Monkeypox Diagnosis in Clinical Settings: A Comprehensive Review of Best Laboratory Practices.

An outbreak of monkeypox (Mpox) was reported in more than 40 countries in early 2022. Accurate diagnosis of Mpox can be challenging, but history, clinical findings, and laboratory diagnosis can establish the diagnosis. The pre-analytic phase of testing includes collecting, storing, and transporting specimens. It is advised to swab the lesion site with virus transport medium (VTM) containing Dacron or polyester flock swabs from two different sites. Blood, urine, and semen samples may also be used. Timely sampling is necessary to obtain a sufficient amount of virus or antibodies. The analytical phase of infectious disease control involves diagnostic tools to determine the presence of the virus. While polymerase chain reaction (PCR) is the gold standard for detecting Mpox, genome sequencing is for identifying new or modified viruses. As a complement to these methods, isothermal amplification methods have been designed. ELISA assays are also available for the determination of antibodies. Electron microscopy is another effective diagnostic method for tissue identification of the virus. Wastewater fingerprinting provides some of the most effective diagnostic methods for virus identification at the community level. The advantages and disadvantages of these methods are further discussed. Post-analytic phase requires proper interpretation of test results and the preparation of accurate patient reports that include relevant medical history, clinical guidelines, and recommendations for follow-up testing or treatment.

U.S. Preparedness and Response to Increasing Clade I Mpox Cases in the Democratic Republic of the Congo - United States, 2024.

Clade I monkeypox virus (MPXV), which can cause severe illness in more people than clade II MPXVs, is endemic in the Democratic Republic of the Congo (DRC), but the country has experienced an increase in suspected cases during 2023-2024. In light of the 2022 global outbreak of clade II mpox, the increase in suspected clade I cases in DRC raises concerns that the virus could spread to other countries and underscores the importance of coordinated, urgent global action to support DRC's efforts to contain the virus. To date, no cases of clade I mpox have been detected outside of countries in Central Africa where the virus is endemic. CDC and other partners are working to support DRC's response. In addition, CDC is enhancing U.S. preparedness by raising awareness, strengthening surveillance, expanding diagnostic testing capacity for clade I MPXV, ensuring appropriate specimen handling and waste management, emphasizing the importance of appropriate medical treatment, and communicating guidance on the recommended contact tracing, containment, behavior modification, and vaccination strategies.

Rapid and highly sensitive colorimetric LAMP assay and integrated device for visual detection of monkeypox virus.

BACKGROUND: The monkeypox virus (MPXV) is a linear double-stranded DNA virus with a large genome that causes tens of thousands of infections and hundreds of deaths in at least 40 countries and regions worldwide. Therefore, timely and accurate diagnostic testing could be an important measure to prevent the ongoing spread of MPXV and widespread epidemics. RESULTS: Here, we designed multiple sets of primers for the target region of MPXV for loop-mediated isothermal amplification (LAMP) detection and identified the optimal primer set. Then, the specificity in fluorescent LAMP detection was verified using the plasmids containing the target gene, pseudovirus and other DNA/RNA viruses. We also evaluated the sensitivity of the colorimetric LAMP detection system using the plasmid and pseudovirus samples, respectively. Besides, we used monkeypox pseudovirus to simulate real samples for detection. Subsequent to the establishment and introduction of a magnetic beads (MBs)-based nucleic acid extraction technique, an integrated device was developed, characterized by rapidity, high sensitivity, and remarkable specificity. This portable system demonstrated a visual detection limit of 137 copies/mL, achieving sample-to-answer detection within 1 h. SIGNIFICANCE: The device has the advantages of integration, simplicity, miniaturization, and visualization, which help promote the realization of accurate, rapid, portable, and low-cost testing. Meanwhile, this platform could facilitate efficient, cost-effective and easy-operable point-of-care testing (POCT) in diverse resource-limited settings in addition to the laboratory.

Genomic Analysis of Monkeypox Virus During the 2023 Epidemic in Korea.

We aimed to characterize the genomes of monkeypox virus isolates from the Far East, providing insights into viral transmission and evolution. Genomic analysis was conducted on 8 isolates obtained from patients with monkeypox virus disease in the Republic of Korea between May 2022 and early 2023. These isolates were classified into Clade IIb. Distinct lineages, including B.1.1, A.2.1, and B.1.3, were observed in 2022 and 2023 isolates, with only the B.1.3 lineage detected in six isolates of 2023. These genetic features were specific to Far East isolates (the Republic of Korea, Japan, and Taiwan), distinguishing them from the diverse lineages found in the Americas, Europe, Africa, and Oceania. In early 2023, the prevalence of the B.1.3 lineage of monkeypox virus identified in six patients with no overseas travel history is considered as an indicator of the potential initiation of local transmission in the Republic of Korea.

Nanopore adaptive sampling of a metagenomic sample derived from a human monkeypox case.

In 2022, a series of human monkeypox cases in multiple countries led to the largest and most widespread outbreak outside the known endemic areas. Setup of proper genomic surveillance is of utmost importance to control such outbreaks. To this end, we performed Nanopore (PromethION P24) and Illumina (NextSeq. 2000) Whole Genome Sequencing (WGS) of a monkeypox sample. Adaptive sampling was applied for in silico depletion of the human host genome, allowing for the enrichment of low abundance viral DNA without a priori knowledge of sample composition. Nanopore sequencing allowed for high viral genome coverage, tracking of sample composition during sequencing, strain determination, and preliminary assessment of mutational pattern. In addition to that, only Nanopore data allowed us to resolve the entire monkeypox virus genome, with respect to two structural variants belonging to the genes OPG015 and OPG208. These SVs in important host range genes seem stable throughout the outbreak and are frequently misassembled and/or misannotated due to the prevalence of short read sequencing or short read first assembly. Ideally, standalone standard Illumina sequencing should not be used for Monkeypox WGS and de novo assembly, since it will obfuscate the structure of the genome, which has an impact on the quality and completeness of the genomes deposited in public databases and thus possibly on the ability to evaluate the complete genetic reason for the host range change of monkeypox in the current pandemic.

Multi-center evaluation of the Research Use Only NeuMoDx monkeypox virus (MPXV) fully automated real-time PCR assay.

The mpox outbreak, caused by monkeypox virus (MPXV), accelerated the development of molecular diagnostics. In this study, we detail the evaluation of the Research Use Only (RUO) NeuMoDx MPXV assay by multiple European and US sites. The assay was designed and developed by Qiagen for the NeuMoDx Molecular Systems. Primers and probes were tested for specificity and inclusivity in silico. The analytical sensitivity of the assay was determined by testing dilutions of synthetic and genomic MPXV DNA. A total of 296 clinical samples were tested by three sites; the Johns Hopkins University (US), UZ Gent (Belgium, Europe), and Hospital Universitario San Cecilio (Spain, Europe). The analytical sensitivity of the assay was 50 copies/mL for both clades I and II. The assay showed 100% in silico identity for 80 clade I and 99.98% in silico identity for 5,162 clade II genomes. Clade II primers and probes showed 100% in silico specificity; however, identity of at least one of the two sets of clade I primers and probes with variola, cowpox, camelpox, and vaccinia viruses was noticed. The clinical validation showed sensitivity of 99.21% [95% confidence interval (CI): 95.66-99.98%] and specificity of 96.64% (95% CI: 91.62-99.08%) for lesion swab samples. The NeuMoDx MPXV Test shows acceptable analytical and clinical performance. The assay improves the laboratory's workflow as it consolidates nucleic acid extraction, PCR, data analysis, and interpretation and can be interfaced. The Test Strip can differentiate clades I and II, which has important laboratory safety implications. IMPORTANCE: In this manuscript, we provide detailed in silico analysis and clinical evaluation of the assay using a large cohort of clinical samples across three academic centers in Europe and the United States. Because the assay differentiates MPXV clades I and II, this manuscript is timely due to the current need to rule out the regulated clade I by diagnostic clinical laboratories. In December 2023, and due to first report of cases of sexually transmitted clade I infections in the Democratic Republic of the Congo, when generic assays that do not differentiate the clades are used, samples are considered regulated. The assay meets the need of full automation and has a marked positive impact on the laboratory workflow.

Rapid detection of monkeypox virus and differentiation of West African and Congo Basin strains using endonuclease restriction-mediated real-time PCR-based testing.

The ongoing multi-country outbreak of monkeypox virus (MPXV) has continuously attracted global attention, highlighting the critical need for timely and accurate methods to detect MPXV and differentiate its clades. Herein, we devised a novel multiplex ET-PCR (endonuclease restriction-mediated real-time PCR) assay that integrates PCR amplification, restriction endonuclease cleavage and real-time fluorescence detection to diagnose MPXV infection and distinguish the Congo Basin and West African MPXV strains. In the MPXV ET-PCR system, three sets of specific primers were designed for MPXV, Congo Basin and West African strains. A short sequence, which could be recognized by restriction endonuclease enzyme BstUI, was added to the 5'end of amplification primers. Then, the modified primers were assigned different reporter dyes and corresponding quenching dyes to each of the three targets, enabling real-time fluorescence reporting of the results and multiplex detection. The designed assay enabled the detection of single or three targets in a single tube, with excellent specificity and analytical sensitivity in terms of plasmid and pseudotyped virus. Moreover, the clinical feasibility of our assay was validated using artificially simulated plasma, nasopharyngeal swab and skin swab samples. In conclusion, the multiplex ET-PCR assay devised here had the advantages of simple primer design, cost-effectiveness, low contamination risk, excellent sensitivity, high specificity and multiplex detection, making it a valuable and dependable tool for curbing the extensive spread of MPXV.

Africa steps up battle against mpox outbreaks.

Research consortium announced as new variant spreads in Congolese mining region.

A rapid and sensitive one-pot platform integrating fluorogenic RNA aptamers and CRISPR-Cas13a for visual detection of monkeypox virus.

The recent global upsurge in Monkeypox virus (MPXV) outbreaks underscores the critical need for rapid and precise diagnostic solutions, particularly in resource-constrained settings. The gold standard diagnostic method, qRT-PCR, is hindered by its time-consuming nature, requirement for nucleic acid purification, expensive equipment, and the need for highly trained personnel. Traditional CRISPR/Cas fluorescence assays, relying on trans-cleavage of ssDNA/RNA reporters labeled with costly fluorophores and quenchers, pose challenges that limit their widespread application, especially for point-of-care testing (POCT). In this study, we utilized a cost-effective and stable fluorogenic RNA aptamer (Mango III), specifically binding and illuminating the fluorophore TO3-3 PEG-Biotin Fluorophore (TO3), as a reporter for Cas13a trans-cleavage activity. We propose a comprehensive strategy integrating RNA aptamer, recombinase-aided amplification (RAA), and CRISPR-Cas13a systems for the molecular detection of MPXV target. Leveraging the inherent collateral cleavage properties of the Cas13a system, we established high-sensitivity and specificity assays to distinguish MPXV from other Orthopoxviruses (OPVs). A streamlined one-pot protocol was developed to mitigate aerosol contamination risks. Our aptamer-coupled RAA-Cas13a one-pot detection method achieved a Limit of Detection (LoD) of 4 copies of target MPXV DNA in just 40 min. Validation using clinical MPX specimens confirmed the rapid and reliable application of our RAA-Cas13a-Apt assays without nucleic acid purification procedure, highlighting its potential as a point-of-care testing solution. These results underscore the user-friendliness and effectiveness of our one-pot RAA-Cas13a-Apt diagnostic platform, poised to revolutionize disease detection and management.

Mpox virus DNA detection in different clinical specimens: tertiary-level laboratory experience, Bosnia and Herzegovina, 2022.

Mpox virus (MPXV) infection is difficult to distinguish from other (non-)infectious diseases. The etiology of rash can be differentiated by real-time polymerase chain reaction (rtPCR) on different types of samples. The study aims to provide experience with emerging MPXV diagnostics in a tertiary-level laboratory in Bosnia and Herzegovina. From July-December 2022, a total of 18 mpox suspected persons were tested. MPXV infection was confirmed by rtPCR in 10/18 (55.56 %) persons. The number of cases reached a peak in October 2022. The lowest median Crossing point (Cp) (x̄ = 29.76) was obtained from a swab of skin lesions in a viral transport medium (VTM). Evaluating the Cp values for the 7/9 mpox cases from whom paired swab samples from different anatomic sites were collected, higher positivity of skin lesion swabs in VTM was observed. In conclusion, our data highlighted the confirmatory role of rtPCR in the diagnosis of MPXV in skin lesion samples.

Validation of a new extraction-free real-time PCR test to detect MPOX virus.

The monkeypox (Mpox) virus has raised significant concerns given its recent spread with an increasing number of confirmed cases worldwide. In this study, we evaluated the performance of a laboratory developed test (LDT) using BioGX Xfree hMPXV/OPXV reagents for the qualitative detection of non-variola Orthopoxviruses and Mpox virus DNA, in swabs from human pustular or vesicular rash specimens. Analytical and clinical testing analysis were carried out on two different platforms: the BD MAX™ System (BD Diagnostics) and the new pixl.16 Real-Time PCR Platform (BioGX), using a synthetic Mpox virus DNA (ATCC VR-3270SD) and residual clinical samples previously identified with an EUA approved Mpox real-time PCR assay. In the end, the Xfree hMPXV/OPXV LDT proved to be a sensitive, specific, and reproducible test for the detection of Mpox on both platforms evaluated with the pixl.16 having an advantage of a small footprint and providing faster TAT facilitated by an extraction-free workflow.

[A 19-year-old male migrant with urethritis and vesicular rash]. / Un migrant de 19 ans consulte pour une urétrite et une éruption vésiculeuse.

We report the case of a 19-year-old Malian patient, who presented with urethritis and a vesicular rash during the summer of 2022, following a probable heterosexual intercourse. The epidemic context among the male homosexual population and the clinical picture without genital lesions or lymphadenopathy allowed us to discuss both chickenpox and mpox, the latter being finally confirmed by the detection of Monkeypox virus DNA from vesicular fluid.

Deadliest mpox outbreak ever sparks a new worry.

Congo cases suggest viral strain there is spreading sexually, like less lethal one that triggered last year's global outbreak.

Development and validation of a nanoplate-based digital PCR assay for absolute MPXV quantification.

Quantification of mpox virus (MPXV) across different human body anatomical sites can provide insights about the most likely transmission routes, so methods able to release absolute and exact quantitative values of MPXV DNA are crucial. Here, we optimized a new QIAcuity digital PCR (dPCR) protocol for the detection and quantification of MPXV DNA in clinical samples and assessed the performance of the assay by comparing the results obtained in 144 biological samples with those resulting from the use of an in-house real-time PCR (qPCR). Overall, the concordance between the two assays was 95%, with samples identified concordantly as MPXV DNA positive and having a mean number of copies per µl of 1708 (95% CI: 107-2830 copies/µl). The remaining samples gave discordant results, with 5 out of 7 detected with the QIAcuity dPCR assay but not with the in-house qPCR. MPXV DNA levels measured by QIAcuity dPCR were strongly correlated with the Ct values detected by in-house qPCR and with those detected by another dPCR assay previously developed in our laboratories. The QIAcuity dPCR assay may be a robust and easy-to-perform method for MPXV DNA quantification in several biological samples.

Implementation and clinical evaluation of an Mpox virus laboratory-developed test on a fully automated random-access platform.

While Mpox virus (MPXV) diagnostics were performed in specialized laboratories only, the global emergence of Mpox cases in 2022 revealed the need for a more readily available diagnostic. Automated random-access platforms with fast nucleic acid extraction and PCR have become established in many laboratories, providing faster and more accessible testing. In this study, we adapted a previously published generic MPXV-PCR as a lab-developed test (LDT) on a NeuMoDx Molecular System and isolated MPXV clones from patient materials. To reduce the handling of infectious material, we evaluated a viral lysis buffer (VLB) for sample pretreatment. We further compared the MPXV-LDT-PCR to conventional real-time PCR, determined its sensitivity and specificity using positive swabs, and assessed its performance using external quality assessment samples. Pretreatment of samples with 50% VLB reduced MPXV infectivity by approximately 200-fold while maintaining PCR sensitivity. The assay demonstrated a sensitivity and specificity of 100% with no cross-reactivity in the samples tested and performed with a limit of detection of 262 GE/mL. In summary, the assay had a turnaround time of fewer than 2 h and can easily be transferred to other automated PCR platforms, providing a basis for developing rapid assays for upcoming pandemics.

Detection of mpox virus in ambient air in a sexual health clinic.

Although transmitted mainly through direct (sexual) contact, mpox virus (MPXV) can be detected in ambient air. We explored the use of air sampling for diagnosis or (genomic) surveillance of mpox in a sexual health clinic. For six out of six patients who were infected with MPXV, all four of our ambient air PCR tests were positive. For 14 uninfected patients, PCR was positive in three ambient air samples, albeit with higher cycle threshold (Ct) values. Genomic sequencing of samples from two positive patients showed matching sequences between air and clinical samples.

Rapid Detection of the Monkeypox Virus Genome and Antigen Proteins Based on Surface-Enhanced Raman Spectroscopy.

The conventional detection methods cannot satisfy the need for early and rapid detection of monkeypox virus (MPXV) infection. This is due to complicated pretreatment, time consumption, and complex operation of the diagnostic tests. Based on surface-enhanced Raman spectroscopy (SERS), this study attempted to capture the characteristic fingerprints of the MPXV genome and multiple antigenic proteins without the need to design specific probes. The minimum detection limit of this method is 100 copies/mL, with good reproducibility and signal-to-noise ratio. Therefore, the relationship between characteristic peak intensity and the protein and nucleic acid concentration can be used to construct a concentration-dependent spectral line with a good linear relationship. Additionally, principal component analysis (PCA) could identify the SERS spectra of four different MPXV proteins in serum. Therefore, this rapid detection method in the current outbreak of monkeypox control and the future response to possible new outbreaks has broad application prospects.

Isolation and characterization of emerging Mpox virus from India.

Mpox (previously known as Monkeypox) has recently re-emerged, primarily through human-to-human transmission in non-endemic countries including India. Virus isolation is still considered as the gold standard for diagnosis of viral infections. Here, the qPCR positive skin lesion sample from a patient was inoculated in Vero E6 cell monolayer. Characteristic cytopathic effect exhibiting typical cell rounding and detachment was observed at passage-02. The virus isolation was confirmed by qPCR. The replication kinetics of the isolate was determined that revealed maximum viral titre of log 6.3 PFU/mL at 72 h postinfection. Further, whole genome analysis through next generation sequencing revealed that the Mpox virus (MPXV) isolate is characterized by several unique SNPs and INDELs. Phylogenetically, it belonged to A.2 lineage of clade IIb, forming a close group with all other Indian MPXV along with few from USA, UK, Portugal, Thailand and Nigeria. This study reports the first successful isolation and phenotypic and genotypic characterization of MPXV from India.

Laboratory assessment of a multi-target assay for the rapid detection of viruses causing vesicular diseases.

BACKGROUND: The recent mpox outbreak has highlighted the need to rapidly diagnose the causative agents of viral vesicular disease to inform treatment and control measures. Common causes of vesicular disease include Monkeypox virus (MPXV), clades I and II, Herpes simplex viruses Type 1 and Type 2 (HSV-1, HSV-2), human herpes virus 6 (HHV-6), Varicella-zoster virus (VZV) and Enteroviruses (EVs). Here, we assessed a syndromic viral vesicular panel for rapid and simultaneous detection of these 7 targets in a single cartridge. OBJECTIVE: The aim of this study was to evaluate the QIAStat-Dx ® viral vesicular (VV) panel and compare with laboratory developed tests (LDTs). Limit of detection, inter-run variability, cross-reactivity and specificity were assessed. Positive and negative percent agreement, and correlation between assays was determined using 124 clinical samples from multiple anatomical sites. RESULTS: The overall concordance between the QIAstat and LDTs was 96%. Positive percent agreement was 82% for HHV-6, 89% for HSV-1 and 100% for MPXV, HSV-2, EV and VZV. Negative percent agreement was 100% for all targets assessed. There was no cross-reactivity with Vaccinia, Orf, Molluscum contagiosum viruses, and a pooled respiratory panel. CONCLUSION: The QIAstat VV multi-target syndromic panel combine ease of use, rapid turnaround, good sensitivity and specificity for enhanced diagnosis, clinical care and public health responses.