Mem Inst Oswaldo Cruz, Rio de Janeiro, VOLUME 119 | 2024
Research Articles

Multiplex PCR assays developed for neglected pathogen detection in undifferentiated acute febrile illness cases in tropical regions

Leidi Carvajal Aristizabal1, Karl Ciuoderis1, Laura S Pérez-Restrepo1, Jorge E Osorio2,3, Juan P Hernández-Ortiz1,4,+

1Universidad Nacional de Colombia, Global Health Institute - One Health Colombia and One Health Genomic Laboratory, Medellín, Colombia
2University of Wisconsin, Global Health Institute, Madison, WI, USA
3University of Wisconsin, School of Veterinary Medicine, Department of Pathobiological Sciences, Madison, WI, USA
4Universidad Nacional de Colombia, Department of Materials and Nanotechnology, Medellín, Colombia

DOI: 10.1590/0074-02760240053
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ABSTRACT

BACKGROUND Undifferentiated acute febrile illness (UAFI) cause by several pathogens poses a diagnostic challenge due to the similarity on the clinical manifestations across these diseases. Precise pathogen detection is vital for appropriate medical intervention, early treatment, and timely outbreak alerts regarding emerging pathogens. In tropical regions, UAFI is predominantly linked to a wide range of viral, bacterial, and parasitic infections. Hence, confirmatory laboratory tests are essential for specific pathogen identification.
OBJECTIVES Our primary goal was to develop two real-time multiplex polymerase chain reaction (PCR) assays for simultaneous detection of six neglected pathogens (Leptospira spp., Rickettsia spp., Borrelia spp., Anaplasma spp., Brucella spp., and Bartonella spp.), known for causing UAFI in tropical regions.
METHODS We rigorously assessed assays parameters including: linearity, efficiency, sensitivity, and reproducibility in both singleplex and multiplex formats.
FINDINGS Our results demonstrated that these multiplex assays are reliable and sensitive methods. They showed good performance with low intra- and inter-variability (< 10%) and consistently high efficiencies (> 90%).
MAIN CONCLUSIONS These assays offer the alternative of streamlining work, reducing processing costs, and minimizing sample volume use. In conclusion, we present two dependable, user-friendly, rapid, and cost-effective methods for simultaneously detecting six neglected bacteria, as a significant laboratory tool in resource-limited tropical settings.

key words: PCR optimization multiplex neglected bacteria febrile illness
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Financial support: This work was supported by GHI One Health Colombia Center of the University of Wisconsin-Madison and the Universidad Nacional de Colombia (Medellín).
+ Corresponding author: jphernandezo@unal.edu.co
ORCID https://orcid.org/0000-0003-0404-9947
Received 29 February 2024
Accepted 13 September 2024

HOW TO CITE
Carvajal Aristizabal L, Ciuoderis K, Pérez-Restrepo LS, Osorio JE, Hernández-Ortiz JP. Multiplex PCR assays developed for neglected pathogen detection in undifferentiated acute febrile illness cases in tropical regions. Mem Inst Oswaldo Cruz. 2024; 119: e240053.

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