Cytomegalovirus sequence variability, amplicon length, and DNase-sensitive non-encapsidated genomes are obstacles to standardization and commutability of plasma viral load results

Background: Cytomegalovirus (CMV) management post-transplantation relies on quantification in blood, but inter-laboratory and inter-assay variability impairs commutability. An international multicenter study demonstrated that variability is mitigated by standardizing plasma volumes, automating DNA extraction and amplification, and calibration to the 1st-CMV-WHO-International-Standard as in the FDA-approved Roche-CAP/CTMCMV. However, Roche-CAP/CTM-CMV showed under-quantification and false-negative results in a quality assurance program (UK-NEQAS-2014). Objectives: To evaluate factors contributing to quantification variability of CMV viral load and to develop optimized CMV-UL54-QNAT. Study design: The UL54 target of the UK-NEQAS-2014 variant was sequenced and compared to 329 available CMV GenBank sequences. Four Basel-CMV-UL54-QNAT assays of 361 bp, 254 bp, 151 bp, and 95 bp amplicons were developed that only differed in reverse primer positions. The assays were validated using plasmid dilutions, UK-NEQAS-2014 sample, as well as 107 frozen and 69 prospectively collected plasma samples from transplant patients submitted for CMV QNAT, with and without DNase-digestion prior to nucleic acid extraction. Results: Eight of 43 mutations were identified as relevant in the UK-NEQAS-2014 target. All Basel-CMV-UL54 QNATs quantified the UK-NEQAS-2014 but revealed 10-fold increasing CMV loads as amplicon size decreased. The inverse correlation of amplicon size and viral loads was confirmed using 1st-WHO-International-Standard and patient samples. DNase pre-treatment reduced plasma CMV loads by > 90% indicating the presence of unprotected CMV genomic DNA. Conclusions: Sequence variability, amplicon length, and non-encapsidated genomes obstruct standardization and commutability of CMV loads needed to develop thresholds for clinical research and management. Besides regular sequence surveys, matrix and extraction standardization, we propose developing reference calibrators using 100 bp amplicons.Peer reviewe

The rate of mother-to-child transmission of antiretroviral drug-resistant HIV strains is low in the Swiss Mother and Child HIV Cohort Study

AIMS OF THE STUDY Combination antiretroviral therapy (cART) has reduced mother-to-child transmissions (MTCT) and improved the prognosis of HIV-infected newborns. However, drug resistance mutations (DRM) in HIV-infected children, either transmitted by MTCT (HIV-tDRM) or selected by suboptimal adherence and drug levels (HIV-sDRM), remain a concern. We sought to determine the rate of HIV-tDRM and HIV-sDRM in MTCT pairs in Switzerland. METHODS We performed a retrospective analysis of prospectively collected clinical data and available stored samples from MTCT pairs participating in the Swiss Mother-Child HIV (MoCHIV) cohort. RESULTS We identified 22 HIV-infected mother-child pairs with delivery between 1989 and 2009 who had 15 years of follow-up (33% white ethnicity). Twenty-one women (96%) were treatment-naïve before pregnancy, 8 (36%) had an unknown HIV status and delivered vaginally, 2 were diagnosed but not treated, and 11 (50%) received antiretrovirals during pregnancy or at delivery, of whom only 6 cases (27%) had cART. HIV subtypes were concordant in all mother-child pairs (subtype B 13/22 [59%]). Using stored plasma (n = 66) and mononuclear cell (n = 43) samples from the children, HIV-tDRM (M184V) was identified in 1 of 22 (4.5%) mothers (1/11 treated, 9%) and was followed by HIV-sDRM at 10 months of age. HIV-sDRM (M184V 23%; K103N 4.5%; D67N 13.6%) occurred in 16/22 (73%) after 4 years, half of whom were treatment naïve. HIV-sDRM were associated with a lower CD4 T-cell nadir (p <0.05) and tended to have higher viral loads and more frequent cART changes. CONCLUSIONS HIV-tDRM were low in this Swiss MoCHIV cohort, making them a minor yet preventable complication of prenatal HIV care, whereas HIV-sDRM are a significant challenge in paediatric HIV care

Development and dynamics of cytomegalovirus UL97 ganciclovir resistance mutations in transplant recipients detected by next-generation sequencing

BACKGROUND: (Val)ganciclovir resistance mutations in CMV UL97 (UL97-GCV-R) complicate anti-CMV therapy in recipients of solid organ and hematopoietic stem cell transplants, but comprehensive data on prevalence, emergence, and outcome are scarce. METHODS: Using next-generation sequencing (NGS; Illumina MiSeq platform), we analyzed UL97-GCV-R in patients with available plasma samples and refractory CMV replication/DNAemia (n = 87) containing viral loads ≥910 IU/mL. Twenty-one patients with CMV DNAemia resolving under antiviral therapy were analyzed as controls. Detected mutations were considered induced and of potential clinical significance if they increased by ≥10% compared with the first detected frequency or if they had a maximum frequency ≥25%. RESULTS: Nineteen of 87 (21.8%) with refractory CMV replication had ≥1 UL97-GCV-R detected by NGS, in comparison to 0/21 of the controls (P = .02). One-third of the recipients had 2 or more induced UL97-GCV-R mutations. The most frequently induced mutations affected codons 595 (42% [8/19]), 594 (32% [6/19]), and 603 (32% [6/19]). C592G was present in all episodes of both cases and controls at frequencies <15%, but never induced. UL97-GCV-R tended to be more frequent in donor/recipient CMV immunoglobulin G mismatch or following failure to complete primary prophylaxis, and many developed invasive CMV disease. CONCLUSIONS: UL97-GCV-R is common among transplant patients with refractory CMV replication. Early testing by NGS allows for identification of major mutations at codons 595, 594, and 603 and excludes a major role of C592G in ganciclovir resistance. Large prospective studies on UL97-GCV-R are warranted