Performance evaluation of a laboratory developed PCR test for quantitation of HIV-2 viral RNA.

Management of Human Immunodeficiency Virus Type 2 (HIV-2) infections present unique challenges due to low viral titers, slow disease progression, and poor response to standard antiviral therapies. The need for a nucleic acid assay to detect and quantify HIV-2 virus has led to the development of a number of molecular-based assays for detection and/or quantification of HIV-2 viral RNA in plasma in order to provide laboratory evidence of HIV-2 infection and viral loads for use in treatment decisions. As HIV-2 is less pathogenic and transmissible than HIV-1 and has resistance to several of the antiretroviral drugs, delay of treatment is common. Cross sero-reactivity between HIV-1 and HIV-2 makes it difficult to distinguish between the two viruses based upon serological tests. As such we developed a quantitative reverse transcription PCR (qRT-PCR) assay targeting the 5' long terminal repeat of HIV-2 for detection and quantification of HIV-2 viral RNA in plasma to identify HIV-2 infection and for use in viral load monitoring. Serial dilutions of cultured HIV-2 virus demonstrated a wide dynamic range (10 to 100,000 copies/ml) with excellent reproducibility (standard deviation from 0.12-0.19), linearity (R2 = 0.9994), and a lower limit of detection at 79 copies/ml (NIH-Z). The assay is highly specific for HIV-2 Groups A and B and exhibits no cross reactivity to HIV-1, HBV or HCV. Precision of the assay was demonstrated for the High (Mean = 6.41; SD = 0.12) and Medium (Mean = 4.46; SD = 0.13) HIV-2 positive controls. Replicate testing of clinical specimens showed good reproducibility above 1,000 copies/ml, with higher variability under 1,000 copies/ml. Analysis of 220 plasma samples from HIV-2 infected West African individuals demonstrated significantly lower viral loads than those observed in HIV-1 infections, consistent with results of previous studies. Slightly more than seven percent of clinical samples (7.3%) demonstrated viral loads above 100,000 copies/ml, while 37.3% of samples were undetectable. The high sensitivity, specificity, precision, and linearity of the WRAIR qRT-PCR assay makes it well suited for detection and monitoring of HIV-2 RNA levels in plasma of infected individuals

Discrepant amplification results during the development of an assay leads to reclassification of two AIDS reagent repository HIV-2 isolates as HIV-1.

The development and verification of HIV-2 assays depends in part on the availability of well-characterized samples, including those from reagent repositories. During the development of an HIV-2 RNA quantification assay, two HIV-2 viral isolates (CDC 301340 and CDC 301342) obtained from the NIAID AIDS Reagent and Reference Repository were not detected leading to an investigation. Two HIV-2 primers/probe sets of known performance in real-time viral RNA quantification assays, targeting different regions of the virus, also failed to generate RT-PCR products for these two isolates. These isolates were tested in the HIV-1 specific COBAS AmpliPrep/COBAS TaqMan HIV-1 Test v2.0 (Roche Molecular Diagnostics) and were quantified at high copy number. Other HIV-2 isolates tested were not amplified in the COBAS HIV-1 TaqMan assay. Furthermore, the discrepant isolates were highly reactive in an HIV-1 p24 antigen test while the other HIV-2 isolates showed very weak, if any, cross-reactivity with the HIV-1 p24 assay. Phylogenetic tree analysis of sequences from the protease-reverse transcriptase regions of the discrepant HIV-2 isolates mapped with HIV-1 Group M, Subtype CRF02_AG confirming these isolates were of HIV-1 origin and had been misclassified as HIV-2. The use of misclassified isolates in the verification of molecular and immunological assays can lead to misinterpretation of test results, misdirection of efforts into assay redesign and increased development costs. The results of this study were shared with the NIAID AIDS Reagent Program, leading to the reclassification of the two discrepant isolates as HIV-1

Performance of two HIV-2 primers/probe sets in real-time RT-qPCR assays.

<p>Two HIV-2 primers/probe sets were assessed for amplification of serial dilutions of an HIV-2 isolate, NIH-Z, under standard ampification conditions that were not optimized for each primers/probe set. Primers/probe sets, designated as PD  =  Primer Design LTD HIV-2 PCR Kit and SM  =  Delarue et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096554#pone.0096554-Delarue1" target="_blank">[12]</a>, showed linear amplification profiles that were parallel. PD  =  •. SM  =  ▴.</p

Phylogenetic Tree Analysis of Protease/Reverse Transcriptase Sequences for CDC 310340 and CDC 310342 Viral Stocks.

<p>HIV-1 sequences were obtained using the Siemens' TRUGENE HIV-1 Genotype Test, analyzed using MegAlign version 9.0.4 (DNASTAR, Inc) and subtyped against the HIV Sequence Database using BLAST (<a href="http://www.hiv.lanl.gov/content/sequence/BASIC_BLAST.html" target="_blank">www.hiv.lanl.gov/content/sequence/BASIC_BLAST.html</a>). Both sequences were found to align with HIV-1 Group M, subtype CRF02_AG. The homology between the two sequences was 98% with 0.3% divergence. Both sequences are designated in the tree with a •. The dotted line indicates a negative branch length, which is a result of averaging.</p

Performance of HIV-1/2 viral stocks in real-time RT-qPCR and HIV-1 p24 antigen tests.

<p>Individual viral stocks identified as HIV-2 were obtained from the NIAID AIDS Reagent and Reference Repository and SeraCare, Inc. The HIV-1 subtype and HIV-2 group identification is based upon data sheets provided by NIAID and from publications. An HIV-1 Subtype B isolate from the United States (91US_4) was used as an HIV-1 control. Viral RNA was extracted and tested in HIV-2 real-time RT-qPCR assays. Viral isolates selected for testing in the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 v2.0 quantitative RNA assay and the Perkin-Elmer p24 Antigen test included the two viral isolates that were not amplified in the HIV-2 real-time RT-qPCR assays, three HIV-2 viral isolates that amplified well and the HIV-1 91US_4 isolate. Although the HIV-2 RT-qPCR assays were not optimized, the HIV-2 amplifications were robust for the majority of the isolates tested as reflected in the HIV-2 RNA concentrations reported based upon relative values extrapolated from the NIH-Z standard for each primers/probe set. The CDC 310340 and CDC 310342 viral stocks were not amplified in HIV-2 RT-qPCR assays but were quantified and reactive in HIV-1 specific tests. PD  =  Primer Design LTD HIV-2 PCR Kit primers/probe. SM  =  Delarue et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096554#pone.0096554-Delarue1" target="_blank">[12]</a> primers/probe set. TND  =  Target Not Detected. UNK is unknown group. Viral isolate was not tested (−).</p

Co-designing Improved Communication of Newborn Bloodspot Screening Results to Parents: Mixed Methods Study.

BACKGROUND: Each year in England, almost 10,000 parents are informed of their child's positive newborn bloodspot screening (NBS) results. This occurs approximately 2 to 8 weeks after birth depending on the condition. Communication of positive NBS results is a subtle and skillful task, demanding thought, preparation, and evidence to minimize potentially harmful negative sequelae. Evidence of variability in the content and the way the result is currently communicated has the potential to lead to increased parental anxiety and distress. OBJECTIVE: This study focused on the development of co-designed interventions to improve the experiences of parents receiving positive NBS results for their children and enhance communication between health care professionals and parents. METHODS: An experience-based co-design approach was used to explore experiences and co-design solutions with 17 health professionals employed in 3 National Health Service Trusts in England and 21 parents (13/21, 62% mothers and 8/21, 38% fathers) of 14 children recruited from the same 3 National Health Service Trusts. Experiences with existing services were gathered via semistructured interviews with health professionals. Filmed narrative interviews with parents were developed into a composite film. The co-design process identified priorities for improving communication of positive NBS results through separate parent and health professional feedback events followed by joint feedback events. In total, 4 interventions were then co-designed between the participants through a web-based platform. RESULTS: Parents and health professionals provided positive feedback regarding the process of gathering experiences and identifying priorities. Themes identified from the parent interviews included impact of initial communication, parental reactions, attending the first clinic appointment, impact of health professionals' communication strategies and skills, impact of diagnosis on family and friends, improvements to the communication of positive NBS results, and parents' views on NBS. Themes identified from the health professional interviews included communication between health professionals, process of communicating with the family, parent- and family-centered care, and availability of resources and challenges to effective communication. In response to these themes, 4 interventions were co-designed: changes to the NBS card; standardized laboratory proformas; standardized communication checklists; and an email or letter for providing reliable, up-to-date, condition-specific information for parents following the communication of positive NBS results. CONCLUSIONS: Parents and health professionals were able to successfully work together to identify priorities and develop co-designed interventions to improve communication of positive NBS results to parents. The resulting co-designed interventions address communication at different stages of the communication pathway to improve the experiences of parents receiving positive NBS results for their children. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1186/s40814-019-0487-5

Impact of Early Antiretroviral Therapy on Detection of Cell-Associated HIV-1 Nucleic Acid in Blood by the Roche Cobas TaqMan Test

The Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test, v2.0 (the CAP/CTM assay), was used to quantify cell-associated HIV-1 (CAH) nucleic acid in peripheral blood mononuclear cells (PBMC) from well-characterized clinical specimens from HIV-1-infected individuals on antiretroviral therapy (ART). Chronically infected individuals on ART with no detectable plasma HIV-1 RNA demonstrated average CAH burdens of 3.2 HIV-1 log10 copies/million cells. Assay sensitivity and specificity were 98.9% and 100%, respectively, with the positive and negative predictive values being 100% and 98.6%, respectively. The CAH burden was also measured at weeks 0, 1, 2, 8, and 60 in 37 participants (RV254/SEARCH010, Bangkok, Thailand) stratified by Fiebig stage (Fiebig stage I [FI] to FVI) at ART initiation. Prior to ART initiation, the average CAH burden was 1.4, 4.1, and 3.6 log10 copies/million PBMCs for individuals who initiated ART at FI, FII, and FIII to FVI, respectively. Initiation of ART resulted in a rapid decline of CAH in all individuals, with the greatest decrease being observed in individuals who initiated ART at FI to FIII. By week 60, 100% (FI), 71.8% (FII/FIII), and 20.5% (FIV to FVI) of samples from individuals initiating treatment were at or near the limit of quantitation. Residual CAH was detectable at 60 weeks in most individuals who initiated ART at later stages (FIV to FVI) and averaged 1.9 ± 0.7 log10 copies/million PBMCs. The modified Roche CAP/CTM assay provides a convenient, standardized approach to measure residual HIV in blood and may be useful for monitoring patients under therapy or those participating in HIV remission studies