Introduction

World Health Organization (WHO) reported mpox (formally known as monekypox) as a global public health challenge on July 23, 2022 [1]. The disease has been reported at least in 111 countries with 179 deaths and more than 93,937 confirmed cases between 06 May 2022 and 03 February 2024 [2]. First time, the monkeypox virus (MPXV) was reported in 1958 in the Democratic Republic of Congo. It is a DNA virus, producing symptoms such as rash, fever, headache, and chills with lymph pain [3]. MPXV DNA has been reported in urine, stool, saliva, semen, spit, or body sweat of clinical symptoms [4]. Many studies have confirmed MPXV DNA in wastewater and environmental samples [5,6,7]. The initial confirmed cases of mpox in 2022 indicated the possibility of a new pandemic, although positive cases decreased later [8]. Wastewater-based surveillance (WBS) has emerged as a supplementary tool to clinical surveillance for the surveillance of various infectious diseases including SARS-CoV-2 [8], poliovirus, hepatitis E virus, rotavirus, influenza A virus, dengue virus, and antimicrobial resistance pathogens circulating at the population level [4]. WBS can be used to inform people and local health authorities about the ongoing outbreak, so they get prepared for management.

WBS of MPXV can be used to monitor activity/trends and hotspots at the population level, which provides evidence even before their detection by clinical-confirmation (Fig. 1) [9]. As this virus is continuously spreading, it could be important for establishing WBS for the virus, in possible risk areas, being prepared for the possible outbreak. Table 1 summarizes the cases of detection of MPXV and their analysis in wastewater globally in many countries.

Fig. 1
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Wastewater-based surveillance of mpox: an overview of the procedure for decision-making

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Table 1 An overview of the countries and/or regions with detected cases of mpox disease and analyses of MPXV in wastewater
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Infectious pathogens detected in wastewater can be result from their shedding in personals from urine and feces, oral or nasal secretions, and sloughing of skin [10]. As a result, wastewater samples can use as a surveillance tool for monitoring trends of public health diseases [9]. MPXV was reported in both animal feces and human feces [11]. It proves that feces either carry excreted MPXV or are contaminated during defecating. In addition, human skin rushes, secretion, and excretion are ending up in wastewater. Moreover, MPXV DNA from positive patient’s crusts, scabs, and lessons may be discharged with rinsing, bathing, washing, or toilet usage with wastewater [1, 4, 10, 12]. On 10 May 2023, WHO announced multicountry mpox outbreak is not a global public health emergency in the 5th International Health Regulations (2005) (IHR). However, further mpox waves and outbreak might appear, and the committee suggested to concern about new, significant cases [13].

Few literature reviews [4, 14], experiment-based has been published in WBS of MPXV (Table 1). We have summarized important information from these articles in Table 1. This article provides viral morphology, epidemiology, and symptoms to detect patients, various possible diagnostic methods, and WBS strategies. We also indicate the biosafety, biosecurity, and laboratory rules to handle this contagious virus. In our notice, a few research laboratories are currently underway to assess vaccines’ activity, feasibility, safety, and appropriateness to prevent and control MPXV.

Outbreak of Mpox Disease

Concurrent with the COVID-19 pandemic, unprecedented outbreaks of orthopoxvirus were reported worldwide repeatedly over time [12, 15, 16]. WBS has proved as an important tool for tracking COVID-19 status and for predicting its trend in any geographical area [9, 10, 17,18,19,20, 21••, 22, 23]. Other orthopoxvirus, for example, cowpox was detected in different countries in Europe, Middle East, and India,camelpox and buffalopox infections were found in India; vaccinia was recorded in South America [24, 25]. Previously, this disease was observed in Central and Western Africa and the Democratic Republic of Congo,sporadic clusters were found outside Africa in 2003 [4]. In May 2022, the UK first confirmed a few cases of mpox in Europe; after that, it was identified in Spain and Portugal [1]. As of 29 June 2023, 88,060 confirmed cases, with 147 deaths of mpox, have been reported from 111 countries [26]. The highest five countries with MPXV confirmed cases are the USA (30,298 cases), Brazil (10,950 cases), Spain (7559 cases), France (4146 cases), and Colombia (4090 cases) by June 21, 2023 [26, 27].

Characteristics of MPXV

MPXV is an enveloped double-stranded (ds) deoxyribonucleic acid (DNA) virus, belongs to the Orthopoxvirus genus of the Poxviridae family [28]. Other notable members of this group are the variola virus causes smallpox and the vaccinia virus [29], camelpox, cowpox, skunpox, taterapox, raccoonpox, and ectromelia virus [30].

MPXV contains 197 kbp genome containing an encapsulated virus, and 6 kbp IRTs (identical reversely terminal reads), a cluster of STRs (short tandem repeats), and nodal hairpin [31]. Poxvirus and vaccinia also belong to the same family of MPXV, while camelpox, cowpox, and volepox are notably from the same family group [30]. The clinical features of MPXV are observed to be similar to smallpox, which is clinically less severe and self-controlled [32]. Other signs and symptoms were chills and/or sweat, body pain, headache, diarrhea, and vomiting. The symptomatic patients are dominant but asymptomatic or subclinical, with 30% possible infection [33]. Despite growing evidence of acute or prior infection, the definite MPXV reservoir species is unknown. Infections with MPXV have been reported in a variety of rodents, including mice (Mus musculus), rodents (Oryctolagus cuniculus), ferrets, woodchucks (Marmota monax), jerboas (Jaculus sp.), and raccoons (Atherurus africanus) [34].

MPXV Transmission

MPXV can be transmitted to any infected persons through close or intimate contact or body fluids, such as rash, scabs, sweat, and secretions (respiratory secretions, vaginal discharges, etc.); contaminated surfaces; and substances (cloths, bed, or towels) [10, 20, 21••]. Sexual contact including anal, vaginal, oral sex, or multi-sex; sex with the same gender, especially men with men; sucking or touching reproductive organs (penis, vagina, labia, and testicles), kissing, hugging, or massage; and face-to-face contact without a mask are considered to be a major concern [35]. Domestic animals such as pets (cats, cows, and dogs) can play as a vector for MPXV transmission, which is not being determined currently, although the first positive MPXV case was reported in a dog [3]. This indicates a reverse zoonosis from humans to animals.

Symptom of Mpox Disease

The clinical signs and symptoms of mpox are fever, headache, weakness found initially, skin rash, and lymph pain. Mostly the infection is self-limiting and observed moderately in the range where incubation time is found 5–21 days sometimes extended up to 5 weeks [36]. Severe conditions were identified in the case of children, young adult patients, pregnant women, and immunocompromised persons. Skin rashes develop in a distinct process macules convert to papules, then vesicles form, pustules, and crusts are found at last. It was also found that the number of rashes is proportional to patients’ severity. The rash was observed maximum in the face, limb, and trunks while the face (in 95% of cases), palm of hands and sole of the feet (in 75% of cases), oral mucous membrane (in 70% of cases), genitalia (30%), and conjunctiva (20%) as well as the cornea [32]. The early invasion stage, which lasts between 0 and 5 days, is characterized by fever, intense headache, lymphadenopathy (swelling of the lymph nodes), back pain, muscle aches, and lack of energy [37]. The latter prodromal stage is characterized by skin eruption, which begins within 1–3 days of a fever.

Detection and Monitoring of MPXV

Many countries, particularly low and middle-income countries have poor sanitation conditions. This condition can more critically different between urban and rural areas. Municipal wastewater treatment plants is often treated in domestic/municipal wastewater treatment plants (WWTPs), mainly based on biological technologies as a secondary treatment process, and the tertiary phase (disinfection) is usually occasional or poor. In one preprint study published in medRxiv, environmental samples from the isolated hospitalized patient with MPXV in the UK, surface swabs, PPE of healthcare workers, and air sampling were used to analyze MPXV qPCR [38]. This research team found 90% (n = 73) samples with surface contamination, with an average CT value of 31.5 [38]. Another preprint has also reported the MPXV DNA from wastewater between May 23 and July 3, 2022, while the sampling country (the Netherlands) had first confirmed human cases on May 20, 2022. According to this study, 64% (58/90) of samples showed positive signals for MPXV genes MPXV DNA from WWTP and wastewater samples. All specimen-related laboratory operations should be carried out in a biological safety cabinet (BSC) or other appropriate primary containment devices, in conjunction with personal protective equipment. All types of specimens must be centrifuged in closed containers put in sealed safety cups or rotors loaded and unloaded in a biological safety cabinet.

Various ways are used to detect mpox based on serological, molecular, immunological, or prepaid diagnostic tests. Wastewater-based epidemiology (WBE) can be alternative to Clinical Diagnosis Tests (CDT) to monitor the prevalence of patients and trends in this disease [39]. Although it is challenging to collect and transport wastewater samples conveniently, a validated method and autosamplers with the help of clinical samples can work well [40]. One may use CDT and WBS synergistically to monitor the current outbreak of mpox. The wastewater samples collection, nucleic acid extraction, qRT-PCR, and whole genome sequencing are the main steps of wastewater-based surveillance, while the sampling spot’s identification and time need to draw attention (Fig. 2). Autosampler or manual specimens, including grab and composite samples, can be collected. However, real-time quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR) are preferred and available as sensitive methods [8]. Apart from taking wastewater samples, additional information about the prevalence of diseases will be collected from different secondary sources, like hospitals and diagnostic centers with wastewater physicochemical properties (e.g., pH, temperature, salinity, BOD, COD, DO, TDS, total coliform form and fecal coliform, E. coli). Wastewater treatment plants can be used as a suitable place for wastewater sampling for detecting MPXV which stands for the entire population from a fixed community or catchment area. Positive patients excrete skin rash, spilt, urine, sweat, feces, and urine mixed with wastewater from their toilets, basins, and bathroom drains. Wastewater samples can be used as a surveillance tool for monitoring public health and tracking the trend of infectious diseases from a sewer network (Table 2). Viruses from the same family, such as smallpox, were also identified in the patient’s urine [25]. Although there is no abundant data regarding wastewater and MPXV DNA, persistence, and ultimate fate, research is ongoing. According to the vaccinia virus, genetic materials found in marine water and fresh water pointed to the persistence of this DNA [41]. This literature-based study indicated the persistency of orthopoxvirus DNA in wastewater even in wastewater treatment plants. McCall et al. reported the presence of poxvirus DNA in untreated samples collected from urban wastewater for metagenomics analysis [42]. Groundwater, sub-Saharan African surface water, dug wells, and spring water samples also showed positive signals for the Poxviridae family virus which was identified by metagenomics analysis [14]. Previously published articles support that poxvirus DNA is resistant in comparatively higher temperature and persist in fomites [43,44,45,46].

Fig. 2
figure 2

The standard procedure adapted for the surveillance of monkeypox virus (MPXV) from wastewater

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Table 2 A summary of the successful WBS tools for tracking the trend of infectious diseases and monitoring public health [71, 72]
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In one preprint version, MPXV DNA level was assessed in liquid and solids both wastewater samples. MPXV DNA was found 103 times larger in the solid samples from the liquid wastewater samples. La Rosa et al. used the polyethylene glycol/sodium chloride precipitation method which was used for SARS-CoV-2 previously for MPXV WBS with little modifications. The aluminum adsorption-precipitation method was used to analyze 312 samples collected from 24 different wastewater treatment plants in Spain between May 9 and August 4, 2022 [47]. This study identified 17% positive samples (56/312), and range of genome copies found between 2.2 × 103 and 8.7 × 104 (gc)/L. Poland detected MPX viral DNA in the Left-Bank WTP mostly from mid-September through the end of October, 2022, and in the Central WTP during weeks 29, 43, and 47. This finding suggested that data from sewage treatment plants differ from hospital data [48]. This finding is consistent with the hypothesis that clinical diagnosis misses many infected MPX-positive patients.

Data Sharing and Research Collaboration

Globally concerted efforts practicing open data sharing and open science priorities are required to generate and gain adequate knowledge on monkeypox are crucial for rapidly tackling the epidemic and reducing long-term human monkeypox virus infection [19, 23, 49, 50]. Several countries such as the USA, France, Italy, Thailand, and the Netherlands successfully identified MPXV DNA from wastewater samples [21••, 51••, 52••, 53••, 54••, 55••]. The purposes of WBS of MPXV are rapidly identified hotspots, clusters, and areas of the source of spreading this virus. Data sharing publicly is crucial in this situation, where worldwide data can be deposited and follow an archive for sequence submission of MPXV. Furthermore, web-based dashboard making should be started for alarming neighboring countries. Inter-laboratory assessment and international quality control system must open to maintain internal and external facilities. In addition, campus-based WBS can play an important role here in the identification of patient numbers earlier than clinical tests. It is also necessary to publish research articles including review papers as early as possible to spread the knowledge to all. Genomics analysis of the MPXV adapted to the epidemic and non-epidemic regions is critical for vaccine development and therapeutic interventions. The genome sequence is an icon of early twenty-first century biology and carries a signature of evolutionary history. The whole genome sequence analysis from wastewater from the epidemic and non-epidemic areas needs to perform as genome sequences of any pathogenic microorganisms can be used to study the comparative genomics patterns.

Wastewater Sample Collection, Processing, and PCR/RT-PCR Analyses

Both composite and grab samples can be collected to detect MPXV DNA using PCR or RT-PCR tests by CDC primers or probes [56]. Real-time/conventional PCR can be used using CDC MPXV sequences are forward 5′-GGA AAA TGT AAA GAC AAC GAA TAC AG-3′ and reverse 5′-GCT ATC ACA TAA TCT GGA AGC GTA-3′ probe 5′-FAM-AAG CCG TAA TCT AGT TGT CTA TCG TGT CC-Spacer C6-3′ [26].

After collection, wastewater samples need to maintain at a cool temperature (2 to 8 °C) or frozen (−20 °C or below) within an hour of collection and transferred to the laboratory as soon as possible. After collecting the sample, a solid sample (without transport medium) or a liquid sample utilizing a virus transport medium (VTM) or universal transport medium (UTM) can be delivered to the laboratory. All types of equipment (sample collection vial, spatula, etc.) need to be sterilized properly, and an autosampler is recommended for wastewater sample collection. There should be correct labeling and paperwork, as well as proper documentation of the sample location, and the required triple packing may be used. Personal protective equipment (PPE) should be used while collecting samples in a specific area [57]. The raw sample may be pre-treated using heat treatment or any other chemical that does not act as an inhibitor for PCR. Although there is no fixed method for wastewater sample concentration, PEG/NaCl can be used followed by nucleic acid extraction using automatically or manually as well as PCR/RT-PCR run followed by sequencing for mutational analysis.

DNA Stability, Correlation with Positive Cases, and Comparison with SARS-CoV-2

Following COVID-19, WBS has become more interested in locating hotspots and estimating patient numbers. WBS accurately predicted COVID-19 cases in San Diego, California, using the same monkeypox technique. The same technique is followed to identify MPX DNA. On July 10, 2022, 10,565.54/L gene copies were used as the limit of detection for the first MPXV-positive case, which escalated to 189,309.81 on August 2. US San Diego and UC San Diego Health began working together in late 2020 to collect wastewater samples in order to detect COVID-19 using autosamplers. A research team, the Sewer Coronavirus Alert Network (SCAN) from the Stanford University, the University of Michigan, and Emory University, recalibrated the WBS for SARS-CoV-2 and expanded to MPX detection [2].

To monitor MPX spreading from wastewater based on DNA concentration, Monte Carlo modeling is used by one study [58]. According to this study, an average of 7.78 log10 genome copies of MPX spread from an infected patient’s stool. Following published articles, this study also predicted 54.7% of infections were found in the UK, where 44.5% in Spain, and 3.1% in Democratic Republic of Congo (DRC). Analyzing the limit of detection these findings directed, 10 gc/L may be a source of seven MPX-positive cases from 100,000. Another study was completed from nine California Communities where authors claimed it was impossible to get the concentration of MPX DNA for two reasons. One of them is the lack of true positive cases from wastewater and a comparatively higher correlation between MPX DNA in wastewater and positive cases [51••].

In Bangkok city, Thailand, 63 sewered and non-sewered used for WBS and second week of June 2022 first 16.4 copies/mL (n = 3) were identified which was 45.92 copies/mL found the first week of July. Wastewater samples were collected from the SARS-CoV-2 wastewater surveillance program from. This study reported MPX cases higher in the community rather than reported positive cases and highly supported that MPX tracing is feasible from wastewater [52••]. MPXV DNA is identified from 24 wastewater treatment plants (n = 312) of various places of Spain from SARS-CoV-2 wastewater surveillance following aluminum adsorption-precipitation method and found 2.2 × 103 to 8.7 × 104 genome copies (gc)/L. This study also reported asymptomatic cases were not counted in the clinical report based on wastewater data when pain a total of 6119 cases have been confirmed as of August 19, 2022 [47].

Prediction of mpox cases from wastewater was comparatively tough rather than SARS-CoV-2 due to environmental cofactors, lower DNA in wastewater, kinetics, asymptomatic patients, lack of diagnosis, and concentration method stability. There is very limited information about the prevalence, decay and stability, and persistence of MPX DNA in wastewater. MPX DNA is detected in untreated wastewater from urban areas by McCall et al. using metagenomics where groundwater, springs, dug wells, and surface water in sub-Saharan Africa. As poxviruses contain lower lipid content in the envelope, they are more resistant to environmental conditions. A total of 179 samples were tested from MPX-positive patients’ room with high efficiency particulate air filter (HEPA) where 1 week contamination found that declined after 3 weeks [59].

Safety Issues

Working with any pathogen is risky, whereas other viruses in wastewater are more dangerous than MPXV [4]. However, all lab workers, staff, and related personnel who come into contact with MPXV wastewater samples should follow the standard infection control procedures [60]. People who may be in danger, like health workers, laboratory personnel, and fast reaction teams, are offered vaccines in several countries [61].

In addition, orthopoxviruses are susceptible to a combination of 0.5% sodium hypochlorite, chloroxylenol-based disinfectants, glutaraldehyde, and formaldehyde. Heat (autoclaving and incineration) is required to render orthopoxviruses inactive. To minimize any risk by the risk group 3 MPXV, laboratory workers must take precautions, acquire proper immunization, and adhere to normal SOPs. Biosafety level 3 (BSL-III) laboratories, equipment for viral DNA extractions, PCR/RT-PCR, ELISA, WGS, cell culture, and expertise are necessary for handling this infectious virus. Staff and workers must change out of their regular attire, usual clothing, and jewelry to wear specific protective laboratory coats, shoes, masks, or full-coverage personal protective equipment (PPE) before entering the laboratory [62].

Biological safety cabinets (BSC) or other appropriate primary containment devices, along with PPE, are used for all laboratory operations involving infected specimens. Closed containers put in sealed safety cups or rotors loaded and unloaded in biological safety cabinets must be used for centrifugation of all specimen types. The use of gloves, masks, goggles, and other protective gear is mandatory in laboratory settings [63].

There are additional biosecurity requirements for MPXV because it is a biological agent with a high-security risk [64, 65]. For safety reasons, it is recommended that all laboratory personnel wash their hands after handling this virus and disinfect the lab area. Medical detergents like 0.1% sodium hypochlorite (1000 ppm available chlorine) will be used to eradicate this infection. Regularly touched surfaces, such as tables, doorknobs, toilet flush handles, and faucets, require extra attention. To the extent possible, carpets and soft furnishings should be steam cleaned. Following regular medical waste guidelines, the PPE used to remove clothing and linens should be placed in a secure bag for burning [66].

Tables, doorknobs, toilet flush handles, and taps get a lot of use, so take extra care with them. Steam cleaning carpets and other soft furnishings are a good option when it is possible. It is important to dispose of medical waste properly after cleansing and disinfection so that it can be disposed of in accordance with the usual medical waste standards. Following removal from rooms, clothing and linens can be washed in a standard washing machine with warm water (above 600 °C), and detergent bleach can be applied, although it is not essential [67]. People should be educated on the dangers of cleaning and disinfecting a home on their own, even if it is only a little space. In coordination, the local HPT and the local government should handle decontamination and rubbish disposal. Even though there are no registered treatments against MPXV, the only approach to avoid this quickly spreading disease is to educate laboratory or health staff [33] and follow proper regulations for sick or dead wild animals that may contain the virus [68]. The monkeypox awareness campaign should focus on timely identification of the disease and adopting protective measures to avoid viral infection [69, 70].

Conclusion and Future Recommendations

During the COVID-19 pandemic, community wastewater from WWTPs has become a premising source of information for epidemiological surveillance. However, there is very little certainty about the treatment type’s influence on removing viral load. Accurate and rapid diagnostic tests are essential for identifying MPXV-positive cases, contact tracing, and making public health decisions. WBS of MPXV can provide timely decisions from surveillance data that might be useful to track current conditions and predict the red zones which should be under follow-up study. Indeed, correlation analysis established that solids, pH, and temperature are the most influential parameters in viral removal from WWTPs. Viral surveillance within WWTPs is a valuable health surveillance tool, as MPXV DNA has already been detected in different countries. In addition, it allows evaluation of various types of viruses that could be a health risk if treated wastewater is recycled.

To control this unwanted quick transmission, we need to follow a swift multidisciplinary rapid action including researchers, experts in public health, policymakers, biologists, and physicians, as well the fast diagnosis of confirmed cases for tracking affected areas using wastewater-based surveillance combined with serological surveillance, the development active prophylaxis vaccines, and antiviral drugs, and follow other preventive and control strategies.