Monkeypox has been declared a “public health emergency of international concern (PHEIC)” by World Health Organization on July 23, 2022. This sudden and unexpected global outbreak of monkeypox virus (MPXV) raises significant concern [1], particularly in view of the on-going COVID-19 pandemic. From January 1 through June 22, 2022, a total of fifty countries with a total of 3413 laboratory confirmed cases have been reported. The majority of confirmed cases were from Europe and the Americas, with total no. and percentage at 2933, 86%, and 381, 11%, respectively (https://www.who.int/emergencies/disease-outbreak-news/item/2022-DON396). These statistics are expected to increase. Furthermore, Singapore and South Korea have reported imported cases of monkeypox, indicating a worsening of global spread by international travels. Laboratory diagnostic tests with high sensitivity and specificity are essential for early detection and case screening to reduce further MPXV spread.
Whereas transmission is thought to be through direct contact [2], recent reports had highlighted the unusual concentration among men who have sex with men (MSM), and among bisexual population, suggesting a unique transmission of the virus in these populations. The mechanism of transmission during sexual contact remains unknown. Although infectious MPXV has been found in semen, and MPXV DNA was detected in urine, feces, rectal swab, nasopharyngeal swab, and saliva in a recent report [3]. However, whether the virus can infect semen cells and reproduce in the genital tract remains unknown.
Monkeypox infection is a viral zoonosis. Infection results in characteristic overt lesions, similar to that of smallpox. Clinical presentations include fever and swollen lymph nodes, followed by a centrifugal evolving rash [2]. Interestingly, there are several unique clinical features of this current monkeypox outbreak, e.g., only few lesions appear, and at different stages of development, the distribution is either exclusively peri-genital and/or peri-anal, and the lesions appear to be ulcerated with umbilicated pustules [4].
In addition to clinical presentations, confirmatory diagnosis of monkeypox requires nucleic acid amplification tests (NAAT), i.e., PCR using various targets in the viral genome, to detect and to differentiate from other poxviruses. Several diagnostic assays had been well established from previous monkeypox outbreaks (Table 1 ). Real-time quantitative PCR (RT-qPCR) alone, or in combination with sequencing, was recommended by the WHO. We had critically examined with respect to their sensitivities three methods listed in the Interim Guidance by WHO [5] (Table 1).
Table 1.
List of assays for detecting monkeypox virus infections. The three assays by the WHO Interim guidance for detecting monkeypox virus infections are in bold.
| Detection technique | Viruses detected | Target viral gene | Limit of detection | Reference | Specimen type |
|---|---|---|---|---|---|
| RT-qPCR | West African strain | G2R | 1.7 fg (∼8.2 genome) | https://linkinghub.elsevier.com/retrieve/pii/S0166-0934(10)00254-5 |
Preference types: the swabs of lesion surface or exudate, roofs from multiple lesion, or lesion crusts. Additional types: the swabs of semen, urine, rectal or genital, venous whole blood collected in EDTA. |
| Congo Basin strain | C3L | 9.46 fg (∼40.4 genomes) | |||
| MPXV generic | G2R | 0.7 fg (∼3.5 genomes) | |||
| RT-qPCR | Variola virus | B12R | 20 copies/reaction | https://linkinghub.elsevier.com/retrieve/pii/S0166-0934(16)30067-2 | |
| Monkeypox virus | F3L | 20 copies/reaction | |||
| Varicella-zoster virus | ORF38 | 50 copies/reaction | |||
| RT-qPCR | Orthopoxvirus | Viral core cysteine proteinase | 22.08 copies/reaction | https://linkinghub.elsevier.com/retrieve/pii/S0890-8508(09)00077-2 | |
| Molluscipoxvirus | MC036R | 9.7 copies/reaction | |||
| Parapoxvirus | Envelope protein | 28.1 copies/reaction | |||
| Digital PCR | Nonvariola (NVAR) orthopoxviruses | E9L | 9.697 copies/mL | https://wwwnc.cdc.gov/eid/article/28/9/22-0917_article#r9 | |
| Monkeypox virus | B7R | 6.359 copies/mL | |||
| Monkeypox virus | Both targets combined | 4.795 copies/mL | |||
| RT-qPCR | Variola virus | A38R | 20 copies/reaction | https://www.sciencedirect.com/science/article/pii/S0166093411001984?via%3Dihub | |
| Monkeypox virus | B7R | 20 copies/reaction | |||
| Cowpox virus | D11L | 50 copies/reaction | |||
| Vaccinia virus | B10R | 70 copies/reaction | |||
| RPA | Monkeypox virus | G2R | 16 DNA molecules/μL | https://www.sciencedirect.com/science/article/pii/S0732889318307466?via%3Dihub | |
| RT-qPCR | Monkeypox virus | F3L | 11-55 fg (50–250 copies of each gene) | https://www.nature.com/articles/3700143.pdf | |
| N3R | |||||
| PCR | Monkeypox virus | ATI | undescribed | https://www.sciencedirect.com/science/article/pii/S0166093498000998?via%3Dihub | |
| RT-qPCR | Variola virus | A27L | 0.7 fg (∼4 copies/assay) | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC404623/pdf/1612-03.pdf | |
| Other orthopoxvirus | A27L | 6 copies/assay | |||
| RT-qPCR | Nonvariola (NVAR) orthopoxviruses | E9L | 2.54 fg viral DNA (∼12.5 genomes) | https://www.sciencedirect.com/science/article/pii/S1386653206001223 | |
| Monkeypox virus | B6R | ∼10 viral copies (2 fg) | |||
| GeneXpert | Monkeypox virus | G2R | undescribed | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303045/pdf/tropmed-96-405.pdf | |
| Orthopoxvirus | E9L | undescribed | |||
| LAMP | West African strain | ATI | 103 copies/reaction | https://onlinelibrary.wiley.com/doi/epdf/10.1002/jmv.21494 | |
| Congo Basin strain | D14L | 102.4 copies/reaction | |||
| Both Congo Basin and West African strain | ATI | 102 copies/reaction | |||
| RT-qPCR | Variola virus | 14-kD protein gene | 0.05 fg (25 copies/assay) | https://academic.oup.com/clinchem/article/53/4/606/5627648?login=true | |
| Monkeypox, cowpox and vaccinia viruses | 14-kD protein gene | 0.05 fg (25 copies/assay) | |||
| Microarray-based assay | Variola virus | C23L/B29R and B19R | undescribed | https://onlinelibrary.wiley.com/doi/10.1002/jmv.20698 | |
| Monkeypox virus | C23L/B29R and B19R | ||||
| Ectromelia virus | C23L/B29R and B19R | ||||
| Camelpox virus | C23L/B29R and B19R | ||||
| Vaccinia virus | B19R | ||||
| Cowpox virus | C23L/B29R and B19R | ||||
| FRET RT-qPCR | Cowpox virus | HA | 2.74–9.88 copies/PCR vial | https://academic.oup.com/clinchem/article/50/4/702/5639914?login=true | |
| Vaccinia virus | |||||
| Camelpox virus | |||||
| Variola major virus | |||||
| Monkeypox virus | |||||
| RT-qPCR | Orthopoxvirus (camelpox, cowpox, monkeypox, virus vaccino, vaiolo) | CrmB | 60 copies/assay | https://www.infezmed.it/index.php/article?Anno=2007&numero=1&ArticoloDaVisualizzare=Vol_15_1_2007_47 | |
| Herpes Simplex 1 virus | DNApol | 990 copies/assay | |||
| Herpes Simplex 2 virus | DNApol | 600 copies/assay | |||
| Varicella-zoster virus | ORF 29 | 160 copies/assay | |||
| PCR | Vaccinia virus | F4L | 20-30 PFU | https://www.sciencedirect.com/science/article/pii/S0890850804000726?via%3Dihub | |
| Cowpox virus | B9R | 20-30 PFU | |||
| Monkeypox virus | E5R | 80-100 PFU | |||
| Variola virus | B11R–B12R | 20-30 PFU | |||
| Vaccinia virus | C9L | 20-30 PFU | |||
| RT-qPCR | Variola virus | J7R | 25 copies/assay (0.1fg) | https://www.sciencedirect.com/science/article/pii/S0166093408002541?via%3Dihub | |
| Camelpox, cowpox, monkeypox and vaccinia virus | J7R | 50 copies/assay (10 fg) | |||
| Nested-multiplex PCR | Orthopoxvirus | VGF | 1 ng | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749831/ | |
| Parapoxvirus | B2L | 1 ng |
These tests, with different target sites in the viral genome, have different sensitivities and limit of detection (LOD) for different strains of monkeypox virus, and for different Orthopoxviruses (OPV). As shown in Table 1, the RT-qPCR test developed by Li et al. has a limit of detection (LOD) for the “generic” monkeypox virus (MPXV) at 0.7 fg, which is equivalent to approximately 3.5 genomes; whereas for West African strain, the LOD is at 1.7 fg (∼8.2 genome); and for the Congo Basin strain, at 9.46 fg (∼40.4 genomes). On the other hand, the RT-qPCR test developed by Maksyutov et al. targets the viral F3L gene. This assay has a LOD of 20 copies/reaction. The test developed by Schroeder et al. for detecting OPV has a LOD of 22.08 DNA copies.
These above RT-qPCR testes had been deployed to monitor MPXV infection in several countries, and in combination with next-generation sequencing (NGS) technology, they provide accurate detection results and at the same time, expands genome sequence information.
A person with characteristic lesions associated with monkeypox is considered infectious until the lesions crusted over and fallen off. Rapid diagnostic tests are important to detect the early stages of infection. Accurate and sensitive testing reduces spreading of the virus by appropriate mitigation strategies, and provides evidence basis for treatments, such as use of antiviral drugs or vaccination. Of note, as more viral genome sequences are available, a larger number of nucleotide polymorphisms have been observed. A revised classification has been proposed, with three main clades, MPXV clades 1, 2 and 3. This new classification scheme or nomenclature avoids discrimination of infected persons, and stigmatizing [6].
Despite cumulating cases, the natural animal reservoir of MPXV remains unknown. However, previous studies have shown that MPXV can infect several species of rodents in Africa [7], suggesting that these animals could be potential reservoirs for monkeypox virus. Also, there may be more MPXV strains in other yet-to-be-identified reservoir hosts, and posing a potential threat to humans. Therefore, continuing surveillance of MPXV from their animal reservoir is important, and more viral clades may be unveiled, further broadening the diversity of MPXV [7]. Therefore, use of previous testing methods should be taken into account of this genetic diversity, as the sensitivity and specificity may be negatively impacted by the genetic diversity of the virus.
The advantages of RT-qPCR include high sensitivity, detection of virus at earlier stages (of infection), usefulness in the surveillance of the virus, including the wildlife, and its application in molecular epidemiology, etc. However, in remote locations or in rural areas of Africa, the infrastructure may not be feasible for these molecular testing. Reducing the cost of testing, increasing the sensitivity and specificity, and developing rapid tests for point-of care (POC) for rural areas will increase the monitoring of monkeypox infection. To control this global outbreak of monkeypox, we need similar strategies as used for mitigating the current COVID-19 pandemic, i.e., early detection, develop antivirals and vaccines, and to fill the knowledge gaps regarding the viral ecology and evolution of monkeypox virus.
Declaration of conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was financially supported by the Fundamental Research Funds for the Central Universities Y0201900459 and the Bioinformatics Center of Nanjing Agricultural University.
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