Performance and workflow assessment of six nucleic acid extraction technologies for use in resource limited settings.

Infectious disease nucleic acid amplification technologies (NAAT) have superior sensitivity, specificity, and rapid time to result compared to traditional microbiological methods. Recovery of concentrated, high quality pathogen nucleic acid (NA) from complex specimen matrices is required for optimal performance of several NA amplification/detection technologies such as polymerase chain reaction (PCR). Fully integrated NAAT platforms that enable rapid sample-to-result workflows with minimal user input are generally restricted to larger reference lab settings, and their complexity and cost are prohibitive to widespread implementation in resource limited settings (RLS). Identification of component technologies for incorporation of reliable and affordable sample preparation with pathogen NA amplification/detection into an integrated platform suitable for RLS, is a necessary first step toward achieving the overarching goal of reducing infectious disease-associated morbidity and mortality globally. In the current study, we evaluate the performance of six novel NA extraction technologies from different developers using blinded panels of stool, sputum and blood spiked with variable amounts of quality-controlled DNA- and/or RNA-based microbes. The extraction efficiencies were semi-quantitatively assessed using validated real-time reverse transcription (RT)-PCR assays specific for each microbe and comparing target-specific RT-PCR results to those obtained with reference NA extraction methods. The technologies were ranked based on overall diagnostic accuracy (analytical sensitivity and specificity). Sample input and output volumes, total processing time, user-required manual steps and cost estimates were also examined for suitability in RLS. Together with the performance analysis, these metrics were used to select the more suitable candidate technologies for further optimization of integrated NA amplification and detection technologies for RLS.

Assessment of eight nucleic acid amplification technologies for potential use to detect infectious agents in low-resource settings.

Nucleic acid amplification technologies (NAATs) are high-performance tools for rapidly and accurately detecting infectious agents. They are widely used in high-income countries to diagnose disease and improve patient care. The complexities associated with test methods, reagents, equipment, quality control and assurance require dedicated laboratories with trained staff, which can exclude their use in low-resource and decentralized healthcare settings. For certain diseases, fully integrated NAAT devices and assays are available for use in environmentally-controlled clinics or emergency rooms where relatively untrained staff can perform testing. However, decentralized settings in many low- and middle-income countries with large burdens of infectious disease are challenged by extreme environments, poor infrastructure, few trained staff and limited financial resources. Therefore, there is an urgent need for low-cost, integrated NAAT tools specifically designed for use in low-resource settings (LRS). Two essential components of integrated NAAT tools are: 1) efficient nucleic acid extraction technologies for diverse and complex sample types; and 2) robust and sensitive nucleic acid amplification and detection technologies. In prior work we reported the performance and workflow capacity for the nucleic acid extraction component. In the current study we evaluated performance of eight novel nucleic acid amplification and detection technologies from seven developers using blinded panels of RNA and/or DNA from three pathogens to assess both diagnostic accuracy and suitability as an essential component for low-cost NAAT in LRS. In this exercise, we noted significant differences in performance among these technologies and identified those most promising for potential further development.

Dynamics of viremia in early hepatitis C virus infection.

BACKGROUND: It is important to characterize viral dynamics in early hepatitis C virus (HCV) infection to further our understanding of viral pathogenesis and the potential for secondary transmission in acute infection through blood transfusion or other routes. STUDY DESIGN AND METHODS: Serial units given by 77 source plasma donors who had evolved from HCV RNA-negative to HCV RNA-positive by nucleic acid amplification technology (NAT) screening with 512-unit pool-NAT or were followed from RNA detection to antibody conversion were tested by individual NAT and quantitative RNA assays. RESULTS: During the ramp-up phase when exponential growth occurs, HCV viral load doubled every 10.8 hours (95% confidence interval [CI], 9.9-12.0). Intermittent viremia was observed before the ramp-up phase in 37 of 50 panels with the earliest detectable viremic bleed occurring 63 days before the estimated onset of ramp-up. The plateau phase or high-titer viremic period that occurs between ramp-up and seroconversion was estimated to last 56.3 days (95% CI, 44.8-67.8). CONCLUSIONS: Intermittent low-level HCV viremia can occur as much as 2 months before the periods of exponential increase in viral load and the high-titer plateau-phase viremia that usually precede seroconversion. Animal inoculation studies are in progress to evaluate if transfusion of low-level viremic plasma can transmit HCV infection.

Relative sensitivities of licensed nucleic acid amplification tests for detection of viremia in early human immunodeficiency virus and hepatitis C virus infection.

BACKGROUND: Screening donors for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNA is primarily performed on minipools (MPs) with one of two commercial nucleic acid amplification tests (NAT; Roche Molecular Systems; or Gen-Probe/Chiron). We compared these assays with respect to detection of RNA in early HIV and HCV infection. STUDY DESIGN AND METHODS: Twelve HIV plasma donor panels (116 serial samples) and 12 HCV panels (180 serial samples) were selected to optimally represent early viremia. Initial testing was performed in singlicate or triplicate on separately coded aliquots, both neat and at dilutions corresponding to MP screening (1:16 for Gen-Probe; 1:24 for Roche); 20 additional replicates were performed when discordant results were observed. Odds ratios (ORs) comparing detection of RNA by different assays were derived with logistic regression models. Differences in window-period closure and yields of assays in MP or individual-donation (ID) format were estimated. RESULTS: Differences in detection rates between Roche and Gen-Probe NAT assays were small and only observed with samples with very-low-level viremia. ORs for detecting RNA by the Gen-Probe versus the Roche assay were significant for HIV if conducted on MPs (1.8; 95% confidence interval [CI], 1.3-2.5) but not neat (1.0; 95% CI, 0.72-1.4). Odds of detecting HCV RNA were higher if the Gen-Probe assay was conducted either neat (2.3; 95% CI, 1.6-3.2) or on MPs (4.0; 95% CI, 2.8-5.8). These differences translated to <1 day window-period closure and

Comparative yield of HCV RNA testing in blood donors screened by 2.0 versus 3.0 antibody assays.

BACKGROUND: Two HCV antibody tests (EIA 2.0 [EIA2], Abbott; and the Version 3.0 ELISA [EIA3], Ortho) are currently licensed for screening of US blood donors. Testing of donors for HCV RNA allows comparison of the sensitivities of the two antibody-screening assays. STUDY DESIGN AND METHODS: All allogeneic blood donations at 13 US test sites were screened for HCV RNA by testing plasma minipools using an investigational assay (COBAS AmpliScreen HCV test, v2.0, Roche Molecular Systems). Some sites screened for HCV antibody by EIA2 and some used EIA3. The frequency of RNA-positive and antibody-negative (RNA-pos and Ab-neg) donations among donors screened by each antibody assay was compared. Antibody appearance was assessed in a donor follow-up study. RESULTS: A total of 5.51 x 10(6) donations were screened for HCV RNA. Of these, 2.27 million were screened for antibody by EIA2, and 3.24 million by EIA3. Twenty-three donations were HCV RNA-pos and Ab-neg. The frequency of RNA-pos and Ab-neg donations was higher among donations screened by EIA2 (1 in 134,000), compared to those screened by EIA3 (1 in 540,000) (p = 0.001). Of the 17 RNA-pos and Ab-neg donations identified by test sites that used EIA2, 14 were retested by EIA3 and 10 (71%) were reactive. Most RNA-pos and Ab-neg donors appear to be in the process of seroconversion. Donors that were initially EIA2-negative and EIA3-reactive showed a more prolonged pattern of seroconversion compared to those that were initially nonreactive by both antibody assays. Four donors were EIA2-negative, EIA3-reactive, and RIBA-indeterminate (c33c) for at least 90 days, 1 for more than 317 days. CONCLUSION: EIA3 would have detected the majority of RNA-positive donations missed by EIA2. Some RNA-positive donors are EIA2-negative and EIA3-reactive for a prolonged period of time.

Absence of HBV and HCV, HTLV-I and -II, and human herpes virus-8 activation after allogeneic RBC transfusion in patients with advanced HIV-1 infection.

BACKGROUND: The Viral Activation Transfusion Study was a prospective, randomized, double-blind comparison of transfusion with WBC-reduced versus non-WBC-reduced RBCs to HIV+ patients. The primary study characterized the effect of transfusion on HIV and CMV activation by monitoring viral load changes. The present study analyzed HBV, HCV, HTLV-I and -II, and human herpes virus-8 (HHV-8) viral load before and after transfusion to evaluate the further hypothesis that global immune stimulation following allogeneic RBC transfusion results in activation and increased viral proliferation of chronic viral infections other than HIV and CMV. STUDY DESIGN AND METHODS: Baseline samples from 519 to 523 subjects were screened for HBV, HCV, HTLV-I and -II, and HHV-8 infection, and baseline, serial weekly, and quarterly blood samples from infected subjects in the non-WBC-reduced arm were evaluated for changes from baseline in viral nucleic acid and ALT levels. RESULTS: Seroprevalence of HBV, HCV, HTLV-I and -II, and HHV-8 was 68, 25, 5, and 30 percent, respectively. No significant induction of HBV, HCV, HHV-8, or HTLV-I and -II viral replication following allogeneic transfusion of non-WBC-reduced blood was observed. A significant, albeit small, association was observed between transfusion and ALT. CONCLUSIONS: Based on these results and our previous finding that no adverse effect on HIV and CMV viral load and disease progression results from allogeneic transfusion, no evidence is found to support the selective use of WBC-reduced blood components for HIV-infected patients.

HCV viral load in anti-HCV-reactive donors and infectivity for their recipients.

BACKGROUND: An attempt has been made to determine the minimum level of HCV nucleic acid in donors associated with infection of recipients. This is important for considerations about assay sensitivity, use of minipool versus single-donation testing, and continued use of serologic testing. STUDY DESIGN AND METHODS: A total of 5387 specimens from the Transfusion-Transmitted Viruses Study in the 1970s were screened for antibody to HCV (anti-HCV). The outcome in recipients of seroreactive donations was examined for viremia and seroconversion. Present techniques for both groups included third-generation EIA, RIBA, quantitative RT-PCR assay, and transcription-mediated amplification (TMA) assay. RESULTS: A total of 156 recipients of components from 180 anti-HCV-reactive donors were identified. One-hundred seven of these were HCV-naïve before transfusion and received a single, confirmed seropositive unit; 94 (88%) became infected. Eighty-five recipients had donors whose HCV RNA level was quantifiable by RT-PCR (range, 182-3,310,000 copies/mL). Eighty-three (98%) seroconverted. Of the remaining 22, a total of 10 received units positive for HCV RNA detected only by TMA; all 10 recipients seroconverted. Of the remaining 12 recipients of anti-HCV+, TMA-negative units, 1 recipient seroconverted. CONCLUSIONS: High rates of transmission were seen at all levels of viremia, and one donor transmitted with undetectable levels in the TMA assay. Current HCV RNA testing will therefore not interdict all infectious units, even with single-donation testing, and serologic screening must be continued.