Rapid Differentiation of Filariae in Unstained and Stained Paraffin-Embedded Sections by a High-Resolution Melting Analysis PCR Assay.
BACKGROUND
Apart from infection with human filariae, zoonotic filariasis also occurs worldwide, and the numbers of cases have been increasing steadily. Diagnosis of intact filariae in tissues or organs depends on histological identification. The morphology of parasites in tissue-embedded sections is poor and shows high levels of homoplasy. Thus, the use of morphological characteristics in taxonomic studies is difficult and may not allow a specific diagnosis.


METHODS
Here we report the use of real-time PCR with high-resolution melting analysis (HRM) to detect and identify Brugia malayi, Brugia pahangi, Wuchereria bancrofti, and Dirofilaria immitis in paraffin-embedded sections. Assay specificity was determined using other tissue-dwelling parasites, Angiostrongylus cantonensis, Gnathostoma spinigerum, and Cysticercus cellulosae. We also developed a quick paraffin removal protocol.


RESULTS
Both human and animal filariae in formalin-fixed paraffin-embedded sections (FFPES) were diagnosed and identified rapidly, whereas other parasites were negative. There was no difference in the melting temperature of products amplified from filarial DNA obtained from unstained FFPES and Hematoxylin & Eosin-stained sections. Therefore, the DNA extraction protocols developed in this study could be used for real-time PCR with HRM.


CONCLUSIONS
We report the successful application of a HRM-PCR assay to differentiate four filarial parasites in FFPES, thus providing the pathologist with an effective alternative diagnostic procedure. Furthermore, the quick paraffin removal protocol developed could shorten the duration and number of steps required for paraffin removal using a standard protocol.
1 Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University , Bangkok, Thailand .
Sirichit
S
Wongkamchai
2 Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University , Bangkok, Thailand .
Benjaporn
B
Mayoon
3 Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University , Bangkok, Thailand .
Namthip
N
Kanakul
2 Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University , Bangkok, Thailand .
Suporn
S
Foongladda
1 Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University , Bangkok, Thailand .
Darawan
D
Wanachiwanawin
1 Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University , Bangkok, Thailand .
Hathai
H
Nochote
4 Filaria Project, Phikulthong Royal Development Study Center , Narathiwat, Thailand .
Sumart
S
Loymek
D000818:Animals; D017178:Brugia malayi / Q000235:genetics / Q000302:isolation & purification*; D017179:Brugia pahangi / Q000235:genetics / Q000302:isolation & purification*; D018509:DNA, Helminth / Q000302:isolation & purification; D004183:Dirofilaria immitis / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D005368:Filariasis / Q000469:parasitology* / Q000473:pathology; D006801:Humans; D016612:Paraffin Embedding; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014958:Wuchereria bancrofti / Q000235:genetics / Q000302:isolation & purification*; D015047:Zoonoses
D018509:DNA, Helminth
Brugia malayi; Brugia pahangi; Dirofilaria immitis; Formalin-fixed paraffin-embedded sections; High-resolution melting; Wuchereria bancrofti