In the future, will microbiology be performed from a purple top blood tube?

With more than 1,000 potential microbes known to cause human disease, the differential diagnosis of pathogens can be complex and challenging. Traditional microbial culture-based methods are the most common and readily available tests for identification, but the turnaround time can be long when organisms are difficult to culture or arduous when testing requires invasive techniques. Other technologies, like polymerase chain reaction (PCR) or mass spectrometric (MS) techniques, which are more rapid, have become extremely popular; however, these methods still have limitations, such as the need for presumptive diagnosis in order to test or the need for isolated pure cultures to perform the test.(1) Metagenomic next-generation sequencing (NGS), by detecting nucleic acids, is showing promise as a means of rapid pathogen identification.

Liquid biopsy technology based on cell-free DNA (cfDNA) is a useful and non-invasive tool using NGS technology. cfDNA originate from dying human cells as a result of cellular necrosis or apoptosis (e.g. tumor cells). cfDNA molecules are generally easily accessible in peripheral blood and have the potential for use as biomarkers for diagnostics, prognostics, and therapeutics, especially in certain types of cancers.(6) Early studies using cfDNA did not focus on nucleic acids of microbial origin (mcfDNA), because these small molecules were less well understood. However, mcfDNA-based tests for the identification of infectious disease have been gaining traction in clinical practice. In addition, an improved understanding of mcfDNA molecules has led to an increased number of studies demonstrating mcfDNA detection.(1,2,4-8) In turn, mcfDNA detection allows sensitive, broad, unbiased pathogen detection and has the potential for clinical application to identify a great variety of pathogens, such as invasive fungal infections, tuberculosis, and sepsis.

Recent reports indicate that NGS measuring mcfDNA are extremely useful in the diagnosis of a number of pathogens and syndromes, including organisms requiring invasive culture techniques in critically ill patients or those that are difficult to grow or take a long time to grow using traditional culture. For example, in a study using 21 patients with endocarditis, results demonstrated a 95% sensitivity with mcfDNA NGS results. mcfDNA testing in these patients showed the same organism as isolated from blood culture in 20 patients and further identified Enterococcus faecalis in one of three patients with definitive culture-negative endocarditis.(2)

Furthermore, patients presenting with severe disease may be administered empiric antibiotics. This treatment plan limits the sensitivity of subsequent microbiologic studies, making it difficult to evaluate prescribed antimicrobial therapies and draw a conclusion with clinical outcomes.(8) Another complication during the recent pandemic was that patients presenting with severe COVID-19 pneumonia may not have undergone standard invasive bronchoscopy due to the risks associated with healthcare personnel exposure to aerosolized SARS-CoV-2. Consequently, secondary infections by respiratory pathogens may have been undetected and negatively contributed to patient outcomes.

A study using mcfDNA isolated from plasma was conducted on fifteen critically ill patients confirmed by qPCR for infection by SARS-CoV-2.(8) Data was compared from survivors versus non-survivors, and non-survivors were found to have had higher mcfDNA molecules per microliter of plasma compared to survivors. In addition, non-survivors showed a trend for a higher number of identified microbes per sample.(8) In most cases, secondary pneumonia was clinically suspected or diagnosed by treating physicians, but in a small subset no secondary infection was suspected or antibiotic therapy was not prescribed or discontinued based on negative blood cultures. In patients who did not receive antibiotic therapy, 50% did not survive. Subsequent mcfDNA testing detected respiratory pathogens in 3/4 of these patients.(8) The study demonstrates an opportunity for integration of mcfDNA testing with clinical data to supplement antibiotic stewardship, which may have the potential to decrease mortality rates in critically ill patients. 

In both studies, the Karius Test®, a rapid commercially available liquid biopsy metagenomic NGS test utilizing cfDNA technology was used. The test shows promise in identifying over 1,200 pathogens: including members from all three domains, Bacteria, Eukarya (including fungi), and Archaea, as well as some common viruses.(2-4,8) The Karius Test® utilizes a simple, non-invasive whole blood draw from a standard plasma or EDTA tube. Results for most samples are attained within one day of testing and available via mobile app or through a web portal, but the cost is still quite high.

However, there are limitations to this type of sample processing. NGS mcfDNA testing is highly sensitive and has the potential to create polymicrobial results reporting, including the presence of commensal or transient non-pathogenic microorganisms depending on the specimen type.(4) Careful interpretation, in conjunction with clinical presentation and other diagnostic tools, may be necessary.(4,8)

Nevertheless, such a powerful diagnostic test can be an extremely useful tool to aid in identification of pathogens using less invasive techniques, or as a backup to culture negative results in critically ill patients when infection is strongly suspected.

Kerry Davies Pierce

References: 1, 2, 3, 4, 5, 6, 7, 8

FLU FACTS

• 21 per 100,000 children under the age of 4 have been hospitalized for the flu; last year it was less than one.

• The Oregon governor declared a state of emergency, since all pediatric ICU beds were full in Portland.

• Nearly 20,000 people in the United States were admitted to the hospital during the week of November 28, almost double the number of admissions from the week before, according to the CDC.

• The CDC estimates there have been at least 8.7 million illnesses, 78,000 hospitalizations and 4,500 deaths from influenza so far this season.

RSV FACTS

• The CDC states the the rate of RSV infections so far this year are ten times that of last year.

• RSV (Respiratory Syncytial Virus) causes bronchiolitis and pneumonia, usually mild in adults, but can be severe in infants and the elderly.

• 171 per 100,000 infants under the age of six months are being hospitalized currently; that's seven times the rate in 2018.

• Many children are coming down with multiple respiratory infections; including RSV, influenza, covid, rhinovirus, and others.

• A New York hospital has seen a 44% increase in admissions, mostly due to RSV.

• There is currently no vaccine for RSV. Almost all children get this infection by the time they are two years of age. However, the cases this year are far more severe.

References: 1, 2, 3,

Get prepared now for this year's flu season with Hardy Diagnostics products to help detect and protect against influenza and COVID-19.

The graph from the CDC below shows RSV detection for the state of California. Other states are available at this link.                                     

Pseudomonas infections are fairly common and dangerous in the United States. Multi-drug resistant Pseudomonas aeruginosa caused an estimated 32,600 infections among hospitalized patients and an estimated 2,700 deaths in the U.S. in 2019. (1)

Pseudomonas is commonly found in the environment in soil and water. These infections are among the most common to occur in hospitalized patients. Symptoms of Pseudomonas infections vary depending upon the site of infection but range from fever and chills, to abscesses and hearing or vision loss. (2) Pseudomonas is also a cause of hot tub rash (as pictured above), since it can commonly be found as a contaminant in hot tub water where it infects the hair follicles.

Pseudomonas aeruginosa’s ability to form biofilms on both biotic and abiotic surfaces makes it particularly resistant to a host’s immune system and particularly difficult to clean from hospital instruments, especially tubing in catheters and endotracheal tubes (ETT) of ventilated patients. (3) Pseudomonas aeruginosa is one of the aggressive organisms at producing biofilm. Biofilm represents a complex microbial community, embedded in an abundant matrix of hydrated extracellular polymeric substance (EPS), primarily composed of polysaccharides, proteins, nucleic acids and lipids. (3) The highest risk groups for these types of infections are individuals on ventilators and catheters, as well as those with surgery wounds, burns or extended hospital stays. (1) Pseudomonas aeruginosa was identified as a common co-infecting pathogen in COVID-19 patients during the pandemic. (4)

As far as mitigation for biofilm production, new antimicrobials like hydrogel, silver salts, or nanoparticles have been used to coat new generation catheters with promising results. Even with these advances, the approach of coating the tubing only produces limited results, as the shape and material composition of these devices is believed to also promote biofilm formation. (3)  Biofilm occurs on more than 90% of endotracheal tubes (ETT) within 7 days of insertion, as standard cleaning practices are not capable of fully clearing formed bio-film. (3) From biofilm, bacterial cells can easily detach and make their way into lower airways through gas flow of the ventilator and aspiration. (3)

Luckily, Pseudomonas aeruginosa can be controlled through aseptic technique, thorough cleaning regimens, and correct isolation procedures.(5)

By: Joseph Tripp

Technical Services

References: 1, 2, 3, 4, 5