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Unexpected Prevalence of High-Risk HPV Types Demonstrates Utility of Zeesan Biotech Assay in Low-Resource Setting


NEW YORK (GenomeWeb) – An evaluation of an HPV typing assay from Zeesan Biotech has shown the firm's test kit can be incorporated successfully into an inexpensive screening protocol and run near patients in a low-resource setting.

Among a population of female factory workers in Honduras, the Zeesan HPV typing test detected a low prevalence of high-risk HPV types 16 and 18, but an unexpectedly high prevalence of high-risk type 58.

The study, which was published this month in the Journal of Global Oncology, was a collaboration between researchers at Dartmouth College and scientists and clinicians in San Pedro Sula, Honduras. It evaluated a PCR-based, CE-marked kit called the MeltPro High Risk HPV Genotyping Assay from Zeesan Biotech.

The kit, which is distributed by QuanDx in Europe as a CE-marked test and in North America as a research-use-only test, consists of a set of lyophilized reagents to detect all high-risk HPV subtypes. The study also incorporated a real-time PCR instrument called the SLAN-96, also from Zeesan Biotech.

Gregory Tsongalis, director of the laboratory for clinical genomics and advanced technology (CGAT) at Dartmouth, said his team initially tried to develop its own HPV genotyping assay for low-resource settings, but then learned about the Zeesan test and decided to assess it.

"This is a very robust assay with relatively high throughput and [it] can differentiate between all high-risk and low-risk HPV types in two separate assays," Tsongalis said in an email. Still, the team put the assay through "a very rigorous validation" before deploying it, he added.

The team's evaluation used the Zeesan kit and SLAN-96 instrument to determine HPV infection status and virus subtype as part of a screening program.

The program evaluated 1,732 female employees at a maquiladora, or manufacturing facility, in San Pedro Sula, Honduras, operated by international apparel manufacturing firm Gildan which makes clothing for American Apparel, among other firms.

Tsongalis noted that the team chose to screen the factory worker in order to be able to "test many women in a convenient setting that had constant electricity and some facilities" for the researchers to use.  

There are 37 types of HPV that typically infect people, but a subset of 14 types are considered to carry a higher risk of causing cervical cancer. These include HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, with types 16 and 18 typically included in molecular cervical cancer screening tests as they are considered to be among the most carcinogenic types.

The QuanDx assay detects and distinguishes all 14 high-risk HPV types, as well as an internal human DNA sequence control.

Specifically, it is a single-tube multiplexed PCR with endpoint multicolor melt-curve analysis. According to the JGO study, the test resolves type-specific melting temperatures for four distinct probes labeled with the dyes ROX (HPV 31, 33, 16, 35, 68, and 18), CY5 (HPV 56, 52, 45, and 39), FAM (HPV 59, 66, 58, and 51), and using HEX for the internal control.

In the study, the researchers also demonstrated a low-cost way to adapt a locally purchased rice cooker for 10-minute nucleic acid sample preparation, and showed it was possible to process 94 cervical cancer screening samples within two and half hours using the workflow.

The researchers also noted certain features that make the workflow particularly amenable to near-patient diagnosis of HPV type in low-resource settings.

For example, unlike other PCR-based assays that use hydrolysis probes, the Zeesan instrument can be run off a generator or variable power sources because it returns results despite power interruptions, the researchers said. The LEDs in the PCR system require little maintenance and the DNA extraction method uses minimal and readily available disposables. Finally, "the combination of lyophilized reagents, crude lysate, and HPV genotyping performed by the instrument makes for an easy-to-perform operation that is suitable for testing in locations without highly trained personnel," the study authors wrote.

Among the intriguing findings of the study was the fact that it found no HPV 16 and HPV 18 coinfections and relatively few single HPV 16 or HPV 18 infections. The authors suggested that the overall results could inform vaccine development, as the current nonavalent HPV vaccine protects against nine HPV types but not all the ones that were prevalent among this particular population.

The study found that 480 of the 1,732 samples from the factory worker screening effort were positive for high-risk HPV, or approximately 28 percent, while 1,199 samples had no detectable HPV, and 53 samples failed to amplify either the internal control or an HPV target and were deemed to be invalid.

By comparison, the prevalence of high-risk HPV among women in the US during a period spanning 2013 to 2014 was approximately 20 percent, according to the US Centers for Disease Control and Prevention, with rates as high as 28 percent among non-Hispanic black women.

The most common genotypes detected in the study were HPVs 58, 35, and 16. Specifically, HPV 58 was detected in 90 samples, or among 19 percent of positive samples, while HPV 35 was detected in 64 samples, or among 13 percent of positive samples, and HPV 16 was detected in 63 samples, also approximately 13 percent of all of positive infections.

The study authors further highlighted the fact that there were only 31 samples positive for HPV 18, which is a subtype that is more common in other parts of the world. For example, HPV types 16 and 18 account for approximately 66 percent of cervical cancers in the US, according to a 2015 assessment.

The nonavalent vaccine protects against high-risk types that cause cervical cancers as well as types that cause genital warts. Specifically, it protects against types 6, 11, 16, 18, 31, 33, 45, 52, and 58.

The study noted that based on the QuanDx genotyping results, 47 percent of the women who are HPV positive in the study could have received some amount of protection from either single or coinfections, and possible progression to cervical cancer, but approximately 53 percent of the participants would not have benefited from the nonavalent vaccine on the basis of genotype prevalence alone.

The main competing molecular technology for low- and middle-income country would be the Digene assay, now part of Qiagen, Tsongalis said. "Some folks propose using [immunohistochemistry], but a problem in LMICs is the lack of pathologists who have the microscopic expertise," Tsongalis said.

And, knowing the more specific HPV type can be important for population and vaccine studies and trials. The group has also previously found the result that HPV 16/18 are not the most prevalent types in Honduras, for example, but rather an unexpected HPV distribution with an abundance of HPV 52 and HPV 72.

The Dartmouth team will now be following up on the study. "Our next steps are to obtain cervical cancer tissues from women and test those to see which HPV types are actually found in the tumor cells," Tsongalis said. "This could give us a lot of information about causality."

Matthew Lei, CEO of QuanDx, noted in an email that the Dartmouth lab purchased the HPV testing kits from QuanDx, and that neither QuanDx nor Zeesan provided materials or funding for the study.

Now, QuanDx hopes to collaborate with more labs to test the MeltPro high-risk HPV genotyping kits, Lei said.

"More data have shown the non-16/18 HPV types play an important role for cervical, head and neck, and genital cancer," he said. The Zeesan kit could potentially help clinicians to "further explore the relationship between the virus and the tumorigenesis," Lei added, as well as to "facilitate new HPV vaccine development in the future."

QuanDx also markets a tuberculosis test from Zeesan that can detect multidrug-resistant (MDR-) and extensively drug-resistant tuberculosis (XDR-TB) in patient sputum samples. Lei also said the company continues to explore the marketing potential for its own molecular diagnostic kits based on a proprietary technology called Yin-Yang Probes. These kits include ones for leukemia fusion gene screening, and two lung cancer panels that test for EGFR/KRAS/BRAF and ALK/ROS1/RET.

The company also intends to launch an integrated and automated system, called Q-Velox, later this year, Lei said.

"This system is a sample-in, results-out platform which can use our current reagents. We hope, by commercializing this automated instrument, that more labs will adopt our assays," he said.