Seven coronaviruses are known to infect humans. Human coronavirus (HCoV) types HCoV-OC43 and HCoV-229E were first identified as causes of the common cold during the mid-1960s.1–3 In 2003, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) was discovered to be the cause of severe acute respiratory syndrome in adults during an outbreak that originated in China,4 reinvigorating interest in this group of pathogens. In 2004, Dutch virologists discovered HCoV-NL63,5 and 1 year later, a group in Hong Kong first identified HCoV-HKU1.6 The cause of Middle Easte respiratory syndrome (MERS), a life-threatening infection first described from Saudi Arabia, was identified in 2012 as MERS-CoV.7 Finally, in late 2019, SARS-CoV-2 was discovered as the cause of coronavirus disease 2019.8 The global SARS-CoV-2 pandemic has again focused attention on this group viruses. Serologic data obtained from archived samples in the United States and the Netherlands demonstrated that HCoV types HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1, unlike MERS-CoV, SARS-CoV-1 and SARS-CoV-2, have circulated widely for decades.5,9,10 All 4 typically cocirculate during any given year, but 1 typically predominates.11,12 Infections caused by HCoV-OC43 and HCoV-NL63 are more common than those caused by HCoV-229E and HCoV-HKU1,11–13 each causing major outbreaks every other winter.14 All 4 of the seasonal HCoVs are known to cause a spectrum of respiratory illnesses ranging in severity from the common cold to bronchiolitis and pneumonia.9,15 When multiplex polymerase chain reaction (PCR) assays are used to evaluate the etiologies of respiratory tract infections, seasonal HCoVs are found to be among the most common.14,16 The detection of more than 1 pathogen from the same respiratory sample is also fairly common using this methodology, particularly among children <5 years of age.17
Surveillance studies describing HCoV epidemiology from developed countries located in temperate regions may not be reflective of the experience in underdeveloped tropical areas of the world. Our study has focused on the etiologic surveillance of pediatric outpatient respiratory infections in Ecuador. To date, published studies from Ecuador have only focused on causes of severe illness in hospitalized children.18–20 In July 2018, we began a 5-year surveillance study of the epidemiology and etiologies of outpatient acute respiratory tract infections among Ecuadorean children <5 years of age. Study efforts were halted temporarily on March 16, 2020, when the country went on lockdown in response to the SARS-CoV-2 pandemic. Here, we report a subset of our study findings to date, focused on seasonal HCoV infections during the 20-month period immediately preceding the countrywide lockdown.
MATERIALS AND METHODS
The project is approved by the State University of New York Upstate Medical University Institutional Review Board, by the Bioethics Committee at the University of San Francisco, Quito, and by the Ministry of Health of Ecuador. The parent project is a 5-year cross-sectional surveillance study of the etiology and clinical presentation of medically attended outpatient respiratory tract infections among Ecuadorean children <5 years of age. Subjects are recruited from pediatric outpatient facilities in Ecuador’s capital city of Quito (elevation 2800 m, latitude 0°), and in the southwestern port city of Machala (sea level, latitude 4°S). Enrollment in the 5-year parent study began in July 2018 and was scheduled to continue through July 2023. Ecuador’s first case of SARS-CoV-2 infection was reported on February 29, 2020.21 A national health emergency was declared on March 11, all schools were closed on March 13 and all entry to the country by air, land and maritime transport was banned starting March 16. Study enrollment was halted on March 16, with plans to resume enrollment when the lockdown is lifted.
Children <5 years of age, presenting to 1 of 2 study-designated ambulatory clinics, located in Quito and Machala, with a suspected acute respiratory tract infection are eligible for enrollment. One team member recruits subjects at each enrollment site on a given day. During recruitment, all eligible subjects are approached; however, site enrollment for a given day is considered complete after 4 hours of recruitment has elapsed, or after 5 subjects have been enrolled at the site that day. A maximum of 15 subjects are enrolled per site, per week. Recruitment efforts are scheduled year-round, for a minimum of 3 days each calendar week, alternating between 9 am–1 pm and 1 pm–5 pm time slots.
For study purposes, acute respiratory tract infection was defined as the presence or parental report of 2 or more of the following signs or symptoms for fewer than 8 days: fever (temperature ≥38°C by any route), nasal congestion or discharge, cough, tachypnea, wheezing, rales, hypoxia or apnea. Children in foster care, with parents or legally authorized representatives of questionable capacity to consent and those hospitalized or treated with antibiotics within the last 30 days are excluded.
Informed consent is obtained, then demographic data, immunization history and details regarding the current illness are gathered and recorded. A single nasopharyngeal swab is collected from the study subject, placed into a collection tube containing 3 mL universal transport media and transported to the laboratory. Respiratory specimens collected at illness visits are tested for the detection of respiratory pathogens, including the 4 seasonal HCoV types HCoV-HKU1, HCoV-OC43, HCoV-229E and HCoV-NL63, using the Biofire Film Array Respiratory Panel (BioFire Diagnostics, LLC; Salt Lake City, UT). All International Classification of Diseases, 10th Revision visit diagnosis code(s) assigned to the patient on the day of the visit are also collected. For study purposes, the following diagnoses were predefined as upper respiratory tract infections (URI): common cold, acute sinusitis, acute pharyngitis, acute tonsillitis, acute laryngitis or tracheitis and otitis media. Study-defined lower respiratory tract infections (LRTI) include croup, bronchiolitis and pneumonia. Patients identified as having an “influenza-like illness” are defined as having an URI unless they were also diagnosed with croup, bronchiolitis or pneumonia at the visit.
Descriptive statistics are used. Symptoms common among subjects were arranged into symptom sets. For comparisons of categorical data, the Fisher exact test and χ2 test are used where appropriate. Significance was set a priori to a P value of <0.05.
Between July 2018 and March 16, 2020, 850 subjects were enrolled in the study, 359 (42%) at site 1, and 491 (58%) at site 2. Of these, 677 (80%) tested positive for the detection of at least 1 pathogen on the multiplex PCR respiratory panel, including 49 (7.2%) who tested positive for at least 1 of the 4 seasonal HCoVs. In total, 51 HCoVs were detected from the 49 samples; 1 sample was positive for both HCoV-229E and HCoV-OC43, 1 was positive for both HCoV- 229E and HCoV-NL63. Overall, HCoV-NL63 was the most frequently detected (20/51; 39%), followed by HCoV-OC43 (14/51; 27%), HCoV-229E (11/51; 22%) and HCoV-HKU1 (6/51; 12%). More than 1 pathogen was detected in 13 of 49 (27%) samples (Fig. 1), with rhinovirus/enterovirus accounting for the majority (8/13; 62%).
The median age of the 49 subjects who tested positive for a HCoV was 17 months (range 14 days to 59 months); 25 (51%) were male. The median duration of symptoms before presenting for medical attention was 2.6 days (range 1–7 days). Nearly all of the subjects who tested positive for a HCoV had symptoms of nasal congestion or secretions (47/49; 96%). A majority also had cough (39/49; 80%) and/or fever (38/49; 78%), while a substantial number (18/49; 37%) had audible wheezing. A diagram of common combinations of symptoms (symptom sets) is shown in Figure 2A. Congestion, cough and fever were often accompanied by wheeze (11/49, 22%) or nasal secretions (11/49, 22%). The next most common symptom set was congestion, fever and nasal secretions (8/49, 16%). The most frequent clinic discharge diagnosis associated with HCoV infection was a common cold (41%), followed by bronchiolitis (27%), influenza-like illness and croup (10% each; Fig. 2B). Overall, 28 (57%) of the HCoV-positive subjects were diagnosed with study-defined URIs, and 21 (43%) with study-defined LRTIs. None of the HCoV-positive patients were diagnosed with sinusitis, pharyngitis, otitis or tonsillitis. We found no association between the infecting HCoV type and the anatomic location (upper vs. lower) of the respiratory tract infection (P > 0.05 using χ2 test; Table 1), no association between the infecting virus genus (α or β coronavirus) and the anatomic location (upper vs. lower) of the respiratory tract infection (P > 0.05 using Fisher exact test; Table 1) and no association between the infecting virus type and the individual’s syndromic diagnosis (P > 0.05 for each condition using χ2 test).
TABLE 1. -
Genus and Types Associated With Study-defined Upper and Lower Respiratory Tract Infections
||Upper Respiratory Tract Diagnosis
||Lower Respiratory Tract Diagnosis
||Any Upper Tract Infection
||Any Lower Tract Infection
*One patient sample tested positive for both HCoV-229E and OC43; another tested positive for both HCoV-229E and NL63. Both subjects had a single International Classification of Diseases, 10th Revision coding diagnoses of common cold. They are included once each in this table as individual cases of common cold caused by HCoV-OC43 and NL63; however, the statistical analysis was performed for each of the 3 possible permutations (229E and 229E; 229E and OC43; 229E and NL63). We found no significant association between HCoV type and location of infection, χ2 (3, N = 49) for 229E and 229E= 1.24, P = 0.74; 229E and OC43 = 0.73, P = 0.87; 229E and NL63 = 1.30, P = 0.72. Similarly, Fisher exact test showed no association between HCoV genus (α or β) and upper vs. lower anatomic location of infection (P = 0.38).
Figure 3 shows the number and types of the seasonal HCoVs detected each week during the study. The majority of HCoV infections (43/49; 88%) were detected between October 1 (week 40) and March 16 (week 12). HCoV-OC43 accounted for 11 of 21 (52%) cases during this time period in 2018–2019 while HCoV-NL63 predominated (15/22; 68%) in 2019–2020. Only 4 (8%) cases were detected between June 1 and October 1.
This is the first published report to describe the epidemiology and clinical manifestations of childhood respiratory infections caused by the 4 seasonal HCoVs in Ecuador. Using diagnostic multiplex PCR, we detected at least 1 pathogen in 677 of 850 (80%) of nasopharyngeal samples collected from children who were diagnosed clinically with a respiratory tract infection, including 49 (7.2%) that were positive for the detection of HCoV. Of the samples positive for a HCoV, 13 of 49 (27%) were also positive for a second pathogen. As a group, URIs were more common than LRTIs (57% and 43%, respectively). The most common syndromic clinical diagnosis associated with seasonal HCoV infection was the common cold (20/49; 41%) followed by bronchiolitis (13/49; 27%), croup (5/49; 10%) and influenza-like illness (5/49; 10%). We found no evidence to suggest that HCoV disease manifestations differed by the infecting HCoV type (HCoV-OC43, HCoV-NL63, HCoV-229E, HCoV-HKU1) or by the infecting coronavirus genus (α or β coronavirus), although others have reported a clear syndromic association between HCoV-NL63 infection and croup.22,23
We identified virus codetection in 13 (27%) of our coronavirus-positive samples. Published studies on the etiology of childhood respiratory tract infections using diagnostic multiplex PCR report a range of pathogen codetection between 17% and 36% overall.24–27 Codetection rates that specifically include at least 1 seasonal HCoVs have been reported as high as 43%.17 Such high rates of codetection complicate the ability to determine whether associations exist between symptom sets or syndromic diagnoses and infecting HCoV type because it is not possible to determine which codetected pathogen(s) are contributing to, and which are primarily responsible for the patient’s respiratory symptoms.
Across our study surveillance period of 20 months, 88% of the HCoVs were detected between October 1 and March 16. The 2018–2019 peak in HCoV activity, due to HCoV-OC43, occurred in October, with cases continuing through the month of December, while peak activity in 2019–2020, due to HCoV-NL63 began at the end of January with ongoing, sustained activity detected on March 16, 2020, the date that enrollment was halted. Our study observation that most seasonal HCoV activity occurred between October and mid-March suggests that the seasonal epidemiology of these viruses in Ecuador parallels that described for temperate regions of the world.13,15,28 Surveillance data on circulating HCoVs in tropical areas of the world are sparse. Among the few published studies that are available, 1 included adults only, 1 included children only and 2 included both.29–31 Only 2 of these reports included data from samples collected for more than a 12-month period.29,31 The longest, a study from Thailand spanning 24 months, conducted in 2012 and 2013, reported that 46 of 5833 (0.8%) samples collected from children and adults, ages 4 months to 93 years, with influenza-like illness were positive for the detection of a HCoV. HCoV activity was greatest between the months of May and October. The low rate of infection is explained in part by the large number of adults included in the study.29 A 12-month surveillance study of 2060 outpatient adults from Kuala Lumpur, Malaysia, that was restricted to HCoVs OC43 and HKU1, reported detection of a β HCoV in 48 (2.4%) samples.30 The majority of positive tests were identified between the months of January and September. Similar seasonality was described in a 7-month long household surveillance study conducted along coastal Kenya 2 years earlier.31 The country of Kenya, like Ecuador, spans the equator by several degrees of latitude in both directions. Of the 16,918 nasal swab samples that were collected from children and adults participating in the household study, 497 (2.9%) were positive for the detection of HCoV with a clear peak in disease activity during the month of May. Finally, a multinational study from 8 tropical and subtropical nations reported the etiologies of acute respiratory tract infections in 3717 children 6 months to 10 years of age over an 18-month period.14 A total of 209 (5.6%) samples were positive for the detection of HCoV, with country-specific rates ranging between 3.3% in the Philippines and 8.2% in Mexico. Seasonality of infection also varied from country to country. Surveillance from Brazil (n = 40), Colombia (n = 40) and Costa Rica (n = 21) indicated that HCoV activity predominated between the months of April and September. In contrast, HCoV activity in Mexico (n = 55), the Philippines (n = 34), Singapore (n = 2) and Thailand (n = 8) predominated between October and March. The mean number of HCoV cases detected per tropical country was only 26 (range 2–55), so it was not possible to identify country-specific peaks in virus activity. Our results from surveillance in Ecuador show that most HCoV activity occurs between October and March. This pattern is similar to that reported from similar numbers of cases in Mexico and the Philippines despite Ecuador’s closer geographic proximity to Brazil, Colombia and Costa Rica where seasonal activity predominated between April and September. The underlying reason(s) for this unexpected finding are unknown. One possibility is that the seasonality of HCoV activity across a given tropical region can shift periodically. Country-specific surveillance spanning multiple seasons is needed to address this possibility. It is also worth exploring whether region-specific meteorologic, climate, air quality or other characteristics influence the seasonality of HCoV (and other viral) infections in the tropics. Additional years of surveillance data are needed from Ecuador and other tropical areas of the world to determine whether our observed variations in peak HCoV activity between 2018–2019 and 2019–2020 represent a consistent difference in seasonal patterns of HCoV activity between tropical and temperate regions of the world.
There are several limitations to this study. First, our plan to enroll subjects year-round on a continuous basis for 5 years was thwarted by the SARS-CoV-2 global pandemic. The interruption limits our ability to reliably assess seasonality of HCoV activity beyond the 20-month period of data collection. Second, although our report includes results from 850 Ecuadorean children, HCoVs were detected in only 49 (7.2%) of all positive samples. The low number limits our ability to draw strong conclusions from our results, but is similar to numbers reported from individual tropical countries included in a multinational report.14
As noted, HCoV-OC43 and HCoV-HKU1 are members of the β coronavirus genus. Genus assignments were originally made based on serologic reactivity, indicating that antibodies directed against 1 β coronavirus have the potential to recognize related viruses in the same genus. For example, some of the antibodies produced in response to infection with HCoV-OC43 also recognize the related β coronavirus, HCoV-HKU1.32 Similarly, antibodies generated in response to SARS-CoV-1 infection can cross-react with HCoV-OC4333 and those generated in response to HCoV-OC43 infection can cross-react with SARS-CoV-1.34 These observations are important because antibodies that cross-react also have the potential to cross-neutralize.29–31 For example, antibodies produced in response to HCoV-OC43 infection afford an estimated 70% heterotypic protective immune response against infection with HCoV-HKU1.32,35,36 Unfortunately, the protective immune response that develops following infection with HCoV-OC43 wanes quickly during the following year,35 perhaps explaining, at least in part, why outbreaks of HCoV-OC43 infections predominate every other year. The degree of heterotypic immune cross-protection against SARS-CoV-2 following recent infection with HCoV-OC43, if any, is not yet known. Our surveillance data from Ecuador, a country very heavily impacted by the SARS-CoV-2 pandemic, show that HCoV-OC43 disease activity last peaked 15 months before the county’s first reported case of SARS-CoV-2 infection. A multinational study comparing the extent and severity of the SARS-CoV-2 pandemic relative to the timing of each region’s most recent preceding peak in HCoV-OC43 activity may offer population-based insights into whether recent prepandemic HCoV-OC43 activity was associated with attenuating effects on the pandemic.
This study provides the first report on seasonal HCoV activity from Ecuador, demonstrating that both α and β coronaviruses circulate regularly among young children, causing respiratory infections associated with congestion, fever, cough and/or wheezing. The most frequent syndromic diagnoses are common colds and bronchiolitis. The vast majority of HCoV activity in Ecuador occurred between the months of October and mid-March, overlapping completely with seasonal HCoV transmission in temperate regions of the world. During the 2018–2019 season, however, peak activity was seen in October and November, suggesting that the seasonal epidemiology of HCoV in Ecuador may not be as consistent as the patterns observed in temperate areas. Additional and longer surveillance studies in tropical regions of the world will provide additional insights into the global epidemiology of HCoV disease. As we gain a better understanding of protective immune responses to HCoV infections during the search for a vaccine against SARS-CoV-2, the degree and duration of cross-protection afforded by a recent infection with HCoV-OC43, if any, may emerge.
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