1. Introduction
Monkeypox is a re-emerging zoonotic disease caused by the monkeypox virus (MPXV), a DNA virus belonging to the Orthopoxvirus genus, part of the Poxviridae family. It is related to the variola virus, which causes smallpox [1], [2], [3], [4], [5]. Two main genetic clades are known to cause illness: the Central African (Congo Basin) clade and the West African clade. The West African clade is generally associated with milder disease compared to the Congo Basin clade. Current outbreaks are primarily linked to the West African subtype [[3](#bib3),5,6]. Despite its name, monkeypox is more commonly found in rodents like squirrels, rats, and mice, which transmit the virus to humans. The name “monkeypox” originates from its initial discovery in monkeys in 1958, where it caused a smallpox-like illness [[7](#bib7)]. The natural reservoir and transmission cycle of the virus are still under investigation. The virus has been isolated from wild animals only twice: first from an African squirrel in the Democratic Republic of Congo in 1985, and second from a deceased mangabey monkey in the Ivory Coast in 2012 [[3](#bib3),4,[8], [9], [10], [11]]. Given the potentially atypical presentation of monkeypox in recent outbreaks, it’s crucial to consider it in differential diagnoses, especially when patients present with rashes resembling sexually transmitted infections (STIs), even if localized [[12](#bib12)]. Monkeypox rashes can be mistaken for conditions like chickenpox, shingles, or herpes. Expert dermatological examination, focusing on rash patterns and location, can help narrow down potential diagnoses. Laboratory testing, specifically PCR (polymerase chain reaction) testing of rash swabs, is essential for confirming monkeypox. The recent global increase in human monkeypox cases and its spread across geographical areas has elevated concerns about this re-emerging zoonosis [[1](#bib1),4,[13], [14], [15]]. Factors contributing to this rise may include decreased herd immunity following the cessation of smallpox vaccination programs in the early 1980s, potential changes in the virus itself, and ecological shifts increasing human-wildlife interactions. Notably, the virus strain in the current non-endemic outbreaks appears to have diverged from the strain causing the 2018-19 Nigerian outbreak and exhibits a higher number of mutations than expected, some potentially enhancing transmission [[16](#bib16)]. In response to the escalating situation, the World Health Organization (WHO) declared the current monkeypox outbreak a Public Health Emergency of International Concern (PHEIC) on July 23, 2022 1. This decision was made amidst cases spreading across over 70 countries, predominantly non-endemic, with many cases lacking clear epidemiological links and presenting with milder, non-specific clinical signs [[17](#bib17)].
2. Transmission Pathways of Monkeypox Virus to Humans
Monkeypox virus (MPXV) can spread through various routes and affect anyone via close, personal contact, often skin-to-skin. Transmission methods include: direct contact with monkeypox rash, scabs, or bodily fluids of an infected person; indirect contact with contaminated objects, fabrics (clothing, bedding, or towels), and surfaces; and respiratory secretions. Vertical transmission from a pregnant person to their fetus via the placenta is also possible. Zoonotic transmission occurs through contact with infected animals, including bites or scratches, or through handling or consumption of improperly cooked meat or animal products from infected animals [[15](#bib15),[18], [19], [20], [21], [22], [23], [24], [25], [26], [27]]. Individuals with monkeypox are contagious from the onset of symptoms until the rash fully heals and a new layer of skin forms, typically lasting 2–4 weeks. Ongoing research aims to clarify: (i) the potential for asymptomatic transmission, (ii) the frequency and conditions under which respiratory transmission occurs, and (iii) the possibility of transmission through semen, vaginal fluids, urine, or feces. Unlike COVID-19, monkeypox virus is not as efficiently transmitted between humans, facilitating isolation and spread prevention. While not definitively confirmed as a sexually transmitted infection, transmission during sexual intercourse is possible due to close physical contact [[11](#bib11),21]. Air travel can also contribute to the international spread of the infection [[11](#bib11),14,15,21,28].
3. Clinical Manifestations and Differential Diagnosis
As an Orthopoxvirus, monkeypox presents clinically similar to smallpox [[13](#bib13),18,21,22,24]. The incubation period in humans typically ranges from 7 to 14 days, but can vary from 4 to 21 days [[8](#bib8),[19], [20], [21], [22],29]. The illness usually begins with a prodromal febrile phase lasting 1–4 days, characterized by fever, headache, muscle aches, backache, and sometimes exhaustion, sweats, and fatigue, preceding the cutaneous phase. Skin rashes typically emerge 1–3 days after fever onset. Rashes can appear on the face, inside the mouth, hands, feet, chest, genitals, anus, and eyes. In some cases, rash may be the initial symptom, followed by systemic symptoms. The number of lesions varies significantly among individuals. Lesions progress through stages: starting as flat macules, evolving into raised papules, then forming vesicles filled with clear fluid. The fluid then becomes yellowish, forming pustules. Eventually, pustules crust over, and lesions resolve as the crusts fall off. Patients are considered non-infectious once all crusts have detached. Disease severity tends to be greater in immunocompromised individuals. Scarring from the rash is a common outcome. However, more serious complications, as documented in a 2009 study of human monkeypox, include pulmonary distress and bronchopneumonia. Severe complications and sequelae are more frequently observed in unvaccinated individuals. Ocular infections can occur, potentially leading to corneal scarring and permanent vision loss [[1](#bib1),5,8,11,12,21,23,26,27,[30], [31], [32]]. Lymphadenopathy is a key differentiating feature of monkeypox from smallpox. It typically occurs around the onset of fever, 1–2 days before rash onset, or rarely concurrently with the rash. Enlarged lymph nodes are commonly found in the submental, submandibular, cervical, and inguinal regions. Exposed individuals may also experience sore throat, cough, and/or mucous membrane rashes in the mouth [[1](#bib1),8,9,[21], [22], [23],25,28,32,33]. Non-specific clinical presentations, lesions, and inflammation of the pharyngeal, conjunctival, and genital mucosae have also been reported [[8](#bib8),[21], [22], [23],25,28,33]. Many of these symptoms overlap with other viral and non-viral illnesses, making differentiation challenging for clinicians unfamiliar with monkeypox or diseases mimicking it. Therefore, laboratory diagnosis is of paramount importance [[9](#bib9),23,[32], [33], [34]].
Image: Progression of monkeypox rash stages, illustrating macules, papules, vesicles, pustules, and crusts for clinical diagnosis.
4. Indications for Monkeypox Testing
Monkeypox presents significant challenges for public health and healthcare workers (HCWs) regarding surveillance and laboratory diagnostic capabilities. Historically endemic, monkeypox has recently spread to non-endemic regions, with cases rising across Europe, North America, and Australia [[1](#bib1),4,6,9,11,21,34]. Contributing factors may include increased international travel, relaxed surveillance measures, and the easing of COVID-19 restrictions. Additionally, smallpox vaccination, which provided approximately 85% cross-protection against monkeypox, has not been routinely administered since 1982. Effective surveillance relies on epidemiological data. Initial cases often reported international travel within 21 days before symptom onset, particularly to countries not known for endemic monkeypox, and participation in events with close contact. However, recent travel history does not definitively confirm the source of infection. Since late June, an increasing number of cases have been linked to local community transmission [[35](#bib35)]. As previously mentioned, clinical signs and symptoms of monkeypox are non-specific. Clinical presentation alone can be insufficient for diagnosis, especially in atypical cases, due to similarities with various rash-causing conditions. Clinical presentation in current outbreaks may be more frequently atypical. Thus, considering other etiologies for similar skin lesions or widespread rashes is crucial. Differential diagnoses include herpes simplex virus, varicella zoster virus, molluscum contagiosum virus, enteroviruses, measles, scabies, syphilis, bacterial skin infections, rickettsialpox, drug allergies, parapoxviruses, and related conditions [[32](#bib32),[36], [37], [38], [39]]. Clinicians should be vigilant for patients presenting with new, characteristic rashes, or those meeting epidemiological criteria with high clinical suspicion for monkeypox. The rash can be easily confused with rashes from herpes, syphilis, and varicella. Co-infections with Monkeypox virus and other infectious agents (e.g., varicella zoster, herpes, syphilis) have been documented [[12](#bib12),18,26,27,32,37,39]. In settings with limited testing capacity, the decision to test should be guided by both clinical and epidemiological factors to assess the likelihood of infection. Monkeypox testing should be offered to anyone meeting the clinical and epidemiological suspected case definition [[13](#bib13),34,36].
5. Specimen Collection, Shipment, and Storage for Monkeypox Diagnosis
5.1. Safety Procedures for Sample Handling
Strict adherence to standard operating procedures (SOPs) is essential for the safe collection, storage, and handling of monkeypox specimens. Laboratory personnel must be thoroughly trained in proper personal protective equipment (PPE) donning and doffing throughout the entire sample handling process. Samples suspected of containing monkeypox virus should be treated as potentially infectious materials. Precautions must be taken to minimize contamination risks. Only trained and competent personnel should handle specimens. Careful PPE use and avoidance of aerosol generation are crucial. Vaccination is recommended for laboratory staff, particularly healthcare workers directly involved in caring for confirmed monkeypox patients, including staff in sexual health clinics assessing suspected cases [[40](#bib40)]. Suitable disinfectants include quaternary ammonium compounds and freshly prepared 0.5% (or 200 ppm) bleach [[8](#bib8),28,36,41]. Pre-exposure vaccination should be prioritized for high-risk workers, such as staff in high consequence infectious disease units expected to care for monkeypox patients.
Recommended Samples for Monkeypox Diagnosis: Surface lesion and skin materials are the preferred specimens for monkeypox diagnosis, including exudate swabs and lesion crusts [[8](#bib8),16,19,20,23,31,32,36,41]. Swabs should be vigorously rubbed against lesions to ensure sufficient viral DNA collection (Table 1). Ideally, samples should be collected from multiple locations and different lesions. Swab samples should be transported to the laboratory in a dry tube or Viral Transport Medium (VTM). Samples from two lesions of the same type can be placed in a single tube, but different lesion types (lesions, crusts, vesicular fluids) should not be combined in the same tube. It’s important to be aware of the potential for false negative or false positive test results in monkeypox diagnosis. The U.S. Food and Drug Administration (FDA) recommends using lesion-derived swab samples for monkeypox virus testing due to limited clinical data supporting the use of other sample types like blood or saliva. Testing non-lesion samples may increase the risk of false results [[37](#bib37),42]. Depending on clinical context, urine, semen, rectal, and/or genital swab samples may also be collected. EDTA blood samples may be helpful, but viremia is typically short-lived, and viral load in blood may be lower than in lesions. Macular stage biopsies should be performed only by highly experienced personnel [[19](#bib19),20,[41], [42], [43], [44]]. Antibody tests (plasma/serum) alone are insufficient for acute monkeypox diagnosis. IgM detection in acute infection or IgG increase in paired samples taken 21 days apart can support diagnosis. Vaccination can affect serological test results [[5](#bib5),19,41].
Table 1. Specimen Types, Collection Materials, and Storage Temperatures for Monkeypox Virus (MPXV) Diagnostic and Differential Testing.
| Sample type | Collection materials | Storage temperature | Collection purpose |
|---|---|---|---|
| Skin lesion material, including: – Swabs of Lesion exudate – Lesion roofs – Lesion crusts |
Dry swab or VTM (Dacron or polyester flocked swabs recommended) | Refrigerate (2–8 °C) immediately after collection until lab transport; maintain cold chain during transport. | Recommended for primary diagnosis. |
| Oropharyngeal swab | Dry swab or VTM (Dacron or polyester flocked swabs recommended) | Refrigerate (2–8 °C) immediately after collection until lab transport; maintain cold chain during transport. | Recommended for diagnosis, ideally in addition to skin lesion material, when feasible. |
| Serum | Serum separator tubes | Refrigerate (2–8 °C) immediately after collection until lab transport; maintain cold chain during transport. | Consider for serology in differential diagnosis and research purposes, not primary acute diagnosis. |
6. Sample Collection and Storage for Monkeypox Diagnosis (Continued)
6.1. Transport of Clinical Specimens
Specimens should be refrigerated or frozen within one hour of collection and transported to the laboratory as promptly as possible. Proper handling and storage during transport are critical for accurate diagnostic testing. Monkeypox specimens should be refrigerated (2–8 °C) or frozen (−20 °C or lower) within 1 hour of collection. If transport time exceeds 7 days, specimens should be stored at −20 °C or lower. For long-term storage (beyond 60 days), −70 °C is recommended [[41](#bib41)]. Sample transport must comply with national and international regulations. International transport of specimens from suspected, probable, or confirmed monkeypox cases, including clinical samples, viral isolates, and cultures, must be categorized as Category A, UN2814 “infectious material affecting humans”. All remotely transported samples require appropriate triple packaging, labeling, and accompanying documentation. Viral DNA in skin lesion material remains relatively stable in dark, cool conditions, which can be considered if cold chain maintenance is challenging. Repeated freeze-thaw cycles should be avoided [[19](#bib19),20,36,[41], [42], [43], [44], [45], [46]].
6.2. Laboratory Testing Methods and Algorithm for Monkeypox Diagnosis
Monkeypox diagnostic tests should be performed in appropriately equipped laboratories by trained technicians following strict technical and safety protocols. Real-time or conventional Polymerase Chain Reaction (PCR) assays are used to detect MPXV DNA and/or specific viral DNA sequences. Post-PCR sequencing can also be conducted. Several validated PCR protocols are available for detecting orthopoxviruses (OPXV), and specifically MPXV, some differentiating between Congo Basin and West African clades (Fig. 1) [[8](#bib8),19,32,41,[44], [45], [46],46,47].
Fig. 1. Algorithms for Orthopoxvirus (OPXV) Initial PCR or Monkeypox Virus (MPXV) Specific Initial PCR.
Figure: Diagnostic algorithm for monkeypox testing using PCR assays, outlining steps for OPXV and MPXV specific detection and confirmation.
* Explanations: A positive result with an OPXV PCR test necessitates MPXV confirmation via a separate PCR and/or sequencing. In suspected cases from both endemic and non-endemic areas, a positive MPXV PCR result is considered confirmatory. Genetic sequence data (GSD) provides valuable information on the origin, epidemiology, and characteristics of monkeypox cases.
6.3. Interpretation of Laboratory Results for Monkeypox Diagnosis
Clinical and epidemiological information are paramount in confirming monkeypox infection. A positive OPXV PCR result requires confirmation of MPXV through further PCR and/or sequencing. Confirmation of MPXV infection relies on integrating clinical, epidemiological, and laboratory findings. A positive OPXV PCR followed by MPXV confirmation via PCR or sequencing, or a positive MPXV PCR in suspected cases, confirms MPXV infection. While MPXV-specific confirmatory testing is preferred, a positive OPXV PCR result is considered sufficient for laboratory confirmation of suspected cases. Member States are obligated to immediately notify WHO of laboratory-confirmed cases [[2](#bib2),32,33,48]. All laboratory-confirmed cases must be reported to WHO. In cases where clinical and epidemiological evidence strongly suggest monkeypox despite negative PCR results, serological testing may be useful for epidemiological investigations of past infection. False-negative PCR results can occur due to factors like sample quality, incorrect test setup, technical issues, or improper sample transport (e.g., DNA extraction failure).
Sequencing plays a crucial role in agent identification and understanding its origin. Genetic sequence data (GSD) aids in determining the virus’s origin, epidemiology, and characteristics, and whether cases originate from a single source or multiple introductions. Currently, sequencing MPXV from as many positive samples as possible from diverse patients and locations is recommended. WHO encourages countries and laboratories to share GSDs, including raw data, via publicly accessible databases. GSD can be generated using Sanger or Next-Generation Sequencing (NGS) methods [[36](#bib36)]. Some protocols are two-step, initially detecting OPXV in the first PCR reaction, followed by a second PCR step with type-specific primers to identify the specific strain. Tests must be performed by trained personnel using validated and/or verified methods [[36](#bib36),41]. Reagents should be stored under appropriate conditions. Published primer and probe sequences for OPXV and MPXV PCR tests are available for in-house test development in adequately equipped laboratories [5–7]. PCR kits for OPXV or MPXV detection are under development, but commercial PCR or serology kits are not yet widely available. Positive control material for PCR tests can be obtained from commercial suppliers [[23](#bib23),41,49]. Best practice includes using a low, yet detectable, concentration of positive control. Quality control steps should assess sample quality, nucleic acid quality, and process integrity. PCR’s high sensitivity necessitates measures to prevent or detect contamination. Sample integrity controls (e.g., RNase P), extraction controls, positive controls, and inhibition controls help differentiate false negatives from true negatives. Controls should be used after laboratory SOP verification. Test repetition is required if any control fails [[41](#bib41),48,49].
Waste Disposal: All potentially MPXV-containing waste must be decontaminated before disposal via approved methods like autoclaving or chemical disinfection, following specific laboratory protocols [[36](#bib36)].
Electron Microscopy: Electron microscopy can detect poxviruses in samples, but it is not routinely used for monkeypox diagnosis due to its technical complexity and facility requirements compared to molecular testing.
Viral Cell Culture and Isolation: Viral cell culture and isolation are generally not recommended for routine monkeypox diagnosis and should only be performed in specialized laboratories with appropriate biosafety facilities and experienced personnel.
6.4. Establishing an Effective Laboratory Network for Monkeypox Diagnosis
Timely and accurate monkeypox diagnosis depends on efficient sample delivery to capable laboratories, proper test execution, and consistent data recording and sharing. All countries, especially those reporting MPX cases, should have access to reliable testing, either nationally or through referral laboratories in other countries with OPXV or MPXV diagnostic capacity.
WHO can assist Member States in accessing referral testing through its Regional Offices. Sample inactivation at local laboratories, when performed safely and correctly, can simplify shipping logistics. All countries with reported cases are encouraged to share ranking data to enhance understanding of the current outbreak with organizations such as: The US Centers for Disease Control and Prevention (CDC), the WHO Collaborating Center for Smallpox and Other Poxvirus Infections (United States), and the Federal Budgetary Research Agency – State Research Center for Virology and Biotechnology, “VECTOR” (Russian Federation), WHO Collaborative Center for Orthopoxvirus Diagnosis and Repository for Variola Virus Strains and DNA [[36](#bib36),50].
7. Conclusion
The recent surge in human monkeypox cases across geographically diverse and non-endemic regions, coupled with the potential for further spread, has raised significant global health concerns. The WHO Director-General declared the current monkeypox outbreak a Public Health Emergency of International Concern (PHEIC) on July 23, 2022. As of August 9, 2022, nearly 32,000 confirmed monkeypox cases had been reported across 82 non-endemic countries [[51](#bib51)]. While the primary transmission routes of monkeypox virus (MPXV) are now well-established, further research is needed to fully understand the potential for asymptomatic transmission, the frequency and conditions of respiratory transmission, the likelihood of symptomatic individuals spreading the virus via respiratory secretions, and other possible transmission routes, such as through semen, vaginal fluids, urine, or feces. Scarring is the most common outcome of monkeypox infection, but severe complications can occur, particularly in unvaccinated individuals, highlighting the need for increased awareness and targeted vaccination of at-risk groups, including healthcare and sexual health workers.
For effective monkeypox diagnosis, lesion-derived swab samples are recommended for PCR testing to minimize false negative results. Specimens should be promptly refrigerated or frozen within one hour of collection and transported under appropriate cold chain conditions, adhering to national and international transport regulations. Confirmation of MPXV infection requires integration of clinical, epidemiological, and laboratory findings. Positive OPXV PCR results should be confirmed with MPXV-specific PCR or sequencing, although a positive OPXV PCR alone is often sufficient for initial laboratory confirmation in suspected cases.
Member States are urged to immediately notify WHO of all laboratory-confirmed monkeypox cases and to report new confirmed and probable cases weekly through established IHR channels, utilizing the WHO Case Report Form (CRF). To enhance global capacity for managing this re-emerging zoonosis, strengthening laboratory capacity and international specimen referral networks is crucial for timely and accurate monkeypox virus infection diagnosis and surveillance, primarily through nucleic acid amplification testing (NAAT) methods like real-time or conventional polymerase chain reaction (PCR).
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