Implementation of Multicolor Melt Curve Analysis for High-Risk Human Papilloma Virus Detection in Low- and Middle-Income Countries: A Pilot Study for Expanded Cervical Cancer Screening in Honduras

Abstract

PurposeCervical cancer is a leading cause of cancer-related mortality in low- and middle-income countries (LMICs) and screening in LMICs is extremely limited. We aimed to implement on-site high-risk human papillomavirus (hrHPV) DNA testing in cohorts of women from an urban factory and from a rural village.MethodsA total of 802 women were recruited for this study in partnership with La Liga Contra el Cancer through the establishment of women’s health resource fairs at two locations in Honduras: a textile factory (n = 401) in the city of San Pedro Sula and the rural village of El Rosario (n = 401) in Yoro. Participants received a routine cervical examination during which three sterile cytobrushes were used to collect cervical samples for testing. hrHPV genotyping was performed using a hrHPV genotyping assay and a real-time polymerase chain reaction instrument.ResultshrHPV status across all participants at both sites was 13% hrHPV positive and 67% hrHPV negative. When hrHPV status was compared across all three testing sites, hrHPV-positive rates were approximately equal among the factory (13%), village (12%), and confirmatory testing at Dartmouth-Hitchcock Medical Center (Lebanon, NH; 14%). hrHPV genotype was compared across sites, with HPV16 showing the highest infection rate (15%), followed by HPV59 (12%), and HPV68 (11%). There was a low prevalence of HPV18 observed in both populations compared with the hrHPV-positive population in the United States.ConclusionIn collaboration with oncologists and pathologists from La Liga Contra el Cancer, we were able to provide a continuum of care once health-fair testing was performed. We established a method and implementation plan for hrHPV testing that is sustainable in LMICs.

Screening for Human Papillomavirus in a Low- and Middle-Income Country

“…We therefore developed a low-cost, simple DNA testing method for hrHPV using a crude cell lysate and real-time PCR instrument. 4,5 In several studies, we showed that hrHPV type distributions were more reflective of those found in Asia than in North America. 4,5,17 This geographically unexpected distribution underscores the need for HPV genotype surveillance to effectively triage hrHPV-positive participants for follow-up care and to strategize the implementation of successful vaccination programs.…”

“…Last, the low-cost DNA lysate and sensitive HPV assay described here allowed us to process 94 samples in less than 2.5 hours and return rapid genotype-specific results. This approach offers the following benefits: in contrast to other PCR-based assays that use hydrolysis probes, the equipment can be run off of a generator or variable power sources, as the system returns results despite power interruptions (Fig 4); the PCR system itself uses LEDs that require little, if any, maintenance; the DNA extraction method uses minimal and readily available disposables; lyophilized reagents were tested without refrigeration for 2 weeks and maintained their efficacy 4 ; and 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. These features allow for near-patient care diagnosis that is of critical importance for combining screening and treatment strategies in LMICs.…”

“…Cell lysates from cervical swabs were made as previously described with several modifications. 4 In brief, dried cervical brushes were cut and placed inside individual tubes that contained 400 µL of cell lysis buffer consisting of 50 mM NaOH and 0.2 μM EDTA in nuclease-free water (Fig 1B). Tubes were boiled for 10 minutes using an inexpensive locally sourced rice cooker, the plastic colander of which had been bored out to accommodate the 1.5-mL screw-cap tubes (Fig 1C).…”

“…To alleviate some of these burdens associated with cervical cancer screening, we developed and validated a process with which to screen women using a simple, high-risk human papillomavirus (hrHPV) DNA test. 4 We demonstrate that DNA testing for hrHPV using crude cell lysates and real-time polymerase chain reaction (PCR) with lyophilized reagents was feasible to perform in a mobile system so that women in various regions of Honduras could be tested. 5…”

Feasibility of Brigade-Style, Multiphasic Cancer Screening in Rural Honduras

“…In 2013, the CLARO team committed to an initial attempt to scale-up a PESCA screening, taking it farther afield into a rural mountainous region in Honduras, where cervical screening had been unavailable. 9 Previous community development work provided existing relationships, and local leaders were eager to host a free, large-scale cancer screening event for women that they determined should be called a "jornada" (Spanish for a day of work). Recognizing that this cancer screening jornada would be the first and potentially only cancer screening for recruited participants, it made sense to make the day as useful as possible, so low-tech breast cancer screening (ie, education and clinical examination) was coupled with cervical cancer screening.…”