Practicability of serological assays for upscaling COVID-19 laboratory testing in Africa

www.jogh.org • doi: 10.7189/jogh.11.03038 1 2021 • Vol. 11 • 03038 Several months after the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of the Coronavirus Disease 2019 (COVID-19), the world has experienced unprecedented economic and health uncertainties. As at November 8, 2020, Nucleic acid amplification tests (NAATs) such as the RT-PCR remain the gold standard adopted and recommended by the World Health Organization (WHO) for confirming COVID-19 diagnosis, treatment evaluation and the discharge of cured patients from isolation and/or hospitalization. As at November 10, 2020 there were 1357, 518 RT-PCR confirmed COVID-19 cases and 24 464 associated deaths out of about 12 million tested persons within a population of over 1.3 billion in 47 affected countries and territories in Africa [1,2]. Even though there are efforts by governments of all African nations to scale-up COVID-19 testing capacity and coverage (via house-to-house testing), the number of reported cases is still not an accurate representation of the actual cases in Africa probably. Consequently, many scientists have attributed the relatively low reported COVID-19 cases in Africa to misdiagnosis or underdiagnosis, probably due to low sampling high-risk persons, poor handling of samples, misdiagnosis or/and underdiagnosis, inadequate molecular testing capacity and insufficient manpower required for adequate SARS-CoV-2 molecular testing [3].

S everal months after the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of the Coronavirus Disease 2019 (COVID- 19), the world has experienced unprecedented economic and health uncertainties. As at November 8, 2020, Nucleic acid amplification tests (NAATs) such as the RT-PCR remain the gold standard adopted and recommended by the World Health Organization (WHO) for confirming COVID-19 diagnosis, treatment evaluation and the discharge of cured patients from isolation and/or hospitalization. As at November 10, 2020 there were 1357, 518 RT-PCR confirmed COVID-19 cases and 24 464 associated deaths out of about 12 million tested persons within a population of over 1.3 billion in 47 affected countries and territories in Africa [1,2]. Even though there are efforts by governments of all African nations to scale-up COVID-19 testing capacity and coverage (via house-to-house testing), the number of reported cases is still not an accurate representation of the actual cases in Africa probably. Consequently, many scientists have attributed the relatively low reported COVID-19 cases in Africa to misdiagnosis or underdiagnosis, probably due to low sampling high-risk persons, poor handling of samples, misdiagnosis or/and underdiagnosis, inadequate molecular testing capacity and insufficient manpower required for adequate SARS-CoV-2 molecular testing [3].
Even though it appears that the European and American nations have the highest recorded incidence and worst mortality rates of COVID-19, the relatively low figures reported in Africa ( Table 1) is not a true reflection of the epidemiology of SARS-CoV-2 infection. This is because many Practicability of serological assays for upscaling COVID-19 laboratory testing in Africa 2 www.jogh.org • doi: 10.7189/jogh.11.03038 Photo: From the author's own collection, used with permission.  [4]. Note: These are ongoing data that could change over time.
African countries have weak health care systems and hence COVID-19 data may be inaccurate. Some parts of the world are experiencing a second wave of the COVID-19 pandemic, of which there is an increasing need for rapid and reliable SARS-CoV-2 antigen-based and antibody testing, for deployment for either largescale population screening or serodiagnosis purposes [5].
Even though SARS-CoV-2 RT-PCR tests provide very accurate results, the availability of their consumables and reagents are usually inadequate in most health care facilities in Africa. Furthermore, RT-PCR tests are quite arduous, costly to operate because they require sophisticated laboratory facilities. Countries with insufficient infrastructure quickly accumulate a backlog of samples for tests and have a long test turnaround time from sample collection (pre-analytical phase) to the availability of tests results (post-analytical phase) [6]. In cognizance of the technical limitations of RT-PCR tests, this article sought to provide supportive data on the performance characteristics and applicability of serology-based assays for upscaling COVID-19 laboratory testing in Africa.

TECHNICAL LIMITATIONS OF THE STANDARD COVID-19 TESTING PROTOCOL
One of the main technical limitations in the use of RT-PCR tests is false-negative results despite individuals present with clinical and radiologic features highly suggestive of COVID-19. This has been associated with wrong sampling by inadequately sample collectors where SARS-CoV-2 might have been present and replicating in the lower respiratory tracts instead of upper respiratory tracts. Hence, samples often collected will yield accurate test results. This condition will indeed hinder proper evaluation and diagnosis of COVID-19 persons concerned.
Other factors include poor sample storage, inadequate transportation conditions, purification and handling, the disintegration of purified COVID-19 RNA, presence of RT-PCR antagonists and viral genetic variants. Besides false-negative reports, RT-PCR has also been reported to be associated false-positive results which can be due to cross-contamination of specimens during sampling, poor pipetting and technical flaws during handling [7][8][9][10]. These factors can negatively influence the efficient diagnosis and management of COVID-19 cases.
Till date, there is no categorical explanation on transmission dynamics of SARS-CoV-2 in Sub-Saharan Africa. Significantly, with the reports of the D64G strain of SARS-CoV-2 that could enhance the transmissibility of the pathogen in the population [11]. This mutant alongside others that occurred on genes (N, ORF1ab, E, RdRP) utilized in synthesis RT-PCR primers could also affect the accuracy of the assay and a cause for concern in the reliability of results in locations with records of widespread SARS-CoV-2 mutants. Diagnostically, the N, E and RdRP, ORF1ab are the major targets used in RT-PCR platform. Of note is that mutations in these genes have been reported all over the world. Indeed, the effectiveness and reliability of diagnostic tests are of paramount importance to avoid a large number of false test results if currently used RT-PCR assays are undermined by mutations on SARS-CoV-2 [11][12][13][14][15].

ACCESSIBILITY AND PRACTICABILITY OF COVID-19 SEROLOGICAL TESTS IN AFRICAN SETTINGS
Aside from the significance of accurate laboratory tests in the diagnosis of COVID-19 index cases, diagnostic testing is also needful for persons who had contact COVID-19 confirmed cases [16]. A certain number of laboratory analytical procedures investigate only contacts that develop symptoms or present with any type of ailment within 14 days after contact. Alternate procedures investigate every contact once detected, irrespective of the presence or absence of symptoms. Researches indicate that a considerable number of infected persons presents no symptoms whatsoever. However, it is worrisome that these asymptomatic persons are nevertheless capable of shedding the virus and infecting susceptible individuals via droplets when they speak, thereby spread infection [17]. Tracing and follow up of every contact of confirmed cases as well as investigating them for SARS-CoV-2 is crucial to contain the pandemic effectively. Serodiagnostics are essential to enable quick surveys to ascertain if and to what magnitude SARS-CoV-2 has spread in a population. This needful in strengthening surveillance systems [16]. This approach has also been useful in monitoring community transmission, identify high-risk individuals and the reliability of preventive strategies.
In the quest to solve the need for sophistication, high-cost running and maintenance, long turn-around-times and an accumulated backlog of samples, some point-of-care molecular tests (such as the GeneXpert equipment) for COVID-19 diagnosis have been developed, calibrated and made accessible for rapid community-based screening. However, these RT-PCR tests have their peculiar limitations, which involve longer cartridges production time and the high demand of these devices by high-income countries have put a strain on the accessibility of the devices, reagents and consumables in low-and middle-income Africa countries [16].
Serological-based tests, which are blood-based have been globally accepted in the evaluation of symptomatic, asymptomatic and recovered cases of COVID-19 by determining the level of exposure of those affected based on the humoral immune response to SARS-CoV-2 infection [16].
Based on applicability, SARS-CoV-2 antigen-based tests have developed and proved to be rapid, easy to perform with a less than 30 minutes turn-around-time when compared with the NAATs [18]. These rapid tests are vital either as alternatives or in supplementing the role of nucleic acid-based tests in the confirmation, management and the signaling of cases to be isolated in order to prevent viral transmission. Furthermore, COVID-19 serological assays are less technical, and the quality of specimen required is relatively lesser than those of RT-PCR tests [18]. Despite these advantages, the applicability of serological-based tests is limited as these kits cannot serve for the screening of COVID-19 due to their low diagnostic performance at the early stage of infection [18].
COVID-19 serological tests are globally accepted in the evaluation of symptomatic, asymptomatic and recovered cases of CO-VID-19. Concerns over false negative antibody results could technically be resolved be increasing the number of tests done in a week (eg, 3-4 times) to improve the chances of getting true positive test. This helps to narrow the chances of missing a truly infected COVID-19 patients during the "window period". Conversely, cases of false-positive results due to cross-reaction with antibodies against other coronaviruses are very rare in Africa.
The early phase of the disease from the time of infection till the period just before the anti-SARS-CoV-2 IgM can be detected in serum (usually from day 3) following infection is the window period. During such period, cases of acute infection are most likely to be missed as the virus remains undetectable within 48 hours from infection [19]. The IgM raised against SARS-CoV-2 continues to rise steadily until the 7th day post-infection when IgG begins to increase while IgM declines [19]. To avoid reporting false negative anti-SARS-CoV-2 IgM or IgG results, these could be resolved by increasing the number of tests done in a week (eg, 3-4 times) to increase the chances of getting true positive antibody test. This helps to narrow the chances of missing a truly infected COVID-19 patients during the "window period" when tested just once. Besides the possible false-negative result during the window period, false-positive can also occur when the SARS-CoV-2 antibodies cross-react with those of other coronaviruses (such as MRES-CoV and SARS-CoV-1) that have high genomic homology with SARS-CoV-2 [20]. For issues of antibodies cross-rection between SARS-CoV-2 and other coronaviruses in 2020, so far there is currently no available report on co-circulation of other coronaviruses with SARS-CoV-2 in sub-Saharan Africa. Hence, the impact of cross-reaction of related viruses on the serological test has little or no effect on the precision of test results. Even though MERS-CoV continues to circulate in the Arabian Peninsula, including countries boarding North Africa, there have not been any confirmed human MERS-CoV infection in Africa, despite the frequent occupational and domestic contact between dromedary animals and humans [21]. However, there is a need for further studies of MERS-CoV at the animal-to-human interface, especially with the recent report on the absence of serological evidence of human MERS-CoV infection in Nigeria [22]. Besides, SARS-CoV-1 has since been exclusively reported in the Asian continent and Europe [23].
Hundreds of serodiagnostic tests devices and kits have so far been developed, and their technical performance characteristics have been made available. Interestingly, the performance characteristics of various serological assays were incredibly encouraging ( Table 2). Several serological test kits and devices have proved to be reliable for clinical diagnosis due to their excellent diagnostic performance characteristics (ie, accuracy, specificity, sensitivity, negative predictive and positive predictive values) especially during the window period where the SARS-CoV-2 Infection ( Table 2).

DRAWBACKS OF SARS-COV-2 SEROLOGICAL TESTS
Sero-diagnostic test kits with high performance characteristics have proved to be mostly reliable especially in areas with high prevalence of the SARS-CoV-2 infection, but they are not recommended for use in isolation for the clinical decision making about SARS-CoV infection in geographical locations with few cases of CO-VID-19 [35]. This is because there is a significant chance in false-positive test outcomes. On the other hand, false-negative results are also anticipated in high-risk individuals (ie, those with asymptomatic acute phase of the infection, those with other respiratory tract illnesses, those who had contact with confirmed cases) due to low antibody titers which are far below the limited of detection (LOD) of most sero-diagnostic kits and devices. These misleading outcomes can give a false impression of the presence of SARS-CoV-2 infection and misinform public health experts on the appropriate non-pharmaceutical approaches suitable for SARS-CoV-2 prevention and control. The decision to ramp-up testing capacity for public health surveillance and risk assessment by using these sero-diagnostic kits and devices must be guided on the availability of data on the severity and prevalence of the infection in the population of a region [36].
Based on economic reasons, the use of the standard protocol can be restricted to high-risk individuals, especially health care workers, with subsequent follow-up using serodiagnostic medical devices. Previous studies have associated the immunoglobulin level raised against the epitome-binding domain of the SARS-CoV-2 structural proteins with their neutralizing capacity which seems to confer passive immunity in the presence of low viral load [35]. However, there is an inadequate data on the duration of such immunity. Hence, the knowledge gap in understanding the immunological interplay during SARS-CoV-2 reinfections [36,37].
Despite the opinions on passive immunity to avert subsequent SARS-CoV-2 reinfection, the WHO has consistently dismissed this notion based on the paucity of sufficient supporting data on the association between immune status and neutralizing immunoglobulins [38]. In support of the WHO's stance, several studies have demonstrated instances where infected individuals have high levels of the SARS-CoV-2 nucleic acid in the presence of elevated levels of neutralizing immunoglobulins raised against viral structural proteins (such as the Spike and Nucleocapsid) [39]. Consequently, this reveals that the neutralizing antibodies are poor biomarkers of protective immunity and thus cannot be useful guide (in isolation) to determine the recovery status of SARS-CoV-2 infected individuals. However, the quest to further improve on the accuracy of serological markers for use in evaluating the time of recovery of COVID-19 patients before discharge from the isolation/ quarantine or hospital is still ongoing.

Limitation of the study
Lassauniere et al [24] Evaluated the sensitivity and specificity of 9 commercially serological tests. These included 3 ELI-SAs and 6 point-POCTs which were validated using serum samples from SARS-CoV-2 PCRpositive patients with a documented first day of disease, archived sera obtained from healthy individuals before the emergence of SARS-CoV-2 in China and sera from patients with acute non-SARS-CoV-2 viral infections Wantai total antibody ELISA had the best sensitivity (93%) and specificity (100%). However, they reported its potential to cross-react with non-SARS-CoV-2. The number of COVID19 confirmed patients was low. The specificity of these assays was not evaluated using samples that were known to be positive for antibodies to the commonly circulating human coronaviruses, to determine cross-reactivity.

REFERENCES CONCLUSION
The inadequate global cooperation and solidarity during the COVID-19 pandemic have caused many African countries to suffer limited access to molecular diagnostic reagents and consumable for SARS-CoV-2 tests. Furthermore, there is still a paucity of information on the immune response kinetics and dynamics to SARS-CoV-2 Infection, as many reports have not been categorically exhaustive. These have led to hesitation in the use of COVID-19 serological assays, even though these tests are commercially available and scalable. To attain adequate laboratory response required for the adequate containment of the COVID-19 pandemic in Africa, biotechnology development firms, In-vitro diagnostics quality assurance laboratories, and regulatory authorities should consider local production, post-production evaluation and standardization of serological tests devices to scale-up COVID-19 testing capacity.