Molecular Diagnostics in the Post-COVID-19 Era: Technological Advances, Lessons Learned, and Future Perspectives in Tracking Emerging Viruses

Introduction

The COVID-19 pandemic, which began in December 2019, represented one of the greatest health challenges of the contemporary era, causing significant impacts on health systems on a global scale. With rapid dissemination, SARS-CoV-2 has led to the collapse of hospital structures, an exponential increase in morbidity and mortality, and the urgent need to reorganize health services. Estimates indicate that, by the end of 2022, more than 6.8 million deaths had been recorded globally, with important economic and social repercussions WHO 2023 [1]. The health crisis has exposed structural weaknesses, especially in low- and middle-income countries, and has required emergency strategies to ensure the diagnosis, isolation, and treatment of cases Silva, et al. 2021 [2].

In this context, the capacity for rapid response to the health emergency was directly related to access to effective diagnostic methods. Early identification of cases was crucial for containing community transmission and for public health decision-making, such as implementing quarantines, border control, and resource distribution. Molecular methods such as RT-PCR have become fundamental in this process, allowing the accurate detection of viral RNA in the early stages of infection Corman, et al. 2020 [3]. Agility in diagnosis has proven to be one of the central pillars for effective management of the pandemic, especially in the initial phase of viral spread Brasil, 2020 [4].

As a result, a redefinition of epidemiological and laboratory surveillance priorities was observed, driving the adoption of molecular diagnostic technologies on a large scale. The pandemic has driven unprecedented investments in laboratory infrastructure, process automation, human resources qualification, and the development of decentralized testing platforms Vasconcelos, et al. 2021[5]. In addition, there was a strengthening of international collaborative networks, such as GISAID, for the sharing of genomic data in real time, contributing to the tracking of variants and the formulation of vaccine and health strategies Shu & McCauley, 2017 [6]. This new configuration represented not only an emergency response, but also the consolidation of a new paradigm in the molecular surveillance of emerging pathogens.

Rise and consolidation of molecular diagnostics as the gold standard

Reverse transcription polymerase chain reaction (RT-PCR) was quickly adopted as the gold standard method for diagnosing COVID-19, due to its high sensitivity and specificity in detecting the genetic material of SARS-CoV-2. This technique allowed the accurate identification of infected individuals, even in the asymptomatic or pre-symptomatic phase, contributing decisively to strategies to contain viral transmission (Corman et al., 2020). RT-PCR, despite being widely used in laboratory settings for decades, has been implemented on a global scale like never before, demonstrating its central role in tackling pandemics Li, et al. 2020 [7].

The massive use of RT-PCR also revealed logistical challenges, such as scarcity of inputs, prolonged processing time in regions with less infrastructure, and the need for specialized labor. These limitations motivated the development of complementary methodologies, such as rapid antigen tests and automated real-time RTPCR platforms, which sought to balance accuracy and agility West, et al. 2020 [8]. The consolidation of RT-PCR as a diagnostic method further boosted investments in molecular biology equipment and fostered the decentralization of testing, with the creation of regional laboratories and public-private partnerships Brasil 2021 [9].

In addition to its diagnostic role, RT-PCR has been used in genomic surveillance strategies, contributing to the tracking of variants of concern and to the evaluation of vaccine efficacy in different populations. Its ability to detect viral loads was also relevant to clinical decisions about isolation, hospital discharge, and return to work, broadening its scope of application Vogels, et al. 2021 [10]. In this way, RT-PCR transcended its initial function and established itself as a strategic tool for health management at different levels of complexity, consolidating molecular diagnosis as an indispensable component in epidemiological crisis scenarios.

Inequality in access and challenges to global laboratory capacity

The COVID-19 pandemic has exposed historical disparities between countries in terms of diagnostic capacity and laboratory infrastructure. While high-income nations rapidly expanded their testing and input production networks, many low- and middle- income countries faced severe difficulties in ensuring continued access to molecular testing and basic diagnostic equipment Fayet-Mello, et al. 2021 [11]. Scarcity of reagents, dependence on imports, and limited testing capacity have resulted in underreporting, delays in diagnosis, and difficulties in controlling viral spread Boum, et al. 2021 [12].

In Brazil, despite important advances, such as the implementation of state central laboratories (LACENs) and the strengthening of the Fiocruz Genomics Network, logistical and regional bottlenecks persisted, especially in the North and Northeast regions, where access to molecular diagnosis was initially limited Lima, et al. 2021 [13]. This inequality was directly reflected in the indicators of mortality and morbidity due to COVID-19, evidencing that diagnostic capacity is not just a technical tool, but a matter of equity in public health. In addition, the centralization of testing in capitals slowed the response in remote areas, increasing the time between collection and result Silva, et al. 2022 [14].

The need for decentralization of testing has led to the adoption of emergency strategies, such as training local staff, installing mobile RT-PCR platforms, and validating more accessible diagnostic technologies, such as isothermal and CRISPR-based tests Ouma, et al. 2021 [15]. Despite the efforts, the pandemic has shown that the sustainability of laboratory capacity depends not only on technological resources, but also on public policies that guarantee national autonomy in the production of inputs and continuous investments in science and technology. This understanding is essential to prepare countries for future health emergencies and mitigate the effects of structural inequalities.

Contributions of laboratories and Brazilian research in the fight against COVID-19

The performance of research and diagnostic laboratories in Brazil was decisive in facing the COVID-19 pandemic, especially in the early stages of the health emergency. The Oswaldo Cruz Foundation (Fiocruz), through the Oswaldo Cruz Institute (IOC) and the Institute of Technology in Immunobiologicals (Bio-Manguinhos), led initiatives for the production of diagnostic kits, training of health professionals, and structuring the National Network of Public Health Laboratories (LACENs), significantly expanding the national testing capacity Fiocruz 2020 [16]. This rapid response was essential for the country to implement more effective epidemiological surveillance measures, even in the face of regional inequalities and logistical bottlenecks faced.

An important milestone in Brazil’s scientific contribution was the sequencing of the first complete genome of SARS-CoV-2 in Latin America, carried out just 48 hours after the first case of the disease was confirmed in the country. This feat was conducted by biomedical scientist Jaqueline Goes de Jesus, linked to the Institute of Tropical Medicine of the University of São Paulo (USP) and the Oswaldo Cruz Foundation of Bahia, using next-generation sequencing (NGS) technology with the MinION platform Jesus, et al. 2020 [17]. The speed and accuracy in generating and sharing this data were essential for monitoring mutations, as well as for Brazil’s integration into global genomic surveillance networks, such as GISAID, which guided vaccine and health decisions in real time Candido, et al. 2020 [18].

In addition to genomic sequencing, Brazilian research stood out on multiple fronts, including the development of molecular and serological assays, clinical studies with drugs and vaccines, and mathematical modeling of the spread of the virus. Several federal universities and research centers, such as UFRJ, Unicamp, and the Butantan Institute, worked collaboratively to expand scientific knowledge about the pandemic and develop technological solutions adapted to the Brazilian reality Zimerman, et al. 2021 [19]. National scientific production related to COVID-19 has grown exponentially, placing Brazil among the countries with the highest number of publications on the subject, highlighting the role of science as a central axis of the response to the health crisis.

Highlight for RT-PCR as the main tool in viral detection

Since the first reports of COVID-19, RT-PCR (reverse transcription polymerase chain reaction) has been rapidly adopted as a central tool in the diagnosis of SARS-CoV-2 infection. The technique, based on the amplification of specific viral RNA sequences, has been shown to be effective in identifying the virus in respiratory clinical samples with high sensitivity and specificity, including in asymptomatic patients or in the first days of symptoms Corman, et al. 2020 [3]. The World Health Organization has recommended the adoption of RT-PCR as the gold standard for laboratory confirmation of COVID-19, and it is widely incorporated into clinical protocols and public health policies around the world WHO 2020 [20].

The reliability of RT-PCR, combined with its consolidated technical base, has allowed the rapid production and validation of diagnostic kits in several countries. In Brazil, institutions such as Fiocruz, the Butantan Institute, and federal universities played an essential role in the development and distribution of RT-PCR-based tests, collaborating to expand testing in a context of health emergency Brasil 2021 [9]. This technique thus became the main viral detection strategy during the first years of the pandemic, sustaining epidemiological surveillance actions and guiding non-pharmacological interventions.

Benefits: high sensitivity, specificity, and early detection

One of the greatest benefits of RT-PCR lies in its high sensitivity, which allows detecting minimal viral loads in the first days after infection, before the appearance of evident clinical symptoms. This characteristic is especially useful in environments with a high risk of contagion, such as hospitals and long-term care institutions, enabling rapid interventions to contain the spread of the virus Wiersinga, et al. 2020 [21]. In addition, the ability to detect infections at an early stage has contributed to contact tracing and isolation of asymptomatic carriers, playing an essential role in containing the pandemic Pan, et al. 2020 [22].

Another important aspect is the specificity of RT-PCR, achieved through the use of primers targeting conserved regions of the viral genome, such as the E, N, and RdRp genes. This specificity reduces the likelihood of false positives and makes the technique reliable even in the face of the emergence of SARS-CoV-2 variants Chan, et al. 2020 [23]. By combining high diagnostic accuracy with analytical stability, RT-PCR has proven to be a robust and adaptable tool, contributing to evidence-based clinical, epidemiological, and health decisions.

Global logistical and structural limitations evidenced by the pandemic

Despite its efficacy, the large-scale application of RT-PCR has revealed several logistical and structural limitations, especially in countries with low installed capacity for molecular diagnostics. The shortage of reagents, the dependence on specialized equipment, and the need for trained professionals have made it difficult to expand testing in remote and vulnerable areas Fayet-Mello, et al. 2021 [11]. These barriers have compromised the early detection and control of transmission in many regions, making evident the importance of laboratory decentralization and the national production of strategic inputs Silva, et al. 2022 [14].

In Brazil, the impact of these limitations was particularly felt in the North and Northeast regions, which had longer waiting times for results and lower diagnostic coverage, which made it difficult to control the pandemic in these areas Lima, et al. 2021 [13]. The centralization of testing in the capitals and the overload of public laboratories showed the vulnerability of the system in the face of an emergency scenario. These challenges have driven the search for complementary technologies, such as rapid tests, RT-LAMP, and CRISPR-based platforms, which offer promising alternatives for expanding access to molecular diagnostics in future health crises Broughton, et al. 2020 [24].

Barriers faced by low- and middle-income countries

In low- and middle-income countries, the barriers faced for the molecular diagnosis of COVID-19 were multiple and interdependent. The scarcity of laboratories with an adequate level of biosafety, the lack of professionals trained in molecular biology, and the absence of integrated logistics systems have compromised the implementation of efficient testing protocols Boum, et al. 2021 [12]. In addition, the high cost of equipment and reagents, combined with the bureaucracy for the international acquisition of supplies, made it difficult to expand testing in a timely manner Fayet-Mello, et al. 2021 [11].

In Latin America, for example, many countries centralized testing in laboratories located in capitals, which implied long distances for sending samples, increased response time, and diagnostic exclusion from remote communities. In Brazil, despite the existence of the National Network of LACENs, regions such as the North and Northeast had a deficit in diagnostic coverage and a longer average time to deliver results, reflecting historical inequalities in investment in health infrastructure Silva, et al. 2022 [14]. These obstacles compromised the effectiveness of the national response and reinforced the urgency of structuring measures.

Importance of decentralization and expansion of diagnostic infrastructure

The decentralization of laboratory services has proven to be a fundamental strategy to expand access to molecular diagnosis and reduce regional inequalities. By distributing testing capacity across multiple regions, it is possible to optimize logistics, reduce response time, and expand coverage in historically neglected areas Uddin, et al. 2021 [25]. The creation of regional laboratories, local training of professionals, and the use of portable technologies, such as isothermal tests and CRISPR platforms, have proven to be viable and efficient alternatives, especially in contexts of low structural complexity Fozouni, et al. 2021 [26].

Structural investments in this area also promote greater technological sovereignty and strengthen epidemiological surveillance at the national level. Decentralization allows outbreaks to be detected more quickly, favoring early interventions and reducing the impact of emerging diseases Castro, et al. 2020 [27]. The strengthening of the Brazilian laboratory network, as seen with the expansion of the capacity of LACENs and the strategic role of Fiocruz in the national production of diagnostic inputs and kits, is an example of how integrated public policies can contribute to a more resilient and equitable health system.

Fundamentals of molecular diagnostics

Molecular diagnostics is an approach based on the identification of specific nucleic acid sequences (DNA or RNA) of microorganisms, using techniques such as polymerase chain reaction (PCR), genetic sequencing, and molecular hybridization. These techniques allow the direct detection of the genetic material of the etiological agent, conferring high sensitivity and specificity to the diagnostic process Mackay, et al. 2002 [28]. The main advantage of this approach over conventional methods, such as microbiological cultivation or immunological testing, is the speed and ability to detect pathogens even at very low loads, which is essential for the containment of communicable diseases.

In addition to RT-PCR, which was widely used during the COVID-19 pandemic, other molecular techniques such as LAMP (Loop-Mediated Isothermal Amplification), microarrays, and methods based on CRISPR-Cas have been incorporated into the diagnostic arsenal, with emphasis on their applicability in the field, including in environments with limited infrastructure Notomi, et al. 2000 [29]; Broughton, et al. 2020 [24]. These technologies have been revolutionizing laboratory diagnosis, allowing for more accurate and real-time response to outbreaks, epidemics, and pandemics.

Role in Infectious Disease Tracking and Control

Molecular diagnostics have played a central role in the screening and control of infectious diseases, since it enables the rapid and accurate identification of the etiological agent, even in the early stages of infection or in asymptomatic individuals. This early detection capacity is vital for interrupting transmission chains, especially in highly contagious diseases such as influenza, tuberculosis, and COVID-19 Wiersinga, et al. 2020 [21]. Access to molecular diagnostics also allows monitoring of response to treatment, detection of mutations associated with antimicrobial resistance, and surveillance of viral variants.

The incorporation of these techniques into the epidemiological surveillance system strengthens public health strategies, contributing to rapid and evidence-based decisions. During the COVID-19 pandemic, the use of molecular diagnostics was decisive for the implementation of social distancing policies, border closures, contact tracing, and evaluation of the effectiveness of vaccines Li, et al. 2020 [7]. The capacity for mass testing, associated with molecular diagnosis, has become an indicator of the resilience of health systems, reinforcing the need for continuous investment in laboratory infrastructure and technical training.

Historical evolution and integration in public health policies

Historically, molecular methods emerged in academic research laboratories in the 1980s, with the discovery and development of PCR, which transformed clinical diagnosis by allowing the amplification of specific DNA fragments in a few hours Mullis & Faloona 1987 [30]. Since then, these methods have been progressively incorporated into laboratory practice, initially in high-complexity institutions and, more recently, in public health networks, including in low- and middle-income countries. The incorporation of these techniques into diagnostic protocols has been a milestone in the modernization of health systems.

In Brazil, the introduction of molecular diagnostics in public health policies intensified with the H1N1 pandemic in 2009 and was strategically expanded during the COVID-19 pandemic, with the support of institutions such as Fiocruz, the Butantan Institute, public universities, and central laboratories (LACENs). The strengthening of diagnostic capacity has come to be seen not only as an emergency demand, but as a fundamental part of permanent health surveillance actions. This evolution was consolidated with the creation of integrated networks and the decentralization of testing, which increased the capillarity of molecular diagnosis in the national territory Castro, et al. 2021 [31].

Biotech innovation catalyzed by the pandemic

The COVID-19 pandemic has acted as an unprecedented catalyst for innovation in biotechnology, forcing the scientific community to accelerate processes of development, validation, and application of diagnostic technologies in record time. The urgent need to curb the spread of SARS-CoV-2 has driven massive investments in research and development, fostering public-private partnerships, and mobilizing scientific and technological resources on a global scale Nature Biotechnology 2021 [32]. This accelerated mobilization has resulted in significant innovations in areas such as molecular biology, nanotechnology, artificial intelligence applied to diagnostics, and point-of-care detection platforms WHO, 2020 [33].

In Brazil, this trend has also manifested itself in the performance of institutions such as Fiocruz, Butantan Institute, USP, and UFMG, which have adapted their structures to meet emergency demand, developing RT-PCR kits, serological tests, and prototypes based on new technologies, such as LAMP and CRISPR Paula, et al. 2022 [34]. The rapid Brazilian scientific response, even in the face of historical funding limitations, highlighted the potential of national research centers to generate innovative solutions in the face of health emergencies. In addition, the pandemic reinforced the importance of public policies that permanently sustain the national innovation capacity.

Accelerating research and development of new methods

The urgency imposed by the pandemic has also broken down regulatory and institutional barriers that, under normal conditions, would slow down the development of new diagnostic methods. Research processes that would normally take years have been condensed into weeks, with simplified protocols for conducting clinical trials, kit validation, and emergency use authorization by agencies such as the FDA, EMA, and ANVISA Tenny & Hoffman, 2021 [35]. This scenario has allowed rapid entry into the molecular and immunological testing market, as well as innovative technologies such as biosensor detection and the use of microfluidics, increasing the diversity and scalability of diagnostic tools.

The most striking example of this acceleration was the development and dissemination of CRISPR-Cas-based platforms for the detection of SARS-CoV-2. Methods such as SHERLOCK and DETECTR were adapted within a few weeks of viral genome release, demonstrating comparable sensitivity to RT-PCR and feasibility for field use Broughton, et al. 2020 [24]. These emerging technologies have not only met the demands of COVID-19, but have also laid the groundwork for diagnostics of future infectious diseases, contributing to global pandemic preparedness. Experience reinforces that flexible and collaborative regulatory environments are essential for innovation in public health.

Emergency adaptation and validation of diagnostic technologies

The global emergence has also forced the adaptation of pre-existing technologies for new uses, with a focus on large-scale clinical applicability. RT-PCR platforms, previously restricted to research and surveillance laboratories, have been automated, miniaturized, and integrated with computerized data management systems to speed up sample processing and result release Chaimayo, et al. 2020 [36]. Rapid antigen tests, initially limited in accuracy, have been reengineered to meet the minimum sensitivity standards required by the WHO, expanding their use in community screenings and low-complexity settings Cubas-Atienzar et al. 2021 [37].

In Brazil, the emergency validation process was also accelerated through programs such as Diagnose to Care, coordinated by the Ministry of Science, Technology, and Innovation (MCTI), which articulated networks of universities and startups to quickly test and approve national diagnostic solutions MCTI 2021 [38]. This articulation reduced dependence on imported products and stimulated the strengthening of the Brazilian industrial and technological base. The legacy of these emergency validations remains a reference for future rapid responses, reinforcing the importance of flexible protocols, without giving up scientific rigor.

CRISPR-Cas technology does not diagnose the virus

CRISPR-Cas technology, originally developed for gene editing, has come to be used as a promising diagnostic tool during the COVID-19 pandemic, with the potential to transform the detection of viral pathogens. The CRISPR-Cas12 and Cas13 systems have been adapted to act as biosensors, capable of identifying specific viral RNA sequences with high sensitivity, activating side reactions that result in detectable visual or fluorescent signals Chen, et al. 2018 [39]; Broughton, et al. 2020 [24]. This approach, employed in platforms such as SHERLOCK and DETECTR, has shown results comparable to those of RT-PCR, being implemented quickly in response to the global health emergency.

One of the biggest advantages of CRISPR-Cas technologies in diagnosis is their adaptability and speed of development. After the publication of the SARS-CoV-2 genome, researchers were able to develop functional prototypes of CRISPR-based tests in a few weeks, evidencing its potential for rapid response to new viral threats Kellner, et al. 2019 [40]. In addition, these platforms offer a highly specific approach, since they can be programmed to recognize unique genomic regions of each virus, reducing the risk of cross-reactivity with other respiratory pathogens Li, et al. 2021 [7].

Portability, speed and potential for low-complexity environments

An important differential of CRISPR-based diagnostic platforms is their applicability in environments with low laboratory infrastructure. The systems can be coupled with portable devices, such as benchtop fluorescent readers or even smartphones, enabling use in remote regions, with little access to specialized equipment Fozouni, et al. 2021 [26]. The simplicity of the protocol, which dispenses with complex amplification and thermocycling steps, reduces costs and facilitates implementation in low- and middle-income countries, being a viable alternative to RT-PCR in scenarios of logistical constraints.

In addition to portability, CRISPR-based tests offer agility in the release of results, with response times of less than one hour under ideal conditions Patchsung, et al. 2020 [41]. This characteristic is strategic for rapid screening in localized outbreaks, emergency care units and airports, where early detection of cases can prevent secondary outbreaks. In Brazil, pilot projects coordinated by federal universities and startups have already been evaluating the incorporation of these tools into the Unified Health System (SUS), reinforcing the role of national biotechnology in reducing health inequalities.

Future applications in arboviruses and other emerging viruses

The success of CRISPR-based tests during the COVID-19 pandemic paved the way for their application in the diagnosis of other emerging viruses, such as Dengue, Zika, Chikungunya, and Yellow Fever, which continue to pose important challenges to public health, especially in tropical regions Silva, et al. 2022 [42]. The possibility of quickly adapting RNA guides for new variants or different viruses makes CRISPR a strategic tool for real-time epidemiological surveillance. Multiplexed tests, capable of simultaneously detecting different arboviruses, are already being developed, with promising results in preclinical studies.

In addition to arboviruses, the CRISPR system can be directed to the monitoring of zoonotic viruses with pandemic potential, such as Nipah virus, Hendra virus, and highly pathogenic influenza viruses. The rapid response capacity and ease of adaptation make the technology an ally in the early detection of outbreaks in rural or forest areas, where these viruses often emerge Joung, et al. 2020 [43]. In this context, Brazil can benefit enormously from the use of these tools, considering its biodiversity and the constant risk of the emergence of new pathogens from human-animal contact.

Genetic sequencing and genomic surveillance

Genetic sequencing has played a central role in the epidemiological surveillance of emerging viruses, allowing not only the identification of pathogens, but also the tracking of their genomic evolution. During the COVID-19 pandemic, the use of next-generation sequencing (NGS) was critical to monitoring the genetic diversity of SARS-CoV-2, identifying relevant mutations, and classifying variants with epidemiological, clinical, or immunological impact Hadfield, et al. 2018 [44]; O’Toole, et al. 2021 [45]. This technological advance has enabled real-time surveillance of viral dynamics, with direct implications for the control of the pandemic.

In addition to its application in pandemics, NGS-based genomic surveillance has been used for tracking local viral outbreaks, arbovirus surveillance, monitoring antiviral resistance, and early detection of zoonotic pathogens with pandemic potential Grubaugh, et al. 2019 [46]. The integration of sequencing with clinical and epidemiological data strengthens evidence-based surveillance, enabling rapid and personalized responses. The ability to detect new variants before they become prevalent is one of the key contributions of genetic sequencing to global health.

Applications of NGS in viral variant tracking

NGS allows the complete sequencing of viral genomes with high depth and speed, being an essential tool for the detection of variants of concern (VOCs-Variants of Concern) and variants of interest (VOIs-Variants of Interest). During the COVID-19 pandemic, this method was used to identify and track the emergence of variants such as Alpha, Beta, Delta, and Omicron, which had mutations with an impact on transmissibility, immune escape, and vaccine efficacy Faria, et al. 2021 [47]. Continuous surveillance of these variants was decisive for adjustments in vaccine strategies and reinforced the importance of real-time sequencing.

In addition to geographic tracking of variants, NGS allows the study of transmission patterns and viral dispersion routes, helping to reconstruct community and interstate transmission chains Candido, et al. 2020 [18]. This information is strategic for containment and resource allocation actions, especially in countries with large territorial dimensions and regional inequalities, such as Brazil. The ability to analyze mutations in different regions of the viral genome also has direct implications for detecting strains associated with greater clinical severity or antiviral resistance.

Supporting public health decisions based on genetic data

The use of genomic data in the formulation of public policies represents an advance in evidence-based health management. The rapid identification of variants of greater transmissibility or pathogenicity allows health managers and authorities to adopt more effective measures, such as mobility restrictions, intensification of vaccination, or adaptation of clinical protocols Gonzalez-Reiche, et al. 2020. This type of decision, driven by genetic evidence, has become an essential practice during the pandemic, especially in response to the rapid spread of variants with global impact.

Sequencing also contributes to the development and updating of vaccines, by monitoring mutations that may compromise the immune neutralization induced by existing vaccine platforms. Laboratories and health agencies around the world use genomic data to decide on the need for vaccine boosters, changes in antigenic targets, and prioritization of vulnerable groups Harvey, et al. 2021. Genomic surveillance, therefore, goes beyond the diagnostic scope and is established as a strategic tool for planning and response in public health.

Brazilian experience with the fiocruz genomics network as a model

In Brazil, the creation of the Fiocruz Genomics Network in 2020 represented a milestone in the molecular surveillance of SARS-CoV-2. Composed of Fiocruz units in different states and institutional partners, the network was responsible for sequencing thousands of viral genomes throughout the national territory, contributing to the mapping of the circulation of variants and to the insertion of Brazil in the global scenario of genetic data sharing Fiocruz, 2022 [48]. Engagement on platforms such as GISAID has enabled the rapid dissemination of information and collaboration with international surveillance centers.

In addition to generating data, the Fiocruz Genomics Network worked to train professionals, standardize laboratory protocols, and strengthen sequencing infrastructure in public research and health institutions Santos, et al. 2022 [49]. This experience has demonstrated the importance of national collaborative networks, which allow for a rapid and coordinated response to health emergencies. The Brazilian model has served as a reference for other Latin American countries, highlighting the potential for integration between science, service, and public policy as a path to sovereignty in genomic surveillance.

Objective of the Article

The main objective of this article is to perform a critical review of the main molecular diagnostic techniques applied to the detection of emerging viruses, with emphasis on the methodologies that have been consolidated during the COVID-19 pandemic, such as RTPCR, next-generation genetic sequencing (NGS), and CRISPR-Casbased technologies. Given the epidemiological relevance of arboviruses and viral zoonoses, mastery of these technologies represents a strategic instrument for public health surveillance and response, especially in health emergency contexts Fari, a et al. 2021 [47]; Broughton, et al. 2020 [24].

It also seeks to contextualize the role of molecular diagnosis not only as a laboratory tool, but as a structuring axis of public policies to combat pandemics, highlighting its importance for early detection, monitoring of variants, and support for decision-making in public health. The COVID-19 pandemic has revealed the potential of these technologies to redefine the parameters of large-scale testing, genomic tracking, and real-time clinical response Wiersinga, et al. 2020 [21]; Hadfield, et al. 2018 [44].

In addition, the present study proposes an analysis of the main lessons learned from the pandemic, especially in relation to the structural gaps evidenced in the diagnostic capacity of low- and middle-income countries. The article discusses inequalities in access to molecular technologies, the challenges faced in testing logistics, and the importance of decentralizing laboratory services as a strategy for health equity Fayet-Mello, et al. 2021 [11]; Silva, et al. 2022 [14].

Based on these learnings, this work points out ways for technological innovation in molecular diagnosis, discussing the role of new detection platforms (such as isothermal and portable tests), integration with artificial intelligence, and the strengthening of collaborative networks for genomic surveillance. In this context, the emerging role of solutions such as CRISPR-based tests and mobile biosensors stands out, which have expanded the possibilities of detection in environments with limited infrastructure Fozouni, et al. 2021 [26]; Paula, et al. 2022 [34].

Another aspect addressed is the need for sustainability and technological sovereignty in national diagnostic systems. The pandemic has highlighted the critical dependence on imported inputs and equipment, reinforcing the urgency of policies that encourage local production, technical training, and continuous investment in research and innovation. The strengthening of networks such as the Fiocruz Genomics Network represents a promising model for articulation between science, service, and public management Fiocruz 2022 [48].

Finally, this article aims to contribute to the debate on the future of molecular diagnostics in a post-COVID-19 scenario, proposing an agenda based on innovation, equity, and intersectoral integration. The objective is to offer technical and strategic subsidies that can guide managers, researchers, and health professionals in the construction of a more resilient, accessible diagnostic system aligned with the emerging epidemiological needs of the twenty-first century.

Methodology

This study consists of a narrative literature review, whose objective was to gather, critically analyze, and synthesize the main advances, limitations, and perspectives of molecular diagnostic techniques applied to the detection of emerging viruses, especially in the context of the COVID-19 pandemic. The narrative format was chosen because it allows for a comprehensive and interpretative approach, integrating different levels of evidence and theoretical, technical and institutional perspectives. The review included national and international studies that address the use of methodologies such as RT-PCR, CRISPR, and genetic sequencing in diagnostic practice, public health, and biomedical research.

Bibliographic searches were carried out in the PubMed, Scopus, Web of Science and SciELO databases, selected for their relevance and scope in the biomedical area. The descriptors in English: “molecular diagnostics”, “RT-PCR”, “CRISPR”, “next-generation sequencing”, “COVID-19” and “emerging viruses” were used, in isolation and in combination with Boolean operators (AND/OR), in order to increase the sensitivity of the searches. Articles published between January 2014 and March 2024 were considered, with an emphasis on the period from 2020 to 2024, as they reflect the most recent advances catalyzed by the pandemic.

The selection of studies included open access and peer-reviewed publications, focusing on original articles, systematic reviews, technical consensuses, and institutional documents with a scientific basis. Case reports, editorials, and abstracts without full text available were excluded. After the initial screening based on titles and abstracts, the full texts were evaluated for their thematic relevance and methodological quality. The information extracted was organized into thematic axes, which structure the critical discussion presented throughout the article.

Lessons Learned from the COVID-19 Pandemic

Expanding global diagnostic capacity

The COVID-19 pandemic has highlighted, in an unprecedented way, the importance of molecular diagnostics as a central tool in the health response. One of the main legacies was the significant expansion of global diagnostic capacity, with significant investments in laboratory infrastructure, acquisition of molecular biology equipment, and training of professionals. Countries that traditionally faced difficulties in accessing advanced diagnostic technologies began to receive international support for the implementation of RT-PCR, LAMP, and genetic sequencing platforms, which raised the technical level of several health systems Peeling, et al. 2021 [50].

This advance was driven not only by government actions, but also by partnerships between universities, pharmaceutical industries, and multilateral organizations, such as the WHO and FIND (Foundation for Innovative New Diagnostics). In countries such as Brazil, there has been an unprecedented expansion of laboratory networks, such as LACENs, and the strengthening of institutions such as Fiocruz and the Butantan Institute, which have started to coordinate the national production of molecular inputs and tests Fiocruz 2022 [48]. This expansion has provided lasting structural gains for infectious disease surveillance.

Integration between diagnostics, tracking, and genomic surveillance

The pandemic has also demonstrated that an effective response to a health emergency depends on the integration of laboratory diagnostics, contact tracing, and genomic surveillance. Large-scale testing allowed early cases to be identified, while genetic sequencing enabled real-time monitoring of the evolution of SARS-CoV-2, including the emergence and spread of variants of concern Grubaugh, et al. 2019 [46]. The articulation between these components strengthened epidemiological surveillance systems and allowed evidence-based interventions.

This integration has materialized in several global and regional initiatives. In Brazil, the creation of the Fiocruz Genomics Network has made it possible to connect molecular diagnosis to genomic surveillance and health response, with data shared on platforms such as GISAID Santos, et al. 2022 [49]. The articulation between public laboratories, epidemiological surveillance, and researchers contributed to the development of a collaborative and replicable model, highlighting the importance of agile and interoperable information systems in the management of health crises.

Barriers faced: unequal access, logistics and technical training

Despite the advances, the pandemic has highlighted persistent structural barriers, especially in low- and middle-income countries. Among the main challenges were the unequal distribution of diagnostic inputs, the lack of professionals trained in molecular techniques, and logistical obstacles to transporting samples and delivering results in a timely manner Fayet-Mello, et al. 2021 [11]. This inequality limited the effectiveness of containment strategies, resulting in underreporting and worsening of morbidity and mortality indicators in more vulnerable areas.

In Brazil, the North and Northeast regions presented significant difficulties in the expansion of molecular testing, reflecting historical inequalities in the financing and distribution of laboratory resources. The centralization of testing in large urban centers increased response time and made it difficult to timely isolate cases Silva, et al. 2022 [14]. These obstacles reinforce the need for decentralization of diagnostic capacity, with continuous investment in regional laboratories, technical training, and autonomy in the production of reagents.

Accelerating innovation by emergency demand

The pressure for rapid responses during the pandemic triggered an unprecedented acceleration of diagnostic innovation, reducing the time to develop, validate, and authorize new tests. Emerging platforms, such as molecular detection by CRISPR-Cas and portable biosensors, have been tested at scale, with promising results in terms of sensitivity, cost, and applicability in low-complexity environments Fozouni, et al. 2021 [26]. This innovation out of necessity has generated technological solutions that are more accessible and adaptable to different epidemiological contexts.

Regulatory flexibility, with the use of emergency authorizations by agencies such as ANVISA, FDA, and EMA, was decisive for the rapid incorporation of diagnostic technologies into the health system. At the same time, the pandemic has highlighted the importance of balanced regulatory environments, which ensure speed without compromising the safety and accuracy of tests Tenny & Hoffman 2021 [35]. As a legacy, a more dynamic innovation ecosystem was consolidated, with greater articulation between research, industry and public policies.

New Frontiers and Future Perspectives

Democratization of access to molecular technologies

One of the main challenges to be overcome in the near future is the democratization of access to molecular technologies, so that they are not exclusive to centers of excellence or developed countries. The expansion of molecular testing during the COVID-19 pandemic has highlighted the technical feasibility of incorporating these tools into the public health system, provided that there is investment, political will, and national production of inputs Fayet- Mello, et al. 2021 [11]. Equity in access to molecular diagnostics is essential to ensure health justice, especially in emerging disease contexts that disproportionately affect vulnerable populations.

Programs such as “Diagnose to Care” in Brazil have demonstrated that it is possible to articulate universities, industries, and the public sector to foster accessible, sustainable, and technically robust solutions MCTI 2021 [38]. In addition to investments in equipment, democratization requires continuous technical training, decentralization of laboratory capacity and regulations that favor innovation without compromising quality. The construction of public policies aimed at expanding molecular diagnostics is, therefore, a strategic path to strengthen global health surveillance.

Point-of-care platforms and wearable devices

Point-of-care (POC) platforms and wearable devices represent one of the most promising frontiers for the decentralization of molecular diagnostics. Technologies such as RT-LAMP, electrochemical biosensors, and CRISPR-Cas systems integrated into smartphones have demonstrated high sensitivity and specificity, with results obtained in less than an hour and without the need for advanced laboratory infrastructure Fozouni et al. 2021 [26]; Nguyen, et al. 2022. These solutions are especially useful in remote regions, in border zones, or in emergency settings such as natural disasters or refugee camps.

The portability of the devices, associated with cost reduction and ease of operation, favors their implementation in primary health care and mass testing programs. In addition, POC molecular tests can act as initial screening tools, integrated into more complex diagnostic flows when needed. The continuous evolution of these devices depends on the miniaturization of components, the automation of steps, and the support for national production, ensuring technological autonomy and scalability Nguyen, et al. 2022.

Integration with artificial intelligence and big data in diagnostic interpretation

The integration between molecular diagnostics, artificial intelligence (AI), and big data represents a disruptive advance for precision medicine and epidemiological surveillance. With the exponential increase in genomic, clinical, and geospatial data generated by diagnostic platforms, it is essential to use machine learning algorithms to interpret patterns, predict outbreaks, and assist in clinical decision-making Topol, 2019 [45]. AI-based tools are already being used to automate digital PCR reading, identify escape mutations, and cross-reference genomic data with clinical outcomes.

In addition, AI can optimize the development of new primers, predict secondary structures of viral RNA, and contribute to the personalization of diagnostic and therapeutic strategies Libbrecht & Noble 2015. The practical application of these systems requires interoperability between databases, information security policies, and technical training of multidisciplinary teams. With the advancement of these technologies, molecular diagnosis will no longer be just an isolated step in health care and will act as a strategic component of intelligent and responsive systems.

Emerging applications beyond virology: oncology, microbiota, and rare diseases

Molecular diagnostic technologies have transcended virology and are increasingly being applied in other critical areas of medicine, such as oncology, the study of the microbiota, and the investigation of rare diseases. In the oncological field, the use of NGS panels allows the detection of somatic mutations and the personalization of treatment based on the molecular profile of the tumor, expanding the possibilities of precision oncogenomics Mardis 2017. Tests based on liquid biopsy, which identify fragments of circulating tumor DNA, are already a reality in many clinical centers.

In the study of the gut microbiota, sequencing platforms have been used to understand the role of microbial communities in immunity, metabolism, and the development of chronic diseases. In rare diseases, whole exome or genome sequencing has allowed early diagnoses and more effective genetic counseling, especially in children with complex syndromic conditions Bick, et al. 2020. These applications reinforce that the future of molecular diagnostics is not restricted to infectious diseases, but extends to all of precision medicine.

Final Thoughts

The COVID-19 pandemic marked a turning point in the history of global public health, consolidating molecular diagnostics as one of the main tools for responding to emerging infectious diseases. Technologies such as RT-PCR, NGS, and CRISPR-Cas have been widely incorporated into the laboratory routine, proving to be essential not only for individual diagnosis, but also for variant monitoring and public health decision-making Wiersinga, et al. 2020 [21]; Broughton, et al. 2020 [24]. This consolidation reflects the technical and political maturation of diagnostic practices and represents a historic opportunity to restructure epidemiological surveillance systems based on evidence.

Technological advances achieved in record time during the pandemic revealed the potential of health demand-driven innovation, especially in the development of portable platforms, pointof- care testing, and integration with artificial intelligence Fozouni, et al. 2021 [26]; Topol, 2019 [45]. These solutions, originally designed to combat COVID-19, have wide applicability in other areas of medicine, such as oncology, genetics, and clinical microbiology. Expanding such innovations beyond the pandemic context is essential to consolidate a more resilient and technologically autonomous health system.

However, the challenges revealed during the pandemic, such as inequality in access to diagnostic inputs, technological dependence on developed countries, and the shortage of specialized professionals, remain current and require strategic confrontation Fayet-Mello, et al. 2021 [11]; Silva, et al. 2022 [14]. Overcoming these barriers involves the decentralization of testing, strengthening the national production of reagents and equipment, and continuous training of the health workforce. The democratization of molecular diagnosis should be understood as a health right, and not as a technological privilege.

In addition, it is essential that the regulatory and institutional advances observed during the pandemic are maintained and improved. The creation of agile normative environments, without compromising the safety and efficacy of tests, can facilitate the incorporation of new technologies into the SUS and expand the reach of molecular surveillance Tenny & Hoffman 2021 [35]. Public policies focused on science, technology, and innovation, when articulated with the demands of the health system, have the potential to transform the response to future health emergencies.

In short, the post-COVID-19 [51-53] era presents a unique opportunity to reorganize health diagnosis based on molecular technology, integrating innovation, equity, and sustainability. The legacies of the pandemic must be used to build a continuous, adaptable, and integrated genomic surveillance model, with immediate response capacity and broad territorial coverage. Investing in this model is investing in health security, scientific sovereignty and the right to health for all.s.

Acknowledgement

None.

Conflict of Interest

None.