Following the rise in monkeypox (Mpox) cases, particularly in countries where the disease had not traditionally been observed, the World Health Organization (WHO) declared a Public Health Emergency of International Concern in June 2022 and again in August 2024. Accurate and timely diagnosis plays a critical role in controlling the infection. However, PCR-based methods—the gold standard for Mpox diagnosis—require complex laboratory infrastructure and trained personnel, making them less accessible in many settings. For this reason, the development of point-of-care diagnostic tools is of great importance.
The review, published in the October 2025 issue of Trends in Biotechnology, was authored by Dr. Defne Yiğci from Koç University School of Medicine; Prof. Dr. Önder Ergönül from the Koç University İşbank Center for Infectious Diseases and the Department of Infectious Diseases and Clinical Microbiology at Koç University School of Medicine; and Prof. Dr. Savaş Taşoğlu from the Department of Mechanical Engineering and Koç University Translational Medicine Research Center (KUTTAM). The article comprehensively examines point-of-care diagnostic platforms used in Mpox diagnosis, the technical approaches behind them, and the barriers limiting their widespread implementation, while also discussing future directions involving the integration of molecular diagnostics with AI- and deep learning-based tools.
The first human case of Mpox was reported in 1970 in the Democratic Republic of Congo. The virus had previously been identified in a macaque species transported from Africa to Denmark. Mpox virus can be found in primates, squirrels, rodents, and prairie dogs. Transmission to humans typically occurs through bites or scratches from infected animals. Human-to-human transmission may occur through close contact, respiratory droplets, contact with infectious lesions or bodily fluids. For many years, the disease remained largely confined to Central and West Africa. However, cases were reported in the United States in 2003. In 2022, a global outbreak occurred, with more than 60,000 cases reported in over 100 countries—most of which had not previously been considered endemic.
Mpox usually begins with nonspecific symptoms. In the early days, patients may experience fever, muscle aches, headache, and flu-like complaints. This is followed by often painful swelling of lymph nodes and the development of skin rashes. The rashes initially appear as small spots, then become raised, turn into fluid-filled blisters, and eventually develop into pus-filled lesions. Because this clinical picture can resemble measles, chickenpox, syphilis, and other poxvirus infections, it is difficult to establish a definitive diagnosis based solely on symptoms.
Currently, the most reliable diagnostic method is PCR testing. In this method, genetic material of the virus is detected in samples taken from skin lesions. PCR provides highly sensitive and accurate results. However, it requires specialized equipment, laboratory conditions, and trained personnel. Therefore, its use directly at the patient’s location is not always feasible.
The researchers also discuss immunological tests used in Mpox diagnosis. Serological tests such as ELISA have limited use in clinical practice. Because Mpox virus shares similarities with other viruses in the same family, false-positive results may occur. This is particularly relevant for individuals previously vaccinated against smallpox. In recent years, rapid antigen tests targeting specific viral proteins have been developed. Some of these tests can produce results within 10–15 minutes. However, they are generally less sensitive than PCR and have limited capacity to quantify viral load. More advanced biosensor systems are under development, but they are not yet widely implemented in field settings.
Among alternatives to PCR are LAMP and RPA techniques. These methods operate at a constant temperature and do not require complex thermal cycling equipment. In some studies, their sensitivity has been found to approach that of PCR. However, they may occasionally produce false-positive results and have limited multiplexing capabilities.
In recent years, CRISPR/Cas technology has also been applied in diagnostics. These systems can recognize viral genetic material with high specificity. When combined with LAMP or RPA, they can achieve very high sensitivity. They also have the potential to differentiate between different Mpox clades. Various designs aim to enable single-tube reactions and portable device integration. Nevertheless, cost, technical complexity, and the need for cold-chain storage of certain reagents remain important limitations.
Another key topic discussed in the review concerns the characteristics of an ideal point-of-care diagnostic test. According to WHO, such tests should be affordable, reliable, rapid, user-friendly, robust, and easily accessible. These criteria were later expanded under the “REASSURED” framework to include real-time connectivity and ease of specimen collection. Current technologies generally meet some—but not all—of these requirements simultaneously.
Promising future directions include electrochemical biosensors, paper-based tests, and microfluidic chip systems. Additive manufacturing through 3D printing and integration with smartphones may help reduce costs and improve scalability. In addition, artificial intelligence-based systems can analyze images of skin lesions for preliminary assessment. Although such tools are not intended to replace molecular testing, they may serve as useful screening tools, particularly in areas lacking specialist physicians.
In conclusion, while PCR remains the gold standard for Mpox diagnosis, there is a growing need for rapid and accessible diagnostic tools, especially in low-resource settings. Technologies such as LAMP, RPA, and CRISPR show strong potential to address this gap. However, challenges related to cost, large-scale production, field applicability, and integration into health systems must still be resolved. The future likely lies in integrated diagnostic platforms that combine molecular testing, biosensor technologies, and artificial intelligence-supported decision systems.