Molecular Diagnostics and Therapeutic Approaches in Zoonotic Infections

Molecular Diagnostics and Therapeutic Approaches in Zoonotic Infections

Dr. Densingh Johnrose., Mihir Sharma., Azaruddin Gohil., Shivam Singh

Department of Microbiology, SDIS

DOI: https://doi.org/10.51584/IJRIAS.2025.10060054

Received: 04 June 2025; Accepted: 14 June 2025; Published: 07 July 2025

ABSTRACT

Zoonotic infections—diseases naturally transmitted between animals and humans—represent over 60% of emerging infectious diseases globally. The increasing frequency of zoonotic outbreaks, driven by factors such as globalization, environmental change, and intensified human–animal interactions, underscores the urgent need for robust diagnostic and therapeutic strategies. Traditional diagnostic tools, while useful, often lack the sensitivity, specificity, and speed required to manage emerging zoonoses effectively. In response, molecular diagnostics have revolutionized the detection and characterization of zoonotic pathogens. Techniques such as real-time polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), next-generation sequencing (NGS), and CRISPR-based assays have enabled rapid, accurate, and high-throughput identification of a broad spectrum of zoonotic agents. These technologies not only facilitate early detection but also support genomic surveillance, antimicrobial resistance profiling, and outbreak tracking.

Parallel to diagnostic advances, therapeutic strategies have evolved significantly. Conventional approaches—including antimicrobial agents and supportive therapy—are now being complemented by innovative modalities such as monoclonal antibodies, host-directed therapies, and next-generation vaccine platforms, including mRNA and viral vector-based vaccines. These therapeutics have shown promise in controlling infections like COVID-19, rabies, and Ebola. However, the development and equitable distribution of such interventions remain a challenge, particularly in low-resource settings.

This review critically examines current and emerging molecular diagnostic tools and therapeutic strategies for key zoonotic infections. It highlights case studies of major zoonotic outbreaks, discusses the translational potential of novel technologies, and emphasizes the necessity of an integrated One Health approach that considers the interconnectedness of human, animal, and environmental health. Strengthening molecular surveillance and treatment capabilities worldwide will be essential to mitigate the global burden of zoonoses and to prevent future pandemics.

Keywords: polymerase chain reaction, mRNA, viral vector-based vaccines

INTRODUCTION

Zoonotic infections—diseases that are transmissible between animals and humans—represent a critical and growing challenge to global public health, accounting for more than 60% of all emerging infectious diseases worldwide. Historical and contemporary outbreaks such as the plague, avian influenza, Ebola virus disease, and the COVID-19 pandemic are striking examples of how zoonotic pathogens can rapidly cross species barriers and cause widespread morbidity, mortality, and socio-economic disruption. The increasing frequency of such events is closely linked to several converging global trends, including deforestation, urbanization, climate change, wildlife trade, agricultural intensification, and increased human encroachment into natural habitats.

Effective management of zoonotic diseases requires timely and accurate detection of the causative agents, alongside the deployment of appropriate therapeutic interventions. Traditional diagnostic methods such as culture techniques, microscopy, and serological assays, while foundational, often suffer from limitations related to sensitivity, specificity, and turnaround time. These challenges can lead to diagnostic delays, contributing to underreporting, inappropriate treatment, and unchecked transmission.

Recent advances in molecular diagnostics have significantly transformed the landscape of zoonotic disease detection. Techniques such as real-time polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), next-generation sequencing (NGS), and CRISPR-based diagnostic platforms offer unprecedented speed, sensitivity, and scalability. These technologies enable not only early and precise identification of pathogens but also real-time genomic surveillance, antimicrobial resistance profiling, and outbreak source tracking capabilities that are essential for effective disease control.

In parallel, the field of therapeutics has undergone rapid innovation. In addition to conventional antimicrobial and antiviral agents, emerging therapies include monoclonal antibodies, host-targeted treatments, and next-generation vaccines, such as mRNA and viral vector-based platforms. These innovations have demonstrated high efficacy in recent zoonotic outbreaks, including COVID-19 and Ebola virus disease, and continue to evolve in response to new threats.

This paper aims to provide a comprehensive review of the current and emerging molecular diagnostic tools and therapeutic strategies for zoonotic infections. By exploring both technological advancements and practical implementation challenges, the review underscores the importance of an integrated, multidisciplinary approach—aligned with the One Health framework—to effectively predict, detect, and respond to zoonotic threats.

Overview of Zoonotic Infections

Classification and Epidemiology

Zoonotic infections encompass a wide array of pathogens that cross the species barrier between animals and humans. These infections can be caused by bacteria, viruses, parasites, or fungi, each with unique transmission dynamics, reservoirs, and geographical distributions. The global burden of zoonotic diseases is particularly high in low- and middle-income countries, where close human–animal interaction, inadequate health infrastructure, and limited diagnostic capacity increase vulnerability to outbreaks.

Bacterial Zoonoses

Bacterial zoonoses are among the most commonly reported globally. Brucellosis, caused by Brucella spp., is typically transmitted through contact with infected livestock or consumption of unpasteurized dairy products. It presents with flu-like symptoms but can progress to chronic illness if untreated. Leptospirosis, caused by Leptospira interrogans, is another widespread bacterial zoonosis, transmitted through contact with water or soil contaminated by the urine of infected animals. Outbreaks are common in tropical climates, particularly after flooding.

Viral Zoonoses

Viral zoonoses pose significant public health threats due to their potential for human-to-human transmission and pandemic spread. Rabies, a nearly always fatal disease if not promptly treated, is transmitted via the bite of infected mammals, especially dogs in endemic regions. Hantavirus infections, transmitted via rodent excreta, cause severe respiratory illness. Notably, coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2 have demonstrated how zoonotic viruses can rapidly evolve and result in global pandemics.

Parasitic Zoonoses

Parasitic infections like toxoplasmosis (caused by Toxoplasma gondii) and leishmaniasis (caused by Leishmania spp.) continue to affect millions worldwide. Toxoplasmosis is typically acquired through ingestion of undercooked meat or contact with contaminated cat feces, while leishmaniasis is transmitted via sand-fly vectors and is endemic in parts of Africa, Asia, and South America.

Fungal Zoonoses

Fungal zoonoses are less common but can be severe, especially in immunocompromised individuals. Histoplasmosis, caused by Histoplasma capsulatum, is contracted through inhalation of fungal spores found in bat or bird droppings. The disease is endemic in specific regions such as the Ohio and Mississippi River valleys in the United States.

Table 1: Classification and Epidemiology of Major Zoonotic Infections

Transmission Pathways

Understanding the transmission pathways of zoonotic infections is crucial for designing preventive strategies and containment policies. These pathways include:

Direct Contact

Many zoonoses are transmitted through direct contact with the bodily fluids, tissues, or excretions of infected animals. This route is particularly relevant for individuals working in animal husbandry, veterinary medicine, or wildlife conservation. Rabies, brucellosis, and anthrax are classic examples of direct-contact transmission.

Vector-Borne Transmission

Some zoonotic diseases require an intermediate arthropod vector for transmission. For example, leishmaniasis is spread by the bite of infected sandflies, while plague is transmitted by fleas. Vector-borne transmission adds complexity to disease control, as it involves ecological and climatic factors influencing vector populations.

Foodborne and Waterborne Routes

Contaminated food and water are major routes for several zoonotic infections. Ingestion of undercooked or raw animal products, such as meat or unpasteurized milk, can transmit pathogens like Salmonella, Campylobacter, Toxoplasma gondii, and Brucella spp. Leptospirosis and cryptosporidiosis are commonly associated with contaminated water sources, especially during natural disasters or in areas with poor sanitation.

Molecular Diagnostic Tools

Accurate and timely diagnosis of zoonotic infections is pivotal for effective clinical management and public health intervention. Traditional diagnostic methods, including culture and serology, often lack the sensitivity and specificity required for early detection, particularly during the incubation period or in asymptomatic carriers. Molecular diagnostic technologies have transformed the landscape of infectious disease diagnosis by enabling the rapid and precise identification of pathogens at the genetic level. This section explores key molecular tools currently employed in the detection of zoonotic pathogens.

Molecular Diagnostic Tools

Accurate and timely diagnosis of zoonotic infections is pivotal for effective clinical management and public health intervention. Traditional diagnostic methods, including culture and serology, often lack the sensitivity and specificity required for early detection, particularly during the incubation period or in asymptomatic carriers. Molecular diagnostic technologies have transformed the landscape of infectious disease diagnosis by enabling the rapid and precise identification of pathogens at the genetic level. This section explores key molecular tools currently employed in the detection of zoonotic pathogen

Polymerase Chain Reaction (PCR) and Reverse Transcription PCR (RT-PCR)

PCR and RT-PCR are among the most widely adopted molecular diagnostic techniques for zoonotic diseases. PCR amplifies specific DNA sequences, making it highly effective for identifying bacterial and protozoal infections. RT-PCR, an adaptation that allows for RNA detection, is particularly useful for diagnosing viral infections. For instance, RT-PCR became the global gold standard for detecting SARS-CoV-2 during the COVID-19 pandemic due to its exceptional sensitivity, specificity, and quantitative capabilities.

These methods are advantageous for their ability to provide results within hours, their scalability in automated platforms, and their compatibility with a wide range of clinical samples. However, they often require laboratory infrastructure and trained personnel, limiting accessibility in low-resource settings.

Loop-mediated Isothermal Amplification (LAMP)

LAMP is a rapid, low-cost, and field-deployable molecular diagnostic technique that amplifies nucleic acids under isothermal conditions, eliminating the need for thermo cycling equipment. It offers comparable sensitivity to PCR and is well-suited for point-of-care applications in rural and remote areas.

LAMP has been successfully employed in detecting pathogens such as Leptospira, Brucella, and Trypanosoma, making it a valuable tool for surveillance of zoonoses in endemic regions. The visual readout (color change or fluorescence) enhances its usability in non-laboratory environments.

Next-Generation Sequencing (NGS)

NGS enables high-throughput sequencing of entire microbial genomes, facilitating pathogen discovery, strain differentiation, and real-time outbreak surveillance. It has played a crucial role in identifying novel zoonotic viruses such as SARS-CoV-2 and tracking their mutation patterns and transmission dynamics.

Table 2: Molecular Diagnostic Tools for Zoonotic Infections

Table 3: Therapeutic Approaches in Zoonotic Infections

NGS also supports metagenomic analysis, which is especially useful in detecting unknown or unexpected pathogens in complex biological samples. While its cost and technical requirements are barriers to widespread use, the development of portable sequencers and cloud-based bioinformatics platforms is rapidly increasing its accessibility.

CRISPR-Based Diagnostics

CRISPR technology, originally developed for gene editing, has been repurposed for ultrasensitive molecular diagnostics. Platforms such as SHERLOCK (Specific High-sensitivity Enzymatic Reporter unlocking) and DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) use CRISPR-associated enzymes (Cas12, Cas13) to detect specific nucleic acid sequences.

These assays can be performed with minimal equipment and provide rapid results, often within 30–60 minutes. CRISPR diagnostics have shown potential for point-of-care testing of zoonotic diseases including COVID-19, dengue, and Zika virus. Ongoing research aims to expand their use to bacterial and parasitic infections.

Serological and Biosensor-Based Assays

Serological assays detect host antibodies or antigens and are commonly used for surveillance and retrospective diagnosis. ELISA (enzyme-linked immunosorbent assay) is a staple in diagnosing infections such as brucellosis, leptospirosis, and toxoplasmosis. It is scalable, cost-effective, and useful for evaluating population-level exposure.

Lateral flow assays (LFAs) offer rapid, visually interpretable results at the point of care. They were widely deployed for COVID-19 antigen and antibody detection and continue to be developed for other zoonoses.

Electrochemical and optical biosensors represent an emerging frontier, combining sensitivity with portability. These devices can detect pathogen-specific biomarkers in blood, saliva, or urine, and are being integrated with smartphone-based technologies to enhance real-time diagnostics in resource-limited settings.

Therapeutic Approaches

The management of zoonotic infections requires a multipronged approach that includes pathogen-targeted treatments, supportive care, immunotherapy, and vaccination. As many zoonotic diseases have no specific cures, therapeutic strategies focus on reducing viral or microbial load, modulating the host immune response, and preventing disease progression. Recent technological advances and lessons from emerging outbreaks have catalyzed the development of novel therapeutics aimed at improving clinical outcomes and curbing transmission.

Antimicrobial and Antiviral Agents

Antimicrobial therapy remains the first-line treatment for bacterial and some protozoal zoonoses. Doxycycline, a broad-spectrum tetracycline antibiotic, is widely used in the treatment and prophylaxis of leptospirosis and rickettsial infections. Similarly, rifampin and streptomycin are used in the treatment of brucellosis, often in combination therapy to prevent relapse.

For viral zoonoses, several antiviral agents have been developed or repurposed. Remdesivir, a nucleotide analog, has shown efficacy against SARS-CoV-2 and was one of the first antivirals approved during the COVID-19 pandemic. Favipiravir, originally developed for influenza, has been used off-label for a range of RNA viruses, including Ebola and Lassa fever. Paxlovid, a combination of nirmatrelvir and ritonavir, has been authorized for early outpatient treatment of COVID-19 and is under evaluation for other zoonotic viruses.

Limitations of these treatments include potential resistance, side effects, and limited efficacy in advanced disease stages. Hence, early diagnosis and timely initiation of therapy are critical.

Monoclonal Antibodies and Immunotherapies

Monoclonal antibody (mAb) therapies represent a major advancement in the treatment of zoonotic viral infections. Rabies immunoglobulin, administered alongside rabies vaccine, provides immediate passive immunity to individuals exposed to the virus. In the context of COVID-19, multiple monoclonal antibody cocktails (e.g., casirivimab and imdevimab) have been developed to neutralize the virus by targeting the spike protein.

Beyond neutralization, mAbs are being designed for post-exposure prophylaxis, disease mitigation, and even as prophylactic agents in high-risk populations. Similar strategies are being investigated for Ebola, hantavirus, and Zika virus, with several candidates showing promise in clinical trials.

Vaccine Development

Vaccination remains the most cost-effective strategy for preventing zoonotic infections. Recent innovations have significantly accelerated vaccine development timelines and broadened the scope of target pathogens.

mRNA vaccines, such as those developed by Moderna and Pfizer-BioNTech, demonstrated the rapid development potential and high efficacy in combating SARS-CoV-2. These vaccines deliver genetic instructions for viral antigen expression, inducing strong humoral and cellular immune responses.

DNA vaccines and viral vector-based vaccines (e.g., Oxford-AstraZeneca’s adenovirus vector) are also being explored for various zoonotic pathogens, including Ebola, Lassa fever, and Crimean-Congo hemorrhagic fever.

Additionally, veterinary vaccines targeting zoonotic reservoirs (e.g., oral rabies vaccines for wildlife) are crucial in breaking transmission cycles and preventing spillover events.

Host-Directed Therapies and Immunomodulation

Host-directed therapies aim to modulate the host immune response rather than directly targeting the pathogen. These strategies are especially relevant in severe zoonotic infections characterized by dysregulated immune responses or cytokine storms.

Agents targeting host cell pathways—such as Janus kinase (JAK) inhibitors, interleukin-6 (IL-6) blockers like tocilizumab, and tumor necrosis factor (TNF) inhibitors—have been employed in severe viral infections, notably in COVID-19 and Ebola virus disease, to reduce systemic inflammation and prevent multiorgan failure.

Other host-targeted approaches include statins, metformin, and autophagy modulators, which have shown immunomodulatory effects and are under investigation for repurposing against various zoonotic pathogens. These therapies hold promise but require careful risk-benefit assessment due to their potential to impair protective immunity.

Case Studies and Global Impact

The global burden of zoonotic infections has come into sharp focus over the last two decades, particularly due to high-impact outbreaks that have tested the resilience of health systems worldwide. Case studies of recent zoonotic disease outbreaks, such as COVID-19 and Nipah virus, underscore the critical role of molecular diagnostics and therapeutic innovations in shaping public health responses and influencing long-term preparedness strategies.

Table 4: Case Studies of High-Impact Zoonotic Infections

COVID-19: Zoonosis and Molecular Response

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, emerged in late 2019 in Wuhan, China. Genomic analyses and epidemiological studies strongly suggest that the virus originated in animals—most likely bats—with a possible intermediate host facilitating spillover to humans. This outbreak represents one of the most significant zoonotic events in modern history.

Genomic Surveillance and Origin Tracking

Advanced molecular tools such as next-generation sequencing (NGS) played a pivotal role in the early characterization of the SARS-CoV-2 genome. Within weeks of the first reported cases, the complete viral genome was sequenced and shared publicly, enabling global research collaboration. Ongoing genomic surveillance has been essential in monitoring the emergence of variants of concern (e.g., Alpha, Delta, and Omicron), guiding public health measures and vaccine updates.

Rapid Vaccine Rollout and mRNA Technology

A major scientific milestone during the pandemic was the rapid development and deployment of mRNA-based vaccines, such as those by Pfizer-BioNTech and Moderna. These vaccines were developed, tested, and authorized for emergency use within a year of the virus’s discovery—unprecedented in the history of vaccinology. Their success not only curbed severe disease and mortality globally but also validated mRNA technology as a platform for rapid-response vaccine development against other zoonotic threats.

Moreover, PCR-based diagnostics became the cornerstone of testing strategies worldwide, allowing early detection, isolation, and contact tracing. The pandemic highlighted the necessity of global molecular diagnostic infrastructure, equitable vaccine distribution, and cross-sectoral collaboration under the One Health framework.

Nipah Virus: Emerging Threat

Nipah virus (NiV) is a highly pathogenic paramyxovirus classified as a priority pathogen by the World Health Organization due to its high case fatality rate (up to 75%), zoonotic origin, and potential for human-to-human transmission. First identified in 1998–99 in Malaysia, it has since caused sporadic outbreaks in Bangladesh, India, and Southeast Asia, often linked to bat-to-human transmission via contaminated date palm sap or contact with infected animals.

Molecular Diagnosis Using NGS and PCR

NiV outbreaks are often detected through real-time reverse transcription PCR (RT-PCR), which allows for early and specific identification of the virus from human or animal samples. In addition, NGS has been critical for whole-genome sequencing of NiV isolates, aiding in source tracking, mutation analysis, and understanding virus evolution.

Molecular diagnostics have also been adapted for field-deployable use, particularly in rural and outbreak-prone areas, using LAMP and portable PCR platforms. These tools improve outbreak response times and reduce reliance on centralized labs.

Therapeutic Gaps and Ongoing Research

Currently, no approved antivirals or vaccines exist for Nipah virus, and treatment is limited to supportive care. However, monoclonal antibody therapies, such as m102.4, have shown efficacy in non-human primate models and have progressed to human clinical trials. Additionally, efforts are underway to develop vector-based and mRNA vaccines against NiV, leveraging platforms used in the COVID-19 response.

Despite its relatively limited spread compared to SARS-CoV-2, Nipah virus remains a pandemic threat due to its zoonotic origin, human-to-human transmissibility, and lack of widely available medical countermeasures. It exemplifies the critical need for sustained investment in zoonotic surveillance, rapid diagnostics, and pre-emptive vaccine development.

Challenges and Future Directions

Despite significant advancements in the diagnosis and treatment of zoonotic infections, several critical challenges hinder the global ability to respond rapidly and equitably to emerging threats. Addressing these limitations is essential for strengthening preparedness and ensuring sustainable health security, particularly in vulnerable populations. This section outlines key barriers and proposes future directions to improve the management of zoonotic diseases.

Limited Access to Molecular Tools in Low-Resource Settings

One of the most pressing challenges is the unequal distribution of molecular diagnostic technologies. Low- and middle-income countries (LMICs)—where zoonotic spillovers are most likely to occur due to high levels of human-animal interaction—often lack the infrastructure, trained personnel, and funding needed to implement and maintain advanced molecular testing platforms such as RT-PCR, NGS, or CRISPR-based diagnostics.

This disparity leads to underdiagnosis, delayed outbreak response, and weaker surveillance systems, creating blind spots that may facilitate the unchecked spread of pathogens. Portable and field-deployable diagnostic tools such as LAMP assays, biosensor-based tests, and affordable point-of-care devices need to be scaled up and adapted for these settings. In parallel, capacity building through workforce training and local manufacturing is crucial.

Need for Global Pathogen Surveillance Networks

Effective surveillance is fundamental for the early detection and containment of zoonotic outbreaks. However, fragmented data systems, limited cross-border cooperation, and underreporting of cases hinder comprehensive surveillance.

A coordinated, global pathogen surveillance network—anchored in the One Health approach—is needed to integrate human, animal, and environmental health data. Initiatives such as the WHO Global Genomic Surveillance Strategy and FAO-OIE-WHO Tripartite Collaboration offer valuable frameworks, but require stronger funding, political commitment, and real-time data sharing mechanisms.

Furthermore, investment in community-based surveillance, including the use of digital tools for outbreak reporting and environmental monitoring (e.g., wildlife sampling, wastewater surveillance), can help detect spillover events before they escalate.

Integration of AI in Diagnostic Interpretation

Artificial intelligence (AI) and machine learning (ML) offer promising solutions to enhance diagnostic efficiency, accuracy, and scalability. AI algorithms can support image-based diagnostics, interpret large-scale genomic data, and predict outbreak trends using epidemiological and environmental inputs.

For instance, AI-driven platforms can assist in identifying zoonotic pathogens in sequencing data more rapidly than conventional methods, enabling real-time risk assessment during outbreaks. Integration of AI in diagnostic workflows can also reduce human error and streamline clinical decision-making, especially in high-throughput testing scenarios.

However, the deployment of AI requires standardized data inputs, robust digital infrastructure, and ethical considerations around data privacy and algorithmic bias.

Development of Broad-Spectrum Antivirals

The reliance on pathogen-specific therapeutics presents a major limitation during emerging zoonotic outbreaks, where there may be no existing treatment or vaccine. The development of broad-spectrum antivirals—agents capable of targeting multiple viruses within a family or across different viral families—could significantly enhance preparedness.

Current research focuses on identifying conserved viral proteins or host-cell pathways essential for viral replication. Promising candidates include protease inhibitors, polymerase inhibitors, and host-targeted agents with pan-viral activity. Broad-spectrum antivirals such as remdesivir, though initially developed for one virus, have shown cross-reactivity against others.

Efforts to develop drug libraries and platform-based antiviral screening systems are critical to accelerate discovery and approval pipelines. Collaborative global initiatives and public-private partnerships will be essential to translate these innovations into deployable therapies. To overcome these challenges, there must be a coordinated global effort that combines technological innovation with political will, equitable funding, and sustainable health systems development. Strengthening diagnostic infrastructure, enhancing surveillance capacity, and promoting research into novel therapeutics—particularly in high-risk regions—are imperative to reduce the global burden of zoonotic diseases and prevent future pandemics.

CONCLUSION

The emergence and re-emergence of zoonotic infections continue to pose significant threats to global health, economies, and ecosystems. The integration of molecular diagnostics and innovative therapeutic strategies has dramatically enhanced our capacity to detect, monitor, and manage these infections with greater speed, accuracy, and precision than ever before. Technologies such as PCR, LAMP, NGS, CRISPR-based diagnostics, and biosensor platforms have revolutionized pathogen identification and outbreak tracking, while novel therapies—including monoclonal antibodies, mRNA vaccines, and host-directed treatments—are reshaping the landscape of clinical management.

Despite these advancements, considerable challenges remain—particularly in ensuring equitable access to molecular tools, expanding real-time surveillance networks, and developing broad-spectrum countermeasures that can rapidly respond to emerging pathogens. The threat of future pandemics, as illustrated by COVID-19 and other high-consequence zoonoses like Nipah virus, underscores the urgent need for sustained global investment and innovation.

A One Health approach, which recognizes the interconnectedness of human, animal, and environmental health, must be central to future strategies. This requires multisectoral collaboration, data sharing, capacity building in low-resource settings, and robust research ecosystems capable of rapid response.

In conclusion, while molecular diagnostics and next-generation therapeutics have transformed our capabilities, their full potential can only be realized through integrated, inclusive, and forward-looking global health frameworks. Continued progress will depend not only on scientific advancement but also on political commitment and international solidarity to preempt and mitigate the impact of zoonotic diseases worldwide.

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