Cowpox virus is an orthopoxvirus, a genus that also includes variola virus, the agent of smallpox, vaccinia virus, and monkeypox virus, now designated mpox. Despite its name, cowpox virus circulates primarily in wild rodents, with domestic cats serving as the most common bridge to human infection. In Europe, reported human cases have been sporadic and typically linked to contact with infected domestic animals, particularly felines that hunt rodents. The virus produces a localized infection at the site of inoculation, characterized by a lesion that progresses through macular, papular, vesicular, and pustular stages before crusting over, accompanied in many cases by fever, malaise, and painful swelling of the lymph nodes draining the affected region. Although most infections in immunocompetent individuals are self-limiting, the lesions can be extensive, slow to heal, and disfiguring, and severe or even fatal outcomes have been described in immunosuppressed patients.

The clinical course in the two Czech patients followed the classic pattern of orthopoxvirus infection but initially defied a precise etiological diagnosis. Both presented with vesiculopustular lesions on the shoulder, firm red swelling at the affected site, and regional lymphadenopathy. Laboratory workup revealed only low levels of inflammatory markers, a finding consistent with a viral rather than bacterial process. Initial microbiological investigations, however, were inconclusive. The patients were empirically treated with acyclovir, targeting a possible herpesvirus infection, and doxycycline, covering atypical bacterial pathogens, before being switched to symptomatic topical treatment once those agents failed to alter the course of disease. Over the following weeks, the lesions evolved into deep ulcers, a hallmark of cowpox that distinguishes it from the more superficial lesions of herpes or varicella-zoster infection, and eventually healed with scarring over a period of six to eight weeks.

The severity of the illness was underlined by the fact that one of the two patients required surgical excision of necrotic lymph nodes. Lymph node involvement in cowpox can be pronounced, and in some cases the draining nodes become so inflamed and necrotic that they mimic bacterial lymphadenitis or even malignancy. The need for surgical intervention in a young, otherwise healthy adult illustrates that cowpox, while rarely life-threatening in immunocompetent hosts, is far from a trivial infection. The prolonged healing time, the residual scarring, and the requirement for an operative procedure all contribute to a disease burden that clinicians in non-endemic regions may not anticipate when evaluating patients with unexplained vesiculopustular eruptions.

The epidemiological investigation quickly centered on the couple’s new pets. Four weeks before symptom onset, consistent with the typical incubation period for cowpox virus, the pair had purchased pet rats. Shortly afterward, all but one of the animals developed respiratory symptoms and died. Respiratory disease in rodents infected with cowpox virus is a recognized phenomenon, and die-offs in colonies of pet rats have been implicated in human cases elsewhere in Europe. The temporal association between the rats’ illness and the patients’ lesions, together with the localized inoculation-type lesions compatible with direct contact with the animals, raised strong suspicion of rat-associated transmission, although the source material notes that this link remains suspected rather than definitively proven through sequencing of virus from the animals themselves.

The laboratory odyssey that followed is perhaps the most instructive element of the report. Initial quantitative polymerase chain reaction testing for Orthopoxvirus DNA, performed on patient samples at the State Veterinary Institute, returned negative results. It was only almost a year later, when preserved specimens were reanalyzed at the Czech National Institute of Public Health, that cowpox virus DNA was detected in nearly all of the samples. This delayed confirmation speaks to several technical and systemic challenges. Orthopoxvirus qPCR assays vary in sensitivity depending on the specimen type, the stage of lesion evolution, and the viral load present. Swabs taken from crusted or healing lesions, or samples handled and stored under suboptimal conditions, can yield false negatives. Moreover, access to specialized orthopoxvirus diagnostics is limited in many European countries, particularly in settings where such infections have never been documented and where testing algorithms are not geared toward them.

The authors argue that these cases expose a critical gap at the interface of human medicine, veterinary medicine, and public health surveillance. Cowpox virus has a wide host range and circulates silently in wild rodent populations across Europe and parts of Asia. Human cases cluster where the reservoir intersects with domestic animals or, as suspected here, with the exotic pet trade. Pet rats have emerged as a recurring source of human infection in several countries, a development that tracks the growing popularity of fancy rats as companion animals. When a clinician in a country with no prior documented cases encounters a patient with an atypical pox-like lesion, cowpox rarely appears on the differential diagnosis, and laboratory infrastructure to confirm it may simply not be in routine use. The result, as this report demonstrates, can be a diagnostic delay measured in months.

The report also carries broader implications in the current era of renewed orthopoxvirus concern. The global mpox outbreaks of recent years have prompted many countries to expand orthopoxvirus testing capacity, and the Czech experience suggests that this capacity should be maintained and made broadly accessible rather than reserved for outbreak response. Distinguishing cowpox from mpox, varicella, disseminated herpes zoster, and other vesiculopustular conditions requires specific molecular diagnostics, and the public health response differs depending on the agent involved. Accurate identification matters not only for individual patient management but also for tracing potential sources, warning other people who may have been exposed to the same batch of animals, and monitoring whether the virus is establishing itself in new reservoir populations.

For clinicians, the practical lessons are concrete. A patient presenting with a hemorrhagic or ulcerating lesion that progresses through vesicular and pustular stages, accompanied by regional lymphadenopathy and only mild systemic symptoms, should prompt a careful occupational and recreational history, including contact with cats, rodents, and exotic pets. Travel history, contact with animals that have died, and the presence of similar illness in household animals are all relevant clues. When orthopoxvirus infection is suspected, collecting material from fresh vesicular lesions and coordinating directly with the reference laboratory can substantially improve the chances of a positive molecular result. Serological methods and additional molecular targets may be needed when initial PCR testing is negative but clinical suspicion remains high.

The Czech cases ultimately tell a story of successful resolution despite systemic friction. Both patients recovered, their lesions healed with scarring, and retrospective testing secured a laboratory diagnosis that had eluded the initial workup. But the authors emphasize that the delayed diagnosis resulted from limited access to appropriate diagnostic testing, and they call for improved laboratory diagnostics and closer collaboration among clinicians, microbiologists, public health authorities, and veterinary professionals. As the exotic pet trade continues to bring wild-derived pathogens into close contact with humans, and as orthopoxviruses command renewed global attention, the experience in the Czech Republic stands as a reminder that rare infections become inevitable somewhere, and that preparedness depends on the unglamorous work of maintaining diagnostic capacity and communication across disciplines before the first case arrives.

Subject of Research: Zoonotic cowpox virus infection transmitted from pet rats to two human patients in the Czech Republic

Article Title: First human cowpox infections in the Czech Republic: suspected transmission from pet rats

Article References: Dzupova, O., Jirincova, H., Gkalpakiotis, S., Bohac, P., Pierzynova, A., & Bartos, H. (2026). First human cowpox infections in the Czech Republic: suspected transmission from pet rats. Virology Journal. https://doi.org/10.1186/s12985-026-03312-0

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03312-0

Keywords: cowpox virus, Orthopoxvirus, zoonotic infections, pet rats, Czech Republic, qPCR diagnostics, vesiculopustular lesions, lymphadenopathy, One Health, virology, case report, public health

The research team, led by Xuetong Zhu and Jiancheng Xu of the First Hospital of Jilin University in Changchun, China, together with colleagues from several Jilin institutions, assembled an unusually large immunological dataset. The analysis included 112 peripheral blood mononuclear cell samples, which allowed the investigators to probe T-cell responses, and 3,202 serum samples, which provided the statistical power needed to characterize antibody profiles across health and disease states. Using enzyme-linked immunosorbent assay to quantify antibody titers and enzyme-linked immunospot assay to measure cellular reactivity, the researchers built a two-pronged picture of what remains of vaccinia-induced immunity years after the last dose was administered.

The cellular arm of the study delivered a sobering message. Among individuals with historical smallpox vaccination, T-cell responses were limited, and — perhaps most strikingly — no significant differences were observed between healthy participants and those living with disease. In other words, the cellular memory that once formed a critical line of defense against variola virus and its relatives has thinned to a low, relatively flat baseline across the population studied. This matters because cell-mediated immunity is generally considered the more durable component of protection against orthopoxviruses, and its scarcity in previously vaccinated adults suggests that historical vaccination status alone cannot be assumed to confer meaningful cellular protection today.

The humoral picture was more nuanced and more clinically revealing. Vaccinated individuals with comorbidities had significantly lower immunoglobulin G titers against the vaccinia virus Tianan strain than their healthy counterparts, a difference that reached strong statistical significance with P values below 0.001. When the researchers broke the disease groups down into chronic disease subgroups, the variations persisted, indicating that the erosion of antibody memory is not a uniform process but one shaped by the specific burden of illness a person carries. Antibody titers are the standard surrogate for neutralizing capacity against orthopoxviruses, so lower titers in comorbid groups translate directly into a plausible reduction in protective buffering.

Regression analyses sharpened the risk stratification. Chronic disease was associated with lower odds of high antibody positivity, with an odds ratio of 0.67 and a 95 percent confidence interval of 0.44 to 0.98. Cancer carried an even stronger signal, with an odds ratio of 0.58 and a confidence interval of 0.37 to 0.90. These figures suggest that people with cancer who were vaccinated decades ago are roughly 40 percent less likely to maintain high-level antibody positivity than comparable healthy individuals. At the same time, the proportion of individuals with negative neutralizing antibody responses decreased with age — consistent with more of the oldest cohort having been vaccinated — but remained higher among disease groups than among healthy participants at every comparison point.

Beyond clinical diagnoses, the study uncovered associations between routine laboratory markers and antibody levels, offering a biochemical window into why some vaccinated individuals retain stronger humoral memory than others. High-density lipoprotein cholesterol, often called good cholesterol, showed a positive association with antibody levels, as did aspartate aminotransferase, an enzyme commonly monitored as a marker of liver and cellular turnover. Cholinesterase, by contrast, displayed an inverse association with antibody titers. While such correlations do not establish causation, they hint that metabolic and hepatic status may modulate the persistence of vaccine-induced humoral immunity, and they raise the possibility that routinely collected blood chemistry could help clinicians estimate residual orthopoxvirus protection without specialized immunological testing.

The second half of the study moved from measurement to simulation. The team constructed an exploratory susceptible-infected-recovered-dead model — a classic epidemiological framework that tracks individuals through states of susceptibility, infection, recovery, and death — parameterized with published epidemiological values rather than observed outbreak data. Within this framework, they simulated hypothetical transmission scenarios among immunocompromised populations to explore how reduced immune competence might alter the shape of an orthopoxvirus outbreak. The authors are careful to frame this modeling as exploratory and hypothetical, but the exercise provides a structured way to think about vulnerability that cannot ethically or practically be studied in real outbreaks.

The simulated scenarios produced an age-stratified pattern that public health planners will recognize as a double burden. In the model, immunocompromised individuals aged 15 to 19 years showed higher simulated infection proportions, suggesting that younger immunocompromised people — many of whom were never vaccinated and may have weakened immune systems due to disease or treatment — could contribute disproportionately to transmission in a hypothetical outbreak. At the other end of the age spectrum, individuals aged 70 and above showed higher simulated mortality proportions, reflecting the compounding of advanced age, waning or absent immunity, and reduced physiological reserve. Together, the two ends of the simulated curve sketch a scenario in which an orthopoxvirus introduction could simultaneously stress pediatric and adolescent transmission control and geriatric intensive care capacity.

The authors conclude that individuals with historical smallpox vaccination do show detectable residual immune responses, but that these responses vary meaningfully across comorbidity groups and correlate with measurable laboratory markers. They further suggest that immunocompromised individuals may warrant further evaluation for potential orthopoxvirus susceptibility — a recommendation that carries practical weight given that mpox clade outbreaks continue to evolve and that smallpox vaccines, including modern non-replicating formulations, are being redeployed in some settings. The study does not claim that previously vaccinated people are unprotected, nor does it establish that the modeled outbreak dynamics would occur in reality; its value lies in quantifying the heterogeneity of residual immunity and in providing a transparent, parameterized starting point for preparedness planning.

What emerges from the combined evidence is a portrait of a partially protected world. The generation vaccinated before routine smallpox immunization ended carries measurable but uneven antibody memory, eroded most in those with chronic disease and cancer, and supported by cellular responses too limited to distinguish the sick from the healthy. Meanwhile, younger immunocompromised people who never received the vaccine represent a structurally susceptible population in whom simulated outbreaks spread more readily. As the authors note, these findings may inform future orthopoxvirus preparedness and prevention strategies — from targeted serological screening of comorbid vaccinees to prioritized vaccination of immunocompromised cohorts — and they underscore a broader lesson of post-eradication immunology: population immunity is not a switch that stays on, but a gradient that shifts with age, health, and time.

Subject of Research: Residual smallpox vaccine immunity and orthopoxvirus outbreak modeling in immunocompromised populations

Article Title: Residual immune responses after historical smallpox vaccination and exploratory outbreak modeling in immunocompromised populations

Article References: Zhu, X., Wang, J., Wang, X., Tu, Z., Zhou, Q., Zhu, Y., & Xu, J. (2026). Residual immune responses after historical smallpox vaccination and exploratory outbreak modeling in immunocompromised populations. Journal of Translational Medicine. https://doi.org/10.1186/s12967-026-08907-3

Image Credits: AI Generated

DOI: 10.1186/s12967-026-08907-3

Keywords: smallpox vaccination, orthopoxvirus, mpox, neutralizing antibodies, immunocompromised, vaccinia virus, humoral immunity, cellular immunity, SIRD model, antibody titers, chronic disease, outbreak modeling

The study was structured as a descriptive cross-sectional investigation of every patient who met the World Health Organization’s suspected-case criteria for mpox and was reported through the Khyber Pakhtunkhwa Integrated Disease Surveillance and Response System, known as KP-IDSRS. Over the study period, 137 suspected cases were flagged by the surveillance network. Of these, 29 were laboratory-confirmed using real-time polymerase chain reaction, the standard molecular diagnostic for orthopoxvirus infection. Samples with sufficiently low cycle threshold values, at or below 23, indicating high viral loads, were selected for whole-genome sequencing on an Illumina iSeq platform, allowing the researchers to reconstruct near-complete viral genomes for phylogenetic analysis.

The demographic profile of the 29 confirmed cases illustrates that mpox in this setting is not confined to any single population group. The median age of patients was 31 years, with ages ranging from an infant of 0.6 years to an adult of 80 years. Twenty patients, or 68.9 percent, were male, eight, or 27.6 percent, were female, and one patient, 3.4 percent, was transgender. This breadth of age and gender stands in contrast to the epidemiology seen in many high-income countries during the 2022 global outbreak, where cases were heavily concentrated among men who have sex with men, and suggests that in Pakistan the routes of introduction and transmission are more varied, driven largely by travel and household contact rather than by a single transmission network.

Classifying each case by likely origin, the investigators found that 13 infections, or 44.8 percent, were imported, meaning the patients acquired the virus abroad and brought it back to Pakistan. Five cases, 17.2 percent, were secondary transmission cases, representing people infected through contact with an imported case. Six cases, 20.7 percent, were classified as locally acquired with no direct epidemiological link to a known imported infection, and a further five, 17.2 percent, were deemed probable local infections. Taken together, the locally acquired and probable local categories account for more than a third of confirmed cases, providing clear evidence that imported mpox viruses have seeded chains of transmission within the province rather than simply arriving and dying out.

Clinically, the illness observed was characteristic of mpox but generally mild. A vesiculopustular rash, the hallmark dermal manifestation of orthopoxvirus infection in which fluid-filled vesicles evolve into pus-filled pustules before crusting over, was present in all 29 confirmed cases, a 100 percent frequency. Fever was recorded in 82.8 percent of patients, musculoskeletal pain in 37.9 percent, genital or perianal lesions in 34.5 percent, and sore throat in 24.1 percent. The presence of genital and perianal lesions in roughly a third of cases is notable because such lesions can be mistaken for other sexually transmitted infections, delaying diagnosis and isolation. Most patients recovered completely, but the study also records two deaths among the confirmed cases, a somber reminder that even in a largely mild outbreak, mpox carries real mortality risk, particularly for vulnerable patients.

The genomic component of the study is where its significance is most pronounced. Whole-genome sequencing of four representative cases identified both Clade IIb viruses, specifically of lineage A.2.1, and Clade Ib viruses. Mpox virus, an enveloped double-stranded DNA virus of the genus Orthopoxvirus, was long divided into two major clades, with the former Congo Basin or Clade I lineage generally associated with more severe disease and the West African or Clade II lineage with milder illness. The nomenclature has since been refined: Clade IIb denotes the lineage responsible for the sustained human-to-human global spread that began in 2022, while Clade Ib is a recently emerged branch of Clade I that has driven outbreaks in the Democratic Republic of the Congo and neighboring countries and prompted the WHO to declare a second mpox Public Health Emergency of International Concern in 2024. Detecting Clade Ib in a non-endemic country is therefore a marker of how far this newer lineage has traveled.

Phylogenetic analysis of the Pakistani genomes revealed close genetic relationships with sequences reported from within Pakistan itself and from the United States, Nepal, and Thailand. This pattern of clustering across widely separated countries is consistent with the well-established role of international travel in moving mpox virus between continents. Pakistan’s particular vulnerability stems from extensive labor migration to Gulf countries, which creates a constant flow of travelers between South Asia and regions where mpox introductions occur. Each returning traveler represents a potential introduction event, and with limited decentralized diagnostic capacity, infections can go unrecognized until secondary cases appear, exactly the pattern the epidemiological classifications in this study document.

The laboratory work underpinning these findings reflects a coordinated public health infrastructure operating under real constraints. Specimens were processed at the Khyber Pakhtunkhwa Public Health Reference Laboratory at Khyber Medical University in Peshawar, with sequencing support from the National Institutes of Health in Islamabad. The workflow, from standardized surveillance forms through real-time PCR confirmation to genome assembly and phylogenetic comparison against international databases, demonstrates that a provincial reference laboratory in a resource-limited setting can generate genomic data of direct global relevance. The authors acknowledge the WHO and the Khyber Pakhtunkhwa Health Department for supplying diagnostic kits and consumables, and note that the study received no formal external funding, underscoring how much of this surveillance was sustained through institutional commitment rather than dedicated program budgets.

The public health implications of co-circulating clades are substantial. When two genetically distinct lineages circulate simultaneously, each introduction event must be traced independently, and genomic surveillance becomes essential for distinguishing imported cases from established local transmission chains. A locally acquired Clade Ib case, in particular, signals that the newer lineage has achieved at least limited autonomous transmission in the province. The authors argue that this evidence makes a case for strengthened genomic surveillance, decentralized diagnostics that can confirm cases outside a single reference laboratory, and improved access to vaccines and therapeutics. Vaccines such as the Modified Vaccinia Ankara Bavarian Nordic product, which is used for mpox prevention in many countries, remain limited in availability across much of South Asia, and expanding access is a policy challenge that extends well beyond laboratory walls.

For the broader scientific community, the study adds an important data point to the global map of the ongoing mpox emergency. It shows that Clade Ib has reached South Asia and established detectable local transmission there, that Clade IIb lineage A.2.1 continues to circulate in the same region, and that a surveillance system built on WHO case criteria, rapid response teams, and provincial laboratory capacity can detect and characterize this diversity. As international travel continues to connect endemic and non-endemic regions, the Pakistani experience illustrates a pattern likely to recur elsewhere: imported cases are inevitable, but whether they fade out or ignite sustained transmission depends on how quickly diagnostics, contact tracing, vaccination, and genomic sequencing can be brought to bear. The findings from Khyber Pakhtunkhwa suggest that in much of the world, that capacity still needs deliberate investment before the next introduction arrives.

Subject of Research: Emergence and co-circulation of Clade Ib and Clade IIb mpox virus in a non-endemic region of Pakistan

Article Title: Emergence and co-circulation of Clade Ib and Clade IIb Mpox viruses in Khyber Pakhtunkhwa Pakistan, 2024–2026: clinical, genomic, and public health insights from a non-endemic region

Article References: Zia, A., Islam, A., Jamal, Z., Khan, M., Khattak, A. A., Manzoor, M., Umair, M., Muhammad, A., Mehmood Noor, S., Rehman, K., Zohaib, A., Khan, M. A., & Yousafzai, Y. M. (2026). Emergence and co-circulation of Clade Ib and Clade IIb Mpox viruses in Khyber Pakhtunkhwa Pakistan, 2024–2026: clinical, genomic, and public health insights from a non-endemic region. BMC Infectious Diseases. https://doi.org/10.1186/s12879-026-14542-6

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14542-6

Keywords: mpox, Clade Ib, Clade IIb, orthopoxvirus, whole-genome sequencing, genomic surveillance, Pakistan, Khyber Pakhtunkhwa, travel-associated infections, public health, epidemiology, emerging infectious diseases

The reference method for mpox diagnosis remains real-time quantitative polymerase chain reaction, or qPCR, which offers high sensitivity and specificity. However, qPCR depends on expensive thermocycling instruments, trained personnel, and biosafety-compliant laboratory facilities, all of which limit its accessibility in decentralized laboratories, outbreak settings, and resource-limited regions. Alternative molecular approaches such as restriction fragment length polymorphism analysis and next-generation sequencing are either labor-intensive, costly, or unsuitable for rapid frontline diagnostics. The World Health Organization has repeatedly emphasized the expansion of laboratory diagnostic capacity and the deployment of rapid, reliable, and accessible assays as a core component of its global mpox preparedness and response strategy, creating an urgent need for tools that can operate outside centralized reference laboratories.

The new assay, described in a study published in New Microbes and New Infections, is built on loop-mediated isothermal amplification, commonly known as LAMP. This nucleic acid amplification technique operates under constant-temperature conditions and enables rapid DNA amplification using a set of primers that recognize multiple regions of the target genome. LAMP-based assays require substantially less equipment than qPCR, offer shorter turnaround times, and allow visual interpretation of results. In the colorimetric format used by the Indian team, amplification is read out through a pH-sensitive dye: the reaction mixture starts pink under alkaline conditions and turns yellow when amplification succeeds, a transition that can be judged by eye without any specialized instrumentation.

To design the assay, the researchers retrieved complete and partial genomic sequences of monkeypox virus and other representative orthopoxviruses, including Vaccinia, Variola, Buffalopox, and Camelpox viruses, from the NCBI Virus database. Multiple sequence alignments were analyzed to identify conserved genomic regions suitable for isothermal amplification. Candidate genes evaluated included H2R, encoding a putative viral transmembrane protein; B6R, encoding an envelope protein; F3L, encoding a double-stranded RNA-binding protein; and F8L, encoding DNA polymerase. After preliminary analytical sensitivity testing and temperature optimization, primer sets targeting the orthopoxvirus genus-specific B6R gene and the monkeypox virus species-specific F3L gene were selected for full assay development. Primers were designed with PrimerExplorer V5, optimized for GC contents of approximately 50 to 60 percent, compatible melting temperatures, and minimal secondary structure or primer-dimer formation.

A defining feature of the assay is its dual-target architecture, which incorporates an internal control targeting the human beta-actin gene to verify sample integrity and amplification suitability. Under the predefined interpretation criteria, a specimen is classified as mpox-positive only if both the genus-specific B6R and species-specific F3L reactions yield positive colorimetric results. Specimens that are B6R-positive but F3L-negative are interpreted as non-mpox orthopoxvirus-positive, while specimens negative for both targets are classified as mpox-negative. This design provides two important advantages: reliable identification of monkeypox virus infections and the ability to differentiate mpox from other circulating orthopoxviruses, a capability that is particularly valuable in regions where multiple orthopoxviruses may co-circulate.

Clinical evaluation was performed using a well-characterized panel of 130 specimens obtained retrospectively from suspected mpox cases received for routine diagnostic testing at the Maximum Containment Facility of ICMR-NIV as part of national surveillance activities. The panel comprised 50 mpox-positive and 80 mpox-negative specimens as determined by qPCR, spanning five specimen categories: nasopharyngeal and throat swabs, vesicle swabs, lesion swabs, crust specimens, and lesion fluid specimens. All specimens had previously been tested by real-time qPCR targeting the B6R and F3L genes, which served as the reference standard. Sample processing and nucleic acid extraction were carried out in biosafety level-3 and level-4 containment laboratories in accordance with national biosafety guidelines, and the study received approval from the Institutional Ethics Committee of ICMR-NIV.

The results were striking. The LAMP assay correctly identified all 50 qPCR-positive specimens as positive and all 80 qPCR-negative specimens as negative, achieving complete concordance with the reference method. Sensitivity, specificity, positive predictive value, negative predictive value, and overall percent agreement were each 100 percent, with a Cohen’s kappa of 1.00 indicating perfect agreement beyond chance. Likelihood ratio analysis yielded a positive likelihood ratio of 160.4 and a negative likelihood ratio of 0.0099, corresponding to a diagnostic odds ratio of approximately 16,261. No discordant results were observed in any specimen category, indicating consistent performance across swabs, crusts, and lesion fluids. Per-target analysis confirmed that both the F3L and B6R assays individually achieved 100 percent sensitivity and 100 percent specificity, underscoring the robustness of the primer design and the dual-target interpretation algorithm.

The assay also demonstrated consistent detection across a broad range of viral loads. Among the 50 qPCR-positive specimens, cycle threshold values ranged from 18.55 to 37.21, with a median of 26.11. The LAMP assay detected every qPCR-positive specimen across this entire range, including the highest Ct specimen evaluated, and maintained 100 percent sensitivity within each Ct category examined, spanning values below 20, between 20 and 25, between 25 and 30, and at or above 30. The authors caution that these Ct-stratified findings should not be interpreted as a formal analytical limit of detection, because quantified monkeypox virus reference material was not available for standardized serial dilution testing. A formal limit of detection assessment will require quantified reference material, replicate testing, and predefined confidence thresholds in future validation studies.

Analytical specificity testing provided further evidence of the assay’s discriminatory power. A dedicated panel of 64 specimens included samples positive for Vaccinia virus, Buffalopox virus, Herpes simplex virus, and Varicella zoster virus, each represented by 16 specimens. Herpes simplex virus and Varicella zoster virus were included because they are clinically relevant causes of vesicular or pustular lesions that overlap with mpox in the differential diagnosis. As expected, the non-orthopox DNA viruses produced no amplification in either target, while Vaccinia and Buffalopox viruses yielded the characteristic B6R-positive, F3L-negative pattern, confirming that the assay can reliably distinguish monkeypox virus from genetically related orthopoxviruses while showing no cross-reactivity with unrelated viral pathogens.

The study’s authors note that several LAMP-based assays for mpox detection have been reported previously, but those efforts focused primarily on analytical sensitivity using synthetic templates or limited clinical evaluation sets. In contrast, the present work emphasizes clinical validation using a structured evaluation panel and comprehensive diagnostic accuracy metrics, including likelihood ratios and agreement statistics, in line with current recommendations for reporting diagnostic accuracy studies. The incorporation of uracil-DNA glycosylase in the commercial master mix further guards against carryover contamination, improving reliability in routine diagnostic settings.

Nevertheless, the researchers acknowledge several limitations. The clinical evaluation was retrospective, used archived specimens from a single reference laboratory, and included a limited sample size of 50 positive and 80 negative specimens, so the performance estimates should not be overgeneralized. A formal analytical limit of detection was not determined, the specificity panel did not include all infectious and non-infectious causes of vesiculopustular lesions, and formal reproducibility testing across multiple days, operators, reagent lots, and low-positive specimens was not performed because of limited specimen volume. Future studies incorporating quantified standards, broader specificity panels, multi-site validation, reproducibility assessment, and prospective field evaluations will be needed before wider clinical implementation.

From a public health perspective, the simplicity of the colorimetric LAMP assay, combined with its minimal equipment requirements, rapid turnaround time, and visual readout, makes it a promising tool for expanding mpox diagnostic capacity beyond centralized laboratories. The diagnostic method is covered by an Indian patent application filed in October 2022 and published in April 2024, held by the Indian Council of Medical Research. While qPCR will continue to play a central role in reference and confirmatory testing, the new assay is particularly relevant for outbreak response, surveillance activities, and diagnostic decentralization in resource-limited settings, aligning with recent WHO mpox preparedness and response frameworks. As mpox transmission continues across multiple continents, accessible tools of this kind may prove decisive in closing the diagnostic gap that has long hampered containment efforts.

Subject of Research: Development and clinical validation of a colorimetric loop-mediated isothermal amplification assay for rapid detection of monkeypox virus

Article Title: Development and clinical validation of a colorimetric isothermal assay for rapid detection of monkeypox virus

Article References: Nandi, S. S., Sawant, S. A., Yadav, P., Shete-Aich, A., Majumdar, T. D., Bavkar, K. C., Mukherjee, A., & Deshpande, J. M. (2026). Development and clinical validation of a colorimetric isothermal assay for rapid detection of monkeypox virus. New Microbes and New Infections, 73, Article 101848. https://doi.org/10.1016/j.nmni.2026.101848

Image Credits: AI Generated

DOI: 10.1016/j.nmni.2026.101848

Keywords: mpox, monkeypox virus, LAMP assay, colorimetric detection, isothermal amplification, qPCR, diagnostic validation, orthopoxvirus, B6R gene, F3L gene, ICMR-NIV, point-of-care testing