ABSTRACT Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae are increasingly reported worldwide, emphasizing the need for rapid diagnostic tools to limit their spread and prevent outbreaks. Although CTX-M enzymes remain predominant, less common ESBLs such as GES, PER, and VEB are emerging and gaining clinical relevance. We developed simplex and multiplex lateral flow immunoassays (LFIAs) for the rapid detection of these ESBLs. Monoclonal antibodies were produced and incorporated into dedicated LFIA strips. The assays were evaluated using 116 WGS-characterized gram-negative bacteria (GNB) isolates expressing diverse β-lactamases and grown on various media. For testing, three colonies were suspended in 150 µL of extraction buffer, applied to the strips, and results were visually interpreted after 15 min. Both simplex and multiplex LFIAs achieved 100% sensitivity and specificity for detecting GES-, PER-, and VEB-producing isolates, irrespective of bacterial species, β-lactamase content, or growth conditions. The panel included prevalent variants of PER (n = 5, including PER-2), VEB (n = 9), and GES (n = 9, including ESBLs and carbapenemases). Detection limits for recombinant PER-1, VEB-1, and GES-1 were 5, 50, and 100 pg/mL, respectively, and 105, 106, and 106 CFU/mL for corresponding Acinetobacter baumannii producers grown in tryptic soy broth. Overall, these simplex and multiplex GES-, PER-, and VEB-targeting LFIAs provide a rapid, accurate, and user-friendly approach suitable for routine use in clinical microbiology laboratories. When combined with the NG-Test CTX-M MULTI assay, which detects the five major CTX-M groups, they may further enhance the overall detection of the most common ESBLs, particularly among non-fermenting GNB. IMPORTANCE Although less prevalent globally than CTX-M-type ESBLs, GES-, PER-, and VEB-type β-lactamases represent an important and often underrecognized mechanism of resistance, particularly in non-fermenting gram-negative pathogens. These enzymes are frequently associated with multidrug-resistant organisms and may significantly compromise available therapeutic options. The detection of the genes encoding these enzymes is often based on homemade PCR. Rapid diagnostic tests based on lateral flow immunoassays (LFIAs) have been successfully used to detect CTX-M-like ESBLs and the five main carbapenemases. Here, we demonstrated that GES, PER, and VEB enzymes can be readily detected by simplex or multiplex LFIAs from Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii colonies. These assays are rapid (≤15 min), highly sensitive (105–106 CFU/mL), requiring no specialized equipment, thus offering a user-friendly alternative to molecular assays in clinical laboratories. These features align well with the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, and Deliverable). Although less prevalent globally than CTX-M-type ESBLs, GES-, PER-, and VEB-type β-lactamases represent an important and often underrecognized mechanism of resistance, particularly in non-fermenting gram-negative pathogens. These enzymes are frequently associated with multidrug-resistant organisms and may significantly compromise available therapeutic options. The detection of the genes encoding these enzymes is often based on homemade PCR. Rapid diagnostic tests based on lateral flow immunoassays (LFIAs) have been successfully used to detect CTX-M-like ESBLs and the five main carbapenemases. Here, we demonstrated that GES, PER, and VEB enzymes can be readily detected by simplex or multiplex LFIAs from Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii colonies. These assays are rapid (≤15 min), highly sensitive (105–106 CFU/mL), requiring no specialized equipment, thus offering a user-friendly alternative to molecular assays in clinical laboratories. These features align well with the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, and Deliverable).

Introduction With their remarkable flight capabilities, wild and captive birds play a pivotal role in the global dissemination of zoonotic pathogens including Chlamydia psittaci, Avian Influenza viruses (AIV), Chikungunya virus (CHIKV), Usutu virus (USUV), and West Nile virus (WNV). They function both as hosts and reservoirs responsible for transporting the mentioned infectious agents across vast geographic regions. Additionally, captive birds and birds inhabiting urban environments, particularly in tourist destinations, present significant public health concerns due to facilitated close interactions with humans. Methods A total of 358 samples originating from fifteen bird species were collected across 21 locations in Sarajevo Canton, over three consecutive years (2022–2024). Upon collection, the samples were subjected to molecular analysis to detect the presence of zoonotic pathogens. For detection of Chlamydia spp., and C. psittaci, real-time PCRs (qPCR) were used following established protocols. Additionally, reverse transcriptase real-time PCR (RT-qPCR) were utilized for the detection of emergent viral pathogens including avian influenza viruses, Chikungunya, Usutu, and West Nile virus. Results Chlamydia spp. was detected in 29.9% (95% CI: 25.2–34.9) of samples. Further, C. psittaci was identified in 10.3% (95% CI: 5.2–17.7) of positive samples originating from captive birds and birds inhabiting urban environments. One sample (0.3%) originating from a wild bird was positive to West Nile Virus. None of the samples tested positive for Avian Influenza viruses, Chikungunya and Usutu virus. Discussion The identification of C. psittaci and West Nile virus highlights the increased likelihood of zoonotic transmission. This underscores the imperative for bolstered biosecurity measures and public health strategies aimed at mitigating the risk associated with both environmental exposure and direct contact, especially in areas characterized by substantial tourist activity.

Bats are a natural host for a number of viruses, many of which are zoonotic and thus present a threat to human health. RNA viruses of the family Filoviridae, many of which cause disease in humans, have been associated with specific bat hosts. Lloviu virus is a Filovirus which has been connected to mass mortality events in Miniopterus schreibersii colonies in Spain and Hungary, and some studies have indicated its immense zoonotic potential. A die-off has been recorded among Miniopterus schreibersii in eastern Bosnia and Herzegovina for the first time, prompting the investigation to determine the causative agent. Bat carcasses were collected and subjected to pathological examination, after which the lung samples with notable histopathological changes, lung samples with no changes and guano were analyzed using metagenomic sequencing and RT-PCR. A partial Lloviu virus genome was sequenced from lung samples with histopathological changes and found to be closely related to Hungarian and Italian virus sequences. Further accumulation of mutations on the GP gene, coding the glycoprotein responsible for cell tropism and host preference, enhances the need for further characterization and monitoring of this virus to prevent spillover events and protect human health.