Laboratory Diagnosis of Mpox, Central African Republic, 2016-2022.

During 2016-2022, PCR testing confirmed 100 mpox cases among 302 suspected cases in the Central African Republic. The highest detection rates were from active lesions (40%) and scabs (36%); cycle thresholds were lower (≈18) than those for blood samples (≈33). Results were consistent for generic primer- and clade I primer-specific PCR tests

Rapid Detection of the Varicella-Zoster Virus Using a Recombinase-Aided Amplification-Lateral Flow System

Varicella-zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (shingles). VZV infections are ubiquitous and highly contagious, and diagnosis is mostly based on the assessment of signs and symptoms. However, monkeypox, an emerging infectious disease caused by the monkeypox virus (MPXV), has clinical manifestations that are similar to those of VZV infections. With the recent monkeypox outbreak in non-endemic regions, VZV infections are likely to be misdiagnosed in the absence of laboratory testing. Considering the lack of accessible diagnostic tests that discriminate VZV from MPXV or other poxviruses, a handy and affordable detection system for VZV is crucial for rapid differential diagnosis. Here, we developed a new detection method for VZV using recombinase-aided amplification technology, combined with the lateral flow system (RAA-LF). Given the prevalence of VZV worldwide, this method can be applied not only to distinguish VZV from other viruses causing rash, but also to foster early detection, contributing substantially to disease control

Development and Characterization of Recombinase-Based Isothermal Amplification Assays (RPA/RAA) for the Rapid Detection of Monkeypox Virus

International audienceMonkeypox is a zoonotic disease caused by monkeypox virus (MPXV), in which outbreaks mainly occurred in West and Central Africa, with only sporadic spillovers to countries outside Africa due to international travel or close contact with wildlife. During May 2022, multiple countries in Europe, North and South America, Australia, Asia, and Africa reported near-simultaneous outbreaks of MPXV, the first time that patient clusters were reported over such a large geographical area. Cases have no known epidemiological links to MPXV-endemic countries in West or Central Africa. Real-time PCR is currently the gold standard for MPXV diagnostics, but it requires trained laboratory personnel and specialized equipment, and results can only be obtained after several hours. A rapid and simple-to-operate point-of-care diagnostic test for MPXV is crucial for limiting its spread and controlling outbreaks. Here, three recombinase-based isothermal amplification assays (RPA/RAA) for the rapid detection of MPXV isolates were developed. These three assays target the MPXV G2R gene, and the limit of detection for these systems is approximately 10 0 copies of DNA per reaction. The assays were found to be specific and non-cross reactive against other pox viruses, such as vaccinia virus, and the results can be visualized within 20-30 min. The assays were validated with DNA extracted from 19 clinical samples from suspected or confirmed MPXV patients from Central Africa, and found to be consistent with findings from traditional qPCR. These results provide a solid platform for the early diagnosis of potential MPXV cases, and will help with the control and prevention of current and future outbreaks

Development of a Novel Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Monkeypox Virus Infections

A recent outbreak of monkeypox virus (mpox) has prompted researchers to explore diagnostics as a means of impeding transmission and further spread. Rapid, sensitive, and specific methods are crucial for accurately diagnosing mpox infections. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the specific detection of mpox. The primer sets were designed to target regions in and around the N4R gene, and results showed a detection limit of 2 × 100 DNA copies, which is comparable to the gold-standard qPCR method currently used to detect mpox. Particularly, the assay provides results visible to the naked eye within 30 min. This test specifically detects mpox DNA with no cross-reactivity to related DNA viruses including Varicella Zoster Virus (VZV), Hepatitis B virus (HBV), Vaccinia virus (VACV), Herpes simplex virus-1 (HSV-1), Herpes simplex virus-2 (HSV-2), Human papillomavirus-16 (HPV-16) and Human papillomavirus-18 (HPV-18). Furthermore, the LAMP assay has been evaluated using clinical samples from laboratory-confirmed mpox patients and found to be consistent with the qPCR results. Our results show that this single-tube LAMP method can contribute to diagnosis of suspected mpox infections in the field and clinic, especially in regions with limited laboratory resources

Intrafamily Transmission of Monkeypox Virus, Central African Republic, 2018

International audienceMonkeypox is a rare viral zoonotic disease; primary infections are reported from remote forest areas of Central and West Africa. We report an investigation of a monkeypox outbreak in Lobaye, southwest Central African Republic, in October 2018

Laboratory Diagnosis of Mpox, Central African Republic, 2016–2022

During 2016–2022, PCR testing confirmed 100 mpox cases among 302 suspected cases in the Central African Republic. The highest detection rates were from active lesions (40%) and scabs (36%); cycle thresholds were lower (≈18) than those for blood samples (≈33). Results were consistent for generic primer– and clade I primer–specific PCR tests

Nanopore sequencing of a monkeypox virus strain isolated from a pustular lesion in the Central African Republic

International audienceMonkeypox is an emerging and neglected zoonotic disease whose number of reported cases has been gradually increasing in Central Africa since 1980. This disease is caused by the monkeypox virus (MPXV), which belongs to the genus Orthopoxvirus in the family Poxviridae . Obtaining molecular data is particularly useful for establishing the relationships between the viral strains involved in outbreaks in countries affected by this disease. In this study, we evaluated the use of the MinION real-time sequencer as well as different polishing tools on MinION-sequenced genome for sequencing the MPXV genome originating from a pustular lesion in the context of an epidemic in a remote area of the Central African Republic. The reads corresponding to the MPXV genome were identified using two taxonomic classifiers, Kraken2 and Kaiju. Assembly of these reads led to a complete sequence of 196,956 bases, which is 6322 bases longer than the sequence previously obtained with Illumina sequencing from the same sample. The comparison of the two sequences showed mainly indels at the homopolymeric regions. However, the combined use of Canu with specific polishing tools such as Medaka and Homopolish was the best combination that reduced their numbers without adding mismatches. Although MinION sequencing is known to introduce a number of characteristic errors compared to Illumina sequencing, the new polishing tools allow a better-quality MinION-sequenced genome, thus to be used to help determine strain origin through phylogenetic analysis

Genomic history of human monkey pox infections in the Central African Republic between 2001 and 2018

International audienceMonkeypox is an emerging infectious disease, which has a clinical presentation similar to smallpox. In the two past decades, Central Africa has seen an increase in the frequency of cases, with many monkeypox virus (MPXV) isolates detected in the Democratic Republic of Congo (DRC) and the Central African Republic (CAR). To date, no complete MPXV viral genome has been published from the human cases identified in the CAR. The objective of this study was to sequence the full genome of 10 MPXV isolates collected during the CAR epidemics between 2001 and 2018 in order to determine their phylogenetic relationships among MPXV lineages previously described in Central Africa and West Africa. Our phylogenetic results indicate that the 10 CAR isolates belong to three lineages closely related to those found in DRC. The phylogenetic pattern shows that all of them emerged in the rainforest block of the Congo Basin. Since most human index cases in CAR occurred at the northern edge of western and eastern rainforests, transmissions from wild animals living in the rainforest is the most probable hypothesis. In addition, molecular dating estimates suggest that periods of intense political instability resulting in population movements within the country often associated also with increased poverty may have led to more frequent contact with host wild animals. The CAR socio-economic situation, armed conflicts and ecological disturbances will likely incite populations to interact more and more with wild animals and thus increase the risk of zoonotic spillover

National Monkeypox Surveillance, Central African Republic, 2001–2021

International audienceWe analyzed monkeypox disease surveillance in Central African Republic (CAR) during 2001-2021. Surveillance data show 95 suspected outbreaks, 40 of which were confirmed as monkeypox, comprising 99 confirmed and 61 suspected monkeypox cases. After 2018, CAR's annual rate of confirmed outbreaks increased, and 65% of outbreaks occurred in 2 forested regions bordering the Democratic Republic of the Congo. The median patient age for confirmed cases was 15.5 years. The overall case-fatality ratio was 7.5% (12/160) for confirmed and suspected cases, 9.6% (8/83) for children <16 years of age. Decreasing cross-protective immunity from smallpox vaccination and recent ecologic alterations likely contribute to increased monkeypox outbreaks in Central Africa. High fatality rates associated with monkeypox virus clade I also are a local and international concern. Ongoing investigations of zoonotic sources and environmental changes that increase human exposure could inform practices to prevent monkeypox expansion into local communities and beyond endemic areas