Diagnostic approaches for monkeypox virus
Guohao Fan and Jiahua Kuang contributed equally.
Abstract
Mpox (formerly Monkeypox) is a zoonotic infection caused by Monkeypox virus (MPXV). Since 2022, Mpox epidemics have occurred in many non-endemic countries and regions, leading the World Health Organization to declare a public health emergency of international concern. With the persistent transmission and evolution of MPXV, symptoms of Mpox have become milder, with some infections being asymptomatic. In addition, MPXV has become more contagious. Therefore, rapid and accurate diagnosis and screening of MPXV is vital to prevent and control MPXV epidemics. Here, we review and summarize the technical details, application scenarios, and the advantages and disadvantages of MPXV-specific diagnostic methods.
Abbreviations
-
- CPE
-
- cytopathic effect
-
- ELISA
-
- Enzyme-linked immunosorbent assay
-
- EM
-
- electron microscopy
-
- LAMP
-
- loop-mediated isothermal amplification
-
- LOD
-
- lowest limit of detection
-
- Mpox
-
- Monkeypox
-
- MPXV
-
- Monkeypox virus
-
- NAAT
-
- nucleic acid amplification assay
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- NGS
-
- next generation sequencing
-
- OPXV
-
- Orthopoxvirus
-
- RPA
-
- recombinase polymerase amplification
-
- rt-PCR
-
- real-time fluorescent polymerase chain reaction
-
- WHO
-
- World Health Organization
1 INTRODUCTION
Mpox (formerly Monkeypox) is a zoonotic infection caused by Monkeypox virus (MPXV). MPXV is a species of the genus Orthopoxvirus (OPXV), subfamily Ridgepoxviridae and family Poxviridae [1-3]. MPXV infections typically result in skin lesions, skin nodules, or disseminated rashes in humans and many other animals. Other OPXVs that can cause disease in humans include cowpox virus and smallpox virus [4, 5]. MPXV was first detected in monkeys transported from Singapore to Denmark in 1958, hence the name MPXV. The first case of human infection with MPXV was reported in 1970 in a 9-month-old infant in the Democratic Republic of the Congo [6]. Subsequently, outbreaks of MPXV occurred in West and Central Africa. Mpox attracted international attention when 71 human cases were reported in the United States of America in 2003 [7]. Several travel-related human cases have been reported in Europe, North America and Asia during the period from 2003 to 2022 [8, 9]. However, beginning on 13 May 2022, a global outbreak spread through communities in many countries around the world. On 23 July 2022, the World Health Organization (WHO) declared Mpox as a public health emergency of international concern [3, 10]. By 22 August 2023, there were 89,529 confirmed cases and 156 deaths in 114 countries worldwide. In China, the number of new confirmed cases in July 2023 was 491, much higher than the 106 new cases in June, indicating that vigilance is necessary [11].
MPXV is a double-stranded DNA virus with a genome length of approximately 197 kb containing approximately 200 non-overlapping coding genes. Based on clinical presentation and genome sequence, MPXV is classified into two clades—the Congo Basin or Central African Clade (Clade I) and the West African Clade (Clade II) [9]. Clade I appears to be more virulent with case-fatality rates ranging from 1% to 10%, whereas the overall case-fatality rate for Clade II is less than 3%. Recent data show a Clade II mortality rate of 1.4%. Clade II contains two branches: Clade IIa and Clade IIb, which evolved from Clade IIa. Clade IIb is further subdivided into lineages A, A.1, A.2, and B.1. The 2022 MPXV epidemic was caused by lineage B.1. Compared with the Mpox epidemics of 2017–2021, the 2022 outbreak cases were less symptomatic, more adapted to humans and more capable of human-to-human transmission [12-14].
MPXV infection can cause a series of clinical symptoms. The initial phase of the disease generally lasts from 1 to 5 days, during which the patient may suffer from fever, headache, backache, muscle pain, malaise and swollen lymph nodes, which are symptoms specific to Mpox. The second phase usually occurs 1–3 days after the fever has subsided when a rash appears. The rash consists of macules, papules, blisters, pustules and indentations on the umbilicus, followed by a crust that falls off after approximately 2–3 weeks. The rash is usually centralized, beginning on the face and gradually disseminating to the palms of the hands and all over the feet. It may involve the oral mucosa, conjunctiva, cornea and/or genitals. Serious life-threatening complications may occur in patients with Mpox [4, 5, 9, 15].
Since May 2022, when cases of Mpox were reported in the United Kingdom, confirmed cases of Mpox have been found in several countries around the world. Currently, MPXV is spreading rapidly in non-endemic countries and regions. Confirmed cases of Mpox have been found in the European region, North America and Asia. While Mpox, unlike SARS-CoV-2, has not led to a pandemic, preventive and control measures for Mpox should not be ignored. MPXV testing, as a vital aspect of epidemic prevention and control, requires a multitude of testing technologies. It is important to further develop various diagnostic technologies to improve the sensitivity, true-positive rate, assay speed and other technical parameters for clinical diagnosis, on-site quarantine and laboratory identification. This article reviews the technical details, application scenarios and the advantages and disadvantages of MPXV-specific laboratory diagnostic methods.
2 MONKEYPOX VIRUS ISOLATION AND CULTURE
The isolation and culture of MPXV is the “gold standard” for its detection. A wide variety of cell types is suitable for growing MPXV, such as cells derived from the kidneys of rhesus monkeys, African green monkeys, guinea pigs, rabbits, and cows; from the livers of mice; and from human tissues [2, 16]. Different cell types infected with MPXV exhibit different cytopathic effects (CPEs). The shared CPEs of all these cell types are rounding, granulation, and condensation. Cells then eventually detach and take on a hollow appearance under the microscope. The time of CPE appearance in cells depends on the amount of virus inoculated. The titer of infection varies from 10−4 to 10−6/mL for a 50% tissue culture infectious dose [17]. Erez et al. recently used Vero cells to culture MPXV. Cells infected by MPXV show typical rounding and detachment within 24 h after infection. Finally, immunofluorescence with specific antibodies enables the observation of MPXV in these cells [1, 18].
MPXV culture should not be used as part of a routine diagnostic strategy because it is time-consuming, labor-intensive, expensive, and demands laboratory facilities. It should only be performed in facilities with dedicated laboratories and experienced personnel. However, MPXV culture is extremely valuable for viral biology and pathobiology research as well as vaccine development.
3 IMMUNOASSAYS
Immunoassays are a key tool for the rapid detection of MPXV, for determining MPXV vaccination status and prevalence in the population, and for the diagnosis, prevention and epidemiological investigation of MPXV.
3.1 Serum antibody testing
The MPXV serum antibody test is a main method of Mpox diagnosis and epidemiological investigation. Enzyme-linked immunosorbent assays (ELISAs) are the most popular serum antibody tests for qualitative or quantitative analysis of the immune response to MPXV through antigen-antibody-specific binding reactions, which can rapidly detect serum IgG and IgM against MPXV. They are suitable for large-scale epidemiological investigations of high-risk populations and can also be used as an auxiliary diagnostic to RT-PCR technology [1, 19, 20]. Specific IgM and IgG antibodies are commonly detected 5–8 days after the appearance of a rash in a person infected with MPXV [19]. IgG/IgM antibodies can be detected in whole blood, serum, plasma, and fingertip blood specimens. The use of antibody testing using plasma or serum alone is not a diagnostic criterion for MPXV. However, if the results of PCR are inconclusive, it can be helpful to test IgM in a recent acute patient or test IgG in a paired serum specimen (first collected during the first week of illness) in the diagnosis. The diagnosis of MPXV requires duplicate serum specimens from both the acute and recovery phases, and a 4-fold or greater rise in IgG antibody titer in the recovery phase compared with the acute phase is required to confirm an infection [9, 20, 21].
ELISA technology is simple, convenient and relatively safe, but it cannot be used for the early diagnosis of pathogens because of the delayed appearance of antibodies in serum. In addition, smallpox vaccination may interfere with the serological diagnosis of MPXV, which results in false-positive results. Therefore, MPXV antibody testing can only be used as an adjunct to clinical diagnosis and plays an important role in epidemiology and vaccine evaluation.
3.2 Rapid MPXV antigen test
During the SARS-CoV-2 pandemic, rapid antigen detection was one of the most commonly used test methods. It played an essential role in the mass screening of populations and self-testing of patients at home or in quarantine. Rapid detection of pathogen antigens is therefore extremely important for outbreak screening and control [22-25]. The chromatographic immunoassay for MPXV has high specificity for the genus Orthopoxvirus, but the sensitivity of this technique is lower than that of nucleic acid assays. This method has the advantage of being easily commercialized and is suitable as a diagnostic aid for use in the field and remote areas. MPXV antigen rapid test kits are provided with a thin swab that allows healthcare workers to easily collect test samples from a patient's skin lesions. The advantages of the lateral flow rapid assay are the speed of diagnosis, which takes less than 15 min, and the simplicity of the procedure. Low sensitivity and erroneous results are potential limitations of this method. Several companies have developed kits based on MPXV antigens and serum antibodies that can be used as supplemental assays for the detection of MPXV [26, 27]; however, few studies have reported on diagnosis through the specific detection of MPXV antigens. Li et al. prepared three mouse monoclonal antibodies (3A1, 8F8 and 2D1) against MPXV A29L. Used in ELISA and lateral flow immunoassay formats, the 3A1 antibody has excellent specificity and can rapidly and sensitively detect MPXV, with the lowest limit of detection (LOD) of 128.00 pg/mL. The antibody-based A29L assay against MPXV takes only 10–15 min and does not require the use of specific equipment [28]. Davis et al. developed two semi-quantitative assays for MPXV A29L, which were standardized using infectious MPXV cell cultures and MPXV-positive non-human primate serum specimens. These detected viral antigens in serum, which were extremely specific for MPXV compared with other infectious orthopoxviruses (cowpox virus and camelpox virus) and correlated with quantitative PCR results [29]. For rapid antigen detection of MPXV, the majority of tests are currently designed to detect the A29L antigen with high sensitivity and specificity.
4 MOLECULAR METHODS
The nucleic acid amplification assay (NAAT) is recommended as a confirmatory method for the detection of MPXV by the WHO [30]. When compared with viral isolation and culture and immunological detection, NAAT has the advantages of high sensitivity, high specificity and shorter detection time. The SARS-CoV-2 pandemic drove the development of the powerful NAAT-based platform. This has greatly improved the accessibility of the method and a large number of personnel have been trained in its use, even in remote areas or areas with scarce medical resources [2, 31].
4.1 PCR methods
The WHO recommends real-time fluorescent polymerase chain reaction (real-time PCR, rt-PCR) as the “gold standard” for the detection of MPXV. rt-PCR is characterized by high sensitivity and specificity and has been widely applied because of its ability to accurately quantify multiple samples [31, 32].
MPXV and other OPXVs can be detected by rt-PCR using the following methods. Specific primers have been designed to target the conserved regions of the viral extracellular envelope protein gene (B6R), the DNA polymerase gene (E9L), the DNA-dependent RNA polymerase subunit 18 gene (rpo18) and the double-stranded RNA-binding protein region F3L gene [33, 34]. A probe designed to target B6R recognizes two branches of MPXV. A probe designed to target E9L recognizes 13 European and Asian OPXVs but was not effective in identifying variola virus or North American OPXV [31]. The double-stranded RNA-binding protein region of the F3L gene was effective in distinguishing between variola virus, cowpox virus and poxvirus-specific gene fragments. The probe designed to utilize the F3L gene could rapidly and effectively detect MPXV. [35].
The sensitivity and specificity of rt-PCR for detecting MPXV is high. It can effectively identify MPXV branches or distinguish other OPXVs. But rt-PCR may be affected by specimen quality, storage and nucleic acid extraction, which may lead to false negative results. Several companies in China have developed in vitro diagnostic test kits for MPXV (using fluorescent PCR methods) and over 10 tests have obtained CE certification from the European Union. Domestic Chinese MPXV nucleic acid detection products provide a guarantee of accurate diagnosis of Mpox. In addition, NAATs can be performed to quantify MPXV load (number of DNA copies) to assess active infection, viral shedding and the course of infection in humans and animal models. In 2006, Li et al. developed a rt-PCR method for the detection of MPXV DNA in skin rash samples. In this method, PCR was performed using DNA polymerase (E9L) and envelope protein (B6R) gene-specific primers [10, 31, 35].
4.2 Loop-mediated isothermal amplification
Loop-mediated isothermal amplification (LAMP) is a commonly used and mature isothermal nucleic acid amplification technique. LAMP has made tremendous contributions to the detection of pathogens, such as bacteria, viruses and fungi. Double-stranded DNA is in dynamic equilibrium at approximately 65°C. LAMP exponentially amplifies the target DNA using 4–6 specific primers and Bst DNA polymerase (a DNA polymerase with potent strand-replacing activity) at approximately 65°C. The target DNA is then detected by a turbidimetric assay, colorimetric assay, flow chromatography strip assay, or fluorescent dye detection. LAMP is a robust assay with high sensitivity and specificity, and an assay time of 20–60 min. Iizuka et al. developed a real-time quantitative amplification system for the MPXV genome using LAMP. Four primers were designed for six specific regions of the viral target gene. LAMP was effective in diagnosing MPXV infection, distinguishing Congo Basin strains and West African strains and evaluating the process of infection [36]. Feng et al. developed a rapid LAMP-based visual assay for the detection of MPXV based on turbidimetric detection of the LAMP product, which enables visual observation of the LAMP reaction results [37].
LAMP is efficient, simple, and specific, and it does not require thermal cycling equipment and is much less expensive than traditional PCR. However, this technology has the disadvantage of complex primer design and requires substantial optimization and validation of specific primers. LAMP allows rapid screening and effective diagnosis of MPXV and differentiation of OPXV species.
4.3 Recombinase polymerase amplification assay
Recombinase polymerase amplification (RPA) is a novel technique for isothermal DNA amplification based on the activities of recombinases, single-stranded binding proteins and polymerases. RPA has several advantages compared to PCR. (1) RPA does not require a complex temperature cycling device; the assay is performed at a constant temperature of 37–42°C. The device is simple and compact, which makes it suitable for remote sites and medical centers. (2) RPA has a rapid detection time of 5–20 min for the real-time fluorescence detection of pathogens, and the miniaturized instrument can be used to detect samples upon arrival. (3) RPA reagent is stored in the form of lyophilized powder, which does not require strict cold chain preservation and short-term freezing. Some studies have shown that the RPA reagent can be stored at room temperature for 30 days and remain effective [23, 38, 39].
Mao et al. developed three recombinase-based isothermal amplification assays for the rapid detection of MPXV. These three assays targeted the MPXV G2R gene. The LOD for these systems was approximately 100 copies of DNA per reaction and the results could be visualized within 20–30 min [38]. According to Davi et al., the sensitivity of the assay was 95% (16 copies/μL), whereas the real-time PCR method had a sensitivity of 100% (the real-time PCR method detected two weakly positive case, whereas the RPA method was negative). The specificity of both methods was 100% [32]. Chen et al. demonstrated a portable CRISPR-Cas-based system for naked-eye detection of MPXV. The system took advantage of the high selectivity of CRISPR-Cas12 and the isothermal nucleic acid amplification potential of RPA. It detected both the current circulating MPXV clade and the original clades [40].
4.4 Sequencing
Since the outbreak of MPXV in 2022, next generation sequencing (NGS) technology has been widely applied in the detection of MPXV gene sequences. The MPXV that caused the current round of outbreaks was shown to belong to the West African mild strain and was closely related to the MPXV outbreaks in Singapore, the United States and other countries in 2018. The genome and molecular evolution explained to a certain extent the reasons for the obvious changes in the biological characteristics of MPXV in this outbreak. In countries and regions with developed medical services, sequencing platforms can be established in clinical or public health laboratories as powerful tools for laboratory testing and molecular characterization of viruses. In the case of the MPXV outbreak, high-throughput sequencing analysis of isolated strains has provided accurate information for virus traceability [41-43].
NGS has a great advantage in the characterization of MPXV or other OPXVs because it can directly sequence and characterize specimens without the isolation of pathogens and without relying on known nucleic acid sequences. Although NGS is not the method of choice for routine diagnosis of MPXV, the technology can contribute to analyzing and providing real-time genomic data on MPXV to determine its phenotype and mutation status, which is critical for elucidating the origin and international spread of MPXV.
5 Electron microscopy
The first application of electron microscopy (EM) to clinical virology research was for the diagnosis of variola and varicella viruses [44]. EM enables direct visualization of the external features and internal structure of viruses. Ultrastructural images of viruses are generated by EM-negative staining techniques and allow visual classification of MPXV. MPXV is similar in size and shape to other OPXVs. Mature viral particles are usually brick-shaped or elliptical with viral particle sizes ranging from 200 × 250 nm [17, 45].
EM technology integrates molecular biology, immunohistochemistry, and modern computer processing techniques to significantly improve the sensitivity of virus detection. The major strength of the EM assay for MPXV is that it does not rely on specific biological reagents and enables direct visualization, whereas molecular biology and serological assays demand specific probes to detect viruses. In addition, EM techniques can be used as a complement to other assays for elucidating the life cycle of viruses during cellular infection, which plays an important role in controlling the safety of biological agents and contributes to the development of antiviral drugs and vaccines. However, sample preparation for EM is complex, time-consuming and cumbersome. EM must be performed by specially trained technicians; therefore, it is not available as a routine laboratory test [2, 33].
6 SPECIMEN SELECTION
Appropriate specimens are key to confirming MPXV infection. The recommended specimen types for laboratory confirmation of MPXV are skin lesion materials, including swabs of lesion surfaces or exudates, superficial layers of multiple lesions, or lesion crusts. Swabs should be vigorously rubbed onto the lesion to ensure that enough viral DNA is collected. Lesions, crusts and vesicular fluid should be collected separately and placed in separate viral storage tubes [21, 42, 43, 46]. Pharyngeal swabs are also an important supplemental diagnostic specimen type for patients with MPXV. These two specimen types can be used for viral isolation and culture, rapid antigen detection, and nucleic acid diagnostics, including PCR, LAMP, RPA and sequencing technologies. Alternative specimen types can be collected for research purposes, including urine, semen, rectal and/or genital swabs and whole blood. These specimens should not be used for routine diagnosis. Serum and plasma are important specimen types to aid in diagnosis; they can be used to test IgM in acute patients or IgG in paired serum samples (collected in the first week of disease and in later weeks). However, serological diagnosis may be confounded by recent vaccinations [43].
7 CONCLUSIONS
Since the outbreak of MPXV in May 2022, it has spread worldwide. MPXV has evolved and became progressively more adapted to humans. The transmission and pathogenicity of the virus are closely related to the evolution of the virus, so it is necessary to establish detection and surveillance of MPXV. The clinical symptoms caused by MPXV are difficult to distinguish from other poxviruses and it is difficult to confirm the diagnosis of MPXV based on clinical manifestations; therefore, laboratory diagnosis is crucial. A variety of detection techniques should be available to provide more detailed and accurate information to cope with mutation of the virus and to predict epidemiological trends.
We have reviewed four MPXV detection methods, including isolation and culture of MPXV, immunological assays, nucleic acid molecular assays, and EM. Among them, nucleic acid detection is the preferred method for early, rapid and accurate diagnosis of MPXV. rt-PCR is the “gold standard” for MPXV detection, RPA and LAMP have wide application potential for rapid detection and point of care testing, and high-throughput sequencing technology is of great significance for researching the evolution and origin of MPXV. Antigen detection is among the most important tools for rapid virus detection. Antibody detection in serum helps to detect the virus and to screen for previous infections and immunization levels in the population. Isolation and culture of MPXV have an irreplaceable role in subsequent vaccine development, basic research and drug development.
For future biosecurity, it is necessary to develop a combined testing platform with different technologies for MPXV and other OPXV assays. Efficient and accurate testing products should be developed to continuously improve the efficiency and accuracy of testing to enable the implementation of strategic technical preparations and preventive measures to prevent the occurrence of serious public health incidents.
AUTHOR CONTRIBUTIONS
Guohao Fan, Jiahua Kuang and Shengjie Zhang drafted the manuscript. Yang Yang, Yingxia Liu and Hongzhou Lu revised and edited the manuscript. All authors read and approved the manuscript.
ACKNOWLEDGMENTS
None. This work was supported by the Shenzhen High-level Hospital Construction Fund (23250G1001 and XKJS-CRGRK-004) and the Sanming Project of Medicine in Shenzhen.
CONFLICT OF INTEREST STATEMENT
Professor Hongzhou Lu is the Editor-in-Chief of the iLABMED. To minimize bias, he was excluded from all editorial decision-making related to the acceptance of this article for publication. The remaining authors declare no conflicts of interest.
ETHICS STATEMENT
Not applicable.
INFORMED CONSENT
Not applicable.
Open Research
DATA AVAILABILITY STATEMENT
No data was generated for this article.
