12. Sampling and Diagnosis of Mpox virus
The PCR test can detect the monkeypox virus (MPXV), which causes monkeypox. Personal protective equipment (PPE) must be worn while working within 1 metre of a suspected monkeypox case in order to secure the safety of healthcare workers. This includes droplet and contact precautions, as well as eye protection, if spills are possible during diagnostic procedures such as pharynx biopsies or lesion samples. When gathering a sample for diagnostic testing, it is essential to obtain a viral biopsy from an exposed lesion or the surface of a vesicle (a small fluid-filled abscess). If there are multiple incisions, it is crucial that the sample be obtained from a vesicle, an ulcer, or a vesicle with a crust. Rub the probe gently over the lesion and position it in a tube containing viral culture medium or viral transport medium. Additionally, a pharynx sample should be taken if pharyngeal lesions are present. As unlabeled tubes cannot be processed, designate the collection tube with the patient’s name, date of birth, and location of the sample. When high-risk contacts of a confirmed or highly probable case of monkeypox develop systemic symptoms but lack a rash or lesions suitable for sampling, a viral pharynx swab can be taken. Notably, even if the throat biopsy yields a negative result, individuals must continue to monitor their symptoms, adhere to isolation protocols as directed by their local health protection team, and be reevaluated and sampled if additional symptoms develop. It is crucial to package the samples separately when investigating other infections, such as sexually transmitted infections. Samples should ideally be sent to a nearby laboratory for examination. However, if MPXV testing is not available locally, the laboratory should send the samples to the other advance laboratory for MPXV PCR, while continuing to follow their normal local procedures for testing other infections such as HSV, varicella-zoster virus (VZV), or syphilis. For monitoring confirmed or highly probable cases of monkeypox, additional samples for follow-up testing are required. These samples should consist of a lesion swab and a pharynx swab, both of which should be stored in viral transport medium. In addition to obtaining a blood sample using an EDTA tube, a urine sample should be collected in a universally sterile container. It is crucial to submit these samples to a facility capable of conducting MPXV testing. By incorporating these samples into the monitoring procedure, healthcare providers can collect valuable information to evaluate clinical deterioration or determine if a patient should be transferred from isolation to an inpatient setting. In summary, appropriate sampling techniques are essential for accurate diagnostic testing in cases of monkeypox. After collecting the specimens, it is important to store them appropriately for testing. Place the specimens in sterile leak-proof containers, ensuring durability and suitability for shipping and temperature conditions. It is recommended to refrigerate the specimens (2-8°C) or freeze them (-20°C or lower) if testing cannot occur promptly after collection. Specimens such as dry swabs, swabs in viral transport medium (VTM), or lesion crusts stored at 2-8°C can be tested within 7 days from collection. If stored frozen (-20°C or lower), swab specimens in VTM and lesion crusts can be tested within 30 days from collection, while dry swabs stored frozen can be tested within 60 days from collection. When shipping specimens for laboratory testing, it is important to follow certain guidelines. The ongoing Mpox outbreak has been linked to Clade II of the mpox virus, which is now classified as the West African clade. To ship specimens suspected or confirmed to contain Clade II of the Mpox virus, they should be labeled and categorized as Biological Substance. It is recommended to individually bag the specimens to prevent the rejection of all specimens in case of a leak. To maintain the required temperature, specimens should be conveyed on dry ice whenever feasible. It is not recommended to ship specimens at room temperature, as specimens received outside the permissible temperature range will be denied. Even if there are temperature fluctuations because of the refrigerant used, both the primary container and secondary packaging should maintain their integrity. The packaging of dry ice must prevent pressure accumulation and container rupture while allowing for gas discharge. The specimens should only be packaged and shipped by personnel trained in hazardous transportation requirements. The outer package must be marked and labelled with the sender’s name and address, the recipient’s name and address, a biological substance label, the correct shipping name (Biological substance), an identification number, and the name and telephone number of the shipper’s responsible party. Following these instructions ensures the safe and correct shipment of mpox samples for laboratory testing.
To confirm Mpox virus infection, diagnostic tests, and clinical and epidemiological data must be correlated. The diagnosis of Mpox(Table 2) virus infection is based on clinical signs, laboratory tests, and medical history2. By checking for lymphadenopathy at the prodromal stage of the illness, it is possible to clinically distinguish Mpox from chickenpox or smallpox53. The specimen samples are collected and forwarded to the lab to verify the diagnosis in compliance with WHO criteria. PCR is the most widely used laboratory diagnostic method to identify Mpox due to its accuracy and sensitivity 54. The fluid that is produced by vesicles and pustules as well as dry crusts are the best samples to use in the diagnosis of Mpox. When a lesion sample needs to be kept cool and kept in a dry, sterile tube without a viral transport medium, a biopsy appears to be an alternative in some circumstances. PCR blood tests are often inconclusive and should not be routinely obtained from patients because viremia typically lasts for a short period compared to the time the material is taken after symptoms start55. The recommended specimen type for diagnosing monkeypox is skin lesion material, including swabs of lesion exudate, roofs from multiple lesions, or lesion crusts. Testing should be offered to individuals meeting the suspected case definition, considering both clinical and epidemiological factors. It can be challenging to differentiate monkeypox based solely on clinical presentation, and other potential causes of skin lesions should be considered. Safety procedures, including proper personal protective equipment and handling of specimens, must be followed. The collection and storage of specimens, including skin lesion material and additional types for research purposes, are needed. Nucleic acid amplification testing (NAAT), particularly polymerase chain reaction, is recommended for confirming monkeypox infection. Reagents, controls, waste disposal, and biosafety measures should be appropriately managed during testing. Serological testing and genetic sequencing are additional tools for diagnosis and surveillance. Overall, adherence to biosafety measures and risk assessments are crucial in handling specimens and conducting laboratory testing for monkeypox. For confirmation of monkeypox virus (MPXV) infection, clinical and epidemiological data should be considered. Positive detection using an orthopoxvirus (OPXV) polymerase chain reaction (PCR) assay, followed by MPXV confirmation via PCR and/or sequencing, or in suspected cases, positive detection using an MPXV-specific PCR, confirms MPXV infection. Although MPXV-specific confirmatory testing is preferred, a positive OPXV PCR assay result is sufficient for laboratory confirmation. In instances where clinical presentation and epidemiology indicate MPXV infection despite negative PCR results, serological testing can be utilised to investigate prior infection. False-negative results can be influenced by factors such as specimen quality, handling, transportation, and technical issues. Genetic sequence data (GSD), obtained through sequencing positive MPXV specimens, can provide valuable information on the virus’s origins, epidemiology, and characteristics. WHO encourages countries and laboratories to share GSD, including raw data, through public access databases to enhance understanding and collaboration. Sequencing can be performed using Sanger or next-generation sequencing methods. Laboratories must adhere to national reporting regulations. Positive or negative test findings should be immediately reported to national authorities. States Parties to the IHR are reminded of their responsibility to communicate relevant public health information with WHO for occurrences that they have notified WHO. Access to quick and reliable laboratory testing of samples from cases under investigation is critical for diagnosing and monitoring this developing virus. All nations should have access to reliable testing, either locally or by referral to laboratories in other countries willing and capable of diagnosing OPXV or MPXV. WHO’s Regional Offices can help Member States gain access to testing through referral. Inactivation of samples at the local laboratory, where suitable and securely conducted, may improve referral and alleviate logistical issues. Countries are invited to exchange their sequencing data so that the present outbreak can be better understood.