Environmental contamination with zoonotic hookworms in urban Bangladesh: molecular evidence from shared human–dog interfaces

Zoonotic hookworms are important but underrecognized contributors to environmental contamination and public health risk in urban slum communities, where free-roaming dogs frequently interact with human populations. Although molecular investigations of hookworms have been reported from Bangladesh and other endemic countries, comprehensive surveillance of environmental canine fecal contamination in urban Bangladesh remains limited. This study investigated the prevalence and species diversity of hookworms in environmentally deposited canine fecal samples collected from urban slums in northeastern Bangladesh. A cross-sectional study was conducted in five urban slum communities of Sylhet City Corporation between January 2025 and April 2026. A total of 280 environmentally deposited canine fecal samples were examined using the formalin–ether concentration technique, modified Baermann technique (MBT), and polymerase chain reaction (PCR) targeting the internal transcribed spacer (ITS) region of ribosomal DNA. All PCR-positive amplicons were bidirectionally sequenced and subjected to phylogenetic analysis for species confirmation. A laboratory soil-spiking assay evaluated the analytical recovery efficiency of the modified Baermann technique for hookworm L3 recovery. Hookworm contamination was detected in 49.6% (139/280; 95% CI: 43.7–55.5) of samples by PCR, compared with 38.2% (107/280) by microscopy and 40.4% (113/280) by MBT. PCR demonstrated superior diagnostic sensitivity, while microscopy and MBT achieved sensitivities of 77.0% and 81.3%, respectively, using PCR as the comparator method. ITS-PCR amplification followed by sequence analysis identified five hookworm species: Ancylostoma caninum (n = 90), A. ceylanicum(n = 26), Necator americanus (n = 12), Uncinaria stenocephala (n = 7), and A. duodenale (n = 4). Sequence identities with GenBank reference isolates ranged from 98.8% to 99.8%. Hookworm contamination was detected in all study locations, with prevalence ranging from 42.1% to 56.9%. The soil-spiking experiment demonstrated increasing larval recovery efficiency with increasing larval density, ranging from 55.7% to 98.1%. Widespread environmental contamination with hookworms was detected in shared human–dog environments in urban Bangladesh, with Ancylostoma caninum and A. ceylanicum identified as the predominant zoonotic species. The detection of Necator americanus and A. duodenale should be interpreted cautiously, as it may reflect environmental contamination, occasional misclassification of fecal origin, or passive passage following coprophagy rather than established canine infection. These findings highlight the value of molecular environmental surveillance for informing One Health strategies to reduce zoonotic hookworm exposure. Future studies incorporating molecular confirmation of fecal host origin and simultaneous human, animal, and environmental sampling are needed to better understand transmission pathways. • Hookworms were detected in 49.6% of environmental dog fecal samples. • Five hookworm species were identified. • First molecular detection of A. duodenale in environmental canine feces in Bangladesh. • A. ceylanicum and A. duodenale indicate zoonotic spillover risk. • High soil larval burdens revealed environmental transmission hotspots.