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Table 1 Combined HCV RNA and cAg TPP for an HCV test

From: Optimising diagnosis of viraemic hepatitis C infection: the development of a target product profile

Characteristic Optimal Minimal Rationale and evidence
Scope
 Goal of test The goal of the test is two-fold:
1. To diagnose active viraemic HCV infection (new or reinfection) and provide baseline virological assessment (quantitative or qualitative);
2. To confirm cure upon treatment completion.
Ideally, the test would be done with the purpose of initiating treatment within the same clinical encounter or the same day.
Not intended for blood screening.
The timeline of development for tests envisioned in the TPP is 5 years.
The goal of the test is two-fold:
1. To diagnose active HCV viraemic infection (new or reinfection) and provide baseline virological assessment (qualitative) with the purpose of initiating treatment;
2. To confirm cure upon treatment completion.
Not intended for blood screening.
The timeline of development for tests envisioned in the TPP is 5 years.
Detection can be performed by HCV RNA test or by HCV cAg detection. Presence of HCV RNA or cAg in a patient is indicative of active HCV infection. Currently, the HCV RNA or cAg test is performed after a positive anti-HCV serological test (i.e. two-step algorithm).
Conceivably, provided the prevalence is substantial and the cost of the HCV RNA or cAg test is low, either test could be used in a one-step algorithm.
 Target population Countries with a medium to high prevalence of HCV (1.5–3.5% and >3.5%)
High-risk populations in low prevalence settings (<1.5%).
High-risk populations include: persons who inject drugs or have used intranasal drugs (PWID), people living with HIV (PLWH), men who have sex with men (MSM), prisoners, people with tattoos, sex workers, people with frequent contact with the health-care system (i.e. chronically ill) and children born to HCV-infected mothers. In order to achieve the long-term goal of HCV elimination, optimally the test should be performed on all patients in primary care settings, antenatal clinics and in community screening programmes.
 Target operator of test Community workers with minimal training Health-care workers or laboratory technicians with limited training (i.e. able to operate an integrated test with minimal additional steps)  
 Lowest level of setting for implementation (public & private) Community centres District hospital (Level II)  
Performance characteristics
 Diagnostic sensitivity* >99% 90%–95% Rationale of optimal test: Ideally a test should be as sensitive and specific as available plasma-based HCV RNA tests. A commonly used reference standard is the VERSANT HCV RNA Qualitative Assay, which is FDA-approved for diagnosis of active HCV infection (although the VERSANT HCV RNA Qualitative Assay is being taken off the market, it remains the most analytically sensitive assay and was used as the gold standard in most instances).
Rationale of minimal: If a test is easier to implement at lower levels of the health care system without requiring substantial technical expertise or complex laboratory infrastructure, and is less costly, then a compromise can be made on sensitivity. A test with a suboptimal sensitivity of 90–95% with improved operational characteristics was considered acceptable by stakeholders as it would improve rates of diagnosis substantially over what is currently possible. However, no studies or modelling have been done on the minimal acceptable sensitivity and the optimal other characteristics needed by a test for HCV diagnosis to lead to substantial improvement in HCV detection on a population level.
Modelling work for TB has provided insights that could potentially be applicable for HCV as well. A model showed that for the WHO Southeast Asia Region a POC biomarker test with a sensitivity of 50% for smear-negative TB, if employed at the most peripheral health-care setting, would result in a similar reduction in TB incidence as a test with 70% sensitivity for smear-negative TB that would be employed at the district level (e.g. Xpert MTB/RIF) [17]. However, the exact trade-off between a lower sensitivity (for smear-negative TB) and an increase in access to testing is setting dependent.
Under the minimal scenario, some patients would be incorrectly diagnosed as not having active HCV infection, What impact that would have on patient and provider behaviour is unclear.
 Analytical sensitivity (comparison with HCV RNA test reference standard) 200 IU/ml 1000–3000 IU/ml Among the majority of infected individuals with chronic HCV infection, HCV RNA viral loads are between 104 and 107 IU/ml [13]. In studies of viral dynamics during acute infection, viral loads as low as 3 log IU/ml (or 1000 IU/ml) were seen during the first four months after infection [13]. The optimal LOD of 200 IU/ml should therefore detect most patients (>99%). At a minimum, analytical sensitivity of 1000–3000 IU/ml or 3 fmol cAg/l (current LOD of the Abbott HCV cAg assay), the corresponding clinical sensitivity should be 90–95%.
Upper limit of the dynamic range should be equivalent to that of current laboratory-based quantitative HCV RNA tests. An HCV RNA test should be standardized with the WHO International Standard for Hepatitis C Virus RNA, as has been done with current FDA-approved and CE-marked qualitative and quantitative HCV RNA assays.
Interestingly, HCV cAg test have been shown to be negative among a portion of untreated individuals with high HCV RNA levels, indicating the likely presence of mutant variants [18]. The limitation of the cAg assay to accurately detect these variants may present a challenge to elimination.
Data from patients who relapsed after treatment with peg-interferon and ribavirin therapy indicate that HCV rebounds quickly to high levels (103 IU/ml and greater) within a few weeks after the end of treatment [19]. Early data from DAA-based therapy suggests that an even more rapid relapse would happen (unpublished data; communication with A. Hill). Given the high correlation between HCV cAg and HCV RNA levels, either test would likely be suitable for monitoring virologic response after treatment completion several weeks after completion of therapy.
 Diagnostic specificity (comparison with HCV RNA reference standard)* >99% >98% Since the test is a test for detection of active HCV infection, it should be as specific as current commercially available and FDA-approved HCV RNA tests to avoid false positive results.
 Analytical specificity – HCV detection No cross reactivity with endogenous substance and exogenous factors (e.g. HIV-1, HIV-2, HBV, HEV, antimalarials, anti-TB, ART) No cross reactivity with endogenous substance and exogenous factors (e.g. HIV-1, HIV-2, HBV, HEV, antimalarials, anti-TB, ART)  
 Polyvalency Ability to detect HIV, hepatitis B on the same instrument   
 Quantitation Quantitative Qualitative Treatment monitoring is not considered necessary or feasible with novel DAA agents [14], therefore a qualitative test result is preferred. According to stakeholder opinion, a quantitative result would be beneficial as it allows research questions to be investigated; however, it cannot come at an increased cost.
Operational Characteristics
 Specimen type Capillary whole blood Venous whole blood or
plasma
The emphasis is for the use of capillary whole blood that can diagnose infection in the clinic without requiring additional laboratory equipment such as a bench top centrifuge.
The need for phlebotomy to draw venous whole blood would limit the applicability of the test in lower settings of care as per stakeholder opinion. If plasma is to be a specimen type (minimal criteria), the plasma separation step should be integrated into the instrument.
 Specimen prep (total steps) Integrated specimen preparation (including plasma separation if needed); less than 2 steps required (no precision volume control and precision time steps) Maximally 2 steps (no precision volume control and precision time steps) Equipment such as a centrifuge or heat block are available only infrequently at level 1 health centres and some district hospitals, and therefore should not be required for novel assays. Expertise to operate a precision pipette is also often lacking [21].
For the detection of cAg, several specimen preparation steps are needed: i) to dissociate antibody-bound cAg; ii) to lyse viral particles and expose cAg; and iii) to inactivate antibody. These should also optimally be integrated with the test of detection.
 Time to result < 15 min < 60 min The need for a rapid turn-around time, the possibility for batching and/or random access for testing, and the testing of multiple specimens at the same time are interrelated. The time to result is probably the most important parameter, as extending the wait time for patients will possibly result in loss to follow-up [22, 23]. Most current immune-chromatographic rapid tests produce results within 20 min.
The ideal time to result has not been studied and might vary largely between countries and between settings where the patient is tested. But in order to be deployable as a test for POC, the result should be available within the same visit.
 Specimen capacity and throughput Multiple at a time; random access/parallel processing One at a time (any external reagents should be aliquoted for one time use) Preferred that one specimen does not occupy the instrument at a time - i.e., random access/parallel analysis. If the platform is multi-analyte, then running different assays should be feasible at the same time.
 Biosafety + waste disposal Mostly simple waste; minimal biosafety waste; no sharps No need for a biosafety cabinet; consumables should be able to be disposed of as biosafety waste; simple trash. Increased biosafety of a novel test will enhance acceptability of the test by providers. Further information provided in WHO Laboratory Biosafety Manual [24].
 Instrumentation Instrument-free Allow for separate specimen preparation device (e.g. mini-centrifuge) The simpler, more portable and durable/robust the test is, the more likely it will be implemented in peripheral settings. Ideally an instrument free test (e.g. immunochromatographic test) would be the preferred optimal solution but this is likely not feasible with the analytical sensitivity that is necessary and a small specimen volume from a fingerstick.
 Power requirements If device necessary then:
battery-operated with recharging solution (e.g. solar) and circuit protector lasting up to 3 days of constant use and able to run off standard electricity
Rechargeable battery or solar power lasting at least 8 h. Continuous power is not always available at the level of a health and microscopy centre and even less likely at primary care clinics, therefore a battery-operated device with charge possibility conceivably through solar power would be most ideal in order for a test to fit into the entire breadth of settings [21, 23]
 Maintenance/ calibration Disposable, no maintenance or calibration required
If device necessary then: preventative maintenance at 2 years or >5000 specimens; include maintenance alert; remote calibration
Preventative maintenance at 1 year or >1000 specimens; only simple tools/minimal expertise required; include maintenance alert. Swap-out of platforms permitted. If a device is anticipated to have a longer lifespan, then a maintenance alert is essential to ensure proper functionality in settings where it is unlikely that the same person will always handle the device and records will be kept on duration of use.
It is essential that only simple tools/minimal expertise are necessary to do the maintenance given that service visits are difficult outside of urban settings.
 Data analysis Integrated data analysis Integrated data analysis (no requirement for PC); exported data capable of being analysed on a separate or networked PC.  
 Connectivity If device necessary then integrated connectivity; if no device necessary, then the test should allow data export via a separate reader.
Full data export (on usage of device, error/invalid rates, and personalized, protected results data) over USB port and network. Network connectivity through Ethernet, WiFi, and/or GSM/UMTS mobile broadband modem. Results should be encoded using a documented standard (such as HL7) and be formatted as JSON text. JSON data should be transmitted through http(s) to a local or remote server as results are generated. Results should be locally stored and queued during network interruptions and sent as a batch when connectivity is restored.
Full data export (on usage of device, error/invalid rates, and personalized, protected results data) over USB port and network. Network connectivity through Ethernet, WiFi, and/or GSM/UMTS mobile broadband modem. Results should be encoded using a documented standard (such as HL7) and be formatted as JSON text. JSON data should be transmitted through http(s) to a local or remote server as results are generated. Results should be locally stored and queued during network interruptions and sent as a batch when connectivity is restored. Data export will enhance surveillance, device and operator management and allow for supply chain management.
 Result capture, documentation, data display If instrument-free: ability to save results via separate reader.
If device necessary: integrated results screen and ability to save and print results; USB port. On-instrument visual readout and the ability to add information (patient ID, operator ID, date location, etc.)
Ability to save results
The test menu should be simple with integrated LCD screen; simple key pad or touch screen.
Results should be simple to interpret (positive/negative for HCV detection).
 Operating temperature/ humidity/altitude Between +5 to +40o C at 90% humidity and at an altitude of 3000 m Between +10o to +35o C at 70% humidity and at an altitude of 2000 m High environmental temperatures and high humidity are often a problem in countries where HCV is endemic.
 Reagent kit transport No cold chain required; tolerance of transport stress for a minimum of 72 h at -15o to +40 °C No cold chain required; tolerance of transport stress for a minimum of 48 h at -15o to +40o C Refrigerated transport is costly and often cannot be guaranteed during the entire transportation process. Frequent delays in transport are commonplace.
 Reagent kit storage/stability 2 years at +5 °C to +40o C at 90% humidity & transport stress (72 h at 50o C); no cold chain required 12 months at +5 °C to 35o C, 70% humidity, including transport stress (48 h at 50o C); no cold chain required High environmental temperatures and high humidity is often a problem in many countries where HCV is prevalent.
 Internal process quality control Internal full-process control, positive control & negative controls External positive control In addition to compatibility with existing external quality assessment schemes
Pricing
 Maximum price for individual test (reagent costs only; at scale; ex-works) < US $ 5 < US $ 15 For a one-step solution, the cost needs to be low, as a trade-off in the ease-of-use/performance for price would not be accepted. Conversely, in a two-step solution, a higher cost is more likely to be accepted, as people would be willing to make a trade-off provided the overall cost of the algorithm remains low. Cost-benefit analyses are needed to explore different options.
Trade-offs between optimal characteristics may be necessary to achieve optimal pricing. Preferences about acceptable trade-offs need to be further defined.
 Maximum price for instrumentation < US $ 2000 < US $ 20,000 The lower the price for instrumentation, the lower the up-front cost to a health-care system would be and thus the lower the barrier to implementation. Further modelling is necessary to confirm the maximal price estimated. Price should include warranties, service contracts and technical support. Alternatively, rental agreements for equipment should be an option.
  1. The TPP was finalized using input from the Delphi-like survey and discussion at the consensus meeting (April 22, 2015, Vienna, Austria). TPPs were combined because characteristics were similar for both, independent of whether the test envisioned was a RNA or cAg-based test. The TPP needs to be considered in the context of different types of testing strategies (one-step versus two-step)
  2. *Compared to a HCV RNA reference test performed on plasma
  3. **Ex-works, including proprietary reagents and consumables cost only (without instrumentation), produced at scale