Pre-analytical phase

The pre-analytical phase is considered the first stage of process development in the clinical laboratory. Deviations or omissions in this stage impact adequate performance, generating failures in the tests’ performance or even affecting the quality of the results. In the case of SARS-CoV-2 detection, this phase is essential for identifying the virus, diagnosing the disease, and the care of healthcare personnel exposed to such processes.

It is crucial to be aware of biosafety measures during all stages of the analytical process considering all the risks involved in handling biological samples of infectious diseases. The minimum essential personal protective equipment (PPE) is described in the Biosafety and Biosecurity Protocol issued by the InDRE to handle patients during sample collection. Further details can be found in the following link: https://www.gob.mx/salud/documentos/lineamientos-vigentes-red-nacional-de-laboratorios-de-salud-publica?state=published) (Table 2).

During the development of molecular tests, errors to identify SARS-CoV-2 in the pre-analytical phase are similar to those in other diagnostic areas. An example is the type of specimen recommended for these tests; the primary specimen should come from the respiratory tract of the individual with suspected disease. Nasopharyngeal (NP) and oropharyngeal (OP) swabs should be collected and transported in the same tube containing viral transport medium or sterile saline. Primary samples should be kept refrigerated (4-8 °C) and processed within 24-72 h after sampling.

Both the process and the timing of sample collection are critical to the outcome. By the process, we mean the swabbing technique and the region where it will be performed, while the timing of sample collection refers to the time elapsed after exposure to the virus. The estimated incubation period of this new virus ranges from 2 to 14 days; however, incubation periods of 21, 24, or 27 days have been observed in some cases. Therefore, sampling at the early stages of exposure may generate false negative or indeterminate results in most cases¹⁹-²⁵.

Kucirka et al. used a model to estimate the false-negative rate and found that sampling suspected individuals before the onset of symptoms show up to a 100% chance of a false-negative result. This value decreases with the time of symptoms onset. For example, on day 1 of symptoms onset, the estimate reaches 67% (95%CI 27-94%), and on day 4, the mean false negative rate is 38% (95%CI 18-65%). This rate decreased to 20% (95%CI 12-30%) on day 8, and by day 21, the false-negative rate increased again²⁶.

Therefore, the sampling moment should be considered decisive since the incubation cycle of the virus and the manifestations of the disease are variable in each patient and may affect the interpretation of the results (Figure 1).

Figure 1 SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection clinical course concerning the reverse transcription-polymerase chain reaction (RT-PCR) test positivity. 

Another critical variable is the difficulty of sampling in newborns and infants due to the characteristics of the material used for sampling. Although small or low-caliber swabs are available for these patients, the sample collected is usually not representative. Therefore, the personnel in charge of sample collection must be trained in patient-management and sampling material.

For upper respiratory tract sampling, including NP and OP swabs, the following recommendations should be considered:

Analytical phase

For molecular biology tests, it is crucial to recognize two stages within the analytical phase. The first phase involves obtaining the genetic material, which will be the primary source of the process, while the second stage corresponds to the RT-PCR. Before implementing the tests, the following elements are required: the qualification of the facilities and the verification of the chosen analytical platform, in addition to a specific documented operating procedure.

When performing these analytical procedures, the PPE used should be the same as in the pre-analytical phase, and the analytical process should be performed in a biosafety level 2 (BSL-II) laboratory at least¹⁰-²⁸.

As previously described, identification of SARS-CoV-2 in the upper and lower respiratory tract is performed by RNA amplification via real-time RT-PCR during the virus infection cycle that allows qualitative detection in vitro. Positive results indicate an active viral infection; however, bacterial infection or co-infections with other viruses are not excluded. Therefore, negative results should be combined with clinical observations, patient history, and epidemiological information to guide treatment and other medical decisions.

We suggest the following recommendations for handling the primary sample and obtaining viral RNA:

The viral RNA obtained is not infectious; however, it should be kept refrigerated (4-8°C) before the RT-PCR test (which should be performed within 2-3 h after obtaining the RNA) and separate from the reagents used for the test. Once used, this biological material can be stored at -80^oC.

For RT-PCR performance, the PPE equipment should be new. It is crucial to consider the following recommendations:

Figure 2 Detection of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) using the Berlin protocol in a positive case (amplification cycles of the three genes). E gene, envelope protein gene; RdRP gene, RNA-dependent RNA polymerase gene; RP, RNase P gene. 

An essential part of the analytical process is diagnostic accuracy. In the tests for SARS-CoV-2 identification, there are different methodological proposals or analytical designs. The most widely used at the international level are the following:

Post-analytical phase

The recommendations during this phase are the following:

In the case of the graphs or the experiments performed to identify SARS-CoV-2, interpretation criteria should consider each gene’s characteristics and, if necessary, the presence, absence, or combinations that can be obtained. For example, the Berlin protocol seeks to identify three genes (a constitutive gene, a generic viral gene, and a specific viral gene) (Table 3).

Currently, no quantitative method can detect the number of virus copies in a sample; however, the number of amplification cycles of a specific gene detected in RT-PCR can provide information on the concentration of viral load (Table 4). It is important to be aware of some considerations to extrapolate these data: a) RNA concentration obtained during the extraction should be homogenized for all tests. Therefore, RNA should be quantified before the RT-PCR test and dilutions should be performed if necessary; b) the gene used for this determination should always be the same; 3) the interpretation will depend on the protocol implemented, the equipment used for the determination, and the brand of reagents selected.

In the case of interpretation errors, the final report delivered to the physician should be clear and concrete. In the case of positive results for COVID-19, unnecessary information (e.g., primers’ sequence, enzymes used, and other methodological details) should not be included in the reports.

Finally, laboratories from public and private institutions that perform SARS-CoV-2 identification tests must generate a report of positive cases to the federal government’s epidemiological surveillance network for the follow-up of the pandemic in the country. Each healthcare center must report the results of the SARS-CoV-2 tests to each country’s competent health authorities. In the HIMFG Laboratory, we generate weekly reports following the current strategies, and we are part of the epidemiological surveillance support laboratories.

Molecular detection of SARS-CoV-2 is critical for taking action against the pandemic. However, it is essential to perform these procedures using protocols that promote patients’ and healthcare personnel’s safety. In Mexico, the Official Standards (NOM, for its Spanish acronym) for the organization and operation of clinical laboratories (NOM-007-SSA3-2011) and for the management of bio-hazardous waste (NOM-087-ECOL-SSA1-2002), among others, allow the establishment of clinical diagnostic tests.

Considering the heterogeneity of detection protocols, we should promote inter-laboratory networks for the appropriate exchange of information and relevant actions, besides following the guidelines issued by the InDRE to strengthen the timely detection of SARS-CoV-2 in Mexico. Furthermore, laboratories should continue to use the influenza laboratory algorithm recommended by PAHO and WHO for routine surveillance, considering that testing for COVID-19 should be performed only for patients who meet the case definition following epidemiological surveillance strategies.