Steev Loyola

Endemic and (re)emerging infectious diseases pose a major threat to public health and military readiness in Central and South America. The high biodiversity and the differing transmission pathways require the use of highly sensitive methods to track the emergence and spread of these diseases. High-throughput sequencing technologies have become a critical resource for the field of infectious disease monitoring, specifically for tracking pathogen evolution, antimicrobial resistance, and transmission. This review provides an overview of the implementation of genomics-informed infectious disease surveillance conducted by the U.S. Naval Medical Research Unit SOUTH, focusing on operational, infrastructural, and logistical challenges in resource-limited settings and outlining strategies for building sustainable regional capacity. Selected case studies focused on prominent biothreats including dengue, Oropouche, malaria, and antimicrobial resistance demonstrate the significance and applicability of targeted and agnostic sequencing approaches to elucidate transmission pathways, pathogen discovery, and guide public health responses and field-deployable applications.

Oropouche virus (OROV) spread across the Americas in 2024, yet Panama’s Darién migration corridor saw no outbreak until nearly a year after Brazil’s January 2024 peak, raising two hypotheses: cryptic circulation masked by diagnostic gaps, or recent introduction under permissive climatic conditions. Here we resolve this paradox using integrated clinical, genomic, and climate-informed surveillance. Among 1,040 individuals tested, 43% were OROV-positive and showed a clinical signature distinct from co-circulating arboviruses, including headache more frequent than in dengue (RR 2.38, 95% CI 1.74-3.24). The household secondary attack rate was 56%, and waste burning independently predicted infection. Phylogeographic reconstruction identified a single recent introduction in October 2024 with no evidence of adaptive evolution, excluding prolonged cryptic persistence. Climate-informed models indicate broad outbreak susceptibility across Panama, with Bocas del Toro and Los Santos as the next highest-risk provinces. These findings identify a Central American foothold for OROV with potential for further northward spread.

Oropouche virus (OROV) spread across the Americas in 2024, yet Panama’s Darién migration corridor saw no outbreak until nearly a year after Brazil’s January 2024 peak, raising two hypotheses: cryptic circulation masked by diagnostic gaps, or recent introduction under permissive climatic conditions. Here we resolve this paradox using integrated clinical, genomic, and climate-informed surveillance. Among 1,040 individuals tested, 43% were OROV-positive and showed a clinical signature distinct from co-circulating arboviruses, including headache more frequent than in dengue (RR 2.38, 95% CI 1.74-3.24). The household secondary attack rate was 56%, and waste burning independently predicted infection. Phylogeographic reconstruction identified a single recent introduction in October 2024 with no evidence of adaptive evolution, excluding prolonged cryptic persistence. Climate-informed models indicate broad outbreak susceptibility across Panama, with Bocas del Toro and Los Santos as the next highest-risk provinces. These findings identify a Central American foothold for OROV with potential for further northward spread.

The COVID-19 pandemic exposed major disparities in global diagnostic capacity, particularly in low- and middle-income countries (LMICs) where reliance on conventional assays and external technologies hindered rapid response. Aptamers offer a promising alternative; however, practical and experimentally validated frameworks that connect aptamer discovery to assay development and evaluation in LMIC settings remain limited. Here, we present an integrated and stepwise framework and its feasibility for developing and validating aptamer-based testing tools using SARS-CoV-2 as a model. Native viral particles served as targets in a nine-round systematic evolution of ligands by exponential enrichment (SELEX) protocol optimized for low- to medium-complexity laboratories. High-throughput sequencing and bioinformatic analyses identified candidate sequences, which were then experimentally assessed under defined experimental conditions. The top-performing aptamer, APT-35b, was incorporated into a DNA aptamer-based qPCR assay (Apta-qPCR). When evaluated using 475 clinical samples, the Apta-qPCR achieved 80.8% sensitivity and 95.3% specificity for Omicron BA.1/BA.1.1, with no cross-reactivity to other human coronaviruses. However, performance decreased for more evolved Omicron subvariants (n = 68). This framework offers a practical and adaptable roadmap that links aptamer discovery with functional assay development and can be applied to diverse pathogens, ultimately supporting enhanced diagnostic preparedness and response capacity.

Introduction: Antimicrobial resistance is a major and growing concern worldwide. The study aimed to describe the occurrence and characterize the antimicrobial resistance profile of carbapenemase-producing Enterobacterales (CPE) isolated from clinical specimens at a private healthcare center in Lima, Peru, between January 2018 and December 2019. Methodology: A total of 59 bacterial isolates were identified as CPE using the modified carbapenem inactivation method. Bacterial identification and antimicrobial susceptibility testing were performed using the Vitek 2 system, and colistin susceptibility was determined using the compact antimicrobial susceptibility panel. Five carbapenemase encoding genes, including blaNDM, blaIMP, blaVIM, blaOXA-48, and blaKPC, were screened using PCR and lateral flow assay. Also, carbapenemases were screened using the boronic acid- and EDTA-based disk synergy methods. The dynamics of CPE were described every month. Results: Carbapenemase-producing Klebsiella pneumoniae was the most prevalent agent (83.1%), followed by Providencia spp. (6.8%), Escherichia coli (5.1%), Enterobacter cloacae (3.4%), and Citrobacter freundii (1.6%). The isolation of blaNDM-carrying CPE was frequent and increased during the second half of 2019, while blaKPC carriers were detected in the second half of 2018. One CPE carrying blaOXA-48 was detected in June 2019. No other carbapenemase-encoding genes were detected. Conclusions: This report is among the first to document CPE in private health care institutions in Lima, Peru. Results suggest that the emergence and spread of carbapenemases are becoming a latent and growing threat in private healthcare settings.

: The BioFire® Global Fever Panel (GFP; BioFire Diagnostics, Salt Lake City, UT) is an assay designed for detecting multiple pathogens, including dengue virus (DENV; Orthoflavivirus denguei ). Although its performance has been primarily assessed using composite reference standards (CRS) or through direct comparisons with other tests, its real-world accuracy remains uncertain. The performance of the GFP in combination with real-time reverse transcriptase polymerase chain reaction (qRT-PCR) testing is evaluated in the present study using dengue surveillance data from 224 individuals with acute fever (≤5 days) in a dengue-endemic area of the Peruvian Amazon. Dengue virus RNA was detected in serum and whole blood using qRT-PCR testing and the GFP, respectively. Two reference standards were developed: one based on CRS and another based on latent class analysis. The best latent class model (LCM) included a viral load surrogate. Using qRT-PCR testing or the LCM as a reference, the GFP exhibited a sensitivity of 94.5% (95% CI: 88.4–98.0%) and a specificity of 75.7% (95% CI: 66.8–83.2%). With CRS, the GFP displayed a sensitivity of 95.6% (95% CI: 90.7–98.4%) and 100% specificity. The simultaneous and sequential two-test algorithms for negative yielded comparable performance and exhibited high sensitivity compared with other two-test sequential algorithms. The sequential testing algorithm, which used qRT-PCR testing followed by the GFP for negative results, offered the best balance in cost, time, and performance (sensitivity: 99.3–100%; specificity: 75.7–100%). Although the GFP is more expensive than qRT-PCR testing, it offers a shorter turnaround time per sample. Overall, the findings suggest that the GFP is a reliable and high-performing tool for DENV testing, but its cost may restrict its use in resource-limited settings.

Background: Bloodstream infections (BSIs) caused by carbapenemase-producing Enterobacterales (CPE) are associated with increased mortality rates. However, limited data exist in Latin America, particularly in regions where blaNDM is predominant. Methods: We conducted a retrospective cohort study of adult inpatients with Escherichia coli or Klebsiella pneumoniae BSI at a Peruvian referral hospital between 2020 and 2023. Patients were classified as CPE or non-CPE based on molecular testing. The outcome was 30-day all-cause mortality. Multivariable Cox regression was used to identify independent predictors of mortality, adjusting for confounders selected via a directed acyclic graph. Results: Among 506 patients, 97 (19.2%) had CPE BSIs, predominantly blaNDM (63.9%) and blaKPC (36.1%). Overall, the 30-day mortality rate was 27.3%, significantly higher in the CPE group (52.6% vs 21.3%; P < .001). In multivariable analysis, CPE infection remained independently associated with increased mortality (adjusted hazard ratio [aHR] 1.88; 95% CI 1.19–2.96). Other predictors included age ≥60 years (aHR 1.53), septic shock (aHR 2.93), pneumonia (aHR 1.70), and immunosuppression (aHR 1.72). Among CPE subtypes, Klebsiella pneumoniae Carbapenemase (KPC)-producers conferred the highest mortality risk (aHR 2.64). Concordant empirical antibiotic therapy was not significantly protective after adjustment. Conclusions: CPE BSIs were independently associated with increased 30-day mortality, with KPC-producing K pneumoniae posing the greatest risk. Despite the predominance of New Delhi Metallo-β-Lactamase (NDM) in our setting, these findings emphasize the clinical severity associated with different carbapenemase types, and the need to tailor interventions accordingly. Strengthening molecular surveillance and ensuring timely access to effective therapies remain critical priorities in NDM-endemic regions, such as Peru.

Rickettsioses, often underreported, pose public health challenges. Rickettsia asembonensis is a potential emerging pathogen that was previously detected in humans, animals, and a variety of arthropods. While its pathogenicity in humans remains unclear, it poses a potential public health threat. Here, we present an extended epidemiological, diagnostic, and genetic analysis of the information provided in a preliminary report on the investigation of rickettsiae in Peru. In particular, we report the detection of R. asembonensis in blood specimens collected from four human patients with an acute undifferentiated fever of a seven- to nine-day duration, all of whom tested negative for other vector-borne pathogens. Additionally, we describe the replicative capacity of the R. asembonensis isolates in cell cultures.