Suspected reinfections of SARS-COV-2 in Khyber Pakhtunkhwa, Pakistan – analysis of province-wide testing database
Authors: Habab Ali Ahmad, Haleema Khan, Muhammad Shahzad, Zia ul Haq, Steve Harakeh, Yasar Mehmood Yousafzai
Journal: Journal of Infection (2021)

•Following three epidemic waves, we obtained results of 2 million SARS-CoV-2 tests.•We identified 317 cases of suspected reinfections, the highest event-rate reported yet.•Large proportion of symptomatic reinfections are picked up through hospital referrals.•Risk of recurring infection is more prevalent in young and middle-aged adults. SARS-CoV-2 reinfection is a relatively new phenomenon in the ongoing coronavirus disease (COVID-19) pandemic. Although, the first confirmed case of reinfection was identified in August 2020, studies reporting reinfection have been scarce, and limited only to single-case studies, or smaller case-series and cohorts.1Babiker A. Marvil C.E. Waggoner J.J. Collins M.H. Piantadosi A. The importance and challenges of identifying SARS-CoV-2 reinfections.J Clin Microbiol. 2021; 59: e02769-e02820Google Scholar Only few studies have determined incidence of reinfection in large, population-based settings based on epidemiological evidence.2Breathnach A.S. Duncan C.J.A. Bouzidi K.E. Hanrath A.T. Payne B.A.I. Randell P.A. et al.Prior COVID-19 protects against reinfection, even in the absence of detectable antibodies.J Infect. 2021; 83: 237-279Google Scholar, 3Hamed E. Sedeeq S. Alnuaimi A.S. Syed M. ElHamid M.A. Alemrayat B. et al.Rates of recurrent positive SARS-CoV-2 swab results among patients attending primary care in Qatar.J Infect. 2021; 82: 84-123Abstract Full Text Full Text PDF Scopus (5) Google Scholar, 4Hanrath A.T. Payne B.A.I. Duncan C.J.A. Prior SARS-CoV-2 infection is associated with protection against symptomatic reinfection.J Infect. 2021; 82: e29-e30Google Scholar, 5Hansen C.H. Michlmayr D. Gubbels S.M. Mølbak K. Ethelberg S. Assessment of protection against reinfection with SARS-CoV-2 among 4 million PCR-tested individuals in Denmark in 2020: a population-level observational study.Lancet. 2021; 397: 1204-1212Google Scholar, 6Lawandi A. Warner S. Sun J. Demirkale C.Y. Danner R.L. Klompas M. et al.Suspected SARS-CoV-2 reinfections: incidence, predictors, and healthcare use among patients at 238 U.S. healthcare facilities, June 1, 2020- February 28, 2021.Clin Infect Dis. 2021; https://doi.org/10.1093/cid/ciab671Google Scholar, 7Tang X. Musa S.S. Zhao S. He D. Reinfection or reactivation of severe acute respiratory syndrome coronavirus 2: a systematic review.Front Public Health. 2021; 9: 1-10Google Scholar, 8Vitale J. Mumoli N. Clerici P. De Paschale M. Evangelista I. Cei M. et al.Assessment of SARS-CoV-2 reinfection 1 year after primary infection in a population in Lombardy, Italy.JAMA Intern Med. 2021; Google Scholar If genotyping is unavailable, Centres for Disease Control (CDC) recently set an investigative criteria, defining SARS-CoV-2 reinfection as a second positive real time reverse transcription-polymerase chain reaction (RT-PCR) test, taken ≥90 days after the primary result.9National Center for Immunization and Respiratory Diseases (NCIRD), Division of Viral DiseasesCommon investigation protocol for investigating suspected SARS-CoV-2 reinfection.Cent Dis Control Prev. 2020; (https://www.cdc.gov/coronavirus/2019-ncov/php/invest-criteria.html#print)https://www.cdc.gov/coronavirus/2019-ncov/php/reinfection.html#printGoogle Scholar However, not all published work follows a uniformly set criteria for what constitutes confirmed reinfection. Studies which follow the CDC criteria indicate high level of protection following recovery from infection, with reinfection suspected in less than 1.0% of those previously infected.2Breathnach A.S. Duncan C.J.A. Bouzidi K.E. Hanrath A.T. Payne B.A.I. Randell P.A. et al.Prior COVID-19 protects against reinfection, even in the absence of detectable antibodies.J Infect. 2021; 83: 237-279Google Scholar,5Hansen C.H. Michlmayr D. Gubbels S.M. Mølbak K. Ethelberg S. Assessment of protection against reinfection with SARS-CoV-2 among 4 million PCR-tested individuals in Denmark in 2020: a population-level observational study.Lancet. 2021; 397: 1204-1212Google Scholar, 6Lawandi A. Warner S. Sun J. Demirkale C.Y. Danner R.L. Klompas M. et al.Suspected SARS-CoV-2 reinfections: incidence, predictors, and healthcare use among patients at 238 U.S. healthcare facilities, June 1, 2020- February 28, 2021.Clin Infect Dis. 2021; https://doi.org/10.1093/cid/ciab671Google Scholar, 7Tang X. Musa S.S. Zhao S. He D. Reinfection or reactivation of severe acute respiratory syndrome coronavirus 2: a systematic review.Front Public Health. 2021; 9: 1-10Google Scholar, 8Vitale J. Mumoli N. Clerici P. De Paschale M. Evangelista I. Cei M. et al.Assessment of SARS-CoV-2 reinfection 1 year after primary infection in a population in Lombardy, Italy.JAMA Intern Med. 2021; Google Scholar However, further detailed prospective studies are needed to assess frequency of recurring infection. There is a possibility that a large proportion of reinfections are undetected, which could explain the low risk of SARS-CoV-2 reinfection reported in literature. This may especially be true in lower-middle-income settings, such as ours, where testing for asymptomatic infection is rare, and carrying out mass-scale genomic sequencing is not always logistically feasible. Pakistan is currently experiencing a fourth epidemic wave of SARS-CoV-2 transmission. This provided us an opportunity to retrospectively determine incidence of suspected reinfections after three global waves in the pandemic. Using epidemiological criteria set by CDC, we undertook this study to identify suspected cases of SARS-CoV-2 reinfection, in the province of Khyber Pakhtunkhwa (KP), using the provincial government's contact tracing database. This descriptive cross-sectional study was approved by the Khyber Medical University (KMU) Ethical Committee. Due to retrospective nature of our analysis, we were granted a waiver from obtaining informed consent. In KP, testing has been extended to symptomatic and suspected cases, those presenting at hospitals, people identified through contact tracing, contacts of confirmed cases, people with travel history, or anyone using the free COVID-19 response hotline. Samples are sent to one of the 28 public and private sector diagnostic laboratories, serving all regions of KP province, which has a population of 35.53 million people. This network of laboratories is regulated by the provincial health department, and all SARS-CoV-2 viral RNA RT-PCR tests are conducted under guidelines from National Institute of Health (NIH), Islamabad. To ensure uniformity in sample processing and data analysis, all laboratories in KP are periodically assessed through an External Quality Assurance program mandated by NIH. Individual-level test results are logged onto, and maintained on a centralised contact tracing database. We extracted retrospective data for all SARS-CoV-2 RT-PCR tests carried out from March, 2020, to July, 2021, in KP. For purpose of this study, primary infection was defined as a positive RT-PCR test for SARS-CoV-2. Suspected reinfection was defined as a second positive test taken for SARS-CoV-2, ≥90 days after the primary positive test result. Analyses were performed using MS Excel. Comparison between demographic variables of primary infections and suspected reinfections were made using Chi-square test. Between March 2020 and July 2021, a total of 2065,611 SARS-CoV-2 RT-PCR tests were conducted in KP. We found 142,787 individuals had tested positive for SARS-CoV-2 once, out of which 58.2% of primary infections were hospital referrals, while 41.8% were identified through KP's comprehensive contact tracing program. A subset of 29,617 individuals were identified, who had received repeat RT-PCR testing for SARS-CoV-2. Results of the primary infection were used as start of the 90 day interval, and multiple positive test results obtained within 90 days of each other were discounted. Overall, we observed 317 (0.22% of total positive cases) suspected cases of SARS-CoV-2 reinfections (Fig. 1). Mean age of individuals with suspected reinfection was slightly lower compared to those infected once (36.94 ± 14.34, vs 38.41 ± 18.59, p-value 0.004). Suspected reinfection was found to be more common in males (74.8%, p-value < 0.001), and in young and middle-aged adults, between 19 and 45 years old (54.1%, p-value < 0.001). Our analysis shows that 73.1% (231/317) of reinfections were detected through hospital referrals, while 26.9% (85/317) reinfections were identified through contact tracing (p-value < 0.001). Higher suspected reinfections were observed in urban centres where more testing is conducted (supplementary Fig. 1), with Peshawar division reporting highest proportion (189, 59.6%) of suspected reinfection cases (p-value <0.001) (Table 1).Table 1Demographic characteristics of SARS-COV-2 primary and suspected reinfection from Khyber Pakhtunkhwa province.CharacteristicsInfection status n (%)P-ValuePrimary infectionReinfectionGenderfemale49,416 (34.7)80 (25.2)<0.001male93,054 (65.3)237 (74.8)Age, YearsMean ± SD38.41 (±18.59)36.04 (±14.340)*calculated using independent samples t- test.0.004Age categories≤ 18 years15,815 (11.4)17 (5.4)<0.00119 - 30 years35,066 (25.3)123 (38.2)31 - 45 years39,903 (28.8)109 (34.4)46 - 60 years30,040 (21.7)46 (14.5)>60 years17,775 (12.8)22 (6.9)Sample typeContact tracing59,592 (41.8)85 (26.9)<0.001Hospital referral82,878 (58.2)231 (73.1)RegionMalakand29,478 (20.8)58 (18.3)<0.001Hazara16,596 (11.7)29 (9.1)Mardan14,270 (10.1)18 (5.7)Peshawar61,706 (43.5)189 (59.6)Kohat12,036 (8.5)16 (5.0)Bannu4307 (3.0)3 (0.9)DI Khan3550 (2.5)4 (1.3) calculated using independent samples t- test. Open table in a new tab These findings suggest that SARS-CoV-2 reinfection could be common in age groups often presumed less likely to suffer from severe, or critical COVID-19. This is in contradiction to evidence which shows symptomatic COVID-19 results in enhanced, long-lasting protective immunity following recovery from primary infection.10Peluso M.J. Takahashi S. Hakim J. Kelly J.D. Torres L. Iyer N.S. et al.SARS-CoV-2 antibody magnitude and detectability are driven by disease severity, timing, and assay.Sci Adv. 2021; 7: eabh3409Google Scholar Our results reveal that more than two thirds (73.1%) of recurring infections were picked up through hospital referrals, which could indicate that people with severe symptoms are more likely to seek medical attention. Studies have demonstrated that a large proportion of recurring infections are asymptomatic, and reinfection is more common in individuals whose primary infections were also asymptomatic.1Babiker A. Marvil C.E. Waggoner J.J. Collins M.H. Piantadosi A. The importance and challenges of identifying SARS-CoV-2 reinfections.J Clin Microbiol. 2021; 59: e02769-e02820Google Scholar,7Tang X. Musa S.S. Zhao S. He D. Reinfection or reactivation of severe acute respiratory syndrome coronavirus 2: a systematic review.Front Public Health. 2021; 9: 1-10Google Scholar In Pakistan, the testing strategy has largely relied on targeting of symptomatic and suspected cases. Therefore, it is impossible to accurately predict the true number of SARS-CoV-2 reinfections, which may be significantly larger than what has been reported in literature. There were several limitations to this work. Since data was gathered retrospectively, we were unable to obtain medical history, clinical demographics, or personal details, regarding individuals whose test results were utilised in our analysis. Thus, we could not correlate role of any pre-existing health conditions with suspected reinfection. Similarly, the proportion of individuals employed in high-risk professions, such as healthcare workers, was unknown. Our future work aims to address these limitations. To best of our knowledge, this is the largest multi-centre analysis (n = 142,787) of its kind carried out to date, reporting one of the highest event-rates (317 suspected reinfections) in published literature.6Lawandi A. Warner S. Sun J. Demirkale C.Y. Danner R.L. Klompas M. et al.Suspected SARS-CoV-2 reinfections: incidence, predictors, and healthcare use among patients at 238 U.S. healthcare facilities, June 1, 2020- February 28, 2021.Clin Infect Dis. 2021; https://doi.org/10.1093/cid/ciab671Google Scholar Our results fall within range of reinfections reported in literature, despite limited testing being carried out in Pakistan for identification of asymptomatic cases. This analysis suggests that recurring infection with SARS-CoV-2 may indeed be a widespread phenomenon, although the risk is known to be low. The authors declare no conflict of interest. Ethical approval of the study was granted by Ethics Board, Khyber Medical University (Reference number: Dir/KMU-EB/PR/000879). None.