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Stanford-led Group Validates Multiplex Tropical Pathogen PCR for Plasmodium Species Detection

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NEW YORK (GenomeWeb) – Identifying the cause of febrile illnesses can be a challenge, particularly in some tropical and sub-tropical areas of the world where patients can be exposed to different pathogens whose symptoms mimic each other.

To help address this, researchers at Stanford University School of Medicine have been developing a single-reaction multiplex PCR assay that can detect and differentiate the causative agents of dengue fever, leptospirosis, and malaria — three pathogens endemic to such regions.

Specifically, the assay is designed to detect dengue viruses DENV-1 to -4 using a pan-DENV design, as well as all species of Leptospira bacteria, and the five Plasmodium parasite species known to be cause malaria in humans, with a specific call-out for P. falciparum.

A study published online earlier this month in the Journal of Clinical Microbiology showed that the test, dubbed the DLM assay, compared favorably to microscopy and the BinaxNow Malaria rapid diagnostic test from Alere specifically in detecting Plasmodium falciparum.

Many molecular assays separately target the individual causative agents of dengue, leptospirosis, and malaria, but the multiplexed detection of all three pathogens makes the DLM assay unique, Benjamin Pinsky, a co-author on the study and an assistant professor of pathology and infectious diseases at Stanford University Medical Center, told GenomeWeb in an email.

Many other molecular tests for malaria require whole blood or dried blood spots, but the current study is among the first to analyze serum and plasma.

The study was conducted using samples obtained by co-author Wellington Oyibo at the University of Lagos College of Medicine in Nigeria, where P. falciparum is the predominant species.

"The DLM assay uses a pan-Plasmodium primer-probe set that detects all five of the Plasmodium species that infect humans but does not distinguish between them; a separate P. falciparum-specific primer-probe set that utilizes a different fluorescent label allows a call-out for P. falciparum," Pinsky explained.

The DLM assay itself was described last year, also in JCM. The goal of the current study was to compare the DLM assay with gold-standard microscopy and rapid antigen testing for P. falciparum.

On serum or plasma from 317 patients, the assay showed 97 percent sensitivity and 93.5 percent specificity for P. falciparum. A rapid immunoassay from Alere was shown to be similarly sensitive, and both were superior to microscopy, which has a sensitivity of 79 percent.

The mean specimen volume was 70 microliters, but when the researchers constrained their analysis to sample volumes  of greater than 75 microliters, the sensitivity and specificity of the DLM test for P. falciparum improved to 99 percent and 97.5 percent, respectively, possibly due to detection of lower parasitemia in higher volumes.

Pinsky says his group plans to compare the test to PCR-based Plasmodium tests in the future. Meanwhile, it has used this assay "to test thousands of samples from around the world, including samples collected from febrile patients in Nicaragua, Brazil, Sri Lanka, Kenya, and Cameroon."

The dengue virus component of the DLM assay has been licensed, Pinsky said, and the licensee intends to use it as a companion diagnostic for a dengue therapeutic that is entering human clinical trials.

"We are working with this company, as well, to license the DLM assay," he said, but noted that the company requested he not reveal its name at the present time.

At this point, the DLM assay would need to be run in a laboratory capable of molecular diagnostic testing because it runs on a standard real-time PCR platform and requires sample preparation.

"However, the goal is to provide this and other assays for undifferentiated systemic febrile illnesses in a format that can be performed near-care or at the point-of-care," Pinsky said.

This may eventually involve isothermal amplification methods like recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP). Furthermore, the group has "already started working with both academic laboratories and diagnostics companies on adapting our existing assays to PCR-based sample-to-answer systems," Pinsky said.

Indeed, for single-plex assays at least, this appears to be feasible. An isothermal Leptospira assay was recently developed using TwistAmp chemistry from Alere subsidiary TwistDx, as reported by GenomeWeb. Another group has also reported using RPA to build a lateral flow P. falciparum assay, and a malaria assay from Eiken Chemical that uses LAMP was made commercially available with CE marking about two years ago.

Pinsky said his group has also developed other tests, including ones for viruses such as chikungunya, Zika, Yellow Fever, West Nile, O'nyong nyong, Mayaro, and Rift Valley Fever.

"These assays have been designed to be compatible in multiplex with each other as well as [with] the components of the DLM assay, so that regional panels can be designed to best detect the pathogens important in a particular area," Pinsky said.

A number of molecular diagnostics makers, including BioMérieux subsidiary BioFire Diagnostics and German assay developer Hahn-Schickard, are currently developing or considering multiplex PCR panels for undifferentiated febrile illness, sometimes also referred to as "traveler's panels." Metagenomic sequencing using Oxford Nanopore MinIon is another possible method to pinpoint the cause of UFI, as recently discussed

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