Diagnostic Accuracy of Assays Using Point-of-Care Testing or Dried Blood Spot Samples for the Determination of Hepatitis C Virus RNA: A Systematic Review.

BACKGROUND: Finger-stick point-of-care and dried blood spot (DBS) hepatitis C virus (HCV) RNA testing increases testing uptake and linkage to care. This systematic review evaluated the diagnostic accuracy of point-of-care testing and DBS to detect HCV RNA. METHODS: Bibliographic databases and conference presentations were searched for eligible studies. Meta-analysis was used to pool estimates. RESULTS: Of 359 articles identified, 43 studies were eligible and included. When comparing the Xpert HCV Viral Load Fingerstick assay to venous blood samples (7 studies with 987 samples), the sensitivity and specificity for HCV RNA detection was 99% (95% confidence interval [CI], 97%-99%) and 99% (95% CI, 94%-100%) and for HCV RNA quantification was 100% (95% CI, 93%-100%) and 100% (95% CI, 94%-100%). The proportion of invalid results following Xpert HCV Viral Load Fingerstick testing was 6% (95% CI, 3%-11%). When comparing DBS to venous blood samples (28 studies with 3988 samples) the sensitivity and specificity for HCV RNA detection was 97% (95% CI, 95%-98%) and 100% (95% CI, 98%-100%) and for HCV RNA quantification was 98% (95% CI, 96%-99%) and 100% (95% CI, 95%-100%). CONCLUSIONS: Excellent diagnostic accuracy was observed across assays for detection of HCV RNA from finger-stick and DBS samples. The proportion of invalid results following Xpert HCV Viral Load Fingerstick testing highlights the importance of operator training and quality assurance programs.

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.

Evaluation of a hepatitis C virus core antigen assay from venepuncture and dried blood spot collected samples: A cohort study.

The global scale-up of hepatitis C virus (HCV) diagnosis requires simplified and affordable HCV diagnostic pathways. This study evaluated the sensitivity and specificity of the HCV Architect core antigen (HCVcAg) assay for detection of active HCV infection in plasma and capillary whole blood dried blood spots (DBS) compared with HCV RNA testing in plasma (Abbott RealTime HCV Viral Load). Samples were collected from participants in an observational cohort enrolled at three sites in Australia (two-drug treatment and alcohol clinics and one homelessness service). Of 205 participants, 200 had results across all samples and assay types and 186 were included in this analysis (14 participants receiving HCV therapy were excluded). HCV RNA was detected in 29% of participants ([95% CI: 22.6-36.1], 54 of 186). The sensitivity of HCVcAg for detection of active HCV infection in plasma was 98.1% (95% CI: 90-100) and 100% (95% CI: 93-100) when compared to HCV RNA thresholds of ≥12 and ≥1000 IU/mL, respectively. The sensitivity of the HCVcAg assay for detection of active HCV infection in DBS was 90.7% (95% CI: 80-97) and 92.5% (95% CI: 82-98) when compared to HCV RNA thresholds of ≥12 and ≥1000 IU/mL, respectively. The specificity of HCV core antigen for detection of active infection was 100% (95% CI: 97-100) for all samples and RNA thresholds. These data indicate that the detection of HCVcAg is a useful tool for determining active HCV infection; to facilitate enhanced testing, linkage to care and treatment particularly when testing plasma samples are collected by venepuncture.

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).

Prevalence, incidence and risk factors for pharyngeal gonorrhoea in a community-based HIV-negative cohort of homosexual men in Sydney, Australia.

BACKGROUND: Pharyngeal gonorrhoea is common in homosexual men and may be important in maintaining community prevalence of anogenital infections. METHODS: From 2003, all participants in the Health in Men cohort of HIV-negative homosexual men in Sydney were offered annual pharyngeal gonorrhoea screening by BD ProbeTec nucleic acid amplification (NAAT) assay with supplementary porA testing. Participants self-reported diagnoses of pharyngeal gonorrhoea made elsewhere between interviews. Detailed sexual behavioural data were collected 6-monthly. RESULTS: Among 1427 participants enrolled, 65 study-visit-diagnosed pharyngeal gonorrhoea infections were identified (incidence 1.51 per 100 person-years, 95% CI 1.19 to 1.93) of which seven infections were identified on baseline testing (prevalence 0.57%, 95% CI 0.23 to 1.17%). Almost 85% of study-visit-diagnosed pharyngeal infections occurred without concurrent anogenital gonorrhoea. The combined incidence of study-visit-diagnosed and self-reported pharyngeal gonorrhoea (n=193) was 4.45 per 100 person-years (95% CI 3.86 to 5.12). On multivariate analysis, incident infection was associated with younger age (p-trend=0.001), higher number of male partners (p-trend=0.002) and reported contact with gonorrhoea (p<0.001). Insertive oro-anal sex ('rimming') was the only sexual behaviour independently associated with incident pharyngeal gonorrhoea (p-trend=0.044). CONCLUSIONS: The majority of pharyngeal gonorrhoea occurred without evidence of concurrent anogenital infection, and the high incidence-to-prevalence ratio suggests frequent spontaneous resolution of NAAT-detected infection. The association of pharyngeal gonorrhoea with oro-anal sex indicates that a broader range of sexual practices are likely to be involved in transmission of gonorrhoea to the pharynx than previously acknowledged. Screening the pharynx of sexually active homosexual men could play a role in reducing the prevalence of anogenital Neisseria gonorrhoeae.

Possible clearance of transfusion-acquired nef/LTR-deleted attenuated HIV-1 infection by an elite controller with CCR5 Δ32 heterozygous and HLA-B57 genotype.

BACKGROUND: Subject C135 is one of the members of the Sydney Blood Bank Cohort, infected in 1981 through transfusion with attenuated nef/3' long terminal repeat (LTR)-deleted HIV-1, and has maintained undetectable plasma viral load and steady CD4 cell count, in the absence of therapy. Uniquely, C135 combines five factors separately associated with control of viraemia: nef/LTR-deleted HIV-1, HLA-B57, HLA-DR13, heterozygous CCR5 Δ32 genotype and vigorous p24-stimulated peripheral blood mononuclear cell (PBMC) proliferation. Therefore, we studied in detail viral burden and immunological responses in this individual. METHODS: PBMC and gut and lymph node biopsy samples were analysed for proviral HIV-1 DNA by real-time and nested PCRs, and nef/LTR alleles by nested PCR. HIV-specific antibodies were studied by Western blotting, and CD4+ and CD8+ T lymphocyte responses were measured by proliferation and cytokine production in vitro. RESULTS: PBMC samples from 1996, but not since, showed amplification of nef alleles with gross deletions. Infectious HIV-1 was never recovered. Proviral HIV-1 DNA was not detected in recent PBMC or gut or lymph node biopsy samples. C135 has a consistently weak antibody response and a substantial CD4+ T cell proliferative response to a previously described HLA-DR13-restricted epitope of HIV-1 p24 in vitro, which augmented a CD8+ T cell response to an immunodominant HLA-B57-restricted epitope of p24, while his T cells show reduced levels of CCR5. CONCLUSIONS: Subject C135's early PCR and weak antibody results are consistent with limited infection with a poorly replicating nef/LTR-deleted strain of HIV-1. With his HLA-B57-restricted gag-specific CD8 and helper HLA-DR13-restricted CD4 T cell proliferative responses, C135 appears to have cleared his HIV-1 infection 37 years after transfusion.

Low positive predictive value of a nucleic acid amplification test for nongenital Neisseria gonorrhoeae infection in homosexual men.

The diagnosis of nongenital Neisseria gonorrhoeae infection by the Becton Dickinson ProbeTec ET Chlamydia trachomatis and N. gonorrhoeae Amplified DNA assay had low positive predictive value among an Australian community-based sample of homosexual men. Only 30.4% of oropharyngeal samples and 73.7% of anorectal samples were positive for N. gonorrhoeae by the porA assay. The accuracy of nucleic acid amplification tests in this context is compromised without supplemental testing.

Incidence immunoassay for distinguishing recent from established HIV-1 infection in therapy-naive populations.

OBJECTIVE: To identify a specific marker of recent HIV-1 infection. DESIGN: The humoral immune response in individuals recently infected with HIV-1 was followed by analysing the antibody isotype-specific response generated to HIV-1 antigens in sequential samples collected during and following seroconversion. METHODS: Antibody isotype-specific HIV-1 Western blots were analysed to identify interactions indicative of recent HIV-1 infection. These responses were further quantified using an antibody isotype-specific enzyme-linked immunoabsorbent assay based on recombinant HIV-1 antigens. RESULTS: During maturation of the immune response to HIV-1 infection, a rapid and enduring IgG1 isotype response was seen to all the major proteins transcribed by env, gag and pol. An early transient peak of IgG3 reactivity to p24 was observed over an interval of approximately 1-4 months following HIV-1 infection. The presence of IgG3 reactivity to p24 permitted established infection to be distinguished from recently infected individuals during this time period. CONCLUSION: An assay for anti-p24 IgG3 reactivity would provide an estimate of the incidence of HIV infection that may be applicable for epidemiological surveys as well as for monitoring new infections during vaccine trials and for managing treatment programmes.

Greater reversal of CD4+ cell abnormalities and viral load reduction after initiation of antiretroviral therapy with zidovudine, lamivudine, and nelfinavir before complete HIV type 1 seroconversion.

In a prospective open-label study, 41 male subjects received nelfinavir, zidovudine, and lamivudine stratified as either: early stage (ES; negative/indeterminate Western blot; n = 19) or late stage (LS; positive Western blot; n = 22) primary HIV-1 infection. Despite higher median baseline HIV-1 RNA levels and lower CD4(+) cell numbers in the ES subjects, a significantly greater decline in viral load (-3.46 vs. -2.83 log(10) copies/ml; p = 0.023) and increase in CD4(+) cell number (+85 vs. +41 cells/month increase, p = 0.01) were observed over the first 3 months of therapy such that both groups had comparable results at 1 year. The proportion with HIV-1 RNA < 50 copies/mL at 1 year was similar (9 of 19 ES subjects and 11 of 22 LS subjects by intention-to-treat analysis). Memory CD4(+) cell numbers, and activated CD4(+) percentages, were also significantly improved in ES subjects. Despite poorer prognostic markers at baseline ES subjects achieved responses similar to those of LS subjects after 1 year of treatment.

Lack of correlation between three commercial platforms for the evaluation of human immunodeficiency virus type 1 (HIV-1) viral load at the clinically critical lower limit of quantification.

BACKGROUND: Concordance in plasma HIV-1 viral load quantification at the lower limit of quantification (LLOQ) is crucial for current commercial assays. OBJECTIVE: To compare the performance of three commercial viral load assays and carry out a correlation study with the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test, the Roche Cobas Amplicor HIV-1 MONITOR test, and the Abbott RealTime HIV-1 assay. STUDY DESIGN: Assay agreement was analyzed using linear regression and Bland-Altman plots. Concordance near the clinically critical LLOQ was measured by Cohen's kappa statistics. Intra-assay precision was assessed, and assay reproducibility was measured at 50copies/mL across all three platforms. RESULTS: While good overall correlation was observed between the assays (r≥0.93), quantitative differences exceeded 0.5log(10)copies/mL among paired results in 3.7 to 8.3% of specimens. The degree of concordance between the assays near the LLOQ was unsatisfactory, with Cohen's kappa ranging from 0.14 to 0.38. The intra-assay precision of the Abbott RealTime HIV-1 assay ranged from 0.04 to 0.15 (SD log(10)) and 1.34% to 8.37% (CV). Reproducibility at 50copies/mL for RealTime HIV-1, TaqMan, and Amplicor was 10.05, 11.04 and 5.07 (% CV), respectively. CONCLUSION: Although good correlation was observed between the assays across their linear range, their concordance at the clinically critical LLOQ was poor. The accurate quantification of low-level viremia remains elusive, and the lack of correlation of these assays presents a challenge to the interpretation of such results and in the clinical management of HIV-infected patients.

Anal sexually transmitted infections and risk of HIV infection in homosexual men.

BACKGROUND: We examined a range of common bacterial and viral sexually transmitted infections as risk factors for HIV seroconversion in a community-based cohort of HIV-negative homosexual men in Sydney, Australia. METHODS: Detailed information about HIV risk behaviors was collected by interview twice yearly. Participants were tested annually for HIV, anal and urethral gonorrhea and chlamydia, herpes simplex virus types 1 and 2, and syphilis. In addition, they reported annual diagnoses of these conditions and of genital and anal warts. RESULTS: Among 1427 enrolled participants, 53 HIV seroconverters were identified, giving an incidence of 0.78 per 100 person-years. After controlling for number of episodes of insertive and receptive nonseroconcordant unprotected anal intercourse, there were independent associations with anal gonorrhea (adjusted hazard ratio = 7.12, 95% confidence interval: 2.05 to 24.79) and anal warts (hazard ratio = 3.63, 95% confidence interval: 1.62 to 8.14). CONCLUSIONS: Anal gonorrhea and anal warts were independently associated with HIV acquisition. The added HIV prevention value of more frequent screening of the anus to allow early detection and treatment of anal sexually transmitted infections in homosexual men should be considered.

High rates of sexually transmitted infections in HIV positive homosexual men: data from two community based cohorts.

BACKGROUND/OBJECTIVES: Higher levels of sexual risk behaviours have been reported in HIV positive than in HIV negative homosexual men. In clinic based studies, higher rates of sexually transmitted infections (STIs) have also been reported. We compared rates of common STIs between HIV positive and HIV negative homosexual men from two ongoing community based cohort studies in Sydney, Australia. METHODS: Participants in the two cohorts were recruited using similar community based strategies. They were interviewed face to face annually after enrollment. Comprehensive sexual health screening, including hepatitis A and B, syphilis, gonorrhoea, and chlamydia (in urethra and anus) was offered to participants in both cohorts. RESULTS: In participants in the HIV positive cohort, 75% were hepatitis A seropositive, 56% had serological evidence of previous or current hepatitis B infection, and 24% had evidence of vaccination against hepatitis B infection. 19% of men tested positive for syphilis and 4% had evidence of recent infections. Compared with men in the HIV negative cohort, after adjustment for age, HIV positive participants had significantly higher prevalence of previous or current hepatitis B infection, syphilis, and anal gonorrhoea. CONCLUSION: This finding supports the need for frequent STI testing in HIV positive men to prevent morbidity and to decrease the risk of ongoing HIV transmission.