In‐field evaluation of Xpert® HCV viral load Fingerstick assay in people who inject drugs in Tanzania

Abstract Background Although novel hepatitis C virus (HCV) RNA point‐of‐care technology has the potential to enhance the diagnosis in resource‐limited settings, very little real‐world validation of their utility exists. We evaluate the performance of HCV RNA quantification using the Xpert® HCV viral load Fingerstick assay (Xpert® HCV VL Fingerstick assay) as compared to the World Health Organisation pre‐qualified plasma Xpert® HCV VL assay among people who inject drugs (PWID) attending an opioid agonist therapy (OAT) clinic in Dar‐es‐Salaam, Tanzania. Methods Between December 2018 and February 2019, consecutive HCV seropositive PWID attending the OAT clinic provided paired venous and Fingerstick samples for HCV RNA quantification. These were processed onsite using the GeneXpert® platform located at the Central tuberculosis reference laboratory. Results A total of 208 out of 220 anti‐HCV‐positive participants recruited (94.5%) had a valid Xpert® HCV VL result available; 126 (61%; 95% CI 53.8‐67.0) had detectable and quantifiable HCV RNA. About 188 (85%) participants had paired plasma and Fingerstick whole blood samples; the sensitivity and specificity for the quantification of HCV RNA levels were 99.1% and 98.7% respectively. There was an excellent correlation (R 2 = .95) and concordance (mean difference 0.13 IU/mL, (95% CI −0.9 to 0.16 IU/mL) in HCV RNA levels between plasma samples and Fingerstick samples. Conclusion This study found excellent performance of the Xpert® HCV VL Fingerstick assay for HCV RNA detection and quantification in an African‐field setting. Its clinical utility represents an important watershed in overcoming existing challenges to HCV diagnosis, which should play a crucial role in HCV elimination in Africa.


| BACKG ROU N D
The advent of highly efficacious direct-acting antivirals (DAAs) has provided a springboard to raise the awareness of hepatitis C virus (HCV) as a global health issue and acted as a catalyst to the development of the 2030 World Health Organisation (WHO) viral hepatitis elimination targets. 1 However, currently the majority of infected individuals (80%) remain undiagnosed especially in low-and middleincome countries (LMICs) and only a minority (2% annually) are accessing treatment. 2,3 As a result, only a handful of countries are currently on track to achieve the ambitious elimination objectives.
Thus, a great deal of emphasis has been placed on scaling-up simplified strategies for HCV screening and linkage-to-care. 4 As LMICs represent 80% of the global HCV epidemic, it is clear that an increased focus is required to address the challenges of case-finding and treatment in these commonly resource-limited settings. 5 Recent times have seen the WHO prequalification of DAAs to ensure the quality control, and fierce competition from generic manufacturers has resulted in a sharp reduction in cost of treatment.
Ironically, in many resource-limited settings the cost of diagnosis exceeds the cost of treatment. 6 As HCV RNA confirmation is a pre-requisite for commencing treatment, it is not surprising that access to simple HCV diagnostics in such challenging settings has been underlined as a key priority in order to achieve HCV elimination.
However, the resounding limited access to HCV RNA nucleic acid testing (NAT) in LMICs was emphasised by a recent WHO survey, which reported that 40% of LMICs do not have access to HCV NAT. 7 One of the novel solutions to mitigate the dependence on skilled laboratories and personnel is the emergence of simple point-of-care tests for HCV RNA. Currently, two CE-in vitro diagnostic point-of-care platforms (Genedrive ® ) and Xpert ® (Cepheid) for HCV RNA NAT in plasma exist, with one (Xpert ® ) receiving WHO prequalification in 2017. 8,9 However, with the existing point-of-care tests there still remains the issue of plasma separation. Whole blood samples of 50-70 µL have been demonstrated as an alternative method to decentralise and simplify sample collection using dried blood spots (DBS). 10 This is particularly beneficial in rural settings and in people who inject drugs (PWID), where venous access is challenging. In addition, the ease of obtaining a sample can allow task-shifting to a diverse range of healthcare and non-clinical staff. 11,12 DBS have also been validated for HCV RNA quantification on the new Hologic Aptima assay as well as HCV core antigen quantification on the Abbott Architect platform, reporting sensitivities of 96.4% and 76.1% respectively. 13,14 Furthermore, a Fingerstick assay using 100 µL has recently been validated for the Xpert ® system (Xpert ® HCV viral load [VL] Fingerstick assay), which has a lower limit of detection of 40 IU/mL and lower limit of quantification of 100 IU/mL. [15][16][17] When compared with the Genedrive ® device, not only does the Xpert ® HCV VL Fingerstick assay have a superior threshold for HCV RNA detection and avoid the need for any sample prepreparation, it also has a faster turnaround time (60 minutes vs 90 minutes) and is half the price. All of which make it quite an attractive proposition to improve the access to HCV RNA testing in resource-limited settings.
Although HCV RNA point-of-care assays have been designed for LMICs, a lack of in-field validation data in such environments exists and addressing this has recently been highlighted as crucial. 18 For example, the urgent need to scale-up resources to address the risk of blood-borne viruses (BBVs) among PWID engaged in harm reduction in Tanzania has previously been highlighted, including the pressing need for locally available HCV RNA confirmation as part of the cascade of care. 19,20 In addition, a lack of access for temperature regulated sample transport and storage, particularly in a tropical climate, provide further justification for the need to confirm the performance of the Xpert ® HCV VL Fingerstick assay in this resource-limited African setting. Thus, this study population, attending a opioid agonist therapy (OAT) clinic in Dar-es-Salaam, Tanzania, was used to assess the performance of onsite HCV RNA quantification with Xpert ® system by comparing the use of finger-stick capillary blood sample (Xpert ® HCV VL Fingerstick assay) to the use of plasma sample (Xpert ® HCV VL) as a WHO prequalified reference.

| Study population
The Muhimbili National Hospital (MNH), Dar-es-Salaam, Tanzania is the national referral hospital for the country. Situated onsite, the OAT clinic is the product of a Tanzanian government and President's Emergency Plan For AIDS Relief supported initiative that has been operational since 2011. 19 In addition to offering daily OAT to all clients, routine serology screening for BBVs (HIV, hepatitis B and HCV) is offered on enrolment to the clinic. Currently, linkage-to-care is limited to HIV and TB, while HBV-negative patients are offered vaccination. Clients can also seek aid from a wide range of services centralised at the clinic including; psychiatry, social welfare, nursing (for eg wound care) and can also enrol in a harm reduction mentorship program to become a community outreach worker. There is also an evolving link with the Department of Gastroenterology and Hepatology at MNH, which has been nurtured as part of this collaboration. Currently, access to generic

Key points
• Availability of tests to confirm active hepatitis C virus (HCV) infection remain a significant barrier to scaling-up care in resource-limited settings.
• We confirm excellent performance of the Xpert ® HCV viral load Fingerstick point-of-care assay among people who inject drugs in Dar-es-Salaam, Tanzania.
• These encouraging results provide a credible solution improve diagnosis and contribute towards efforts to HCV elimination in this vulnerable population. DAA therapy is available on a private basis only, which is not readily accessible to HCV-infected PWID. In order to assess the performance of HCV RNA quantification, all clients with a known positive result for anti-HCV antibody attending the clinic for OAT, irrespective of concomitant illicit drug use, were invited to participate in the study between December 2018 and February 2019.

| Ethical considerations
This study received clearance from the respective institutional

| Clinical assessment and sample collection
All recruited individuals completed a comprehensive demographic and risk factor-based questionnaire, which was administered by a trained fieldworker. All participants then provided paired blood samples. Venous samples were obtained to fill a 4 mL ethylenediamine tetraacetate (EDTA) bottle, while a 100 µL finger-prick sample was collected in an EDTA-coated capillary (Minivette ® ; Sarstedt) (www.sarst edt.com) and applied directly into the Xpert ® HCV VL Fingerstick assay cartridge in line with the manufacturer's specifications (Cepheid). 15 All samples were then delivered to the onsite Central TB Reference Laboratory at MNH for processing on the day of sample collection.

| Laboratory processing and virological assessment
Laboratory staff prioritised processing Xpert ® HCV VL Fingerstick assay samples on the same day as receipt on the GeneXpert ® II platform in accordance with the manufacturer's guidance, to provide a quantitative HCV RNA result within 60 minutes. All venous samples were kept at 4°C for a maximum of 72 hours prior to plasma separation, 1mL was applied to the Xpert ® HCV VL cartridge before being processed for HCV RNA quantification on the GeneXpert ® II platform in accordance with the manufacturer's guidance. All residual plasma was stored at −80°C. In the event that either plasma or finger sample failed, where possible a repeat paired sample was collected and run on the Xpert ® platform.

| HCV genotyping
Where available historic HCV genotype was used as previously reported. 13 Previously described methods were used to perform sequencing of the HCV genome NS5B region and subsequent phylogenetic analysis. 21

| Sample size calculation
We aimed to demonstrate that a lower boundary of a 95% confidence interval of the diagnostic sensitivity of Xpert ® HCV VL Fingerstick assay is at least higher than 90% with a power of 80%. Assuming that the prevalence of chronic HCV infection is 60%-80% in anti-HCV antibody-positive patients, and the true sensitivity of Xpert ® HCV VL Fingerstick assay is 97%, we needed to recruit at least 184 anti-HCV-positive patients.

| Statistical analysis
Characteristics of the study participants were presented by mean,

| Participant characteristics
During the study period, 220 anti-HCV positive patients agreed to participate. The cohort was overwhelmingly male (98%) and had a median age of 41 years (IQR: 37-45). All had a past history of injecting drug use, on average clients started to inject drugs at the age of 23 years with 38% admitted to having ever-shared needles in the past. The median length of time enrolled in the OAT at the time of recruitment was 5 years. The study population characteristics are presented in Table 1.

| HCV RNA assessment
About 208 of the 220 (95%) of clients enrolled in the study had a valid plasma Xpert ® HCV VL result. Of the 12 (5%) without a valid reference HCV RNA result, a venous sample could not be obtained in 8 (4%) clients owing to difficult venous access, three (1%) clients failed owing to inadequate sample volume and one failed owing to a power cut during sample processing. One hundred and twentysix clients had quantifiable HCV RNA, resulting in a prevalence of chronic HCV infection of 61% (95% CI 54-67). All viraemic patients had quantifiable HCV RNA, with a median HCV RNA level of 6.1 log IU/mL (range; 1.4-7.5). Genotype was available for 53 (42%), with genotype 1a identified in 35 of 53 cases (66%) and genotype 4a in 18 of 53 (34%) cases respectively (Figure 1).
In the case of plasma Xpert ® HCV VL, 21 of 41 (51%) patients were related to sample volume errors and 20 of 41 (49%) were related to technical failure, which in most cases was owing to instrument interruption after a power shortage. In comparison, all 43 (100%) of the Xpert ® HCV VL Fingerstick assay errors were related to inadequate sample volume. Consequently, 32 of 220 (14.5%) patients did not have a paired sample; 4 of 220 (2%) had no sample, 20 of 220 (9%) had a plasma sample only and 8 of 220 (4%) had an Fingerstick sample only (Figures 1 and 2).

| D ISCUSS I ON
The challenges of accessing NAT for HCV RNA confirmation has been raised as a key barrier to improving diagnosis in resourcelimited settings in sub-Saharan Africa and more specifically in the PWID population in Tanzania. 13,19,22,23 In this study we report the quality control but also is often a prerequisite for donor funded programmes. 6 However, despite having a lower limit of detection significantly below recommendations (40 IU/mL) from a whole blood sample, the Xpert ® HCV VL Fingerstick assay is yet to receive WHO pre-qualification.
There are a number of advantages specific to the GeneXpert ® platform that makes it an attractive option to improving the diagnosis of communicable infections across sub-Saharan Africa.
Firstly, it is already a well-established technology used as the firstline diagnostic test for Tuberculosis (TB) in sub-Saharan Africa. In response to the WHO 2010 recommendations for TB diagnosis, 30 an exponential rise in Xpert ® modules to 21 549 was recorded between 2010 and 2015 in 122 'high-burden developing countries' (HBDC). 31 Furthermore, recent data from Nigeria confirm that there are over 1500 modules distributed across the nation. 32 Thus, it is not only a familiar technology but also widely disseminated across the continent. This is particularly key in resource-limited settings as in most cases it would avoid the potentially prohibitive USD$17 000 instrument cost. 33  However, in our study one-third of patients required re-testing owing to test errors, which were attributed to both inadequate sample volume and also loss of electrical supply while samples were being analysed. The rate of error appears to be fair greater than that has previous been reported in the literature (1%-3%). 16,17  HCV VL assay was used as the gold standard as there was no local access to conventional laboratory-based NAT testing. However, it is unlikely that this would have introduced any significant discrepancy as this assay is WHO pre-qualified and has demonstrated excellent performance in previous validation studies. 25,26 Thirdly, as local genotyping was not available, only those with historic genotypes were reported in this study. However, though it is relevant from an epidemiological perspective to determine genotype, its clinical relevance has been minimised by the availability of pangenotypic DAAs, as proposed in a recent algorithm for screening in LMIC settings. 39 Finally, our conclusions are limited to PWID engaged in harm reduction and determining whether Xpert ® HCV VL Fingerstick assay testing is reproducible and feasible in marginalised rural settings is important.
Cepheid has recently launched a portable module (Cepheid Edge), which could serve to address this in the future. 40 Until recently, access to HCV NAT confirmation was considered a major obstacle to improving access to care in resource-limited settings. We provide evidence here that the Xpert ® HCV VL Fingerstick assay is a technically feasible, cost-amenable and ultra-sensitive assay to simplify HCV NAT diagnosis in resource-limited settings and paves the way to facilitate the much-needed HCV treatment intervention in our challenging PWID setting in sub-Saharan Africa. to the design of the study and the analysis of the results. We are also grateful to Rita Carvalho, project manager of the PROLIFICA programme (www.proli fica.africa).

CO N FLI C T O F I NTE R E S T
The authors do not have any disclosures to report.