Genomic characterization of selected influenza A-positive specimens and an A(H1N1)pdm09/SARS-CoV-2 co-infection in the Republic of Moldova
TL;DR
This descriptive laboratory-based study characterized influenza A-positive respiratory specimens collected through routine surveillance in the Republic of Moldova and selected for sequencing to provide a descriptive genomic characterization of a laboratory-selected subset and illustrates a workflow linking molecular testing to sequencing.
Abstract
Background Genomic sequencing can complement routine influenza surveillance by enabling clade-level characterization of selected specimens and the genomic characterization of mixed viral specimens. This descriptive laboratory-based study characterized influenza A-positive respiratory specimens collected through routine surveillance in the Republic of Moldova and selected for sequencing. Methods Between October 2025 and February 2026, 1,081 respiratory specimens were screened by multiplex real-time reverse transcription polymerase chain reaction for influenza A, influenza B, and SARS-CoV-2. Of 291 influenza A-positive specimens, 174 had cycle-threshold values ≤27, and 38 were selected for hybrid-capture sequencing based on repeat cycle-threshold values and technical suitability. Consensus sequences were analyzed with Nextclade, and the SARS-CoV-2 component of one mixed specimen was additionally assessed using Pangolin. Results All 38 selected specimens yielded analyzable influenza A hemagglutinin sequences, with hemagglutinin-segment coverage ranging from 87.6% to 100%. The dataset comprised 32 A(H3N2) specimens, five A(H1N1)pdm09 specimens, and one mixed A(H1N1)pdm09/SARS-CoV-2 specimen. All A(H3N2) hemagglutinin sequences were assigned to clade K, subclade 2a.3a.1. The six A(H1N1)pdm09 hemagglutinin components were assigned to clade D.3.1 (n = 2) or D.3.1.1 (n = 4). The SARS-CoV-2 consensus was assigned to Nextclade clade 25I; poor overall sequence quality limited confidence in fine-scale lineage assignment and precluded robust interpretation of private mutations. Conclusion The study provides a descriptive genomic characterization of a laboratory-selected subset and illustrates a workflow linking molecular testing to sequencing. The findings should not be interpreted as estimates of national clade prevalence, geographical distribution, or A(H1N1)pdm09/SARS-CoV-2 co-infection frequency.