Keywords

16.1 Introduction

Monkeypox (Mpox), a zoonotic infection, is caused by a virus of the genus orthopoxvirus (Farahat et al. 2022a, c). Mpox is an enveloped cytoplasmic virus that enters the host cell by using apoptotic mimicry, a common trait seen also in other members of the genus. The virus is then known to replicate cytoplasmically within the infected cells (Lansiaux et al. 2022). Two clades of Mpox viruses have been described—or Congo Basin or Central African clade (Clade I) and or West African clade (Clade II). The latter can be subdivided into Clades IIa and IIb. The clades can be further characterized by specific lineages. Unlike Clade II, which is seldom fatal, Clade I has a reported case mortality rate of about 10% (Americo et al. 2023).

In early 2022, an outbreak of Mpox with nearly 2,000 laboratory-confirmed cases was reported in more than 40 countries. While the majority of the cases were identified in the men who have sex with the men community, anyone was at risk who come in contact with an infected person. By July 2022, the spread of the disease was ongoing, with more than 5000 cases in the European region (Centers for Disease Control and Prevention 2023). Though Clade I is usually more common, the 2022 outbreak was predominated by the lineage B.1 of Clade II (Gigante et al. 2022). These findings have been thought to be suggestive of a possible sustained human-to-human transmission prior to the current outbreak. As of March 2023, more than 86,000 cases have been reported in more than 100 nations worldwide (Centers for Disease Control and Prevention 2023; Umar et al. 2022).

Mpox’s reservoir has not been identified, but rodents are a likely candidate. Transmission from animals to humans occurs through contact with the bodily fluids (and contaminated materials) of an infected animal or a bite (Nolen et al. 2015; Farahat et al. 2022b). Viral transmission is more likely when there is an invasive contact, such as through an animal bite, rather than simply touching an infected animal (Reynolds et al. 2003). Human-to-human transmission occurs primarily through contact with infected skin lesions or body fluids. Indirect contact, especially when touching objects used by someone who has had Mpox, is considered low risk. Due to the virus’ ability to cross the placenta, transmission can also occur from mother to fetus. Although cases of Mpox are associated with sexual contact, they likely result from direct contact with skin lesions during sex, not from sexual transmission (Centers for Disease Control and Prevention 2023).

The incubation period of Mpox ranges from 7 to 14 days, but it can also vary from 5 to 21 days. Prodromal symptoms such as fever may occur in the first 1–4 days with malaise, sweats, and fatigue. Following viral entry, the virus replicates at the inoculated site and spreads to local lymph nodes, causing initial viremia. Cutaneous lesions then follow, starting from the oropharynx, followed by skin rash (Kalthan et al. 2018). This makes lymphadenopathy a distinguishing feature of Mpox infection from similar viruses like smallpox. The onset of lymphadenopathy usually coincides with the fever, typically 1–2 days before the rash appears. Cutaneous lesions may not appear until 1–3 days after the onset of fever. Skin lesions begin as flat rashes (macules), become raised (papules), then vesicular. Vesicles may turn yellow and become pustules. As the crust falls off, the pustules, crust, and lesions disappear. During all stages, patients are considered infectious and direct contact increases spread. Only after the crust falls away are patients considered non-infectious (Altindis et al. 2022).

During the 2022 outbreak, strangely, most individuals with Mpox reported no prodromal symptoms and presented with a rash. Most patients presented with rash and constitutional symptoms like fever, malaise, chills, sore throat, and headache. The rash, characteristically described as predominantly anogenital, was relatively sparing on the face and extremities (Bunge et al. 2022). In immunocompromised individuals, serious complications such as lung damage and bronchopneumonia may occur. Diagnosis is somewhat challenging because of common signs and symptoms in Mpox and other viral infections. This underscores the importance of patient history, clinical findings, and laboratory diagnosis in establishing the diagnosis of Mpox (Bragazzi et al. 2023). Healthcare providers should maintain a high index of suspicion if there is a history of having recently traveled to endemic countries. Once an epidemiologic link has been established, patients presenting with rash and lymphadenopathy should be evaluated for Mpox (Harris 2022; Girometti et al. 2022).

16.2 Pre-analytical Phase

Early and rapid detection of Mpox is critical to care, both in terms of treatment and public health. To avoid delays in management, appropriate testing becomes paramount. Specimen collection knowledge is needed to improve testing protocol implementation. The pre-analytical phase includes the test request, patient and specimen identification, specimen collection, handling, and transport to the laboratories.

16.2.1 Sample Collection

Patients typically present with genital, perianal, and perioral skin lesions, with proctitis, tonsillitis, and penile edema as the predominant complications (Wang and Lun 2023). Thus, viral yields have been reported to be highest in skin lesion specimens, including lesion surface/exudate swabs, either from genitals, perineal region, or other areas like face, upper/lower limbs, trunk (Pan American Health Organization 2022; León-Figueroa et al. 2022). Collection of specimens from skin lesions should begin with disinfection with an appropriate antiseptic or disinfectant. If skin lesion is still intact, remove roof lesion with lancet or scalpel, collect fluid at lesion base and place in sterile container. The swab must be thoroughly applied to ensure adequate collection of viral DNA (Pan American Health Organization 2022).

The specimen site should be swabbed with a Dacron or polyester-flocked swab containing a viral transport medium (VTM) (Altindis et al. 2022). A single tube should contain two lesions of the same type, preferably from different sites and with different appearances. Mixing lesions, crusts, and vesicular fluid in the same tube is not recommended. For example, if two lesion roof and two crust are collected, roofs are put in one container and crust in the other (Hong et al. 2023). If resources permit, collect two tubes to reduce the risk of sampling errors and inhibiting factors, but test only one and test the second only if the first is equivocal (World Health Organization 2022). Swab skin lesion vigorously back and forth followed by rotating swab. The swab applicator should then be broken off and placed in a sterile plastic container, preferably with an O-ring screw cap (Centers for Disease Control and Prevention 2022a). In addition to the lesion sample, throat swabs should be taken (Gul et al. 2022). However, limited accuracy data limits clinical utility. Meanwhile, using sterile collection tubes to collect urine, semen, and blood is recommended (Sánchez-Romero et al. 2019).

Oropharyngeal and rectal swabs and blood constituents (using ethylenediaminetetraacetic acid/EDTA) can also be used to obtain samples (World Health Organization 2022). It is recommended that both blood plasma and throat swabs be taken if skin lesions are not present, but the clinical history suggests that a person may have Mpox. The virus can be detected in these two specimens during the prodromal period. However, because viremia occurs early in infection and before skin lesions become apparent, blood samples may not contain the high levels of virus found in lesion samples (Billioux et al. 2022). Reportedly, urine and semen analyses were done by some laboratories; however, these specimens are not commonly used for diagnosis and are primarily for research purposes, as long as there is appropriate ethical review board approval and sufficient resources to safely collect, handle, and store them (World Health Organization 2022).

Sampling needs to be timed to be optimal to collect a sufficient amount of viruses or antibodies (Rahmani et al. 2020). A specific guideline for the most effective timeframe in this Mpox situation is also needed from the World Health Organization (WHO). The specimens are subsequently processed for viral DNA identification using nucleic acid amplification tests (NAATs), either real-time or polymerase chain reaction (PCR), with or without sequencing (Antunes et al. 2022). The test can be used to confirm suspected cases, to discharge individuals from the hospital, or to screen individuals with close contacts (World Health Organization 2022). Enzyme-linked immunosorbent assay (ELISA) serology is also available as a means of determining antibody levels and may serve as a means of establishing a history of infection or other epidemiological studies. In the case of equivocal results, IgM in recent acute patients or IgG in matched serum specimens collected 21 days apart, with initial collection during the first week of illness, may aid in diagnosis (World Health Organization 2022).

The San Luis Obispo County Public Health recommends that Mpox samples be obtained within three days of the onset of blistering (Borenstein 2022). In addition, Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) remains positive 21 days after symptom onset (Thornhill et al. 2022). In contrast, a recent study (Peiró-Mestres et al. 2022) found that RT-PCR was 100% positive (mean quantification value 20.48 Cq) when samples were collected a median of 5 days (range: 1–14 days) after the onset of skin lesions. Several detection windows are recommended for ELISA serology, including IGM titers (> 5 days after rash appearance) and IgG titers (> 14 days after rash appearance) (Karem et al. 2005). Mpox DNA was found in several follow-up samples collected between 4 and 16 days after the onset of symptoms, including saliva, rectal swab, sperm, urine, and stool (Peiró-Mestres et al. 2022). A summary of sample collection and transportation is presented in Table 16.1.

Table 16.1 Type of collected specimens, their appropriate storage, and transportation guidelines
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16.2.2 Self-sampling

Although trained health workers should collect specimens, there is emerging evidence of acceptable quality self-sampling strategies where the patient collects clinical specimens for diagnosis (Feng et al. 2022b; Ubals et al. 2022). The rationale is related to experiences of prior diagnoses of many sexually transmitted infections (e.g., chlamydia and gonorrhea) and COVID-19, where self-samples performed on par with physician-collected samples (Ogale et al. 2019; Würstle et al. 2021). In comparison with physician swabbed samples, self-administered swabs demonstrated similar accuracy and Cq values with the highest overall agreement (98%) for lesional skin swabs (Ubals et al. 2022).

The agreement in oropharynx and rectum samples was 79% and 90%, respectively (Ubals et al. 2022). Lower value in the oropharynx is related to variable sampling quality compared to easily visible skin lesions or rectal swabs (Lee et al. 2021; Ubals et al. 2022). Self-collection strategies have many potential benefits for patients and disease control (Rozemeijer et al. 2015; Del Mistro et al. 2017). This may enable the inclusion of Mpox in routine screening schemes for at-risk populations (Ubals et al. 2022).

16.2.3 Storage and Transportation of Samples

The collected specimens need to be stored in the first hour of collection, followed by prompt transportation to the laboratory (Altindis et al. 2022). Skin, oropharyngeal, rectal, genital, urine, serum, and plasma specimens can be stored in the refrigerator (2–8 °C) or freezer (− 20 °C or below) during the first hour after collection. Storage at -20°C allows longer storage up to seven days. Semen and whole blood samples can be kept at room temperature for up to one hour prior to transport to a freezer (− 20 °C or below). For long-term preservation (> 60 days), storage at – 70 °C is recommended (World Health Organization 2022). If the cold chain is unavailable, storing skin lesions in a dark and cool environment preserves viral DNA integrity (Sah et al. 2022). Avoiding multiple freeze and thaw cycles is crucial as it may reduce sample quality (Shao et al. 2012; Kellman et al. 2021).

Proper sample handling and storage during transport are critical to ensure accurate diagnostic testing (Munir 2015; De Plato et al. 2019). All applicable regulations must be followed when transporting specimens. Specimens from Mpox patients, including clinical samples, and isolates and cultures, should be shipped as Category A UN2814 (infectious substance affecting man) for international shipments (World Health Organization 2022). Triple packaging, labeling, and documentation are needed for all specimens transported. Triple packaging, labeling, and documentation are needed for all specimens transported. Triple packaging system Category B (UN P650) should be used for clinical specimens and Category A (UN P620) for cultured virus isolates (World Health Organization 2021). A shipper with hazardous materials certification is required for transport (Gordy et al. 2019). A label containing information such as name, age, sex, date of specimen collection, and specimen type must be attached to each specimen container. In particular, for crust specimens, it is necessary to add information on the location of the crust, for example, on the back, on the hands, and so on (Centers for Disease Control and Prevention 2022a).

16.2.4 Laboratory Biosafety

It is important to ensure adherence to appropriate Standard Operating Procedures (SOPs) when specimens are collected. Adequate training on donning and doffing personal protective equipment (PPE), such as disposable gowns, rubber gloves, protective glasses or goggles, laboratory goggles, and booties/boots, as well as how to collect, store, package, and transport specimens is a must (Kim et al. 2020; World Health Organization 2022). Specimens collected for laboratory testing should be considered highly contagious and handled with care to avoid aerosolization (Verreault et al. 2013; World Health Organization 2022). A risk-based strategy is advised for laboratory handling of Mpox samples from suspected, probable, or confirmed cases. Basic biosafety requirements, similar to those referred to as biosafety level 2 are a must. Enhanced measures may be implemented as necessary (World Health Organization 2022). Prior to the inactivation of the samples, the samples must be placed in a fully functional Class I or Class II biosafety cabinet (Pawar et al. 2021). Virus isolation is too complex and has low sensitivity, and the requirement for biosafety level (BSL-3) or higher facilities makes this method impractical (Hong et al. 2023).

Safety buckets or sealed rotors must be used (Iwen et al. 2020; Lippi et al. 2020) when using a centrifuge. Workplace decontamination should include quaternary ammoniums and 0.5% (or 200 ppm) freshly prepared bleach (Feliciano et al. 2012; Yoo 2018). Laboratory waste potentially containing the Mpox virus should be sanitized by acceptable methods, such as autoclaving or chemical disinfection, according to laboratory procedures before disposal (World Health Organization 2022). Laboratories must comply with national reporting requirements and report all positive or negative test results to the authorities without delay. States Parties are reminded of their obligation to report relevant public health data to WHO (Koenig et al. 2022; World Health Organization 2022).

16.2.5 Avoiding Errors

Pre-analytical errors include those that occur during specimen entry, centrifugation, aliquoting, pipetting, dilution, and batching of specimens for entry into automated analyzers. Most studies have shown that pre-analytical and post-analytical errors account for most laboratory errors, while analytical errors account for fewer errors (Bonini et al. 2002). The common pitfalls have been summarized in Table 16.2.

Table 16.2 Summary of common pitfalls and errors during the pre-analytical phase
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16.3 Analytical Phase

The analytical phase involves the utilization of diagnostic tools for determining the presence of virus. These tools are critical to controlling and monitoring infectious diseases. NAATs, sequencing, and serologic studies have been conducted for Mpox. Besides NAATs and sequencing, complementary isothermal amplification methods have been designed. However, their clinical utility remains unproven. Although isothermal amplification does not require heat cyclers, saving diagnostic costs, it has limitations in selectivity and operational simplicity, leaving potential for future advances.

16.3.1 Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) is the gold standard for nucleic acid detection. According to WHO standards, PCR (conventional or real-time) is a standard technique for Mpox laboratory validation. The usual poxvirus diagnostic method combines clinical signs of the disease with a generic poxvirus PCR assay, followed by a poxvirus-specific PCR assay. These pan-pox real-time PCR assays are useful in accurately diagnosing poxvirus infection. Although real-time PCR (RT-PCR) is the most widely used molecular test for confirmation of Mpox, conventional PCR can also be used. Multiplex PCR that tests for Mpox and other pathogens or clade-specific PCR that is able to differentiate between two clades of Mpox could also serve as confirmatory test (Shchelkunov et al. 2005; Li et al. 2006).

Traditionally, Mpox detection uses PCR amplification and restriction digestion of PCR-amplified fragments by restriction fragment length polymorphism (Paniz-Mondolfi et al. 2023). Based on this technique, a hemagglutinin PCR (HA-PCR) assay was developed that employed Mpox-specific primers and TaqI (Thermus aquaticus) restriction digestion. However, this approach could not distinguish between Mpox clades. Subsequently, to improve the detection accuracy of the PCR assay, the A-type inclusion body protein (ATI) gene was used to identify Mpox clades and other orthopoxviruses using PCR-based gene amplification and XbaI-based (Xanthomonas badrii) restriction digestion (Huggett et al. 2022). Another breakthrough was the discovery, sequencing, and comparison of the ATI gene open reading frame (ORF) with other related poxviruses (Vivancos et al. 2022). Unique deletions in the Mpox ORF were detected and used for specific identification of the Mpox ATI gene. Nineteen Mpox lineages were distinguished using this PCR technique. Specificity was validated by BglII (Bacillus globigii) restriction digestion.

The real-time PCR is faster and has higher sensitivity than conventional PCR. Due to the low GC content and almost 90% genomic similarity to other Eurasian orthopoxviruses, the development of a TaqMan test specific to Mpox is challenging. Researchers have developed a real-time PCR assay using probes based on the minor groove binding protein (MGB) (Cheema et al. 2022). MGB improves the sensitivity and specificity of the assay by stabilizing the probe-template interactions and allowing the use of short probe sequences for the detection of single nucleotide polymorphisms (SNPs). At a concentration of 10 ng, 15 isolates of Mpox were detected by the technique. Test efficiency was 97% when using freshly diluted DNA but dropped to 67% after multiple freeze/thaw cycles. These results indicated that a fresh sample should be used for optimal assay efficiency (Gul et al. 2022).

The development of a clade-specific real-time PCR detection method is a problem due to the lack of unique sequences. The terminal genomic sequences of Mpox clades were studied for distinguishing isolates from the two distinct clades. The G2R protein gene was chosen to construct primers and probes for the G2R-WA West African Mpox-specific assay because the terminal sequences have higher sequence diversity than the central genomic region, and the G2R protein gene is located in the terminal genomic region (Huang et al. 2022; Hraib et al. 2022). The G2R protein gene of the Congo Basin clade did not contain unique sequences. Consequently, Congo Basin Mpox targets another gene, the C3L protein gene.

16.3.2 Sequencing

Genome sequencing is the gold standard for identifying new or modified viruses. In addition to the identification of the target virus, sequencing may also reveal the presence of additional viruses in the sample, which may be useful in the development of a treatment strategy for a particular disease. There have been reports of Mpox detection using quantitative PCR and genome sequencing (Erez et al. 2018). Genome sequencing was used when a mild variant of human monkeypox was first identified outside of Africa during an outbreak in the USA in 2003. This outbreak was extremely worrisome because the culprit virus was traced back to imports of West African rats infected with monkeypox virus. Genomic sequencing confirmed the existence of two clades of monkeypox virus and allowed viral proteins to be predicted that could account for the observed variations in human pathogenicity (Likos et al. 2005).

So far, more than 600 Mpox genome sequences from recent outbreaks in non-endemic countries have been published (Gao et al. 2023). As this is the only way to provide conclusive evidence of a deliberate release, the advances in sequencing technology will undoubtedly make this procedure an important forensic tool in the event of a re-emergence of smallpox. It should be noted that sequencing is a time-consuming technique requiring expensive equipment, experienced personnel, and competent computational bioinformatics. These constraints will need to be addressed in order for the full potential of genome sequencing methods to be realized (Cohen-Gihon et al. 2020; Ejaz et al. 2022).

16.3.3 Isothermal Amplification

Over ten different isothermal amplification techniques have been demonstrated for nucleic acid detection. Loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) are well-studied viral detection techniques based on the isothermal amplification of nucleic acids (Gul et al. 2022). The LAMP technique uses two internal primers, the forward internal primer (FIP) and the backward internal primer (BIP), two external primers, the forward external primer (F3) and the backward external primer (B3), and a strand displacing DNA polymerase. The reaction is performed at 60–65 °C. To accelerate the amplification process (LB), the forward loop (LF) and backward loop (BF) primers are used (Gul et al. 2022; Feng et al. 2022a).

The F3 primer then displaces the FIP strand, resulting in a single-stranded DNA (ssDNA) strand that is used as a template by the BIP. The BIP, which also contains two target sequences complementary to the template DNA at two separate locations, initiates strand elongation of the ssDNA template, which is later displaced by the B3. The 5′ and 3′ ends of the template DNA contain inwardly complementary sequences, resulting in a stem-looped DNA that is exponentially amplified by loop primers (Feng et al. 2022a; Saied et al. 2022). LAMP-based Mpox clade-specific assays have been established that preferentially identify West African (W-LAMP) and Congo Basin Mpox (C-LAMP) clades. The LAMP reaction is analyzed by turbidimetry, and the LAMP products are confirmed by restriction digestion. A LAMP-based approach for rapid Mpox identification has been developed for the identification of Mpox clades. The approach has good sensitivity and response time (Gul et al. 2022).

Although the LAMP is very promising, it requires a reaction time of 60 min and six primers. In addition, primer design is rather complicated. To overcome these limitations, RPA has been presented as an attractive solution. Identification of the RPA signal can be done by gel electrophoresis, real-time monitoring, or lateral flow assays. In the case of real-time detection, the fluorogenic probe and primers are introduced into the reaction system. The exonuclease cleavage results in a fluorescent signal. Detecting Mpox with RPA provides good results with shorter assay times and lower reagent costs (Ejaz et al. 2022).

16.3.4 Oligonucleotide Microarray Analysis

Methods for the differentiation of PCR-amplified, fluorescence-labeled DNA fragments by hybridization, and orthopoxvirus species-specific DNA immobilized on a microchip have recently been published (Lapa et al. 2002). The published research identifies and distinguishes four orthopoxvirus species that are harmful to humans (variola virus, vaccinia virus, cowpox virus, and Mpox virus) from the varicella zoster virus (Laassri et al. 2003). The microchip contains a large number of different oligonucleotide probes that are tailored for each type of virus to ensure redundancy and robustness (Huang et al. 2022).

16.3.5 Immunodiagnostics

The hemagglutination test and agar gel immunodiffusion are simple and inexpensive methods for detecting viruses. Both assays do not require a secondary anti-species antibody, making them ideal for antibody detection in any host species. Hemagglutination relies on erythrocyte agglutination in the presence of a virus (Erez et al. 2018). Based on this mechanism of hemagglutination, another test has been developed, known as the hemagglutination inhibition (HI) assay. In the HI method, virus-specific antibodies are used for the detection of viral antigens. As a result, HI was chosen as the technique of choice for screening mammalian blood samples in the quest for natural reservoirs of the Mpox and cowpox viruses. Both the hemagglutination and the HI tests are used to evaluate strains of Mpox. The tests do not distinguish Mpox from variola or vaccinia viruses. However, they can distinguish cowpox from Mpox and can be used to determine the evolutionary relationships of viral strains or species (Erez et al. 2018; Hraib et al. 2022). HI is generally more sensitive than agar gel immunodiffusion, although it has the same low specificity. The creation of a precipitation line or zone in solidified 1% agar where viral antigen and particular serum antibodies meet is the readout of the latter. If a precipitation line forms with the serum sample that is confluent with that formed by the positive control antigen, the test material is positive. This test was also extensively used as a screening tool for the diagnosis of avipoxviruses, orthopoxviruses, and Leporipoxviruses, but it was discontinued due to its low sensitivity and the time required (2–3 days) to produce results.

For protein detection, ELISA is a popular technique. The results of an antigen-capture ELISA are generally available within three hours. Because of its low cost, ease of use, and relatively quick performance, ELISA is the method of choice in the field when fully equipped laboratory facilities are not available. ELISAs are most utilized in epidemiological research or in disease monitoring programs for endemic and exotic illnesses. In the 2003 US epidemic, an orthopoxvirus-specific IgM test was devised and used to measure acute-phase humoral immunity to Monkeypox virus. IgM antibody testing opened a wider window for collecting samples beyond the rash stage of sickness, which is useful for proving disease retrospectively and/or from remote places.

The Orthopox BioThreat® Alert Assay is a commercially available technology for the detection of the Orthopoxvirus (Tarín-Vicente et al. 2022). This antibody-based lateral flow assay collects viral antigens and measures viral load at a level of 104 PFU/mL (plaque forming units/milliliter). As a result, this test is approximately ten times more sensitive than electron microscopy. Surface protein A27 is the most immunogenic protein for viral particle capture and detection. After a thorough screening of antibodies that bind to A27, an ELISA technique for orthopoxviruses, including Mpox, has been established. The technique’s detection limit is 1 × 103 PFU/mL. An Antibody Immuno Column for Analytical Processes (ABICAP) immunofiltration device is used in similar work (Hraib et al. 2022).

T-cell response testing is not practical for everyday use even though cell-mediated immune responses are thought to be essential for long-term immunity and play a significant role in poxvirus infections. Historically, serological testing in poxvirus diagnoses has only measured humoral immune responses. A fourfold increase in antibody titer between acute and convalescent serum tests in the neutralization tests is typically deemed positive for a poxvirus infection. Antibodies, however, cross-react across members of each poxvirus genus, rendering serology generic for a single virus species. Although this reduces specificity for serological diagnosis, it has the advantage of selecting the least pathogenic member of a genus as the test antigen. This might be a vaccination strain or a member that is known to be incapable of infecting humans. Another important aspect of the neutralization tests to remember is that, in contrast to a simple antigen-antibody binding as in an ELISA, the plaque reduction neutralization tests offer biological information regarding a specific serum’s potential to neutralize virus. As a result, it is the tool of choice for monitoring immunization success.

16.3.6 Electron Microscopy

The search for biomarkers for a newly emerging virus is difficult, and the immediate use of conventional diagnostic methods may be hampered. One powerful option in this area (Kim et al. 2022) is whole-particle detection using electron microscopy (EM). Because it provides information on the shape and amount of viral load using a limited sample volume, transmission electron microscopy (TEM) is an excellent first step in detecting viruses (Feng et al. 2022a). TEM was widely used during the eradication of smallpox, as it became a standard method in diagnostic virology in the 1950s. Due to the characteristic shape of the virion and the often high number of particles present in lesions caused by poxviruses, electron microscopy may offer one of the earliest hints to the source of an unidentified rash disease. The diagnostic accuracy of EM detection can be enhanced by using virus-specific antibodies in immunoelectron microscopy (IEM).

EM has been used to detect Mpox and other orthopoxviruses. Biopsy specimen from infected individual can be examined under electron microscope with negative staining. Mpox appears as intracytoplasmic brick-shaped with lateral bodies and a central core measuring about 200–300 nm (Alakunle et al. 2020). Scabbed or minced material from lesions can be crushed in a mortar with sterile sand or pulverized after freezing in liquid nitrogen. Commercially available systems are advantageous because they allow for method uniformity. Alternatively, the steel shot technique using BB-sized projectiles can be used. It should be noted that these treatments generate kinetic energy that may result in thermal inactivation of the virus. Though irrelevant for electron microscopy, this inactivation may interfere with attempts to isolate the virus in cell culture (Gelderblom and Madeley 2018).

The minimum number of virus particles required for the diagnosis is set at 105 particles. Electron microscopy can provide results within 2 h of sample receipt, as inspection can take up to 30 min depending on the amount of particles. Although excellent for validating laboratory virus detection results, EM has some disadvantages, including high instrumentation costs, needing trained personnel, and poor sample throughput (Tarín-Vicente et al. 2022). Furthermore, morphologically, Mpox is like other species of orthopoxvirus; thus, this test cannot provide definitive diagnosis. It however provides evidence that the infectious agent belongs to poxviridae family which can aid in distinguishing herpes and parapox viruses (Alakunle et al. 2020; Ferdous et al. 2023).

16.3.7 Wastewater Fingerprinting

The current infectious epidemics have highlighted the need for smart diagnostic tools which harness the potential of AI (artificial intelligence), Internet of things (IoT), machine learning (ML), and other related approaches in global healthcare (Mao et al. 2022). Wastewater fingerprinting (WFP) is a relatively new method that can accomplish many noble goals, including determining community exposure to illegal drugs, persistent contaminants, or other hazardous chemicals. During epidemics, since people share sewerage, water collected from common sampling locations could provide useful information on virulence within specific communities (de Jonge et al. 2022).

Mpox has been found in body fluids, including urine, seminal secretions, saliva, nasopharyngeal fluid, serum, plasma, stool, and vaginal fluid (Farahat et al. 2022b). The virus can get into the environment from infected people by shedding skin, bathing, urinating, defecating, or by releasing seminal fluid into water. Based on this idea, Dutch researchers recently tested wastewater samples for Mpox using PCR. Although the authors found the virus in multiple samples, it is not clear how the virus got introduced into the water. Furthermore, it cannot be ruled out that animal reservoirs of Mpox in nearby waters could be shedding the virus. Further research is necessary to determine if the Mpox DNA findings are from a human source (Yu et al. 2022).

In both studies, Mpox DNA was found to be more abundant in the solid fractions of the wastewater compared to the liquid fractions, suggesting that it could be used to identify the virus (Uhteg and Mostafa 2023). Since wastewater collection is a complicated matrix, establishing a standard approach for identifying viral in wastewater seems demanding. Furthermore, it remains to be seen whether Mpox is persistent and contagious in water. These difficulties will have to be considered in the development of detection systems for WFP applications.

16.3.8 Viral Isolation and Culturing

First documented in 1937, the use of chick embryos for poxvirus diagnosis has become an important technique. Only four Orthopox species—variola, Mpox, cowpox and vaccinia—produced chorio-allantoic membrane pocks (CAM) in chicken eggs (Paniz-Mondolfi et al. 2023). Differences in poxvirus morphology observed in two-week-old embryos incubated at 34.5–35 °C helped in distinguishing the species. Successful results from inoculation of CAM with sample from smallpox patients made the assay widely successful during the smallpox eradication campaign (Erez et al. 2018). However, because of the availability of alternative methods (cell cultures) for identifying the appropriate agent, inoculation of animals should be avoided. When all other procedures have failed, the use of the putative natural host animal for virus isolation could be considered.

Cell culture-based virus isolation continues to be the gold standard to calibrate all newer methods. In addition, it can be used to identify a previously unknown pathogen. This is especially important when routinely testing biological samples for viral presence. Moreover, infected viral cell cultures are the sole method for ensuring an immortal supply of live viruses for future studies. However, it is noteworthy that viral isolation sensitivity in cultured cells is still only partially achieved by modern approaches.

16.3.9 Avoiding Errors

Though relatively rare, there are a few common errors that may result in incorrect or unreliable test results (Plebani 2010). These errors can be caused by a variety of factors, including specimen or environmental contamination, use of incorrect reagents or expired reagents, inadequate quality control, or human error. Careful adherence to proper laboratory procedures, the use of fresh and properly stored reagents, and regular training are important to minimize the risk of these errors. Furthermore, test results must be carefully reviewed and interpreted to ensure an accurate diagnosis. Adequate aseptic procedures, use of controls, and internal and external proficiency testing (Agarwal 2014) are essential for minimizing analytical errors. It is also important to use equipment that is properly calibrated and maintained and to consult with experts when necessary to ensure the accuracy of the test results (Hollensead et al. 2004). If any of the assay controls fail, the procedure should be repeated. This is critical because several factors can lead to false-negative results, such as poor specimen quality, improper handling or shipping, or technical reasons inherent in the test, such as DNA extraction failure.

16.4 Post-analytical Phase

A diagnosis of poxvirus infection should be made or ruled out based on the results and the patient’s symptoms. Consultation with specialists or additional testing to confirm or clarify the diagnosis may be necessary. The post-analytical phase involves the interpretation of the test results, provision of medical records, and reporting to the relevant health authorities (national and regional) for surveillance.

16.4.1 Preparing Medical Records

Proper interpretation of test results is an important aspect of the post-analytic phase of poxvirus molecular diagnostics. Interpretation of test results requires that all relevant information be reviewed and considered ensuring the results are accurate and complete. This involves confirmation of the medical history, the specific diagnostic test, and all relevant clinical guidelines (Bolboacă 2019). Another critical part of the post-analytical phase is the preparation of accurate and understandable patient reports. Patient reports should be an accurate description of the test results and their clinical significance. Avoiding technical jargon or ambiguous terms that may confuse patients or other healthcare providers is essential. It should include information about the patient’s complaints, the diagnostic test(s) performed, and the test(s) results (Meyer et al. 2021). Any recommendations for follow-up testing or treatment should also be included.

16.4.2 Reporting and Surveillance

Pursuant to Article 6 of the International Health Regulations (IHR) 2005, it is mandatory for WHO member countries to report probable and confirmed cases to the WHO for surveillance purposes (Panag et al. 2023). In this regard, the WHO has provided the case reporting form (CRF) that acts as a template for data collection and reporting. However, variations in case definitions and diagnostic modalities lead to heterogeneity in the collected data making data modeling challenging (Panag et al. 2023). In the USA, guidelines for reporting results of diagnostic tests for orthopoxvirus, non-variola orthopoxvirus, and Mpox virus have been issued by the CDC. According to these recommendations, all laboratories that perform diagnostic testing for Mpox are required to report all test results, including positive, negative, and equivocal results, to state, tribal, local, or territorial public health departments (STLTs). Laboratories are also encouraged to provide as much information as possible about the patient and specimen, including demographic information, to facilitate a rapid public health response and stop the spread of the virus. All information received by CDC from STLT health departments is de-identified to protect patient privacy (Centers for Disease Control and Prevention 2022b). It is important to note that the surveillance guidelines are different from clinical management guidelines issued by CDC and WHO.

16.4.3 Avoiding Errors

During the post-analytic phase, several common errors may occur. These errors may include misinterpretation of test results due to lack of understanding of diagnostic test principles, inadequate training, or other factors. Errors in data entry, transcription, or communication between healthcare providers can also result in inaccurate patient reports or diagnoses. A lack of follow-up care is another type of error at this stage that can lead to the inadequate management of patients’ conditions. Careful evaluation and interpretation of test results, improved reporting format, consultation with specialists when necessary, and accurate and precise communications between healthcare providers are critical to reducing the possibility of errors in the post-analytic phase (Hickner et al. 2014; Blendon et al. 2002).

16.5 Conclusion

Early and accurate diagnosis is critical to preventing and managing Mpox. Therefore, awareness of signs and symptoms is essential. Mpox diagnosis should rely upon a combination of clinical, epidemiological, and laboratory investigations. Although RT-PCR is the first line and gold standard test to confirm Mpox, other tests like immunodiagnostics or electron microscopy can be used if strong clinical suspicion remains despite negative PCR. Laboratory professionals should take a holistic approach to laboratory diagnosis and work closely with physicians to provide accurate diagnostic services.

Quality control must be implemented in all phases of the diagnostic flowchart, along with periodic reviews and audits. Protecting patient interests and providing quality services is paramount. For an accurate and rapid laboratory test to identify Mpox, specimen collection, storage, and shipment are critical. Healthcare workers are advised to use personal protective equipment and hand hygiene during specimen collection. Cutaneous lesions, whether swabs, exudates, or crusts, are recommended for laboratory testing of Mpox. Depending on the stage of the rash, specimen collection procedures may vary. Research into the detection of Mpox is continuously evolving with potential for future advances to truncate the further transmission of this virus.