Tracking the pandemic through molecular and sequencing tools: a story of SARS- CoV-2 over five years, lessons learned, and further directions.

The COVID-19 pandemic posed unprecedented challenges to public health surveillance frameworks. In response, the Provincial Public Health Laboratory (PPHL), Sindh, contributed to early detection, large-scale diagnostic testing, and genomic surveillance from February 2020 to June 2025. This study reviews five years of PPHL's role in COVID-19 diagnosis, variant tracking, and public health response in Sindh. Diagnostic data included positive cases, while genomic epidemiology was assessed using commercial RT-PCR kits and in-house S-gene dropout assays as proxy tools for variant detection, supplemented by whole genome sequencing (WGS). A total of 194,415 specimens were tested, with five major peaks observed: wild-type (33.3%), Alpha (26.9%), Delta (22.0%), and Omicron (23.1%). WGS was performed on 124 selected samples, which identified Delta (AY.127, B.1.617.2), Omicron (BA.1, BA.5.2, XBB.1.9.1), and, more recently, JN.1, BA.2.86.1, and XEC lineages in May 2025. Reports of NB.1.8.1 from neighboring countries were noted, but this lineage was not detected in Karachi samples. While study limitations like low portion of sequenced samples and potential bias toward low Ct specimens exist, these findings still highlight the value of sustained laboratory-based surveillance in documenting SARS-CoV-2 evolution and supporting public health decision-making in Sindh.