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Published on: February 4, 2018
Comparison of PCR-Based Diagnosis with Centrifuged-Based Enrichment Method for Detection of Borrelia persica in
S R Naddaf1, M Kishdehi, Mr Siavashi
1Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
Background:
The mainstay of diagnosis of relapsing fever (RF) is demonstration of the spirochetes in Giemsa-stained thick blood smears, but during non fever periods the bacteria are very scanty and rarely detected in blood smears by microscopy. This study is aimed to evaluate the sensitivity of different methods developed for detection of low-grade spirochetemia.
Methods:
Animal blood samples with low degrees of spirochetemia were tested with two PCRs and a nested PCR targeting flaB, GlpQ, and rrs genes. Also, a centrifuged-based enrichment method and Giemsa staining were performed on blood samples with various degrees of spirochetemia.
Results:
The flaB-PCR and nested rrs-PCR turned positive with various degrees of spirochetemia including the blood samples that turned negative with dark-field microscopy. The GlpQ-PCR was positive as far as at least one spirochete was seen in 5-10 microscopic fields. The sensitivity of GlpQ-PCR increased when DNA from Buffy Coat Layer (BCL) was used as template. The centrifuged-based enrichment method turned positive with as low concentration as 50 bacteria/ml blood, while Giemsa thick staining detected bacteria with concentrations ≥ 25000 bacteria/ml.
Conclusion:
Centrifuged-based enrichment method appeared as much as 500-fold more sensitive than thick smears, which makes it even superior to some PCR assays. Due to simplicity and minimal laboratory requirements, this method can be considered a valuable tool for diagnosis of RF in rural health centers.
Insights
A new centrifuged-based enrichment method significantly improves detection of low-grade spirochetemia, outperforming microscopy and some PCR assays for relapsing fever diagnosis.
Area of Science:
- Medical Microbiology
- Infectious Diseases
- Diagnostic Technologies
Background:
- Diagnosis of relapsing fever (RF) relies on detecting spirochetes in blood smears, which is challenging during non-fever periods due to low bacterial numbers.
- Microscopy often fails to detect scanty spirochetemia, necessitating more sensitive diagnostic approaches.
Purpose of the Study:
- To evaluate the sensitivity of various diagnostic methods for detecting low-grade spirochetemia in animal blood samples.
- To compare the efficacy of PCR assays, a centrifuged-based enrichment method, and Giemsa staining for relapsing fever diagnosis.
Main Methods:
- Polymerase Chain Reaction (PCR) assays targeting flaB, GlpQ, and rrs genes were performed.
- A centrifuged-based enrichment method and Giemsa thick blood smear staining were also conducted on samples with varying spirochetemia levels.
Main Results:
- PCR methods, particularly flaB-PCR and nested rrs-PCR, detected spirochetes even when dark-field microscopy was negative.
- The centrifuged-based enrichment method demonstrated high sensitivity, detecting as few as 50 bacteria/ml, a 500-fold improvement over Giemsa staining (≥ 25000 bacteria/ml).
Conclusions:
- The centrifuged-based enrichment method is highly sensitive and superior to traditional Giemsa staining for diagnosing relapsing fever.
- Its simplicity and minimal laboratory requirements make it a valuable tool for RF diagnosis in resource-limited settings like rural health centers.
