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

Abstract

Purpose: Low- and middle-income countries have high incidences of cervical cancer linked to human papillomavirus (HPV), and without resources for cancer screenings these countries bear 85% of all cervical cancer cases. To address some of these needs, brigade-style screening combined with sensitive polymerase chain reaction-based HPV testing to detect common high-risk HPV genotypes may be necessary.

Methods: We deployed an inexpensive DNA extraction technique and a real-time polymerase chain reaction-based HPV genotyping assay, as well as Papanicolaou testing, in a factory in San Pedro Sula, Honduras, where 1,732 women were screened for cervical cancer.

Results: We found that 28% of participants were positive for high-risk HPV, with 26% of HPV-positive participants having more than one HPV infection. Moreover, the most common HPV genotypes detected were different than those routinely found in the United States.

Conclusion: This work demonstrates a deployable protocol for HPV screening in low- and middle-income countries with limited resources to perform cytopathology assessment of Pap smears.

FIG 1

Flowchart of rice cooker boiling alkaline lysis DNA extraction. (A) Cervical brushes were obtained and accessioned. (B) Brushes were cut to fit a tube that contained 400 μL lysis buffer. (C) Tubes were placed in the colander of a rice cooker and boiled for 10 minutes. (D) Twenty-five microliters of the supplied and resuspended positive and negative controls were added to two reaction tubes and 17 μL of 70 mM Tris pH 8.0 was added to the remaining reaction tubes. (E) Eight microliters of each sample was added to reaction tubes for a final volume of 25 μL. (F) Real-time polymerase chain reaction (RT-PCR) and multicolor melt-curve analysis were performed. (G) A report was compiled from the results provided by the instrument software. HPV, human papillomavirus; n, patient accession number.

FIG 2

Summary of human papillomavirus (HPV) infections. (A) Distribution of HPV infections among the 1,732 tested samples. (B) Genotype distribution among all 480 HPV-positive samples. Genotypes of the HPV strains present in the nonavalent vaccine are shown in black, whereas those strains not included in the vaccine are shown in gray. The total number of occurrences is listed above each histogram.

FIG 3

Examples of melt-peak outputs. (A) Negative. (B) Human papillomavirus (HPV) -39 positive (arrow). (C) HPV-58 (arrow). (D) HPV-58, HPV-45 coinfection (arrows). (E) HPV-16, HPV-56 coinfection (arrows). (F) HPV-31, HPV-52, HPV-59 coinfection (arrows). Internal control (green/Hex); HPVs 31, 33, 16, 35, 68, 18 (orange/ROX); HPVs 56, 52, 45, 39 (red/CY5); HPVs 59, 66, 58, 51 (blue/FAM).

FIG 4

Instrument thermal profiles. (A) Reaction proceeding without power interruption and (B) with various interruptions to instrument power. Thermal cycler lid temperature (red); block temperature (blue).