In a recent study posted to the medRxiv* preprint server, researchers examined sample collection practices, epidemiological characteristics, and cycle threshold (Ct) values for polymerase chain response (PCR) tests utilized in two reference laboratories in america (U.S.) to detect monkeypox virus (MPXV).

Study: Clinical Performance and Trends Through the First Two Months of Monkeypox Virus PCR Testing at Two United States Reference Labs. Image Credit: Salov Evgeniy / Shutterstock

Background

The recent outbreak of monkeypox in non-endemic countries similar to the U.S. has resulted in increased MPXV PCR diagnostic testing. Monkeypox is attributable to a virus within the Orthopoxvirus genus, much like the smallpox-causing Variola virus. With the virtually complete eradication of smallpox, most countries discontinued the smallpox vaccination, leaving gaps in immunity.

The substantial decrease within the spread of orthopoxviruses had also resulted in clinicians being unfamiliar with orthopoxvirus infections and fewer diagnostic testing facilities. Before the 2022 outbreak, MPXV testing was carried out only in U.S. Food and Drug Administration (FDA) approved laboratories or by the Centers for Disease Control and Prevention (CDC). Between June and July 2022, laboratory-developed and FDA-authorized testing of monkeypox expanded to clinical laboratories.

While clinical and reference laboratories can process substantially higher numbers of cases than public health facilities, and report qualitative test results to the CDC and state health departments, data similar to Ct values and test performances usually are not usually reported. Monitoring these data can assist discover emerging variants and improve testing practices.

In regards to the study

The current study investigated MPXV PCR testing data from two reference laboratories within the U.S — the University of Washington Virology Lab (UW) in Seattle, Washington, and ARUP Laboratories (ARUP) in Salt Lake City, Utah. The examined data included information for greater than 10,000 specimens collected between July and August 2022.

Descriptive epidemiological aspects similar to age and sex distribution in MPXV PCR testing were compared across the 2 laboratories. Moreover, the researchers examined the typical Ct values in each laboratories to match the viral loads detected within the PCR tests. Finally, the test result concordance was compared using the proportion of patients who submitted multiple swabs.

Results

The outcomes reported a rise in testing amongst males aged 18 to 40, considered to be at greater risk of monkeypox. The authors observed that positivity rates decreased with the rise within the variety of submitted specimens. In addition they detected a more significant variety of positive test results amongst males than amongst females, which is consistent with other study results reporting a better incidence of monkeypox infections amongst men who’ve sex with men.

The Ct values between the 2 reference laboratories were comparable, with ARUP laboratory tests detecting a rather higher viral load. The gathering and extraction methods, nonetheless, differed significantly. The Chemagic nucleic acid extractor utilized by ARUP has higher analytical sensitivity than the viral inactivation and lysis protocol utilized in UW. The authors imagine that the difference in specimen processing and extraction procedures could account for the variation in viral loads from the 2 laboratories.

Collecting multiple swabs from the identical individual resulted in an overall test concordance of greater than 95%. A really low percentage (lower than 1.5%) needed three or more tests for a positive result, and their Ct values were significantly high, indicating a low viral load. Dry swabs suspended in phosphate-buffered saline revealed higher viral loads than swabs in viral transport media (VTM), which the authors attribute to the dilution of the viral load in VTM.

In line with the authors, while discordant positive results may very well be as a consequence of low viral loads, cross-contamination can’t be ruled out as a reason. The CDC recently released an advisory concerning the risk of false positives and urged laboratories to check two specimens for results with Ct values greater than 34.

Conclusions

Overall, the study showed that monitoring MPXV PCR testing aspects aside from qualitative test results across clinical and reference laboratories is amazingly useful in understanding the differences in and improving the processes of diagnostic tests.

The authors report that while multiple swabs from one individual do improve the general test concordance, a substantial portion of PCR testing facilities and resources are used for one individual, which is inefficient when it comes to resources and time. Subsequently, they as a substitute propose a technique of mixing multiple swabs from one individual in VTM to enhance diagnostic performance while reducing overall costs.

*Essential notice

medRxiv publishes preliminary scientific reports that usually are not peer-reviewed and, due to this fact, mustn’t be considered conclusive, guide clinical practice/health-related behavior, or treated as established information