽

Claudio Prestigiacomo ◽

Giuliano Alessio ◽

Laura Marcellini ◽

...

Keyword(s):

High Risk ◽

Teaching Hospital ◽

Predictive Value ◽

Diagnostic Performance ◽

Health Workers ◽

Central Italy ◽

Nasopharyngeal Swab ◽

Index Test ◽

Rt Pcr ◽

Pcr Assay

(1) Background: Health workers (HWs) are at high risk of acquiring SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) infections. Therefore, health authorities further recommend screening strategies for SARS-CoV-2 infection in exposed or high-risk HWs. Nevertheless, to date, the best/optimal method to screen HWs for SARS-CoV-2 infection is still under debate, and data on the prevalence of SARS-CoV-2 infection in HWs are still scarce. The present study aims to assess the SARS-CoV-2 infection rate amongst HWs in a teaching hospital in Central Italy and the diagnostic performance of SARS-CoV-2 serology (index test) in comparison with the SARS-CoV-2 RNA PCR assay (reference standard). (2) Methods: A cross-sectional study on the retrospective data of HWs tested for SARS-CoV-2 by RNA-RT-PCR on nasopharyngeal swabs and by an IgM/IgG serology assay on venous blood samples, irrespective of exposure and/or symptoms, was carried out. (3) Results: A total of 2057 HWs (median age 46, 19–69 years, females 60.2%) were assessed by the RNA RT-PCR assay and 58 (2.7%) tested positive for SARS-CoV-2 infection. Compared with negative HWs, SARS-CoV-2-positives were younger (mean age 41.7 versus 45.2, p < 0.01; 50% versus 31% under or equal to 40 years old, p < 0.002) and had a shorter duration of employment (64 versus 125 months, p = 0.02). Exposure to SARS-CoV-2 was more frequent in positive HWs than in negatives (55.2% versus 27.5%, p < 0.0001). In 44.8% of positive HWs, no exposure was traced. None of the positive HWs had a fatal outcome, none of them had acute respiratory distress syndrome, and only one required hospitalization for mild/moderate pneumonia. In 1084 (51.2%) HWs, nasopharyngeal swabs and an IgM/IgG serology assay were performed. With regard to IgM serology, sensitivity was 0% at a specificity of 98.99% (positive predictive value, PPV 0%, negative predictive value, NPV 99.2%). Concerning IgG serology and irrespective of the time interval between nasopharyngeal swab and serology, sensitivity was 50% at a specificity of 99.1% (PPV 28.6%, NPV 99.6%). IgG serology showed a higher diagnostic performance when performed at least two weeks after testing SARS-CoV-2-positive at the RNA RT-PCR assay by a nasopharyngeal swab. (4) Conclusions: Our experience in Central Italy demonstrated a low prevalence of SARS-CoV-2 infection amongst HWs, but higher than in the general population. Nearly half of the positive HWs reported no previous exposure to SARS-CoV-2-infected subjects and were diagnosed thanks to the proactive screening strategy implemented. IgG serology seems useful when performed at least two weeks after an RNA RT-PCR assay. IgM serology does not seem to be a useful test for the diagnosis of active SARS-CoV-2 infection. High awareness of SARS-CoV-2 infection is mandatory for all people, but especially for HWs, irrespective of symptoms, to safeguard their health and that of patients.

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