High-risk human papillomavirus (hrHPV) may cause more than 99% of cervical cancers worldwide. Little is known about performance differences in tests for hrHPV.
This study analysed agreement for detection of hrHPV between the established, clinically validated Xpert HPV assay and the novel isothermal amplification-based AmpFire HPV genotyping assay.
This study was nested in a larger project on cervical cancer screening among approximately 5000 women living with HIV in Kigali, Rwanda. This sub-study included 298 participants who underwent initial screening for cervical cancer using the Xpert HPV assay and visual inspection with acetic acid in 2017 and tested positive by either or both. Participants were rescreened using colposcopy, and cervical samples were collected between June 2018 and June 2019. Samples were then tested for HPV using the Xpert HPV assay and AmpFire HPV genotyping assay. Agreement between results from both tests was analysed using an exact version of McNemar test and chi-square test.
Overall agreement and kappa value for detection of hrHPV by Xpert and AmpFire were 89% and 0.77 (95% confidence interval: 0.70–0.85). AmpFire was marginally more likely to diagnose hrHPV-positive than Xpert (
Overall, there was good to excellent agreement between the Xpert and AmpFire when testing hrHPV types among women living with HIV. AmpFire was more likely to test extra cases of HPV16, the most carcinogenic HPV type, but the clinical meaning of detecting additional HPV16 infections remains unknown.
High-risk human papillomavirus (hrHPV) causes virtually all cervical cancer-related cases worldwide.
Evidence to date indicates that HPV genotyping can help with risk stratification for further triage and management of hrHPV-positive women.
Xpert is a World Health Organization pre-qualified test,
The AmpFire is a promising new lower-cost assay, using isothermal amplification along with a real-time fluorescence system in four channels to individually detect 15 types of hrHPV, including HPV16, 18, 31, 33 35, 39, 45, 51, 52, 56, 58, 59 and 68, plus intermediate-risk HPV53 and 66. The AmpFire assay uses a simple processing protocol to detect HPV directly without prior DNA extraction from clinical samples, including dry samples. The assay has a small footprint and a sample-to-result time of approximately 1 h.
We recently completed a cervical cancer screening study of approximately 5000 Rwandan women living with HIV. In a subset of women who underwent colposcopy, we compared the analytic performance for HPV detection by the Xpert and AmpFire systems.
This sub-study derived from a larger study on cervical cancer screening among Rwandan women with HIV,
The participants included 298 women living with HIV who were recruited from a larger study of cervical cancer screening, which was carried out among approximately 5000 Rwandan women living with HIV. All women were initially screened by either the Xpert HPV assay (Cepheid, Sunnyvale, California, United States) or visual inspection with acetic acid and those positive by either or both methods were referred for colposcopy. A detailed protocol related to the study population and recruitment was further developed in the parent study.
The participants included women who were lost to follow-up in the parent study for a period of 6–38 months. After retrieving them, they were enrolled in the present study, for which specimens were collected between June 2018 and June 2019. These women with a delay in colposcopy attendance underwent colposcopy, during which cervical specimens were collected using a LuckMedical Cervical Brush (Luck Medical Consumables Co., Jiangsu, China). These samples were put into 20 mL of the PreservCyt medium (Hologic, Bedford, Massachusetts, United States).
All women included in the study lived in Kigali, which is the capital and biggest city of Rwanda. The latest Population and Housing Census, dated 2012, indicated that Kigali was populated with 1 132 686 inhabitants.
Cervical specimens were mixed with PreservCyt before being tested for hrHPV. A volume of 1 mL of the mixed sample was added to the Xpert cartridge, which was then placed in the Xpert assay for the analysis. Results from this analysis were available within an hour.
The sample of cervical specimens mixed with PreservCyt was also tested for hrHPV using the AmpFire (Atila Biosystems, Mountain View, California, United States). A volume of 1 mL of this sample was added to a 1.5 mL-micro-centrifuge tube and centrifuged for 10 min at 10 000 revolutions per minute.
The cut-off point values of positivity applied when using Xpert and AmpFire assays were defined according to the manufacturers’ instructions.
Agreement statistics (positive, negative and overall agreement, and kappa values) for 14 HPV types, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, detected by both tests, were calculated. Results for HPV53, detected by AmpFire but not by Xpert, were excluded. Results from AmpFire were grouped according to the HPV groups as detected by the Xpert HPV assay. Firstly, HPV groups were considered non-hierarchically, recognising that a given specimen could test positive for more than one group. Secondly, the two tests were also compared using risk-based hierarchical HPV group types, considering that different HPV groups are associated with different risks of cervical cancer: HPV16 positive, else positive for HPV18/45, else positive for HPV 31/33/35/52/58, else positive for other high-risk HPV types (HPV51/59/39/56/66/68), or else negative; HPV51/59 and HPV39/56/66/68 have similarly lower risk for invasive cervical cancer and were grouped together. McNemar test and chi-square test was applied to assess significant differences (
Overall agreement between the two test results for the 14 HPV types was 89% and the kappa value was 0.77 (95% confidence interval: 0.70–0.85). AmpFire was marginally more likely to test positive for the 13 types of HPV (
Agreement for detection of human papillomavirus overall and HPV types grouped according to the Xpert HPV assay between Xpert HPV assay and AmpFire HPV genotyping assay, Rwanda, June 2018 - June 2019.
HPV results AmpFire/Xpert | +/+ |
−/+ |
+/− |
−/− |
Positive agreement (%) | Negative agreement (%) | Overall agreement (%) | Exact |
Unweighted kappa | 95% confidence interval | ||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
% | % | % | % | |||||||||||
HPV16 | 35 | 12.4 | 1 | 0.4 | 18 | 6.4 | 229 | 80.9 | 65 | 92 | 93 | < 0.001 | 0.75 | 0.64–0.85 |
HPV18/45 | 27 | 9.5 | 3 | 1.1 | 6 | 2.1 | 247 | 87.3 | 75 | 96 | 97 | 0.510 | 0.84 | 0.74–0.94 |
HPV31/33/35/52/58 | 66 | 23.3 | 10 | 3.5 | 12 | 4.2 | 195 | 68.9 | 75 | 90 | 92 | 0.830 | 0.80 | 0.72–0.88 |
HPV51/59 | 13 | 4.6 | 1 | 0.4 | 6 | 2.1 | 263 | 92.9 | 65 | 97 | 98 | 0.130 | 0.77 | 0.61–0.94 |
HPV39/56/66/68 | 23 | 8.1 | 6 | 2.1 | 12 | 4.2 | 242 | 85.5 | 56 | 93 | 94 | 0.240 | 0.68 | 0.55–0.82 |
HR13 |
134 | 47.3 | 10 | 3.5 | 22 | 7.8 | 117 | 41.3 | 81 | 79 | 89 | 0.050 | 0.77 | 0.70–0.85 |
Note:
HPV, human papillomavirus.
, HR13 includes HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.
On the hierarchical analysis, the two assays had good agreement with an overall unweighted kappa value of 0.77 (95% confidence interval: 0.70–0.85) (
Pairwise AmpFire and Xpert results, categorised hierarchically according to HPV risk groups, Rwanda, June 2018 - June 2019.
Xpert AmpFire | HPV16 | HPV18/45 | HPV31/33/35/52/58 | HPV51/59 + HPV39/56/66/68 | Negative | Total |
---|---|---|---|---|---|---|
HPV16 | 2 | 5 | 4 | 7 | 53 | |
Row % | 3.8 | 9.4 | 7.5 | 13.2 | 100.0 | |
Column % | 7.1 | 8.5 | 19.0 | 5.0 | 18.7 | |
HPV18/45 | 0 | 3 | 1 | 2 | 30 | |
Row % | 0.0 | 10.0 | 3.3 | 6.7 | 100.0 | |
Column % | 0.0 | 5.1 | 4.8 | 1.4 | 10.6 | |
HPV31/33/35/52/58 | 0 | 0 | 0 | 8 | 52 | |
Row % | 0.0 | 0.0 | 0.0 | 15.4 | 100.0 | |
Column % | 0.0 | 0.0 | 0.0 | 5.8 | 18.4 | |
HPV51/59 + HPV39/56/66/68 | 0 | 0 | 0 | 5 | 21 | |
Row % | 0.0 | 0.0 | 0.0 | 23.8 | 100.0 | |
Column % | 0.0 | 0.0 | 0.0 | 3.6 | 7.4 | |
Negative | 1 | 2 | 7 | 0 | 127 | |
Row % | 0.8 | 1.6 | 5.5 | 0.0 | 100.0 | |
Column % | 2.8 | 7.1 | 11.9 | 0.0 | 44.9 | |
Total | 36 | 28 | 59 | 21 | 139 | |
Row % | 12.7 | 9.9 | 20.8 | 7.4 | 49.1 | |
Column % | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
Note:
Unweighted kappa = 0.77 (95% confidence interval = 0.70–0.85).
HPV, human papillomavirus.
More than 80% of the HPV18/33/35/45/52/58/59 detected by AmpFire were positive in the Xpert channel that tested for that type (as a group of types) (
Concordance of individual HPV types detected by AmpFire HPV genotyping assay with HPV groups detected by Xpert HPV assay (non-hierarchical), Rwanda, June 2018 - June 2019.
AmpFire positive | AmpFire positive confirmed on the Xpert channel for respective types |
|
---|---|---|
% | ||
HPV16 ( |
35 | 66.0 |
HPV18 ( |
16 | 84.2 |
HPV45 ( |
13 | 81.3 |
HPV31 ( |
11 | 78.6 |
HPV33 ( |
19 | 86.4 |
HPV35 ( |
16 | 100.0 |
HPV52 ( |
20 | 87.0 |
HPV58 ( |
16 | 84.2 |
HPV51 ( |
6 | 54.4 |
HPV59 ( |
7 | 87.5 |
HPV39 ( |
3 | 42.9 |
HPV56 ( |
10 | 71.4 |
HPV66 ( |
6 | 75.0 |
HPV68 ( |
6 | 75.0 |
Negative ( |
117 | 92.1 |
Note: Combining the AmpFire results to match the Xpert HPV groups, in 283 women living with HIV undergoing colposcopy.
HPV, human papillomavirus.
This study sought to compare the Xpert and AmpFire among women living with HIV in Rwanda. We found an overall good to excellent agreement between the two methods for the detection of hrHPV. Notably, AmpFire was more likely to test positive cases for HPV16 than Xpert but the clinical meaning of the detection of these additional HPV16 infections was unknown.
The comparable findings between Xpert and AmpFire HPV regarding the detection of hrHPV may be explained by the well-known performance of these tests. Previous studies have shown that AmpFire could detect all HPV genotypes with sensitivity and specificity of more than 95%.
Further, Xpert has been widely used for the detection of various types of HPV, including hrHPV types. In Zimbabwe, an overall good concordance (77.2%, kappa = 0.698) between Xpert and Seegene Anyplex II HPV HR detection kit was found when testing hrHPV among women aged from 30 to 60 years.
Despite the overall agreement between the Xpert and AmpFire, differences in the detection of some types of hrHPV were noticed. These differences could be reflected by the extra cases of HPV16 and other hrHPV, including HPV18 and 45, which were detected by the AmpFire compared to Xpert. However, we could not find any scientific reason that could be attributed to the extra positivity by AmpFire, especially for HPV16. In contrast, Xpert has shown greater potential of detecting more cases of HPV31/33/35/52/58 than AmpFire. This extra detection of some hrHPV by Xpert compared to AmpFire could probably be explained by its greater performance of detecting concurrent infections by hrHPV.
As explained earlier, both assays perform as expected and use simple procedures with less equipment, and a rapid turnaround time, which make them highly recommendable as point-of-care screening for cervical precancer and cancer in low- to middle-income countries.
This was the first study to have analysed the agreement between Xpert and AmpFire for the detection of hrHPV among women living with HIV in Rwanda. Thus, our findings may provide new insights regarding the performance of the two assays when applied in low-resource settings.
The authors could not determine why there was extra positivity by AmpFire, especially for the detection of HPV16. Further studies, involving larger numbers of specimens and multiple sites, or histopathologic analyses could perhaps be relevant to address this knowledge gap.
This study revealed good to excellent agreement between Xpert and AmpFire when testing hrHPV types among women living with HIV in a Rwandan setting. It has also shown an extra positivity by AmpFire compared to Xpert, especially for HPV16, which requires further studies or adjudication by histopathologic tests.
Authors thank the study participants for having consented to take part in the study. They also acknowledge the data collectors who have been involved in the fieldwork. Authors are also indebted to the facilitation by the management of the health centres where the initial cervical cancer screening took place.
This study received Xpert HPV tests at a reduced cost from Cepheid (Sunnyvale, California, United States).
A. Murangwa and P.E.C. conceived the study and received feedback from K.T.D. and J.C.G. A. Musafili and K.T.D. analysed and interpreted data with substantial inputs from P.E.C., J.C.G., K.A., L.M., G.M., G.K., P.T., F.K. and H.-Y.K. A. Murangwa and K.T.D. drafted the manuscript, which was critically reviewed by other authors. All authors approved the final version of the manuscript before submission.
This study was funded by grants from the National Cancer Institute of the United States National Institutes of Health (5U54CA19016304, 5U54CA254568).
The data sets used or analysed during the current study are available from the corresponding author, A. Murangwa, on reasonable request.
The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors.