SARS-CoV-2: Challenges in Reconverting Diagnostic Laboratories to Combat the Pandemic

Abstract

Coronavirus disease 2019 (COVID-19) was first detected in Mexico in February 2020. Even though health authorities did not perceive then the value of viral detection tests, we anticipated the demand for them. We set up to develop an expeditious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) molecular diagnostic service through the implementation of standardized protocols for biospecimen sampling, transportation, biobanking, preanalytical validation, and nucleic acids (NA) testing (NAT). Nasopharyngeal and oropharyngeal swabs collected in a special transportation medium were the biospecimens from which NAs were purified either manually or automatically. Viral RNA genome presence was determined using commercial SARS-CoV-2 detection kits (based on reverse transcription coupled with real-time PCR [RT-PCR]). Improvements in laboratory processing speed and reliability resulted from semi-automatizing laboratory processes and adopting a quality control/quality assurance system (QC/QA), respectively. NAs that were purified, either manually or automatically, were validated by preanalytical spectrophotometric characterization. Automated purification was less prone to contamination and reduced the processing time. The following six RT-PCR kits were evaluated for their convenience, specificity, sensitivity, time consumption, and required materials (in order, starting with the kit with the best results): RIDA gene and Viasure (tied), Vircell, LightMix, 1copy, and Logix Smart. Redesigning the laboratories' working areas, equipment, fluxes of personnel and material, and personnel skills, and overemphasizing biosafety safeguards were major challenges encountered in the middle of the sanitary crisis. Adopting a QC/QA system, utilizing automatization processes, and working closely with health authorities were key factors in our success. IMPORTANCE Rearranging our diagnostic laboratories to improve the fight against a new unexpected, unpredictable, and sudden public health threat demanded that we move quickly to redesign not only the laboratory processes but also the distribution of space, personnel activities, and fluxes of material coming in and out. We also had to work closely with governmental health authorities to gain their trust in our technical competence. Gaining the confidence of the clients, i.e., mainly individuals, the human resource departments of factories and corporations sending employees for testing, and medical institutions, and implementing as much automatization as possible of processes, in which only officially approved reagents (for extraction and analysis of NA) were used to generate opportune trustable testing results, were key factors. Our laboratories have gathered a considerable amount of experience and significant number of solutions, considering our geographic contexts alongside this continuously morphing pandemic, validating many techniques that might help other laboratories find a better and more precise workflow.

Keywords: RT-PCR; SARS-CoV-2 diagnosis; commercial RT-PCR kits; laboratory techniques.

FIG 1

UV spectrum profiles of NA isolated using the automatic method (A) and the manual procedure (B). The y axis represents the relative absorbance units, and the x axis represents the wavelength.

FIG 2

Variations in the detection rate and C_T values in the six kits used. Nine samples (eight positives and one negative) were subjected to the various RT-PCR assays, according to the respective manufacturer’s instructions for use. The solid dots in the graph indicate C_T values obtained for all clinical samples (n = 9) using all RT-PCR assays. The open circles above the dotted line are negative, for data purposes indicated by a C_T value of 43.5. The results for the negative sample and the positive one displaying the highest viral load were concordant in all kits evaluated.

FIG 3

Results of the PCR analyses of reverse-transcribed RNA samples purified from the naso- and oropharyngeal swabs. (A) Representative results of initial PCRs run without optimization. (B) Representative results of their optimization. (C) Representative results of amplification of an internal control. (D) Representative results of an atypical curve detection. RFU, relative fluorescence units.