The Mpox virus (MPXV) was first isolated in 1959 from an infected monkey, while the first human monkeypox (Mpox) infection was detected in 1970 in a nine-month-old baby from the Democratic Republic of the Congo.
MPXV is transmitted through direct contact with infected respiratory secretions, skin lesions, and body fluids like blood. The recent widespread outbreak of Mpox infection in 2022 was declared a public health emergency of international concern by the World Health Organization (WHO).
Study: Wastewater surveillance suggests unreported Mpox cases in a low-prevalence area. Image Credit: Africa Studio / Shutterstock.com
*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Background
As of May 2023, approximately 86,000 Mpox cases were reported from 110 countries worldwide. Of these nations, 103 countries did not have any history of Mpox infection. Unfortunately, the prevalence of Mpox in Texas has been considerably high, with more than 3,000 confirmed cases.
A promising technique for monitoring community outbreaks of infectious diseases is wastewater surveillance, as wastewater contains viral signals excreted by infected individuals across the whole spectrum of disease symptoms, including asymptomatic, pre-symptomatic, and symptomatic.
In addition, wastewater analysis can detect the shedding of viral particles prior to the onset of symptoms. Therefore, wastewater surveillance allows researchers to assess epidemic progression, estimate the magnitude of infections, and provide early warning for disease outbreaks in a population.
MPXV DNA was detected in several wastewater samples collected from specific sites in California, even prior to the identification of cases. As a result, the United States Centers for Disease Control and Prevention (CDC) has recommended two real-time polymerase chain reaction (PCR) assays to confirm the detection of non-variola orthopoxvirus (F8L) and MPXV (C22L).
About the study
A recent study posted to the medRxiv* preprint server characterized the analytical sensitivity of F3L, F8L, and C22L_m, which are three assays used to detect MPXV in wastewater samples. Samples were collected from four wastewater treatment facilities (FH, HS, JT, and RB) in El Paso, Texas, between February 20 and March 27, 2023.
These wastewater treatment facilities serve a total of 751,982 individuals in the city. The crude sample was centrifuged and subsequently separated into pellets and supernatants. The pellet was collected for DNA extraction, while supernatants were used to isolate viral particles.
This study synthesized all primers, probes, and synthetic gene fragments for MPXV, horsepox, and cowpox virus using integrated DNA technologies. All three assays were used to test the presence of MPXV in the supernatant and pellet of each sample.
Three PCR replicates were performed for each assay. For negative control, the same volume of nuclease-free H2O with no DNA template was used for each PCR cycle.
To minimize the risk of cross-contamination, DNA was added for positive controls. A cycle threshold (Ct) value above 42 was considered negative.
Complete genomic sequences of human Mpox, Horsepox, Cowpox, Smallpox, Buffalopox, and Vaccinia viruses were obtained from the National Center for Biotechnology Information (NCBI).
Oligos sequences, such as a probe, forward primer, and reverse primer, were matched with sequences obtained from the database. Non-matching oligo sequences were extracted and aligned using SnapGene to detect specific nucleotides that differ between genomic sequences.
Study findings
This study investigated the analytical sensitivity and limit of detection of F3L and F8L assays based on real-time quantitative PCR (RT-qPCR) with synthetic gene fragments associated with the genomic regions of MPXV and other orthopoxvirus viruses.
For the F3L assay, serial tenfold dilutions of MPXV DNA were used. This analytical technique was able to detect MPXV DNA across seven-log concentrations from 3.64~3.64*106 copies/µl, with an amplification efficiency of 97.62% and a limit of detection with 22 replicates. Thus, the F3L assay is 100% sensitive to MPXV.
The F8L assay was designed to detect a wide range of orthopoxviruses. The F8L assay exhibited differential sensitivity to various orthopoxvirus species. For example, this assay exhibited 89.49% sensitivity to MPXV, 88.40% to cowpox, and 88.39% to horsepox DNA. Thus, F8L was most efficient in detecting MPXV genome fragments as compared to other orthopoxviruses.
All three assays were used to detect MPXV from wastewater samples collected from FH, HS, JT, and RB. F3L detected 70.8%, C22L_m detected 58.3%, and F8L detected 12.5% of MPXV in samples. Samples collected from the HS facility exhibited the highest F3L assay positivity frequency.
Viral concentrations in the pellet were higher as compared to the supernatants. An increase in the viral concentration was recorded from February 27, which peaked in the week of March 6.
The difference in the timelines between wastewater peak time and new Mpox case reporting dates indicates the possibility of using wastewater surveillance for detecting new infections.
Conclusions
MPXV was detected using three molecular assays in wastewater samples. Interestingly, a rise in the viral concentration was found between one and two weeks prior to the clinical reporting of the new Mpox case.
By comparing the findings of the wastewater analysis and clinical reports, the authors highlighted the prevalence of unreported Mpox cases within the city during the study period. This emphasizes the importance of using multiple molecular assays to improve Mpox detection rates using wastewater samples.
*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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