THE PERFORMANCE OF CEPHEID XPERT HPV
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1 THE PERFORMANCE OF CEPHEID XPERT HPV Zizipho Z.A Mbulawa, Timothy Wilkin, Bridgette J. Goeieman, Eefje Jong, Pamela Michelow, Avril Swarts, Jennifer S. Smith, Patricia Kegorilwe, Cynthia S. Firnhaber, Anna-Lise Williamson Institute of Infectious Disease and Molecular Medicine, Division of Medical Virology, Department of Pathology, University of Cape Town, South Africa. Center for HIV and STIs, National Institute for Communicable Disease, National Health Laboratory Service SAMRC/UCT Gynaecological Cancer Research Center for HIV and STIs. Weill Cornell Medicine, Division of Infectious Diseases, New York, NY USA. Right to Care, Johannesburg, South Africa, Julius Clinical Research, Zeist, The Netherlands. Cytology Unit, Department of Anatomical Pathology, Faculty of Health Sciences, University of the Witwatersrand Clinical HIV Research Unit, Faculty of Health Sciences, Department of Internal Medicine University of the Witwatersrand, Johannesburg, South Africa. Department of Epidemiology, University of North Carolina, Gillings School of Global Public Health, Chapel Hill, NC, USA. National Health Laboratory Service, Groote Schuur Hospital, Cape Town, South Africa
2 HPV Types There are >200 HPV types. They can infect mucosal and cutaneous epithelia. HPV genotypes that infect mucosa are grouped as either high-risk or low-risk according to their link with cancer. HPV types classified as high risk HPV16, 18, 31, 33, 35, 39, 45, 51, 56, 58, 59, 68, 73, 82 HPV types classified as probable high-risk types HPV26, 53, 66 HPV types classified as low risk HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, CP6108. Munoz et al., 2003; Dillner, 2015
3 Cancers caused by HPV Persistent infection with high-risk HPV type(s) is essential for the development of cancer. Cancers caused by HPV Percentage Cervical 100% Anal 95% Oropharynx 70% Vaginal 65% Vulva 50% Penis 5% Munoz et al., 2003; Parkin et al., 2006; Dillner, 2015
4 Age-standardized incidence rate of cervical cancer
5 Role of HPV testing Persistent infection with HR-HPV is necessary for the development of cervical cancer and therefore HR-HPV testing is considered an alternative test to cytology or VIA or used in various combinations for cervical cancer screening VIA: Visual Inspection with Acetic acid. Luhn and Wentzensen, (2):76-85
6 HPV detection Most HPV assays are batch tests, requiring skilled laboratory personnel and taking several hours to complete. The Cepheid Xpert HPV assay (Cepheid Sunnyvale, CA) is a qualitative, real time polymerase chain reaction assay detecting 14 HR-HPV DNA. Single Xpert HPV test can be completed in one hour, thus allowing same day screening, diagnosis and treatment strategy; thus making Xpert HPV assay a point-of-care test. Toliman et al., 2016; Mbulawa et al., 2016; Cuzich et al., 2015
7 Objectives of the study Investigate anal and cervical HPV prevalence in South African HIV infected women using Cepheid Xpert HPV assay Compare Xpert HPV assay performance with that of hybrid capture-2 (hc2, an FDA approved test) for detection of high grade squamous intraepithelial lesion (HSIL).
8 HR-HPV types detected by Xpert & Hybrid Capture 13 HR-HPV types associated with ~ 94% cancer cases. Xpert and hc2 detect these 13 HR-HPV types. Xpert also detect HPV-66, hc2 is known to detect HPV66 due to cross-reactivity IARC POOLED-ANALYSIS OF 3,085 CASES Estimates adapted from Munoz N, et al. Int J Cancer 2004;111(2): Smith et al. Br J Cancer, 2007
9 Performance of Cepheid Xpert HPV on cervical specimens
10 Methodology for cervical HPV study Stored cervical specimens (n=1193) from HIV-infected women that had previously been tested using hc2 were tested using Xpert HR-HPV test. Cervical specimens were collected with a Digene cervical sampler and stored at -80 C. A total of 100µl cervical sample was transferred to 950µl phosphate buffered saline and 1000µl of diluted specimen was used to run Cepheid Xpert HR-HPV according to manufacturer s instructions.
11 Cervical Results Overall agreement was 90% between two tests. The agreement beyond chance (Cohen s kappa) was 0.78 (95% CI: ) indicating good agreement. Samples with discordant hc2 and Xpert results had lower HPV viral load compared to samples where both tests were positive (P<0.0001). (Mbulawa et al., 2016, Papillomavirus research 2: 56-60).
12 Cervical Results The relationship between the amount of DNA and the prevalence of CIN2+ was investigated. Women infected with HPV16, HPV18/45 or HPV31/33/35/52/58 were found to have significantly higher HPV viral load for those with CIN2+ compared to those without CIN2+, P<.0001 for each. CIN: cervical intraepithelial neoplasia (Mbulawa et al., 2016, Papillomavirus research 2: 56-60).
13 Xpert performance for CIN2 Prevalence, N (%) CIN2+ prevalence (test positive vs test negative) Univariate association with CIN2+, OR [95% CI] Multivariate analysis hc2 positive 727 (61%) 42.0% vs. 6.2% 11.0 [ ]* n/a Xpert HR-HPV 720 (62%) 39.2% vs. 8.8% 6.9 [ ]* n/a positive P1 (HPV16) 158 (13.6%) 68.3% vs. 22.3% 7.5 [ ]* 6.8 [ ]* P2 (HPV18/45) 218 (18.8%) 45.1% vs. 24.2% 2.6 [ ]* 2.5 [ ]* P3 (HPV31/33/35/52/58) 434 (37.3%) 45.5% vs. 17.5% 3.9 [ ]* 3.5 [ ]* P4 (HPV51/59) 147 (12.7%) 42.1% vs. 26.1% 2.1 [ ]* 1.3 [ ], P=.19 P5 (HPV39/68/56/66) 270 (23.3%) 37.2% vs. 25.1% 1.8 [ ]* 1.2 [ ], P=.20 *P< CIN: cervical intraepithelial neoplasia (Mbulawa et al., 2016, Papillomavirus research 2: 56-60).
14 Xpert performance for CIN2/3 hc2 positive Sensitivity Specificity CIN2/3 Positive predictive value Negative predictive value 91.5% ( ) 51.0% ( ) 42.1% ( ) 93.9% ( ) Xpert HR-HPV positive 88.3% ( ) 48.4% ( ) 40.1% ( ) 91.3% ( ) P1 (HPV16) 31.5% ( ) 93.5% ( ) 65.5% ( ) 77.7% ( ) P2 (HPV18/45) 30.1% ( ) 85.6% ( ) 45.0% ( ) 75.8% ( ) P3 (HPV31/33/35/52/58) 61.1% ( ) 71.9% ( ) 45.9% ( ) 82.6% ( ) P4 (HPV51/59) 18.8% ( ) 89.7% ( ) 41.7% ( ) 73.9% ( ) P5 (HPV39/68/56/66) 33.6% ( ) 80.7% ( ) 40.7% ( ) 75.6% ( ) CIN: cervical intraepithelial neoplasia (Mbulawa et al., 2016, Papillomavirus research 2: 56-60).
15 Xpert performance for CIN3 Sensitivity Specificity CIN3 Positive predictive value Negative predictive value hc2 positive 97.9% ( ) 42.8% ( ) 14.4% ( ) 99.5% ( ) Xpert HR-HPV positive 95.8% ( ) 41.4% ( ) 13.9% ( ) 99.0% ( ) P1 (HPV16) 43.0% ( ) 89.4% ( ) 28.8% ( ) 94.0% ( ) P2 (HPV18/45) 32.3% ( ) 82.5% ( ) 15.5% ( ) 92.5% ( ) P3 (HPV31/33/35/52/58) 63.9% ( ) 65.3% ( ) 15.4% ( ) 94.8% ( ) P4 (HPV51/59) 16.2% ( ) 87.7% ( ) 11.5% ( ) 91.4% ( ) P5 (HPV39/68/56/66) 36.7% ( ) 78.0% ( ) 14.2% ( ) 92.6% ( ) CIN: cervical intraepithelial neoplasia (Mbulawa et al., 2016, Papillomavirus research 2: 56-60).
16 Cervical Results Among women with CIN2+, 20 were hc2 negative and 29 were Xpert negative. Indicate false negative HR-HPV results, histology false positive or indicate that the observed CIN2+ are due types that are not detected by Xpert or hc2. (Castle et al., 2008; Einstein et al., 2014)
17 Summary Xpert HPV results were found to be comparable to the results by hc2 as demonstrated by high HR-HPV detection agreement between the two assays. Discordant results between the assays were due to the influence of lower HPV DNA amounts All the samples that were HPV negative by Xpert their SAC passed, indicating that the samples were adequate. Xpert is sensitive and specific in detecting CIN2+ and CIN3; and its performance is similar to the one demonstrated by hc2, an FDA approved test. Xpert is a promising cervical cancer screening test
18 Performance of Cepheid Xpert HPV on anal specimens
19 Limited data on anal HPV infection in S.A Data on anal HPV infection among women are very limited in sub-saharan Africa. In South Africa there are no standard guidelines on anal cancer prevention in either HIV-infected or -uninfected women and men. According to the National Cancer Registry which collects statistics for histologically diagnosed cancers among SA women 2011, anal cancer Age Standardized Incidence was 0.48 per (95% CI: ).
20
21 Investigation of anal cancer screening methods among HIVpositive women may be necessary given the high anal cancer burden among HIV infected individuals in the Unites States of America and Europe and the high prevalence of HIV in South Africa.
22 Study population for Anal HPV study Anal ThinPrep specimens obtained from 196 HIV-infected women aged years that were recruited from Themba Lethu Clinic, Helen Joseph Hospital, Johannesburg, South Africa. Stored ThinPrep anal swabs that had previously been tested using Digene hybrid capture-2 were tested for HPV using Xpert HPV according to manufacturer s instruction. Anal cytology and high resolution anoscopy had previously been performed.
23 Results All women were HIV infected with a median CD4 count of 430 cells/mm 3 (interquartile range (IQR): ), 97% (193/200) were on ART and 89% (166/200) had plasma HIV RNA levels of less than 400 copies/ml. A total of 196 women had anal Xpert HPV results (4 specimens were missing). The overall HR-HPV prevalence was 40.8% (80/196) by Xpert and 41.8% (82/196) by hc2
24 Anal HPV prevalence among HIV infected women HPV39/68/56/66 16,3 HPV51/59 11,2 HPV31/33/35/52/58 23,0 HPV18/45 8,7 HPV16 7,1 Xpert HPV 40,8 hybrid capture-2 41, %
25 Agreement between Xpert and hc-2 HR-HPV hc-2 HR-HPV positive negative Overall agreement of 86.7% Xpert HR-HPV Positive negative Cohen s kappa was 0.73 (95% CI: ) indicating substantial agreement CERVICAL SPECIMEN: Overall agreement was 90% between two tests, Cohen s kappa was 0.78 (95% CI: ) indicating good agreement (Mbulawa et al., 2017, Am J Clin Pathol 148:
26 Association of anal HPV with HSIL Univariate analysis Multivariable analysis OR 95% CI P-value OR 95% CI P-value hybrid capture < Xpert HPV < HPV < < HPV18/ HPV31/33/35/52/ HPV51/ HPV39/68/56/ HSIL by anal cytology or high resolution anoscopy directed biopsies was found in 15.8% (31/196). HSIL: High grade Squamous Intraepithelial Lesion (Mbulawa et al., 2017, Am J Clin Pathol 148:
27 The association of HSIL with number of Xpert HPV channel positivity Xpert channels Xpert channels number with HSIL Prevalence of HSIL % n P trend for positivity <0.001 number of channels is strongly related to HSIL, Cochran-Mantel-Haenszel P < HSIL was associated with an increasing number of multiple HPV infection (P<0.001) It has been suggested that in population with high HPV burden, detection of multiple infections could be used as a marker to identify women at risk of developing precancerous lesions (Patel et al., International journal of STD & AIDS 24(3): ) HSIL: High grade Squamous Intraepithelial Lesion (Mbulawa et al., 2017, Am J Clin Pathol 148:
28 The performance of HPV Xpert and hc2 for HSIL Sensitivity (95% CI) Specificity (95% CI) PPV (95% CI) NPV (95% CI) hybrid capture-2 77%, 59%-90% 65%, 57%-72%) 29%, 20%-40% 94%, 88%-98% Xpert HPV 77%, 59%-90% 66%, 58%-73% 30%, 20%-41% 94%, 88%-98% HPV16 29%, 14%-48% 97%, 93%-99% 64%, 35%-87% 88%, 82%-92% HPV18/45 16%, 5%-34% 93%, 88%-96% 29%, 10%-56% 85%, 79%-90% HPV31/33/35/52/58 48%, 30%-67% 82%, 75%-87% 33%, 20%-49% 89%, 83%-94% HPV51/59 29%, 14%-48% 92%, 87%-96% 41%, 21%-64% 87%, 81%-92% HPV39/68/56/66 32%, 17%-51% 87%, 81%-91% 31%, 16%-50% 87%, 81%-92% PPV: positive predictive value. NPV: negative predictive value. Xpert considered positive if any channel is positive HSIL: High grade Squamous Intraepithelial Lesion (Mbulawa et al., 2017, Am J Clin Pathol 148:
29 Conclusion for Anal HPV study Xpert HPV is a promising anal cancer screening test in HIV-infected women that performs similarly to hc2. The typing information provides additional data for determining HSIL risk and may be able to risk stratify who should proceed to HRA alone or in combination with cytology. Xpert performed well in comparison to hc2, and we demonstrated substantial agreement between the two tests.
30 Final remarks The GeneXpert technology is been widely available in South Africa public health sector using it could be cost effective and practical for HPV detection. Point- of-care HR-HPV testing may improve the management and control of cervical cancer. Future studies should investigate Xpert HPV as a primary or adjunctive test for cervical and anal cancer screening programs.
31 Acknowledgements Cepheid donated 25% of cartridges used in this study and sponsored travel grant to attend SA AIDS conference. Supported by Right to Care, Johannesburg, South Africa. President s emergency plan for AIDS relief, and cancer for AIDS Research, University of North Carolina. Funding Source: University of North Carolina Center for AIDS Research (P30-AI50410), USAID PEPFAR (674-A ), PHE ZA South African Research Chairs Initiative of the Department of Science and Technology; PHE ZA with Right to Care. This work was supported by the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation. Internal funds from the Weill Cornell Medicine Division of Infectious Diseases were used to support the Xpert HPV testing. The parent study was funded in part by a 2011 developmental grant from the University of North Carolina at Chapel Hill Center for AIDS Research (CFAR), an NIH funded program P30 AI50410 and funded in part through a supplement to AI Jack Hu and Bruce Allan (Medical Virology, University of Cape Town)
32 Thank you
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