COVID-19 Infection: Data Gaps for Diagnostic Laboratory Preparedness and Tasks on Hand

Abstract

Emergence of the 2019 novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) and its spread, with life-threatening outcomes, have caused a pandemic burden worldwide. Studies of emerging diseases under outbreak conditions have focused on the complete spectrum of pathogens, transmissibility, shedding kinetics in relation to infectivity, epidemiological causes, and interventions to control emergence. During the initial stages of an outbreak, laboratory response capacity focuses on expansion of efficient diagnostic tools for rapid case detection, contact tracing, putting epidemiological findings into sources, mode of transmission, and identification of susceptible groups and reservoirs. It is important for public health diagnostic laboratories to have a fundamental knowledge of viral shedding, antibody response kinetics, assay validation, interpretation, and uncertainties of test results. This study reviewed currently published data from available literature on SARS-CoV-2 infection and compared this with data on viral shedding and antibody response kinetics of other human coronaviruses. Also described are current challenges and comments on some biases and significant data gaps that have limited laboratory preparedness to SARS-CoV-2. Consistent documentation of progress and data gaps from standardized reporting of methods utilized, sampling date, details of test results by specimen type, risk assessments, and symptoms can all be used strategically and provide incentives to governments and their partners to prioritize the development, detection, and response to outbreaks.

Introduction

In December 2019, several cases of patients with pneumonia of unknown etiology were reported in Wuhan City, Hubei Province, China (41,43,60). Isolation of the new virus from patient specimens, subsequent sequencing, and detailed phylogenetic analysis of the viral genome demonstrated its proximate relationship with other β-coronaviruses that were the causative organisms of two recent outbreaks, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). SARS-CoV first emerged in China in 2002 and spread to other countries, resulting in thousands of deaths and a mortality rate of 11% (15,38). In contrast, the MERS-CoV outbreak was first reported in Saudi Arabia and quickly spread worldwide, with a 37% fatality rate (20,39,62).

Currently, the estimated fatality rate of the 2019 novel coronavirus (SARS-CoV-2) infection is 6.6%, relatively lower than those of SARS-CoV and MERS-CoV; however, the numbers of infected and dead are much higher (46). With the explosive increase of confirmed infection, the World Health Organization (WHO) declared this outbreak a public health emergency of international concern by the end of January 2020 (48). As of August 30, 2020, there were 24,854,140 laboratory-confirmed cases of SARS-CoV-2 infection reported to WHO from 215 countries and territories across the globe, with a total of 838,924 deaths (49). Based on the latest available data and updated models, a majority of countries are currently experiencing or yet to reach their peak number of cases and deaths, which are predicted to continue to rise in the coming weeks and months (28). Early case detection and diagnosis are the cornerstone of effective control of this emerging epidemic for which diagnostic laboratories are of crucial importance (21).

Laboratory Preparedness

The laboratory response to any outbreak needs to be timely and accurate, with a high level of sensitivity and specificity (Table 1) (8,11,31,36). Preparedness plans during outbreaks require looking beyond reference and state-of-the-art laboratories to include first-line on-site hospital testing facilities and development of plans for strengthening local capability and capacity as required, depending on the nature of outbreak (37). The laboratory response in the initial stages of an outbreak should emphasize development of immunological and microbiological techniques for diagnosis of patients, comprehensive contact tracing, and putting public health studies into possible sources, modes of transmission, identification of populations at risk, and understanding of potential animal reservoirs. For a precise response, the important basic questions for laboratory diagnostic triage necessitates bridging of any knowledge gaps, which require systematic identification (Table 2). However, optimal use of this public health laboratory capacity involves embedding of data needs for researchers and scientists within outbreak investigations to obtain information needed for accurate interpretation of test outcomes and assay validation (8,11,31,36).

Table 1.

The Basic Diagnostic Laboratory Preparedness Requirements Tolls and Guidelines Consistent with CDC and WHO Recommendations

Needed tolls and information	Comments
1. A written sample collection criteria and transportation policy	To be available in sample collection site and field workers with a prerequisite training in its contents
	Should be prepared to avoid any sources of errors reported by others during preanalysis step
2. Sample collection swabs and triple packaging transport system if needed	Containing viral transport media
	Indicated the allowed time and conditions for sample transport and storage
3. Real-time PCR machine	Two target genes including nucleocapsid targets or E, RdRp/Hel, and S targets should be performed for confirmation purposes as per WHO and CDC recommendation
3. Real-time PCR machine	A preferred closed system to be used to minimize false positive result reporting
4. Antigen and antibody screening kits	IgM and IgG ELISA kits
	Lateral flow assay for antigen and antibody detection
	To be used for research as per WHO recommendation. However, this can assess in rapid and on-site detection, asymptomatic, and population screenings.
	Must be chosen with high sensitivity and specificity ranges
5. Reporting policy	Should include a reference laboratory data collection center
5. Reporting policy	Should be made available to all associated health care and research workers
6. Infection control policy and training along with personal protective equipment and required disposable tolls	To be made available with the corresponding required training to avoid accidental nosocomial infection
	Bio-risk hazard and assessment training program

CDC, Centers for Disease Control and Prevention; PCR, polymerase chain reaction; WHO, World Health Organization.

Table 2.

Crucial Questions for Selection of Sampling and Subsequent Interpretation on Laboratory Diagnosis

Time-point during the infection course when specimens shall be collected?

Viral shedding kinetics and antibody responses with different disease states (initial phases of symptom onset and among asymptomatic cases, during and after symptomatic period, during clinical recovery)

Infection kinetics influenced by various host factors (comorbidities and immunosuppression)

What are various types of adequate specimen for the suspected pathogen and essential for the available diagnostic tests?

Virus concentration in different body compartments, secreta, and fluids during the disease progression

Viral loads associated by host factors (comorbidities and immunosuppression)

What are available in-house and/or commercial laboratory testing methods to confirm or rule out a diagnosis?

Sensitivity and specificity of different nucleic acid (PCR, NAATs, and rapid tests), antigen and antibody tests of the various methods used for the different specimens and related to stage of illness

NAATs, Nucleic Acid Amplification Tests.

Preparedness and response for SARS-CoV-2 infection emergence is especially challenging for several reasons (32). Clinical manifestations of SARS-CoV-2 infection, collectively termed coronavirus disease (COVID)-2019, overlap and are nonspecific in the early phase of disease. A recent publication from China reported that sampling among 1099 positive cases revealed common clinical manifestations of fever (88.7%), cough (67.8%), fatigue (38.1%), sputum production (33.4%), shortness of breath (18.6%), sore throat (13.9%), and headache (13.6%) (16). Second, diagnostic equipment and human resources are inadequate for timely identification of SARS-CoV-2 infection. This gap has influenced most countries' health authorities to prescribe confirmatory laboratory testing only for those patients who show severe COVID-19 symptoms. However, some governments are turning to mass screening of the population (16). Third, despite advanced technical and scientific capacity, a lack of essential information, such as sampling time, viral shedding, and antibody response kinetics, has severely hindered use of the aforementioned techniques in the present outbreak. Herein, we consider these overarching challenges to diagnostic laboratory preparedness during an outbreak and demonstrate potential solutions considering existing systems (Table 3).

Table 3.

Key Challenges to Diagnostic Laboratory Preparedness During Outbreak and Potential Solutions

Challenges	Proposed solutions
Limitation of diagnostic equipment and supply chain interruption during major outbreaks	Preselect suppliers to ensure suitable capacity during outbreak situations
	Establish production lines for diagnostic production and ensure supply chain
	Pooled testing shall be part of a larger strategy based on granular data
Limitation of diagnostic capacity in many countries	Strengthen surveillance capacities through implementation of laboratory networks as well as increasing point of heath care testing facility
Limitation of diagnostic capacity in many countries	Educate health care personnel on the importance of reporting on real time
Lack of essential knowledge about progression kinetics of viral shedding and host serological responses	Develop comprehensive diagnostic algorithm that can be used in association with clinical manifestations and host factors
	Expand and integrate networks of expert personnel and allow more prompt response during outbreak and enhance knowledge sharing
Delays in sharing of diagnostic data affecting containment times and response	Create a diagnostic laboratory network with enabling real-time data reporting. Ensure and establish referral laboratory for quality control of the test results.
Lack of prediction model for disease progression	Comprehensive mapping of clinical manifestation in various stages of disease progression, including asymptomatic cases

Shedding Profiles of Various Specimens for Coronavirus Detection

Diagnosis of viral pneumonias, such as in COVID-19, is comprising collecting the right specimens from infected persons at the correct time as well as accurate and fast laboratory testing. Viral shedding kinetics, defined as viral growth patterns that include a timed growth curve with peaks and declines in viral load and disease progression during the course of infection at multiple body sites, is one of the most important determinants for sampling strategies and ensuing interpretation of laboratory diagnosis. Other determinants include viral load in different body compartments during disease progression, kinetics of infection influenced by host factors, and limits of detection methods used for diagnosis with various specimen types (11,36).

Respiratory shedding

Endemic human coronaviruses have been spotted primarily from a variety of upper and/or lower respiratory specimens and sources, including endotracheal aspirate, sputum, and bronchial fluid (7,13,14). The recommended site for sample collection by the Centers for Disease Control for initial diagnostic testing is from the upper respiratory tract, including oropharyngeal and nasopharyngeal swabs or wash (3). As specimen sampling is not standardized, comparison of data among studies is difficult, with descriptions varying from oronasal swab, nasopharyngeal secretion or swab, nasal swab, or throat swab, all of which reflect “upper respiratory tract” sampling. Nevertheless, based on the hypothesis that viral load correlates with high levels of viral replication (2), the understanding of important information needs to be expanded from time-course analyses. Literature review shows that for SARS-CoV infection, viral loads slowly elevate until the 10th day among 60–95% of patients after symptom onset and then gradually decrease after 13 days in 42–90% of patients (10,34,44). The positive rate for respiratory specimens using reverse-transcriptase polymerase chain reaction (RT-PCR) increased slightly from 7 to 14 days after illness onset and then dropped to lower levels at 21 to 28 days (5). A review on respiratory shedding of MERS-CoV published by de Sousa demonstrated that using upper respiratory specimens for MERS-CoV diagnosis may not be as sensitive as use of lower respiratory tract specimens (11,12,17). This finding demonstrates that even for viruses within the same family, interpretation of testing results may vary even when using a sensitive test such as RT-PCR. The same diagnostic technique applied to samples from an infected person during the early stage of infection would most possibly report to be false-negative outcome as low viral loads are expected during this phase.

Clinical manifestations of SARS-CoV-2 infection cases range from asymptomatic carriers to development of respiratory disease with mild to severe pneumonia, frequently accompanied by acute respiratory distress syndrome and/or disseminated intravascular coagulation (24,29). However, data on respiratory shedding for SARS-CoV-2 have only been reported a few times to date (26,47,61). Prolonged viral shedding has also been observed among patients with SARS-CoV-2 infection up to 63 days after resolution of clinical symptoms and emergence of specific antibodies (26). That study reported that among respiratory specimens from different anatomical sites, sputum showed superior sensitivity to throat swabs and gargling wash. In the early stage, gargling wash specimens exhibited significant results compared with throat swabs or sputum. However, due to higher viral load, RT-PCR examinations from sputum still outweighed throat swab and gargling wash after fever diminished.

Better performance in detecting SAR-CoV-2 has been shown with clinical specimens aspirated from the lower respiratory tract (47), which is dissimilar from another report (61). A productive cough is generally common in the initial stage of illness, but in patients who produce sputum, this specimen provides a high diagnostic yield. A recent study also demonstrated the clinical usefulness of tongue, nasal, or midturbinate versus nasopharyngeal specimens especially since their collection reduces personal protective equipment use and provides a more comfortable patient experience (45). Therefore, to accurately detect viral infection, more efforts should be taken to correlate viral kinetics or symptom onset with sample collection criteria at sample collection sites.

Gastrointestinal shedding

Even though maximum clinical symptoms exhibited by patients with human coronavirus infections are closely associated with the respiratory tract, gastrointestinal distress, including diarrhea, during the stages of illness are also frequently detected (35%) (12,18,30,33). Progressive viral load has been observed in the stool specimens of >70% of patients with SARS-CoV infection within a maximum of 10 days after symptom onset, irrespective of respiratory symptoms (10,34). These findings have important implications for assessing transmission and protecting health care workers, as well as highlight the importance of effective treatment for COVID-19 patients.

Recent publications have demonstrated that SARS-CoV-2 can be identified in stool specimens of up to 53% of COVID-19 patients (54,55,57,58). Although about 23% of cases were no longer positive for the virus in respiratory samples, stool samples were still positive, clearly underlining the importance of fecal testing (54). Data from 98 COVID-19 patients showed viral shedding in stool samples for nearly 5 weeks after respiratory samples were negative (52,56). Even asymptomatic carriers may show elevated SARS-CoV-2 in stool samples (42). Hence, routine RT-PCR of stool specimens is recommended to determine when it is safe to discontinue precautions in recovered COVID-19 patients to prevent viral transmission and best allocate medical resources (52,55).

Other specimens used to detect SARS-CoV-2

To date, no other publications on other human coronavirus infections have reported testing of urine samples, which does not allow for consistent conclusions. Moreover, serological testing indirectly estimates the host response to infection and is best utilized retrospectively during outbreak investigation. These techniques are rapidly being established and have demonstrated to be useful in confirming SARS-CoV-2 infection (59). Serology testing has also previously played an important role in the epidemiology of coronavirus epidemics (4,9). For both immunoglobulin IgG and IgM antibodies, rapid lateral flow assays will undoubtedly play an important role in SARS-CoV-2 outbreaks, helping to estimate infection burden, the role of mild or asymptomatic infections, basic reproduction number, and the overall mortality rate. After SARS-CoV-2 infection, IgM levels have been observed to gradually increase during the first week, reach a peak after 2 weeks, and then disappear in most patients. Meanwhile, IgG levels are detectable after 1 week, reach a peak in 3 weeks, and can maintain heightened levels for >48 days. Therefore, serological diagnosis is less likely to have a role in active case supervision except to determine late diagnosis of SARS-CoV-2 infection and estimate the immunity of health care providers as the outbreak progresses. However, with the development of a specific IgG antibody test, large-scale seroepidemiological studies can be conducted during the current outbreak to improve understanding of the true scale of human-to-human transmission (18).

Factors Inducing Viral Shedding Kinetics and Viral Loads

Although COVID-19 largely appears to be mild, with most lethality among the elderly male patients with comorbidities, it is contagious (19,25). Its viral transmission may arise from contact with subclinical patients or “immediate recovery” patients. A study from China indicated the shedding window continued after clinical recovery in 50% of patients up to 8 days (6). The virus persisted for 12 days in another study from Singapore, in which all of the patients survived the infection (57). These findings demonstrate that recovered patients with low viral load do not spread the virus. In addition, delayed viral clearance, such as in aged populations, the immunodeficient, or those receiving immunosuppressive therapies, might be an alternative explanation for observed extended shedding. In COVID-19, the clinical course among young children was comparatively mild compared with teenage and adult populations. Viral load kinetics and shedding patterns can vary in these groups of patients and impact interpretation of diagnostic test results.

Outbreak control involves comprehensive mapping of the various clinical presentations, including mild symptoms and asymptomatic cases. Assessment of shedding kinetics and immunological response in those categories are extremely difficult, as such study involves recruitment of targeted populations and consent from healthy volunteers during an outbreak. Results are not conclusive without appropriate studies, especially since asymptomatic individuals can contribute to transmission. There is a significant difference among viral respiratory infection between symptomatic and asymptomatic individuals. A previous study reported that infections instigated by human metapneumovirus and respiratory syncytial virus were frequently associated with clinical illness in children up to 6 years old (1). Contrary to non-SARS-CoVs, human bocavirus and rhinovirus are commonly reported in asymptomatic children (23).

Antibody Response Kinetics

Knowledge of the kinetics and clinical correlates of serological responses to SARS-CoV-2 infection is crucial for diagnosing the disease, understanding seroepidemiological data to define prevalence and risk factors for infection, and assessing a potential role for passive immunotherapy. Published literature on antibody response kinetics in SARS-CoV-2-infected patients has demonstrated some contradictory outcomes regarding initiation and time of onset. In a recent study from 173 patients with SARS-CoV-2 infection in Singapore, the median seroconversion time for total antibodies, IgM, and IgG were on day 11, 12, and 14, respectively. The occurrence of antibodies was observed among <40% of patients within 1 week of onset and then sharply elevated among 100% of patients 15 days after onset. Among them, 94.3% and 79.8% were found to have IgM and IgG, respectively, in their sera. In contrast, detectability of viral RNA reduced from 66.7% in infected individuals after the first week to 45.5% during weeks 3–5 after onset. Antibodies against SARS-CoV-2 can be detected generally in the middle and later stage of the illness (53).

It has also been demonstrated that combination RNA and antibody measurement significantly enhance the sensitivity of diagnosis for SARS-CoV-2 infection even 1 week after infection onset (initial phase) (22). According to some reports, the incubation period of the infection and constrained use of nucleic acid detection by quantitative PCR affect confirmation of infection during early diagnosis, thereby underestimating prevalence rate. In contrast, most of the existing SARS-CoV-2 infection serology data were presented and reported from hospitalized patients with severe infection (40,51). In another study, ∼90% of patients who were admitted to the hospital with severe infection developed IgG antibodies within the first 2 weeks of symptomatic infection, and this appearance overlaps with fading of the virus, demonstrating a causal association between these events (51). However, there is very little published data on SARS-CoV-2 infection in individuals with no or mild symptoms who have not been hospitalized (27). A study on SARS-CoV infection in asymptomatic patients who are health care workers has been previously published (50). That study suggested that the extent of exposure to asymptomatic patients with SARS-CoV infection may have been limited to lower antibody levels and associated with less severe illness. Therefore, different test cutoffs may be required for use of diagnostic laboratory tests during outbreak investigations and determination of transmission extent. However, the hypothesis remains debated as the progress of severe respiratory distress symptoms is also supposed to be due to an irresistible immunological response (34,35,50).

Conclusions

According to the natural history of the infection, shedding and viral load kinetics in various anatomic sites of individuals with SARS-CoV-2 infection, sampling procedures largely contribute to the false-negative results. Optimization of specimen types and viral load progression with respect to time during SARS-CoV-2 infections remain to be completely understood and determined accordingly. Detailed data analysis on sampling procedures, testing algorithms, laboratory analyses, and interpretations, as well as clinical diagnosis of patients is required for understanding the epidemiology and clinical significance of SARS-CoV-2 better. Improvement of quality of diagnostic laboratory support during outbreaks and combined use of serological and molecular approaches is recommended; however, complete validation of laboratory assays has been essential. Evidence on the antibody response kinetics to SARS-CoV-2 infection is crucial to understanding seroconversion rates from different pools of antibody response cross-sectional measurements as well as estimations used to avoid misdiagnosis due to reduced sensitivity in initial infection stages and nonspecific reactivity management.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding received from any source.

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