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A molecular toolbox for fast and convenient diagnosis of emerging and reemerging bacterial pathogens causing fever of
Elva Vega-García1,2, Génesis Palacios1, José A Pérez3,4
1Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias (IUESTPC), Universidad de La Laguna (ULL), Avenida Astrofísico Francisco Sánchez S/N, 38200, La Laguna, Tenerife, Spain.
Purpose:
Fever of intermediate duration (FID) is defined as a fever in the community without a specific origin or focus, with a duration between 7 and 28 days. FID is often caused by pathogens associated with animal contact or their arthropods parasites, such as ticks, fleas, or lice. The purpose of this work is to design a collection of molecular tools to promptly and accurately detect common bacterial pathogens causing FID, including bacteria belonging to genera Rickettsia, Bartonella, Anaplasma, and Ehrlichia, as well as Coxiella burnetii.
Methods:
Reference DNA sequences from a collection of Rickettsia, Bartonella, Anaplasma, and Ehrlichia species were used to design genus-specific primers and FRET probes targeted to conserved genomic regions. For C. burnetii, primers previously described were used, in combination with a newly designed specific probe. Real-time PCR assays were optimized using reference bacterial genomic DNA in a background of human genomic DNA.
Results:
The four real-time PCR assays can detect as few as ten copies of target DNA from those five genera of FDI-causing bacteria in a background of 300 ng of human genomic DNA, mimicking the low microbial load generally found in patient's blood.
Conclusion:
These assays constitute a fast and convenient "toolbox" that can be easily implemented in diagnostic laboratories to provide timely and accurate detection of bacterial pathogens that are typical etiological causes of febrile syndromes such as FID in humans.
Insights
This study developed molecular tools for rapid detection of bacterial pathogens causing fever of intermediate duration (FID). The real-time PCR assays accurately identify common causes like Rickettsia and Coxiella burnetii, aiding prompt diagnosis.
Area of Science:
- Molecular biology
- Infectious diseases
- Bacteriology
Background:
- Fever of intermediate duration (FID) is a prolonged fever without a clear source, often linked to zoonotic pathogens.
- Common causes include bacteria from Rickettsia, Bartonella, Anaplasma, Ehrlichia, and Coxiella burnetii, frequently transmitted by arthropod vectors.
Purpose of the Study:
- To design a molecular diagnostic toolkit for the rapid and accurate identification of key bacterial pathogens responsible for FID.
- Focus on developing genus-specific real-time PCR assays for common FID-causing bacteria.
Main Methods:
- Genus-specific primers and FRET probes were designed for Rickettsia, Bartonella, Anaplasma, and Ehrlichia, targeting conserved genomic regions.
- Established primers and a new probe were used for Coxiella burnetii.
- Real-time PCR assays were optimized using bacterial DNA in a human DNA background.
Main Results:
- Four real-time PCR assays were successfully developed and optimized.
- These assays can detect as few as ten copies of target DNA from the five bacterial genera.
- Detection is effective even within a background of 300 ng of human genomic DNA, simulating clinical samples.
Conclusions:
- The developed real-time PCR assays provide a fast and reliable method for detecting bacterial causes of FID.
- This molecular toolbox can be readily adopted by diagnostic laboratories for timely patient diagnosis.
- Enables prompt identification of zoonotic bacterial pathogens contributing to febrile illnesses in humans.
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