Researchers surprised that process designed to detect SARS-CoV-2 also identifies monkeypox in wastewater
Early information about an outbreak in a geographical region can inform local clinical laboratories as to which infectious agents and variants they are likely to see when testing patients who have symptoms. To that end, wastewater testing has become a rich source of early clues as to where COVID-19 outbreaks are spreading and how new variants of the coronavirus are emerging.
Ongoing advances in genetic sequencing and digital technologies are making it feasible to test wastewater for infectious agents in ways that were once too time-consuming, too expensive, or simply impossible.
“Before wastewater sequencing, the only way to do this was through clinical testing, which is not feasible at large scale, especially in areas with limited resources, public participation, or the capacity to do sufficient testing and sequencing,” said Knight in a UCSD press release. “We’ve shown that wastewater sequencing can successfully track regional infection dynamics with fewer limitations and biases than clinical testing to the benefit of almost any community.” (Photo copyright: UC San Diego News.)
Same Process, Different Virus
Following August’s declaration of a state of emergency by California, San Diego County, and the federal government, UCSD researchers added monkeypox surveillance to UCSD’s existing wastewater surveillance program.
“It’s the same process as SARS-CoV-2 qPCR monitoring, except that we have been testing for a different virus. Monkeypox is a DNA virus, so it is a bit of a surprise that our process optimized for SARS-CoV-2, which is an RNA virus, works so well,” said Rob Knight, PhD, Professor of Pediatrics and Computer Science and Engineering at UCSD and one of the lead authors of the study in the press release.
According to the press release, RNA sequencing from wastewater has two specific benefits:
It avoids the potential of clinical testing biases, and
It can track changes in the prevalence of SARS-CoV-2 variants over time.
In 2020, at the height of the COVID-19 pandemic, scientists from the University of California San Diego and Scripps Research looked into genetic sequencing of wastewater. They wanted to see if it would provide insights into levels and variants of the SARS-CoV-2 within a specific community.
Individuals who have COVID-19 shed the virus in their stool.
The UCSD/Scripps researchers deployed commercial auto-sampling robots to collect wastewater samples at the main UCSD campus. They analyzed the samples for levels of SARS-CoV-2 RNA at the Expedited COVID-19 Identification Environment (EXCITE) lab at UCSD. After the success of the program on the campus, they extended their research to include other facilities and communities in the San Diego area.
“The coronavirus will continue to spread and evolve, which makes it imperative for public health that we detect new variants early enough to mitigate consequences,” said Knight in a July press release announcing the publication of their study in the journal Nature, titled, “Wastewater Sequencing Reveals Early Cryptic SARS-CoV-2 Variant Transmission.”
Detecting Pathogens Weeks Earlier than Traditional Clinical Laboratory Testing
In July, the scientists successfully determined the genetic mixture of SARS-CoV-2 variants present in wastewater samples by examining just two teaspoons of raw sewage. They found they could accurately identify new variants 14 days before traditional clinical laboratory testing. They detected the presence of the Omicron variant 11 days before it was first reported clinically in the community.
During the study, the team collected and analyzed 21,383 sewage samples, with most of those samples (19,944) being taken from the UCSD campus. They performed genomic sequencing on 600 of the samples and compared them to genomes obtained from clinical swabs. They also compared 31,149 genomes from clinical genomic surveillance to 837 wastewater samples taken from the community.
The scientists distinguished specific viral lineages present in the samples by sequencing the viruses’ complete set of genetic instructions. Mutational differences between the various SARS-CoV-2 variants can be minute and subtle, but also have notable biological deviations.
“Nothing like this had been done before. Sampling and detection efforts began modestly but grew steadily with increased research capacity and experience. Currently, we’re monitoring almost 350 buildings on campus,” said UCSD’s Chancellor Pradeep Khosla, PhD, in the July press release.
“The wastewater program was an essential element of UC San Diego Health’s response to the COVID pandemic,” said Robert Schooley, MD, Infectious Disease Specialist at UC San Diego Health, in the press release. Schooley is also a professor at UCSD School of Medicine, and one of the authors of the study.
“It provided us with real-time intelligence about locations on campus where virus activity was ongoing,” he added. “Wastewater sampling essentially allowed us to ‘swab the noses’ of every person upstream from the collector every day and to use that information to concentrate viral detection efforts at the individual level.”
Monkeypox Added to UCSD Wastewater Surveillance
In August, UCSD officially added the surveillance of the monkeypox virus to their ongoing wastewater surveillance program. A month earlier, the researchers had discerned 10,565.54 viral copies per liter of wastewater. They observed the levels fluctuating and increasing.
On August 2, the scientists detected 189,309.81 viral copies per liter of wastewater. However, it is not yet clear if the monitoring of monkeypox viral loads in wastewater will enable the researchers to accurately predict future infections or case rates.
“We don’t yet know if the data will anticipate case surges like with COVID,” Knight said in the August UCSD press release announcing the addition of monkeypox to the surveillance program. “It depends on when the virus is shed from the body relative to how bad the symptoms are that cause people to seek care. This is, in principle, different for each virus, although in practice wastewater seems to be predictive for multiple viruses.”
Utilization of genetic sequencing of wastewater sampling will continue to develop and improve. “It’s fairly easy to add new pathogens to the process,” said Smruthi Karthikeyan, PhD, an environmental engineer and postdoctoral researcher in Knight’s lab who has overseen wastewater monitoring at UC San Diego. “It’s doable on short notice. We can get more information in the same turnaround time.”
Thus, clinical laboratories engaged in testing programs for COVID-19 may soon see the addition of monkeypox to those processes.
Officials also worry about diminishing smallpox vaccinations, which offered people protection against the infectious disease
Monkeypox challenges from the current outbreak have dogged public health agencies even though the disease was first identified more than 50 years ago. That is because the virus has found new avenues of infection. These developments will be relevant for the nation’s clinical laboratories, which are often the first healthcare providers to confirm a suspected case is positive for monkeypox and notify a public health laboratory about the positive test result.
The latest monkeypox numbers from the federal Centers for Disease Control and Prevention (CDC) indicate that, as of September 6, the US has identified 19,962 cases in the 2022 outbreak, while worldwide the case number is 52,037.
In “When It Comes to Monkeypox Testing, Clinical Laboratories Should Be Aware of Five Significant Developments,” Dark Daily wrote about steps being taken to identify and control infections in America as well as trends in medical laboratory testing for monkeypox. This included reports of phlebotomists refusing to draw monkeypox blood samples and how social stigma surrounding the disease can affect who gets a medical laboratory test.
Workers at clinical laboratories and anatomic pathology groups will gain from understanding why monkeypox has spread beyond its traditional geography.
“Monkeypox symptoms include swollen lymph nodes, fever, and body aches that result in red bumps on hands, feet, mouth, and genitals,” Bodhraj Acharya, PhD (above), of the Laboratory Alliance of Central New York, told Dark Daily. “It spreads by close contact, respiratory droplets, lesions, and bodily fluids.” Clinical laboratories engaged in testing for monkeypox will want to stay alert to patients presenting with such symptoms. (Photo copyright: Laboratory Alliance of Central New York.)
African Public Health Officials Saw New Monkeypox Challenges Coming
Researchers and public health experts have been perplexed about how and why the latest monkeypox outbreak has occurred so aggressively beyond its origin in rural Central Africa.
“Monkeypox is caused by the pox virus, with a close resemblance to smallpox,” said Bodhraj Acharya, PhD, Manager of Chemistry and Referral Testing at the Laboratory Alliance of Central New York, in a conversation with Dark Daily. “Unlike COVID-19, this is an old enemy which has roots in the 1970s from Congo, when the disease was erratically endemic in Africa.”
According to the World Health Organization (WHO), most monkeypox cases since 1970 have been reported from rural rainforest regions in Central and Western Africa.
Thus, a monkeypox outbreak occurring in Europe and the United States in 2022 has puzzled virologists and microbiologists because it does not follow the historical pattern of the virus’ spread. For example, the first monkeypox case in the US arrived in May from a Massachusetts patient who had traveled to Canada, a state press release noted.
Before the Nigerian outbreak, the virus rose from rural areas where hunters came in close contact with animals. The illness resulted in lesions on the face, hands, and feet, Nature wrote of Yinka-Ogunleye’s recollections.
However, after 2017, she and other epidemiologists warned peers that the virus was spreading in new ways and in urban settings. For example, infected people sometimes had genital lesions, suggesting that the virus might spread through human sexual contact.
Now, in 2022, “the world is paying the price for not having responded adequately” in 2017, Yinka-Ogunleye told Nature.
Lack of Smallpox Vaccination Increases Monkeypox Challenges
The waning effects of smallpox vaccinations, which ended in 1980 after smallpox was basically eradicated from the world, may have opened the door for monkeypox to spread earlier this year. Smallpox vaccines provided some protection against monkeypox, but by now three generations of people have not received smallpox inoculations.
“Eyebrows were raised when multiple cases of monkeypox were reported from various non-endemic countries starting in May of 2022,” Acharya said. “Due to genetic similarity, smallpox vaccination provided some cross-protection, but the termination of smallpox vaccination could have provided ground for the recent insurgence and spread of monkeypox.”
Trying to jumpstart a new monkeypox vaccination campaign on the heels of COVID-19 shots may be met with resistance from a virus-weary public. But other options at preventing the current spread of monkeypox may present challenges as well, such as trying to curtail sexual activity among affected population, the BBC reported.
“The easiest way to prevent it is to close down all highly active sexual networks for a couple of months until it goes away, but I don’t think that will ever happen. Do you?” Paul Hunter, PhD, Professor of Medicine at the University of East Anglia in Norwich, England, told the BBC.
For medical laboratory workers and others who may find themselves testing for the disease in the future, the biggest lessons from current monkeypox challenges are twofold: The virus has invaded new geography, and discontinued smallpox vaccination campaigns may have left younger people exposed to monkeypox.
There are reports of phlebotomists refusing to draw monkeypox blood samples and social stigma surrounding the disease can affect who gets a medical laboratory test
Cases of monkeypox are increasing in the US—14,115 as of Aug. 19, up 1,400 from the prior week—and clinical laboratories around the country are bracing for a potential increase in monkeypox testing orders.
Several factors, however, are affecting the testing. Chief among them:
Lab workers refusing to take blood draws from potential monkeypox patients, and
Community clinics in some cities having to delay other care to deal with an onslaught of monkeypox test orders.
Here are five trends clinical laboratory leaders should be aware of that are influencing the state of monkeypox testing in the country.
Trend 1: Some Phlebotomists Refuse to Draw Possible Monkeypox Specimens
CNN reported that phlebotomists at two of the largest commercial laboratories—Labcorp and Quest Diagnostics—were either refusing or being told not to draw blood samples from suspected monkeypox patients.
“Labcorp and Quest don’t dispute that, in many cases, their phlebotomists are not taking blood from possible monkeypox patients,” according to CNN. “What remains unclear, after company statements and follow-ups from CNN, is whether the phlebotomists are refusing on their own to take blood or if it is the company policy that prevents them. The two testing giants say they’re reviewing their safety policies and procedures for their employees.”
In “Medical Laboratories Respond to Monkeypox Outbreak Using CDC-Developed Diagnostic Test,” Dark Daily noted that skin lesion swabbing, such as that necessary to perform the OrthopoxvirusPCR test, is the preferred method to check for monkeypox because of higher viral counts in the lesions. However, physicians may order follow-up blood tests for confirmed monkeypox patients, and suspected patients may need bloodwork as part of other routine care.
In an update posted on its website, Quest noted it has been testing swab specimens of skin lesions for monkeypox, but those swabs are performed by providers and not Quest. However, the company was also preparing to take blood draws of possible monkeypox patients in its patient service centers.
“Given that monkeypox has been declared a national public health emergency and the most recent CDC guidance, we are now implementing procedures to safely enable patients with suspected or confirmed monkeypox infection into our patient service sites for phlebotomy blood draws and other non-swab specimen collections,” Quest stated. “This approach will enable patients with suspected or confirmed monkeypox infections to receive additional testing they may need.”
Trend 2: Guidance Is Available to Help Lab Workers Avoid Monkeypox Infection
The CDC has posted guidance to maintain infection control around suspected monkeypox specimens. Among the steps noted by the agency:
Lesion specimens from patients suspected of having monkeypox will carry the highest quantity of the virus. When possible, lab workers that have a smallpox vaccination from within the last three years should handle these specimens. Smallpox vaccination also protects from monkeypox in many cases. Unvaccinated workers who test suspected monkeypox specimens need to take extra precautions, such as wearing a buttoned lab coat, gloves, and face protection, and avoiding splashes, the CDC stated.
Blood specimens draw from suspected monkeypox patients will have a low quantity of the virus. Lab workers testing these specimens do not need to be vaccinated for monkeypox, but standard precautions should be followed.
Before using automated testing platforms with suspected monkeypox specimens, labs should conduct a risk assessment to identify potential hazards.
Trend 3: Monkeypox Testing Gains an Early Social Stigma
Some people who need to be tested for monkeypox may be hesitant to seek out a medical laboratory or patient service center because of a stigma being attached to the disease.
Although it does not match the early hysteria associated with HIV infections in the 1980s—in a 1987 poll, 60% of respondents said AIDS patients should carry a card identifying them as such, Gallup noted—there have been clear instances where people and agencies have associated monkeypox infection with men having sex with other men.
“The focus for all countries must be engaging and empowering communities of men who have sex with men to reduce the risk of infection and onward transmission, to provide care for those infected, and to safeguard human rights and dignity,” Tedros Adhanom Ghebreyesus, PhD, the Director-General at the World Health Organization, said in a July 27 media briefing.
Ghebreyesus added that while 98% of monkeypox infections have been among men who have sex with men, anyone can get the disease, including children.
“Stigma and discrimination can be as dangerous as any virus, and can fuel the outbreak,” said Tedros Adhanom Ghebreyesus, PhD (above), Director-General at the World Health Organization (WHO), in a media briefing. Clinical laboratories would be wise to prepare for a marked increase in demand for monkeypox testing. (Photo Copyright: WHO/Christopher Black.)
“Men who have sex with men have been hit the hardest by monkeypox to date, but LGBTQ+ health advocates say improper messaging risks branding monkeypox as a ‘gay disease,’ eroding effective preventive measures and allowing the virus to spread,” Bloomberg Law reported.
For example, 66% of respondents either were not sure or did not believe there is a vaccine for monkeypox.
Trend 4: Workers Who Refuse to Test Patients for Monkeypox Face Possible Backlash
Some medical professionals have raised concerns about healthcare workers being unwilling to test monkeypox patients.
“This is absolutely inexcusable. It’s a grave dereliction of duty,” David Harvey, Executive Director of the National Coalition of STD Directors, told CNN. The group represents sexually transmitted disease (STD) directors at public health departments in the US.
“For every single patient that walks [through] your door, you use universal precautions because every disease doesn’t have a phenotype or outward appearance, so you have to treat everyone exactly the same,” Garfield Clunie, MD, president of the National Medical Association and Assistant Professor of Obstetrics, Gynecology, and Reproductive Science at the Icahn School of Medicine at Mount Sinai, told Bloomberg Law. “You can’t treat someone differently because of their sexual orientation, or race, or gender, or for any other reason.”
Trend 5: Public Clinics Show Early Signs of Monkeypox Testing Pressure
A survey of 80 public health departments conducted by the National Coalition of STD Directors indicated that some sites may already be getting overwhelmed by demand for monkeypox testing.
According to the survey results, 79% of public health clinics saw an increased demand for monkeypox testing over the past four weeks. In a troubling aspect, 28% of clinics said they could not meet testing demand if it increases.
Further, 22% of clinics have reduced screenings for other STDs to prioritize monkeypox testing. Such moves likely delay patients from receiving other care they need.
Clinical laboratories may want to take note of the survey findings. The pressure public health clinics currently face could be a precursor to similar problems at labs if demand for monkeypox testing grows.
Experts say it is time ‘to restore our confidence in vaccines’ as many medical laboratories take steps to support testing for the polio virus
Clinical laboratories and microbiologists in the state of New York will want to know that, in July, a man in New York was diagnosed with polio and subsequently the virus was detected in the wastewater of two New York counties.
The area, Rockland County, N.Y., just north of New York City, was also at the forefront of a measles outbreak that occurred in 2018 and 2019. The outbreak was attributed to low vaccination rates within the community.
The unidentified, immunocompetent young man was admitted to a New York hospital after experiencing a low-grade fever, neck stiffness, back and abdominal pain, constipation, and lower extremity weakness. He eventually developed paralysis from the disease, which is irreversible.
Poliomyelitis, commonly known as polio, is a disabling and life-threatening disease that is caused by the poliovirus. Though it rarely surfaces in the United States, there is now confirmation of the first US case since 2013.
“The polio vaccine is safe and effective, protecting against this potentially debilitating disease, and it has been part of the backbone of required, routine childhood immunizations recommended by health officials and public health agencies nationwide,” said Mary T. Bassett, MD (left), Health Commissioner at the New York Department of Health, in a press release. Clinical laboratories and microbiologists in New York may want to prepare for an increase in vaccination requests. (Photo copyright: Time.)
Is Polio Back in America? Clinical Laboratories Will Want to Be Prepared
“I think it’s concerning because it can spread,” epidemiologist Walter Orenstein, MD, Professor, Department of Medicine, Division of Infectious Diseases at Emory University School of Medicine told STAT. “If there are unvaccinated communities, it can cause a polio outbreak.”
According to the federal Centers for Disease Control and Prevention (CDC), public health experts are working diligently to discover how and where the infected individual contracted polio. The CDC website states that the risk for people who have received the polio vaccine is very low, but there is concern for those who have not received the recommended doses of the vaccine.
“Most of the US population has protection against polio because they were vaccinated during childhood, but in some communities with low vaccine coverage, there are unvaccinated people at risk,” the CDC noted. “Polio and its neurologic effects cannot be cured but can be prevented through vaccination.”
The US uses an injectable polio vaccine for the poliovirus which contains killed viruses. The vaccine “instructs” the immune system to recognize and combat the virus. This inactivated polio vaccine (IPV) is administered to children as a shot in the arm or leg and is typically given in four separate doses.
“The inactivated polio vaccine we have is very effective and very safe and could have prevented this,” Orenstein told STAT. “We need to restore our confidence in vaccines.”
“Based on what we know about this case, and polio in general, the (New York) Department of Health strongly recommends that unvaccinated individuals get vaccinated or boosted with the FDA-approved IPV polio vaccine as soon as possible,” said Mary T. Bassett, MD, Health Commissioner at the New York Department of Health in a press release.
Poliovirus Found in Wastewater via Use of Gene Sequencing
Poliovirus is very contagious and is transmitted through person-to-person contact. The virus lives in an infected person’s throat and intestines and can contaminate food and water in unsanitary conditions. According to the CDC, typical symptoms of the illness include flu-like symptoms such as:
Sore throat
Fever
Tiredness
Nausea
Headache
Stomach pain
Most of these symptoms will disappear within five days, but polio can invade the nervous system and cause more serious complications, such as meningitis, paralysis, and even death.
After confirmation of the new case of polio, wastewater surveillance detected the presence of the poliovirus in Rockland and Orange counties, New York.
Wastewater analysis can uncover pathogens within a community and has been used in the fight against other infectious diseases, including:
“In some regards, wastewater is a public health dream scenario,” said Mark Siedner, MD, an infectious disease doctor at Massachusetts General Hospital and associate professor at Harvard Medical School, in an interview with Fortune. “Everyone poops, and most people poop every day. It provides real-time data on infection rates. In that regard, it’s an extremely powerful tool, particularly good at detecting early warning signs. Before people get sick, we might get a signal.”
Wastewater analysis can provide insights regarding the types of viruses that people within a community are shedding and if the volume of those viruses are increasing. This information can provide scientists with an early marker for an outbreak of an illness that is on the verge of spreading.
Microbiologists and clinical laboratories should be aware of the specific types of infectious agents public health authorities are detecting in wastewater, even as they perform screening and diagnostic tests on their patients for similar infectious diseases.
Polio is Appearing Worldwide
The Global Polio Eradication Initiative (GPEI) has announced that new cases of polio have been reported in Israel and the United Kingdom. These are countries where polio cases are extremely rare.
This indicates that microbiologists and clinical laboratories managers will want to be on constant alert for uncommon infectious diseases that may appear suddenly, even if those illnesses are rare. Accurate and immediate diagnoses of such infectious diseases could play a major role in triggering a public health response to control potential outbreaks while they are in their earlier stages.
Findings could lead to new clinical laboratory involvement in diagnostics targeted at overweight patients
Does the SARS-CoV-2 coronavirus make us fat so it can better take over our bodies? It sounds like the plot for a science fiction horror movie! But a team of scientists in the Pacific Northwest say that is exactly what the virus does, and their findings could lead to clinical laboratories playing a role in evaluating how the virus highjacks fat cells to aid in its invasion of humans.
They found that certain types of lipids support replication of the COVID-19 virus. Their study illustrates how lipids may play a more important role in the human body than scientists previously understood.
“This is exciting work, but it’s the start of a very long journey,” said Fikadu Tafesse, PhD (left), Assistant Professor of Molecular Microbiology and Immunology, OHSU School of Medicine and corresponding author of the study in an OHSU press release. “We have an interesting observation, but we have a lot more to learn about the mechanisms of this disease.” Clinical laboratories may eventually be part of a new diagnostic process for overweight COVID-19 patients. (Photo copyright: Oregon Health and Science University.)
Does Obesity Promote COVID-19 Infection?
The OHSU and PNNL scientists performed their research by examining the effect of SARS-CoV-2 on more than 400 lipids in two different cell lines. They observed that individuals with a high body mass index (BMI) appear to be more sensitive to the COVID-19 virus.
The researchers discovered there is a tremendous shift in lipid levels in those cell lines when the virus was present, with some fats increasing by a massive 64 times! Nearly 80% of the fats in one cell line were changed by the virus and more than half of the fats were altered in the other cell line.
The lipids that were most affected by the COVID-19 virus were triglycerides which are critical to human health. Triglycerides are basically tiny bundles of fat that allow the body to store energy and maintain healthy cell membranes. When a body needs energy, these fat parcels are broken up into useful, raw materials to provide the required energy.
“Lipids are an important part of every cell. They literally hold us together by keeping our cells intact, and they’re a major source of energy storage for our bodies,” said Jennifer Kyle, PhD, in the OHSU press release. Kyle is a research scientist at PNNL who specializes in all stages of lipidomic research. “They are an attractive target for a virus,” she noted.
Stopping SARS-CoV-2 Replication
The scientists discovered that SARS-CoV-2 alters our fat-processing system by boosting the number of triglycerides in our cells and changing the body’s ability to utilize stored fat as fuel. The team also analyzed the effects of lipid levels in 24 of the virus’ 29 proteins. They identified several proteins that had a strong influence on triglyceride levels.
The team then searched databases and identified several compounds that interfered with the body’s fat-processing system by cutting off the flow of fatty fuel. They found that several of these compounds were successful at stopping the SARS-CoV-2 virus from replicating.
A synthetic organic compound known as GSK2194069, which selectively and potently inhibits fatty acid synthase (FAS), and a weight-loss medication called Orlistat, were both able to stop viral replication in the lab.
Although the scientists believe their work is an important step in understanding the SARS-CoV-2 coronavirus, they also note that their results occurred in cell culture (in vitro) and not in people (in vivo). Therefore, more research is needed to determine if the compounds will work in the same manner in human trials.
“As the virus replicates, it needs a continuous supply of energy. More triglycerides could provide that energy in the form of fatty acids. But we don’t know exactly how the virus uses these lipids to its advantage,” Tafesse said in the press release.
“Our findings fill an important gap in our understanding of host dependency factors of coronavirus infection. … In light of the evolving nature of SARS-CoV-2, it is critical that we understand the basic biology of its life cycle in order to illuminate additional avenues for protection and therapy against this global pandemic pathogen, which spreads quickly and mutates with ease,” the OHSU/PNNL scientists wrote in Nature Communications.
More research is needed to validate the findings of this study and to better understand the dynamic between lipids and SARS-CoV-2 infection. However, it is reasonable to assume that, in the future, some COVID-19 patients may require a clinical laboratory work-up to determine how the coronavirus may be hijacking their fat cells to exacerbate the illness.
