Testing, diagnosis, and treatment following the implementation of a program to provide dried blood spot testing for HIV and hepatitis C infections: the NSW DBS Pilot.

BACKGROUND: Dried blood spot (DBS) testing provides an alternative to phlebotomy and addresses barriers to accessing healthcare experienced by some key populations. Large-scale evaluations of DBS testing programs are needed to understand their feasibility. This study evaluated the implementation of a state-wide DBS HIV and hepatitis C virus (HCV) testing pilot. METHODS: The New South Wales (NSW) DBS Pilot is an interventional cohort study of people testing for HIV antibody and/or HCV RNA from DBS samples in NSW, Australia. Participants at risk of HIV/HCV participated in testing via: 1) self-registration online with a DBS collection kit delivered and returned by conventional postal service; or 2) assisted DBS sample collection at 36 community health sites (including drug treatment and harm-minimisation services) and prisons. Participants received results by text (HIV antibody/ HCV RNA not detected) or a healthcare provider (HIV antibody/ HCV RNA detected). The RE-AIM framework was used to evaluate reach, effectiveness, adoption, and implementation. RESULTS: Reach: Between November 2016 and December 2020, 7,392 individuals were tested for HIV and/or HCV (21% self-registration, 34% assisted in community, and 45% assisted in prison). EFFECTIVENESS: Of 6,922 people tested for HIV (19% men who have sex with men, 13% living outside major cities, 21% born outside Australia), 51% (3,521/6,922) had no HIV test in the past two years, 0.1% (10/6,922) were newly diagnosed with HIV, and 80% (8/10) initiated HIV treatment within six months. Of 5,960 people tested for HCV (24% women, 35% Aboriginal and/or Torres Strait Islander, 55% recently injected drugs), 15% had detectable HCV RNA (878/5,960), and 45% (393/878) initiated treatment within six months. Adoption: By the end of 2020, DBS via assisted registration was available at 36 community sites and 21 prisons. IMPLEMENTATION: 90% of DBS cards arriving at the laboratory had the three full spots required for testing; the proportion was higher in assisted (94%) compared to online (76%) registration. CONCLUSIONS: This study demonstrated the feasibility of DBS testing for HIV and HCV in key populations including Aboriginal and Torres Strait Islander peoples, men who have sex with men, people who inject drugs, and demonstrated the utility of DBS in the prison setting.

Clinical signs of trachoma and laboratory evidence of ocular Chlamydia trachomatis infection in a remote Queensland community: a serial cross-sectional study.

OBJECTIVES: To compare the findings of standard clinical assessments and of complementary clinical and laboratory methods for determining whether community-wide treatment for trachoma is warranted in a remote Queensland community. DESIGN: Three cross-sectional screening surveys, 2019-2021, complemented by laboratory pathology testing. SETTING: Small community in northwest Queensland with geographic and cultural ties to Northern Territory communities where trachoma persists. PARTICIPANTS: Children aged 1-14 years; opportunistic screening of people aged 15 years or more. MAIN OUTCOME MEASURES: Prevalence of clinical signs of trachoma, Chlamydia trachomatis infection, ocular non-chlamydial infections, and seropositivity for antibodies to the C. trachomatis Pgp3 protein. RESULTS: During the three surveys, 73 examinations of 58 children aged 1-4 years, 309 of 171 aged 5-9 years, and 142 of 105 aged 10-14 years for trachoma were undertaken, as were 171 examinations of 164 people aged 15 years or more; 691 of 695 examinations were of Aboriginal or Torres Strait Islander people (99%), 337 were of girls or young women (48%). Clinical signs consistent with trachomatous inflammation-follicular were identified in 5-9-year-old children 23 times (7%), including in eleven with non-chlamydial infections and one with a C. trachomatis infection. One child (10-14 years) met the criteria for trachomatous scarring. Two of 272 conjunctival swab samples (all ages) were polymerase chain reaction-positive for C. trachomatis (0.7%). Two of 147 people aged 15 years or more examined in 2019 had trichiasis, both aged 40 years or more. Seven of 53 children aged 1-9 years in 2019 and seven of 103 in 2021 were seropositive for anti-Pgp3 antibodies. CONCLUSIONS: Despite the prevalence of clinical signs consistent with trachomatous inflammation-follicular among 5-9-year-old children exceeding the 5% threshold for community-wide treatment, laboratory testing indicated that childhood exposure to ocular C. trachomatis is rare in this community. Laboratory testing should be integrated into Australian trachoma guidelines.

Evaluation of the Aptima HCV Quant Dx Assay for Hepatitis C Virus RNA Detection from Fingerstick Capillary Dried Blood Spot and Venepuncture-Collected Samples.

BACKGROUND: Simplified diagnostic strategies are needed increase hepatitis C virus (HCV) testing to determine active infection and link people into treatment. Collection methods such as dried blood spots (DBS) have advantages over standard phlebotomy, especially within marginalized populations. METHODS: We evaluated the diagnostic performance of the Aptima HCV Quant assay for the quantification and detection of HCV RNA from paired DBS and venepuncture samples. Specimens were collected from participants enrolled in an Australian observational study. We compared HCV RNA detection from DBS against venepuncture samples (gold standard). RESULTS: One hundred sixty-four participants had paired samples and HCV RNA was detected in 45 (27% [95% confidence interval, 21%-35%]) by the Aptima assay in venepuncture samples. Sensitivity of the Aptima assay for HCV RNA quantification from DBS (≥10 IU/mL in plasma) was 100% and specificity was 100%. Sensitivity for HCV RNA detection from DBS was 95.6% and specificity was 94.1%. A small bias in plasma over DBS was observed with good agreement (R2 = 0.96). CONCLUSIONS: The Aptima HCV Quant assay detects active infection from DBS samples with acceptable diagnostic performance and is clinically comparable to plasma. These data will strengthen the case for the registration of a DBS kit insert claim, enabling future clinical utility.

Preservation of functionality, immunophenotype, and recovery of HIV RNA from PBMCs cryopreserved for more than 20 years.

Background: Many research laboratories have long-term repositories of cryopreserved peripheral blood mononuclear cells (PBMC), which are costly to maintain but are of uncertain utility for immunological studies after decades in storage. This study investigated preservation of cell surface phenotypes and in-vitro functional capacity of PBMC from viraemic HIV+ patients and healthy seronegative control subjects, after more than 20 years of cryopreservation. Methods: PBMC were assessed by 18-colour flow cytometry for major lymphocyte subsets within T, B, NK, and dendritic cells and monocytes. Markers of T-cell differentiation and activation were compared with original immunophenotyping performed in 1995/1996 on fresh blood at the time of collection. Functionality of PBMC was assessed by culture with influenza antigen or polyclonal T-cell activation, to measure upregulation of activation-induced CD25 and CD134 (OX40) on CD4 T cells and cytokine production at day 2, and proliferative CD25+ CD4 blasts at day 7. RNA was extracted from cultures containing proliferating CD4+ blast cells, and intracellular HIV RNA was measured using short amplicons for both the Double R and pol region pi code assays, whereas long 4-kbp amplicons were sequenced. Results: All major lymphocyte and T-cell subpopulations were conserved after long-term cryostorage, except for decreased proportions of activated CD38+HLA-DR+ CD4 and CD8 T cells in PBMC from HIV+ patients. Otherwise, differences in T-cell subpopulations between recent and long-term cryopreserved PBMC primarily reflected donor age-associated or HIV infection-associated effects on phenotypes. Proportions of naïve, memory, and effector subsets of T cells from thawed PBMC correlated with results from the original flow cytometric analysis of respective fresh blood samples. Antigen-specific and polyclonal T-cell responses were readily detected in cryopreserved PBMC from HIV+ patients and healthy control donors. Intracellular HIV RNA quantitation by pi code assay correlated with original plasma viral RNA load results. Full-length intracellular and supernatant-derived amplicons were generated from 5/12 donors, and sequences were ≥80% wild-type, consistent with replication competence. Conclusions: This unique study provides strong rationale and validity for using well-maintained biorepositories to support immunovirological research even decades after collection.

Utilising Integrated Bio-behavioural Surveillance (IBBS) to investigate declining hepatitis C antibody prevalence among people who inject drugs in the Australian Needle and Syringe Program Survey.

BACKGROUND: Prevalence of hepatitis C virus (HCV) antibody (Ab) on dried blood spot (DBS) samples in the Australian Needle and Syringe Program Survey (ANSPS) decreased nationally from 57% in 2015 to 32% in 2022. We aimed to investigate potential explanations for this decline. METHODS: Changes in DBS HCV Ab prevalence were investigated by redefining positive cases as those with those with either a positive HCV Ab test result or a self-reported history of ever having HCV treatment (modified prevalence), examining HCV Ab prevalence by birth and age cohorts, and assessing trends in key risk behaviours. RESULTS: Overall prevalence of DBS HCV Ab declined rapidly and significantly from 57% in 2015 to 32% in 2022 (p<0.001) however modified HCV Ab prevalence remained stable over time (85% and 88% in 2015 and 2022, respectively, p=0.357). The proportion of participants with negative HCV Ab and self-reported HCV infection increased from 20% in 1995 to 40% in 2022 (p<0.001) and the proportion with negative HCV Ab and lifetime HCV treatment increased from 3% in 1999 to 67% in 2022 (p<0.001). We also observed a decreasing trend in DBS HCV Ab prevalence in all birth and age cohorts with a noticeable acceleration in the decline commensurate with the advent of HCV DAA treatment. A long-term decreasing trend was also observed for key risk behaviours (p<0.001) however the short-term trend was not significant for recent receptive syringe sharing. CONCLUSION: The temporal decline in HCV Ab prevalence appears related to reduced sensitivity of DBS HCV Ab detection with viral clearance following treatment. Since 2016, HCV treatment uptake has increased markedly including among people who inject drugs. In this context, continuing to monitor HCV Ab prevalence by DBS testing is problematic, with a shift to surveillance of active infection the most relevant to guide policy and practice in this setting.

Utilising integrated bio-behavioural surveillance (IBBS) to investigate declining hepatitis C antibody prevalence among people who inject drugs in the Australian Needle and Syringe Program Survey.

BACKGROUND: Prevalence of hepatitis C virus (HCV) antibody (Ab) on dried blood spot (DBS) samples in the Australian Needle and Syringe Program Survey (ANSPS) decreased nationally from 57 % in 2015 to 32 % in 2022. We aimed to investigate potential explanations for this decline. METHODS: Changes in DBS HCV Ab prevalence were investigated by redefining positive cases as those with those with either a positive HCV Ab test result or a self-reported history of ever having HCV treatment (modified prevalence), examining HCV Ab prevalence by birth and age cohorts, and assessing trends in key risk behaviours. RESULTS: Overall prevalence of DBS HCV Ab declined rapidly and significantly from 57 % in 2015 to 32 % in 2022 (p < 0.001) however modified HCV Ab prevalence remained stable over time (85 % and 88 % in 2015 and 2022, respectively, p = 0.357). The proportion of participants with negative HCV Ab and self-reported HCV infection increased from 20 % in 1995 to 40 % in 2022 (p < 0.001) and the proportion with negative HCV Ab and lifetime HCV treatment increased from 3 % in 1999 to 67 % in 2022 (p < 0.001). We also observed a decreasing trend in DBS HCV Ab prevalence in all birth and age cohorts with a noticeable acceleration in the decline commensurate with the advent of HCV DAA treatment. A long-term decreasing trend was also observed for key risk behaviours (p < 0.001) however the short-term trend was not significant for recent receptive syringe sharing. CONCLUSION: The temporal decline in HCV Ab prevalence appears related to reduced sensitivity of DBS HCV Ab detection with viral clearance following treatment. Since 2016, HCV treatment uptake has increased markedly including among people who inject drugs. In this context, continuing to monitor HCV Ab prevalence by DBS testing is problematic, with a shift to surveillance of active infection the most relevant to guide policy and practice in this setting.

Improvement of immune dysregulation in individuals with long COVID at 24-months following SARS-CoV-2 infection.

This study investigates the humoral and cellular immune responses and health-related quality of life measures in individuals with mild to moderate long COVID (LC) compared to age and gender matched recovered COVID-19 controls (MC) over 24 months. LC participants show elevated nucleocapsid IgG levels at 3 months, and higher neutralizing capacity up to 8 months post-infection. Increased spike-specific and nucleocapsid-specific CD4+ T cells, PD-1, and TIM-3 expression on CD4+ and CD8+ T cells were observed at 3 and 8 months, but these differences do not persist at 24 months. Some LC participants had detectable IFN-γ and IFN-ß, that was attributed to reinfection and antigen re-exposure. Single-cell RNA sequencing at the 24 month timepoint shows similar immune cell proportions and reconstitution of naïve T and B cell subsets in LC and MC. No significant differences in exhaustion scores or antigen-specific T cell clones are observed. These findings suggest resolution of immune activation in LC and return to comparable immune responses between LC and MC over time. Improvement in self-reported health-related quality of life at 24 months was also evident in the majority of LC (62%). PTX3, CRP levels and platelet count are associated with improvements in health-related quality of life.

Hepatitis C Treatment Uptake Following Dried Blood Spot Testing for Hepatitis C RNA in New South Wales, Australia: The NSW DBS Pilot Study.

Background: Dried blood spot (DBS) testing for hepatitis C virus (HCV) RNA provides a sampling option that avoids venepuncture and can be carried out in a nonclinical setting. Large-scale evaluations are needed to understand how DBS testing can reduce HCV burden. This study estimated prevalence of, and factors associated with, HCV RNA and treatment initiation among people enrolled in a state-wide pilot of people testing in the NSW DBS Pilot in New South Wales, Australia. Methods: People at risk of HIV/HCV could participate via (1) self-registration online with a DBS collection kit delivered and returned by conventional postal service; or (2) assisted DBS sample collection at a community site or prison. Logistic regression was used to identify factors associated with detectable HCV RNA and treatment initiation within 6 months of testing. Results: Between September 2017 and December 2020, 5960 people were tested for HCV (76% men, 35% Aboriginal and/or Torres Strait Islander, 55% recently injected drugs): 21% online self-registration, 34% assisted registration in the community, 45% assisted registration in prison. Fifteen percent had detectable HCV RNA (878/5960). Overall, 44% (n = 386/878) of people with current HCV initiated treatment within 6 months (13% online self-registration, 27% assisted registration in the community, 61% assisted registration in prison). Testing in prison compared with the community (adjusted odds ratio [aOR], 4.28; 95% CI, 3.04-6.03) was associated with increased odds of treatment initiation. Being a woman compared with a man (aOR, 0.68; 95% CI, 0.47-0.97) was associated with reduced treatment initiation. Conclusions: The NSW DBS Pilot demonstrates the feasibility of using DBS to promote HCV testing and treatment in community and prison settings.

Evaluation of serological assays for SARS-CoV-2 antibody testing from dried blood spots collected from cohorts with prior SARS-CoV-2 infection.

Background: Dried blood spot (DBS) specimens are a useful serosurveillance tool particularly in hard-to-reach populations but their application for detecting SARS-CoV-2 infection is poorly characterised. Objectives: To compare detection of naturally acquired SARS-CoV-2 antibodies in paired DBS and serum specimens using commercially available serological immunoassays. Study Design: Specimens were collected through St Vincent's Hospital observational post COVID-19 cohort study (ADAPT). Laboratory spotted DBS from venepuncture were initially tested on seven assays, a DBS validation completed on three with clinically collected fingerstick DBSs tested on one. Results: Sensitivity for Euroimmun nucleocapsid (NCP) IgG ELISA from laboratory spotted DBS (n=145), Euroimmun spike, IgG ELISA from laboratory spotted DBS (n=161), and Binding Site total antibody ELISA from clinically collected fingerstick DBS (n=391) was 100% (95% CI: 95.8-100%), 100% (95% CI: 95.8-100%) and 92.9% (95% CI: 89.5-95.5%), respectively. Specificity was 66.2% (95% CI: 53.6-77.0%), 96% (95% CI: 88.7-99.1%) and 98.8% (95% CI: 93.3-99.9%), respectively. All three assays' results displayed a strong positive correlation between DBS compared to paired serum. Conclusions: The Binding Site™ spike total antibody and Euroimmun™ spike IgG ELISAs provided good analytical performance, demonstrating that DBS specimens could facilitate specimen collection in the epidemiological surveillance of SARS-CoV-2 infection. This is highly applicable in populations and settings where venepuncture is problematic (including community based regional/remote settings, nursing homes, prisons, and schools).

A national survey integrating clinical, laboratory, and WASH data to determine the typology of trachoma in Nauru.

BACKGROUND: The epidemiology of trachoma in several Pacific Islands differs from other endemic settings, in that there is a high prevalence of clinical signs of trachoma, particularly trachomatous inflammation-follicular (TF), but few cases of trichiasis and limited evidence of ocular chlamydial infection. This so-called "Pacific enigma" has led to uncertainty regarding the appropriate public health response. In 2019 alongside Nauru's national trachoma population survey, we performed bacteriological and serological assessments of children to better understand the typology of trachoma and to determine whether there is a need for trachoma interventions. METHODS: We used two-stage cluster sampling, examining residents aged ≥1 year and collecting household-level water, sanitation, and hygiene (WASH) variables. Children aged 1-9 years provided conjunctival swabs and finger-prick dried blood spots to investigate the presence of Chlamydia trachomatis nucleic acid and anti-Pgp3 antibodies, respectively. PRINCIPAL FINDINGS: In 818 participants aged 1-9 years, the age-adjusted TF prevalence was 21.8% (95% CI 15.2-26.2%); ocular C. trachomatis prevalence was 34.5% (95% CI 30.6-38.9), and anti-Pgp3 antibody prevalence was 32.1% (95% CI 28.4%-36.3%). The age- and gender-adjusted prevalence of trichiasis in ≥15-year-olds was 0.3% (95% CI 0.00-0.85), but no individual with trichiasis had trachomatous scarring (TS). Multivariable analysis showed an association between age and both TF (OR per year of age 1.3 [95% CI 1.2-1.4]) and anti-Pgp3 positivity (OR 1.2 [95% CI 1.2-1.3]). There were high rates of access to water and sanitation and no WASH variable was associated with the presence of TF. CONCLUSIONS: TF, nucleic acid, and age-specific antibody prevalence collectively indicate that high levels of C. trachomatis transmission among children present a high risk of ocular damage due to trachoma. The absence of trichiasis with trachomatous scarring suggest a relatively recent increase in transmission intensity.

High titre neutralizing antibodies in response to SARS-CoV-2 infection require RBD-specific CD4 T cells that include proliferative memory cells.

Background: Long-term immunity to SARS-CoV-2 infection, including neutralizing antibodies and T cell-mediated immunity, is required in a very large majority of the population in order to reduce ongoing disease burden. Methods: We have investigated the association between memory CD4 and CD8 T cells and levels of neutralizing antibodies in convalescent COVID-19 subjects. Findings: Higher titres of convalescent neutralizing antibodies were associated with significantly higher levels of RBD-specific CD4 T cells, including specific memory cells that proliferated vigorously in vitro. Conversely, up to half of convalescent individuals had low neutralizing antibody titres together with a lack of receptor binding domain (RBD)-specific memory CD4 T cells. These low antibody subjects had other, non-RBD, spike-specific CD4 T cells, but with more of an inhibitory Foxp3+ and CTLA-4+ cell phenotype, in contrast to the effector T-bet+, cytotoxic granzymes+ and perforin+ cells seen in RBD-specific memory CD4 T cells from high antibody subjects. Single cell transcriptomics of antigen-specific CD4+ T cells from high antibody subjects similarly revealed heterogenous RBD-specific CD4+ T cells that comprised central memory, transitional memory and Tregs, as well as cytotoxic clusters containing diverse TCR repertoires, in individuals with high antibody levels. However, vaccination of low antibody convalescent individuals led to a slight but significant improvement in RBD-specific memory CD4 T cells and increased neutralizing antibody titres. Interpretation: Our results suggest that targeting CD4 T cell epitopes proximal to and within the RBD-region should be prioritized in booster vaccines.

Concordance between self-reported and current hepatitis C virus infection status in a sample of people who inject drugs in Sydney and Canberra, Australia.

INTRODUCTION: Awareness of hepatitis C virus (HCV) status among people who inject drugs is critical to ensure linkage to care and reduce transmission risk. Testing pathways, confusion about results and possible reinfection raise potential for discordance between perceived and actual HCV status among people who inject drugs. We evaluated self-reported and serologically confirmed HCV status concordance among a sample of Australian people who inject drugs. METHODS: Data were collected in May-June 2018 from participants in Canberra and Sydney, Australia, who had injected drugs at least monthly in the past 6 months. Participants completed a structured interview assessing self-reported HCV status and provided a dried blood spot sample for HCV RNA testing. RESULTS: Of 103 participants, 95% self-reported ever receiving antibody testing, 58% of whom reported having received RNA testing. Seventy-three percent of participants reported never having been told that they had HCV, 18% reported current infection and 9% did not know their current status. According to dried blood spot RNA testing, 20% were currently infected. Over a quarter of the sample (28%, n = 29) did not accurately report their HCV status, half of whom were unaware of a current infection. DISCUSSION AND CONCLUSIONS: With over one-quarter of the sample in our study not accurately reporting their current HCV status, our findings reinforce the importance of regular testing for active infection, and the need for improved health literacy on HCV antibody and RNA test results, HCV status post-treatment and reinfection risk.

HIV-1 viral blips are associated with repeated and increasingly high levels of cell-associated HIV-1 RNA transcriptional activity.

OBJECTIVE: Some HIV+ patients, virally suppressed on ART, show occasional 'blips' of detectable HIV-1 plasma RNA. We used a new highly sensitive assay of cell-associated HIV-1 RNA to measure transcriptional activity in PBMCs and production of infectious virus from the viral reservoir, in patients with and without 'blips'. DESIGN/METHODS: RNA and DNA extracted from cells in 6 ml of peripheral blood, from suppressed patients with one to two 'blip' episodes over the past 2 years of ART (n = 55), or no 'blips' (n = 52), were assayed for HIV-1 RNA transcripts and proviral DNA targeting the highly conserved 'R' region of the LTR. Follow-up samples were also collected. Purified CD4+ T cells were cultured with anti-CD3/CD28/CD2 T-cell activator to amplify transcription and measure replication competent virus. RESULTS: HIV-1 RNA transcripts ranged from 1.3 to 5415 copies/106 white blood cells. 'Blip' patients had significantly higher levels vs. without blips (median 192 vs. 49; P = 0.0007), which correlated with: higher levels of inducible transcripts after activation in vitro, sustained higher HIV-1 transcription levels in follow-up samples along with increasing HIV-1 DNA in some, and production of replication-competent HIV-1. CONCLUSION: Viral 'blips' are significant reflecting higher transcriptional activity from the reservoir and contribute to the reservoir over time. This sensitive assay can be used in monitoring the size and activity of the HIV-1 reservoir and will be useful in HIV-1 cure strategies.

Estimating the Consensus hepatitis C Cascade of Care among people who inject drugs in Australia: Pre and post availability of direct acting antiviral therapy.

Background Monitoring the hepatitis C virus (HCV) cascade of care (CoC) among people who inject drugs (PWID) is an essential component of the response to World Health Organisation's (WHO) hepatitis elimination goals. This study aimed to estimate the Consensus hepatitis C CoC among PWID using data collected in Australia prior to and after the introduction of unrestricted direct-acting antiviral (DAA) therapy in March 2016. Methods The Australian Needle Syringe Program Survey is a cross-sectional bio-behavioural surveillance system that recruits >2000 PWID annually. Using data from 2015 and 2019, HCV antibody and ribonucleic acid (RNA) test results from dried blood spots were combined with self-reported data on HCV diagnostic testing and treatment to project HCV Consensus CoC indicators at a population-level among Australian PWID. Results Among an estimated 75,000 people who inject drugs on a regular basis in Australia, the number with active HCV infection declined from 32,619 (44%) in October 2015 to 12,679 (17%) in October 2019. The majority (78% in 2015 and 2019) of PWID reported HCV diagnosis, while the proportion of those diagnosed who were treated increased from 3% in 2015 to 47% in 2019. Among those treated, the proportion who were HCV RNA negative and assumed to have been successfully treated (cured), increased from 27% in 2015 to 88% in 2019. Conclusion This study demonstrates remarkable HCV CoC progress among PWID in Australia following availability of DAA therapy. There was a substantial increase in the proportion of HCV diagnosed PWID who initiated treatment and were cured, while the number of PWID with active HCV infection more than halved over a 3.5 year period. Estimates of the Consensus hepatitis C CoC among PWID is required to monitor progress toward WHO HCV elimination goals.

Performance evaluation of the Hologic Aptima HCV Quant Dx assay for detection of HCV RNA from dried blood spots.

BACKGROUND: The availability of effective direct-acting antiviral therapy for hepatitis C virus (HCV) has led to a need for simplified diagnostic pathways. Barriers to treatment uptake, specifically in people who inject drugs and in remote and resource limited settings, may be overcome by utilizing novel collection methods, such as dried blood spots (DBS). However, there are currently no registered assays for HCV RNA testing from DBS samples. OBJECTIVES: To evaluate the sensitivity and specificity of the Aptima HCV Dx Quant assay for HCV RNA detection in DBS samples STUDY DESIGN: 107 paired venepuncture and DBS samples from HCV antibody positive individuals were analyzed for HCV RNA on the Aptima HCV Dx Quant and Roche CAP/CTM (gold standard) HCV assays. RESULTS: 78% (n=83) had detectable HCV RNA in plasma. Sensitivity of the Aptima assay for HCV RNA detection in DBS was 96.4% (95% CI 89.8-99.3%) and specificity was 95.8% (95% CI 78.8-99.9%). Sensitivity for HCV RNA detection in DBS using a quantitative threshold of ≥15 IU/mL in plasma was 95.1% (95% CI 88%-98.7%) and specificity was 96.0% (95% CI 79.7%-99.9%). The sensitivity of HCV RNA detection in DBS using a quantitative threshold of ≥1000 IU/mL (based on a clinically relevant threshold) was 100% (95% CI 95.3-100%) and specificity was 100% (95% CI 88.4-100%). CONCLUSIONS: Our data indicates that the Aptima HCV Dx Quant can detect active HCV infection from a DBS sample with good sensitivity and specificity, particularly when using a threshold of ≥1000 IU/mL.