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Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse
Heekyoung Kang1, Gi-Sang Seong1, Hae-Jin Sohn1
1Department of Microbiology, Ajou University School of Medicine, Suwon 443-721, Republic of Korea; Department of Biomedical Science, Graduate School of Ajou University, Suwon 443-721, Republic of Korea.
Abstract:
Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6.
Insights
Primary Amoebic Meningoencephalitis (PAM), a neglected tropical disease, is increasing. A new PCR method rapidly detects Naegleria fowleri in clinical samples, offering a crucial diagnostic tool for this rare but fatal infection.
Area of Science:
- Medical Parasitology
- Molecular Diagnostics
- Neglected Tropical Diseases
Background:
- Primary Amoebic Meningoencephalitis (PAM) caused by Naegleria fowleri is a severe, often fatal, neglected tropical disease (NTD).
- Rapid and accurate diagnostic tools are crucial for timely intervention and management of PAM cases.
Purpose of the Study:
- To develop and evaluate a rapid and effective PCR-based diagnostic technique for Naegleria fowleri detection.
- To optimize DNA extraction methods for efficient amplification from clinical samples.
Main Methods:
- Development of a PCR assay using four primer pairs (Nfa1, Nae3, Nf-ITS, Naegl).
- Optimization of genomic DNA extraction from Naegleria fowleri trophozoites using a simple boiling method.
- Testing the PCR assay on cultured trophozoites and experimentally infected mouse models (brain tissue and CSF).
Main Results:
- A simple boiling method proved efficient for DNA extraction, enabling detection of as few as 2.5x10^2 trophozoites with the Nfa-1 primer.
- Primers Nfa1 and Nae3 specifically amplified Naegleria fowleri DNA.
- Naegleria fowleri DNA was detectable in experimentally infected mouse brain tissue 4 days post-infection using the Nfa1 primer.
- The Nae3 primer demonstrated superior performance in detecting Naegleria fowleri DNA in mouse CSF 5 days post-infection compared to Nfa1 at day 6.
Conclusions:
- A rapid and efficient PCR-based diagnostic method for Naegleria fowleri has been established.
- Optimized DNA extraction and specific primer pairs (Nfa1, Nae3) enhance diagnostic sensitivity.
- This technique holds significant potential for the early diagnosis of Primary Amoebic Meningoencephalitis.
