Advances in genome sequencing give virologists and microbiologists new tools for tracking SARS-CoV-2 variants to their sources
Wastewater surveillance has emerged as an essential tool in the detection and tracking of the SARS-CoV-2 coronavirus within communities. Though COVID-19 infections are decreasing in the United States—and clinical laboratories are performing fewer diagnostic tests for the disease—researchers continue to monitor populations for the presence of the coronavirus and its variants to be prepared for the next outbreak.
Genetic sequencing of samples extracted from sewer systems throughout the country have revealed dozens of strains of the coronavirus containing multiple mutations in unusual combinations called cryptic genetic variants (CVG), also known as cryptic lineages. A recent study has indicated that wastewater may also provide answers to questions about long COVID as these mutations can be traced back to individuals who are living with chronic COVID-19 infections.
“Because increases in wastewater [viruses] generally occur before corresponding increases in clinical cases, wastewater surveillance serves as an early warning system for the emergence of COVID-19 in a community,” said Amy Kirby, PhD (above), CDC program lead for the National Wastewater Surveillance System, during a media telebriefing. Wastewater testing for viruses and bacteria may eventually lead to the implementation of systems to alert clinical laboratories in a region whenever infectious agents are detected in wastewater. (Photo copyright: Center for Global Safe Water, Sanitation, and Hygiene.)
Humans Found to Be Primary Source of Cryptic Lineages
To conduct their study, scientist at the University of Wisconsin-Madison and the University of Missouri School of Medicine examined the evolution of a SARS-CoV-2 Omicron subvariant found in wastewater coming from a single facility in Wisconsin that employed about 30 people. The researchers discovered the mutation had been present in the wastewater for more than a year.
Dark Daily originally covered their findings in “New, Cryptic COVID-19 Lineage Found in Ohio Wastewater by Molecular Virologist Tracking Spread of SARS-CoV-2 Variants.” We reported how scientists had tracked the lineage of the cryptic strain to Ohio, where it appeared to have originated from one individual who travels regularly between the cities of Columbus and Washington Court House. They believed the person had a form of long COVID and was unaware that he or she was infected with the coronavirus.
According to Marc Johnson, PhD, Professor of Microbiology and Immunology at the University of Missouri and one of the authors of the study, the individual who shed the mutations had been shedding at least a thousand times more COVID-19 virus than an average infected person sheds. The scientists examined other wastewater monitoring data and identified 37 related cases in the US. They concluded that humans are the main source of the cryptic lineages.
“The fact that someone can have this kind of infection—and there’s every indication that they are still an active member of society and not just lying in the hospital—it’s just amazing,” Johnson told CNN.
Wastewater Surveillance Not an Exact Science, CDC Says
Although not directly involved in this study, through its National Wastewater Surveillance System (NWSS) the US Centers for Disease Control and Prevention (CDC) has been tracking wastewater surveillance programs. The federal agency believes cryptic lineages do not pose a threat to public health.
“The signal we really look for is specific variants increasing in frequency in a community, because that’s what happens at the beginning of a variant surge,” Amy Kirby, PhD, Health Scientist, National Wastewater Surveillance System Lead for the CDC, told CNN. “And it’s not what we’re seeing with these cryptic lineages.”
Kirby also noted that wastewater surveillance is not an exact science and that many factors can impede the interpretation of the data. The mutations observed for this study could be from people with long COVID or even from an infected animal. To be certain of the results, she said, researchers would have to directly link the genetic sequence from a clinical test to a specific wastewater sample.
“Best-case scenario is you find the person, they have long COVID but had no idea they had this infection, and you get them with a doctor who can get them on medicines that will actually give their immune system a bit of an upper hand, and they get better,” Johnson told CNN. “But we only know about the ones we can find, and we don’t know what the implications are, because we still don’t know who those people are.”
Public health messaging in local communities is needed to raise awareness. But though tracking down specific infected individuals could help them receive medical attention, it may not be the most desirable course of action.
“Part of the power of wastewater surveillance is that it is inherently anonymous. It’s a community-level surveillance method,” Kirby said. “And so, tracking back through the wastewater system to identify a person is not what the system is intended for.”
Clinical Laboratories Play Key Role in Public Health
The cryptic lineage that Johnson and his team identified was a mutation that appeared in two watersheds in Ohio—one located in southern Columbus and one in the town of Washington Court House, which is about 40 miles south of Columbus. The researchers hypothesized that an individual living in that area had COVID for more than two years and did not know it, was most likely asymptomatic, and lives in one area and spends a lot of time (perhaps working) in the other area.
“There is almost zero chance the patient in Ohio knew about their infection. There is almost zero chance their doctor would figure it out. It is very likely the infection is causing long term damage,” Johnson wrote in a Twitter tweet. “I’m glad that there is a chance now that they might get appropriate care.”
Wastewater surveillance has materialized as a common method of identifying and monitoring strains of COVID-19 and other infectious diseases. And clinical laboratories play a key role in that process. With genetic sequencing technologies becoming more advanced, lower in cost, faster and more accurate, it’s feasible that those technologies will be utilized more to direct public health initiatives.
Microbiology team has tracked 37 unique strains of the coronavirus since they began researching lineages two years ago
Microbiologists and clinical laboratory scientists will be interested to learn about the discovery of a new strain of SARS-CoV-2, the coronavirus that caused the COVID-19 pandemic, in wastewater sampled in Ohio.
According to an article published in Nature Reviews Genetics, a CVG is “a genetic variation that normally has little or no effect on phenotype but that—under atypical conditions that were rare in the history of a population—generates heritable phenotypic variation.”
Johnson tracked the lineage of the cryptic strain to Ohio, where it appears to have originated from one individual who travels regularly between the cities of Columbus and Washington Court House. He believes this person may have a form of long COVID and is unaware that he or she is infected with the coronavirus.
“This person was shedding thousands of times more material than a normal person ever would,” Johnson told The Columbus Dispatch. “I think this person isn’t well. … I’m guessing they have GI issues.”
“If someone has this infection, the chances are nil that they’re going to figure out what it is,” Marc Johnson, PhD, Professor of Molecular Microbiology and Immunology at the University of Missouri School of Medicine, told Insider. Microbiologists and clinical laboratory scientists in the Columbus, Ohio, area may be able to help locate this person. (Photo Copyright: University of Missouri.)
Other Cryptic COVID-19 Lineages
This isn’t the only “Cryptic COVID” case identified by Johnson and his team. In Wisconsin, another unique strain was discovered and narrowed down to a single facility and about 30 individuals. Two thirds of the employees were tested but, unfortunately, all tests came back negative. The cryptic strain seemed to have disappeared.
“We don’t know why,” Johnson told The Hill. “Either [the infected person] left the job, or got better, or is in remission—we don’t know. But we’re still monitoring it. And we’ve actually now gotten started collecting stool samples from the company.”
“We systematically sampled [sewer] maintenance holes to trace the Wisconsin lineage’s origin. We sequenced spike RBD [receptor-binding domain] domains, and where possible, whole viral genomes, to characterize the evolution of this lineage over the 13 consecutive months that it was detectable.
“The high number of unusual mutations found in these wastewater-specific cryptic sequences raises the possibility that they originate from individual prolonged shedders or even non-human sources. The Wisconsin lineage’s persistence in wastewater, single-facility origin, and heavily mutated Omicron-like genotype support the hypothesis that cryptic wastewater lineages arise from persistently infected humans.”
Johnson and his team have tracked 37 unique strains of the COVID-19 virus, including one in New York City, The New York Times reported.
In a statement to The Columbus Dispatch regarding the Columbus strain, the federal Centers for Disease Control and Prevention (CDC) noted that, “The virus lineage in question is not currently spreading or a public health threat.
“Unusual or ‘cryptic’ sequences identified in wastewater may represent viruses that can replicate in particular individuals, but not in the general population,” the CDC noted. “This can be because of a compromised immune system. CDC and other institutions conduct studies in immunocompromised individuals to understand persistent infection and virus evolution.”
In identifying these lineages, and the individuals who shed them, scientists can learn more about how COVID-19 mutates and spreads.
Mitigating Consequences of COVID-19 Variants
Although the CDC says that particular strain is not a threat to the public it could pose a long-term health risk for the individual suffering. And this individual may hold clues for the future of how the COVID-19 virus mutates and grows. Therefore, locating these people is a priority.
“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,” Rob Knight, PhD, Founding Director of the Center for Microbiome Innovation and Professor of Pediatrics, Computer Science and Engineering at the University of California San Diego (UCSD).
“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.”
Although tracing the individuals shedding cryptic COVID-19 lineages may not have an immediate effect on public health, it could lead to future discoveries about the SARS-CoV-2 coronavirus that can help shape public health goals in fighting future pandemics.
At the very least, one individual in Columbus may learn how to treat long COVID’s adverse symptoms. Microbiologists and clinical laboratory scientists involved in COVID-19 wastewater research can learn much from following these research investigations.
CDC’s findings are a setback for the national effort to encourage hospitals and their clinical laboratories to reduce the number of nosocomial infections and practice better antimicrobial stewardship
Nosocomial infections—also known as hospital-acquired infections—increased during the COVID-19 pandemic. That’s according to a Centers for Disease Control and Prevention (CDC) report that showed increases in several HAIs, including a 14% jump in Methicillin-resistant Staphylococcus aureus (MRSA) from 2020 to 2021.
Clinical laboratory testing is part of a concerted effort in the US to reduce HAIs in acute care hospitals. Additionally, diagnostic testing is vital to antimicrobial stewardship, which is designed to help physicians prescribe to patients only those antibiotics that are appropriate and reduce the chance for antimicrobial resistance (AMR).
So, it’s disturbing to see a setback in both HAIs and antimicrobial stewardship in the wake of the COVID-19 pandemic. Burda called the CDC’s findings a “regression” that “gives new meaning to the term long COVID.”
“I think, without any proof, doctors, nurses, medical technicians, and other clinicians who provide direct patient care regressed in terms of infection control best practices,” wrote healthcare journalist David Burda in his column for 4Sight Health. Clinical laboratories that processed COVID-19 tests during the pandemic can attest to the burnout. (Photo copyright: 4Sight Health.)
CDC Report Reveals Increase in Hospital Acquired Infections
The CDC used standardized infection ratios (SIRs) in its report to detail changes in nosocomial infections. CDC calculates SIRs by dividing the number of observed infections by the number of predicted infections.
“In 2021, the nation and the world continued to experience unprecedented challenges due to the COVID-19 pandemic, which impacted surveillance for and incidence of HAIs,” the CDC explained in its report.
“Compared to pre-pandemic years, hospitals across the nation experienced higher than usual hospitalizations and shortages in healthcare personnel and equipment, which may have resulted in deterioration in multiple patient safety metrics since the beginning of the pandemic,” the CDC added.
In his 4Sight Health article, Burda noted that physicians and other care providers may have “regressed” in their infection control practices due to severe pressures during the COVID-19 pandemic. “I also think the traveling nurse and temporary staff situation had something to do with it. Who has time to learn or follow the infection control policies and protocols at every hospital when you’re moving from one hospital to the next every few weeks?” he added.
The CDC explored HAIs in acute care hospitals, critical access hospitals, inpatient rehabilitation facilities, and long-term acute care hospitals. According to the federal agency’s report, at acute care hospitals, increases in nosocomial infections from 2020 to 2021 include the following:
27 states performed better on at least two types of infection.
30 states performed worse on at least two infection types.
In response to the CDC’s report, the American Hospital Association (AHA) wrote, “In acute care hospitals, the increases seen in some HAIs in 2021 contrast with the success in reducing these infections prior to the pandemic. Despite the challenges of the COVID-19 pandemic, acute care hospitals performed significantly better than the 2015 national baseline in preventing CLABSI, CAUTI, SSIs following colon surgeries, and C. difficile infections.”
The AHA recommended that hospitals “continue to reinforce prevention practices and review HAI surveillance data to identify areas for improvement.”
Dangers of Antimicrobial Resistance
According to CDC data, in the US there are 2.8 million antimicrobial infections each year, and more than 35,000 people die as a result. Dark Daily has reported extensively on the growing danger of antibiotic resistance and outlined the importance of clinical laboratory involvement in hospital antimicrobial stewardship programs.
In “During Pandemic, Clinical Laboratories Should Be Alert for Drug Resistant Infections That Pose High Risk to COVID-19 Patients,” we covered a study conducted at the University of Minnesota which highlighted the continuing need for microbiologists and clinical laboratories to stay alert for COVID-19 patients with drug-resistant infections following a CDC report on 941 confirmed and probable Candida auris cases that had been reported in 13 states, with an additional 1,830 patients that had been found to be colonized with the multidrug-resistant fungus.
The Joint Commission’s expansion of antibiotic stewardship standards, which went into effect on January 1, 2023, could help hospitals reduce nosocomial infections and fight antimicrobial resistance.
Pew conducted research related to the requirements and found “significant room for improvement in adoption and implementation of stewardship practices” in acute care hospitals, Hyun wrote.
Allocate financial resources for staffing and IT to support the antimicrobial stewardship program.
Implement evidence-based guidelines to improve antibiotic use for infections such as urinary tract c. diff. community-acquired pneumonia.
Evaluate the program using evidenced-based criteria.
“New antibiotic stewardship standards should help limit the emergence and spread of new drug-resistant superbugs,” Hyun noted.
Clinical Laboratories Need to Deepen Involvement
By testing patients and quickly reporting results to physicians, hospital-based and independent medical laboratories play an important role in appropriate antibiotic use and elimination of HAIs.
Heightened involvement by microbiologists and other medical laboratory professionals is key to success in light of recent setbacks in elimination of HAIs and antimicrobial resistance due to the SARS-CoV-2 outbreak.
Understanding why some people display no symptoms during a COVID-19 infection could lead to new precision medicine genetic tests medical labs could use to identify people with the mutated gene
New research from the University of California San Francisco (UCSF) may explain why some people could get COVID-19 but never test positive on a clinical laboratory test or develop symptoms despite exposure to the SARS-CoV-2 coronavirus.
According to the UCSF study, variations in a specific gene in a system of genes responsible for regulating the human immune system appears to be the factor in why about 10% of those who become infected with the virus are asymptomatic.
These scientific insights did not receive widespread news coverage but will be of interest to clinical laboratory managers and pathologists who oversee SARS-CoV-2 testing in their labs.
“Some people just don’t have symptoms at all,” Jill Hollenbach, PhD (above), Professor of Neurology atUCSF’s Weill Institute for Neurosciences and lead researcher in the study, told NBC News. “There’s something happening at a really fundamental level in the immune response that is helping those people to just completely wipe out this infection.” Identifying a genetic reason why some people are asymptomatic could lead to new precision medicine clinical laboratory diagnostics for COVID-19. (Photo copyright: Elena Zhukova /University of California San Francisco.)
Fortunate Gene Mutation
According to the Centers for Disease Control and Prevention’s (CDC) COVID Data Tracker, as of April 5, 2023, a total of 104,242,889 COVID-19 cases have been reported in the United States. However, according to a CDC Morbidity and Mortality Weekly Report (MMWR), “Traditional methods of disease surveillance do not capture all COVID-19 cases because some are asymptomatic, not diagnosed, or not reported; therefore, [knowing the true] proportion of the population with SARS-CoV-2 antibodies (i.e., seroprevalence) can improve understanding of population-level incidence of COVID-19.”
She also participates in the COVID-19 HLA and Immunogenetics Consortium, a group of academic researchers, clinical laboratory directors, journal editors, and others who examine the role of HLA variations in determining COVID-19 risk.
Hollenbach’s research identified an HLA variant—known as HLA-B*15:01—that causes the human immune system to react quickly to SARS-CoV-2 and “basically nuke the infection before you even start to have symptoms,” she told NPR.
“It’s definitely luck,” she added. “But, you know, this [gene] mutation is quite common. We estimate that maybe one in 10 people have it. And in people who are asymptomatic, that rises to one in five.”
“HLA variants are among the strongest reported associations with viral infections,” the UCSF study notes. So, the researchers theorized that HLA variations play a role in asymptomatic SARS-CoV-2 infections as well.
To conduct their study, shortly after the SARS-CoV-2 outbreak in 2020, the researchers recruited approximately 30,000 volunteer bone marrow donors from the National Marrow Donor Program to respond to periodic questions via a smartphone app or website. Because HLA variations can determine appropriate matches between donors and recipients, the database includes information about their HLA types.
Each week, respondents were asked to report if they had been tested for SARS-CoV-2. Each day, they were asked to report whether they had symptoms associated with COVID-19. “We were pretty stringent in our definition of asymptomatic,” Hollenbach told NBC News. “[The respondents couldn’t] even have a scratchy throat.”
The researchers eventually identified a cohort of 1,428 people who had tested positive for SARS-CoV-2 between February 2020 and April 30, 2021, before vaccines were widely available. Among these individuals, 136 reported no symptoms for two weeks before or two weeks after a positive test.
“Overall, one in five individuals (20%) who remained asymptomatic after infection carried HLA-B*15:01, compared to 9% among patients reporting symptoms,” the researchers wrote in their medRxiv preprint. Study participants with two copies of the gene were more than eight times more likely to be asymptomatic.
The UCSF researchers also looked at four other HLA variants and found none to be “significantly associated” with lack of symptoms. They confirmed their findings by reproducing the HLA-B association in two additional independent cohorts, one from an earlier study in the UK and the other consisting of San Francisco-area residents.
Individuals in the latter group had either tested positive for SARS-CoV-2 or reported COVID symptoms, and their DNA was analyzed to determine their HLA types.
Pre-existing T-Cell Immunity May Reduce Severity of COVID-19 Infection
The UCSF researchers also attempted to determine how HLA-B*15:01 plays a role in knocking out SARS-CoV-2 infections. They noted previous research that indicated previous exposure to seasonal coronaviruses, such as common cold viruses, could limit the severity of COVID-19. The scientists hypothesized that pre-existing T-cell immunity in HLA-B carriers may be the key.
The COVID-19 HLA and Immunogenetics Consortium website describes how HLA and T-cells work together to ward off disease. HLA “proteins are found on the surface of all cells except red-blood cells.” They’re “like windows into the inner workings of a cell,” and T-cells use the molecules to determine the presence of foreign proteins that are likely signs of infection. “Activated T-cells can kill infected cells, or activate B-cells, which produce antibodies in response to an infection,” the website explains.
Hollenbach’s research team analyzed T-cells from pre-pandemic individuals and observed that in more than half of HLA-B carriers, the T-cells were reactive to a SARS-CoV-2 peptide. The scientists corroborated the hypothesis by examining crystal structures of the HLA-B*15:01 molecule in the presence of coronavirus spike peptides from SARS-CoV-2 and two other human coronaviruses: OC43-CoV and HKU1-CoV.
“Altogether, our results strongly support the hypothesis that HLA-B*15:01 mediates asymptomatic COVID-19 disease via pre-existing T-cell immunity due to previous exposure to HKU1-CoV and OC43-CoV,” the researchers wrote.
Can Genes Prevent COVID-19 Infections?
Meanwhile, researchers at The Rockefeller University in New York City are attempting to go further and see if there are mutations that prevent people from getting infected in the first place. NPR reported that they were seeking participants for a study seeking to identify so-called “superdodger” genes.
Study participants identified as possibly having superdodger genes receive a kit designed to collect saliva samples, after which the researchers sequence the respondents’ genomes. “We hope that in a group of 2,000 to 4,000 people, some people will have genetic mutations that tell us why they’re resistant to infection,” Casanova told NPR.
All this genetic research is in very early stages. But results are promising and may lead to new precision medicine clinical laboratory tests for identifying people who are predisposed to having an asymptomatic response to COVID-19 infection. That in turn could help scientists learn how to moderate or even eliminate symptoms in those unfortunate people who suffer the typical symptoms of the disease.
