Performance evaluation of the Hologic Aptima HCV Quant Dx assay for detection of HCV RNA from dried blood spots Beth Catlett In this study we evaluated a new testing method for detecting active HCV from a dried blood spot

BACKGROUND: 71 million people infected with HCV worldwide1 Only 20% of chronically infected individuals diagnosed Well tolerated and effective therapies broadly available in Australia on the Pharmaceutical Benefit schedule >95% cure rate Understanding barriers and addressing low rates of HCV testing and diagnosis High HCV incidence, highly marginalised populations (people who inject drugs and indigenous populations) Chronic Hepatitis C is a major public health concern with 71 million people estimated to be infected worldwide. However around only 20% of chronically infected individuals are diagnosed and there is no effective vaccine The good news is that treatment options have vastly improved recently with the availability of well tolerated therapies that can achieve a cure in more than 95% of individuals and in Australia these are now broadly available on the PBS. However innovative solutions that are simple, reliable, accessible and affordable to enhance HCV testing, linkage to care and treatment uptake are urgently needed. This is crucial in settings with high HCV incidence and populations who are highly marginalised, such as people who inject drugs (PWID) and Indigenous populations where low treatment rates are often attributed to additional barriers at patient, provider, structural, cultural, and health systems levels. 1Lange B, Cohn J, Roberts T, Camp J, Chauffour J, Gummadi N et al BMC Infect Dis. 2017

BACKGROUND: Grebely J, et al Exp Rev Mol Diag 2017 HCV diagnostic pathways have traditionally required multiple visits to health care providers including general physicians, phlebotomists and specialist clinicians. Simplifying this pathway can be achieved through using alternative sampling and diagnostic options such as dried blood spots (DBS) and point of care testing that take the sampling or testing to the patient. Grebely J, et al Exp Rev Mol Diag 2017

WHY DRIED BLOOD SPOTS? BACKGROUND: DBS exempt from IATA regulations for the transport of dangerous goods and biological materials Allows for remote sample collection, without the need for laboratory infrastructure BACKGROUND: Easy and inexpensive sample to collect as assisted or self Helps remove barriers to testing, provides privacy So why dried blood spots and what are they. DBS are collected from a fingerprick using a lancet and around fifty microlitres are spotted onto a protein saver card. The main advantages to DBS include simple collection – which can be assisted or in the privacy of your own home, minimal processing, inexpensive shipping at room temperature or through the mail and the ability to test for other viral analytes from the same card. Reflex viral testing and other viral analytes of interest such as HIV

AIMS & METHODS: Study aim: Evaluate the performance of the Aptima® HCV assay for HCV RNA detection with dried blood spots (DBS) Methods: Paired EDTA plasma and DBS (N=107) samples prepared from de-identified remnant samples of HCV antibody positive individuals. Statistical analysis Sensitivity and Specificity Bland Altman and Deming Regression analysis Moving onto the aim, we evaluated the performance of the Aptima assay for HCV RNA detection with paired venepuncture and spotted whole blood DBS. We used samples that were de-identified, HCV antibody positive repository samples, with consent from St Vincent’s Human research ethics committee. These samples were then analysed on the Hologic Panther platform with the Aptima assay All results were then statistically analysed to assess diagnostic sensitivity and specificity and bias and agreement.

RESULTS: RESULTS: Table 1. Quantitation of HCV RNA positive results in DBS >LLOQ Plasma Total Not Detected/ Unquantifiable Detected ≥10 IU/mL DBS Not Detected/ Unquantifiable 27 2 29 1* 77 78 28 79 107 *Less than <10 detected on PL, 338 for DBS   Table 2. Quantitation of HCV RNA in DBS versus Plasma samples at ≥1000 IU Plasma Total Not Detected/ Unquantifiable Detected ≥ 1000 IU/mL DBS 31 Detected ≥1000 IU/mL 76 107 (Detectable only) (n= 86), Y = 0.9228*X - 0.07554: R2 = 0.929 Looking at all quantitative results, the assay sensitivity for HCV RNA in DBS was 97.5% and specificity was 100%. The sensitivity increased to 100% when a more clinically relevant threshold above 1000IU/ mL was applied. Encouragingly the correlation between both sample types was strong with a R2 value of 0.929. And the Bland Altman plot showed only a small bias of 0.508 log between plasma and DBS and HCV RNA in DBS could be quantified as low as 525 IU/mL. Sensitivity Specificity 97.5% (95%CI 91-100%) 100% (95%CI 87-100%) Sensitivity Specificity 100% (95%CI 95-100%) (95%CI 89-100%)

DISCUSSION: Applicable for implementing simplified diagnostic strategies in people who inject drugs Home self collection - https://www.hivtest.health.nsw.gov.au Assisted collection through registered decentralized services Further evaluation needed to evaluate real world performance enabling registration of a kit insert claim Drug and alcohol clinics Primary health care / GPs\ Prisons Community health centres NSP services Sexual health Decentralized services shift Centralized testing (patient recall) Central lab Clinics POC RNA Single visit “Test and treat” + or DBS – Home collect or assisted Small labs To summarise the Aptima assay detects active infection in DBS with good sensitivity, specificity, and correlation to plasma especially at a clinically relevant threshold. Which makes it applicable for implementing into simplified diagnostic strategies to increase testing uptake in people who inject drugs. To achieve this, as shown in the diagram, it’s highly important to shift towards decentralized services where Point of care, finger stick single visit testing or DBS home or assisted collection can be offered as an alternative to standard phlebotomy. However dried blood spot testing in Australia is currently conducted under a research use only banner. Further evaluation is needed to determine real world performance which would potentially enable the registration of a DBS kit insert claim and lead to clinical diagnostic use. + +

ACKNOWLEDGEMENTS: St Vincent’s AMR A/Prof. Philip Cunningham Alex Carrera Leon McNally Joanne Sherring Charles Crew UNSW Sydney A/Prof. Jason Grebely Dr. Tanya Applegate Collaborators Peter Lowe Nadika Atapattu Here are my acknowledgements