Diagnosis of canine Echinococcus multilocularis infections by copro-DNA tests: comparison of DNA extraction techniques and evaluation of diagnostic deworming

Abstract Epidemiological studies of Echinococcus multilocularis infections in definitive hosts require a reliable and economic diagnostic method. In this study, the current copro-DNA examination technique was modified by increasing the faecal amounts tested and adding a step to neutralize the faeces before DNA extraction. Reliability of the modified method was evaluated using rectal faecal samples from red foxes and comparing them with intestinal worms detected using the sedimentation and counting technique (SCT) following necropsy. The modified copro-DNA examination method demonstrated 93.9% sensitivity (138/147) on the SCT. Its detectability increased depending on the worm burden, and the sensitivity was 100% in cases harbouring over 1000 worms. From 111 SCT-negative cases, six (5.4%) were copro-DNA-positive, and all were confirmed as E. multilocularis via sequencing analysis. Five of the remaining 105 SCT-negative cases (4.8%) retained polymerase chain reaction (PCR) inhibitors in the extracted solution, suggesting that approximately 5% of the red fox faeces retained these inhibitors after treatment with the present copro-DNA extraction method. Although further evaluation is needed for faeces deposited in the wild, the present copro-DNA examination technique will help monitor the E. multilocularis prevalence in definitive hosts. When used for detailed evaluations of endemicity (e.g. changes in infection pressure or spread in non-endemic areas), the absence of PCR inhibitors should be confirmed, and multiple trials on faecal subsamples are recommended.

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Comparison of Four Rapid DNA Extraction Techniques for Conventional Polymerase Chain Reaction Testing of Three Mycobacterium spp. that Affect Birds

Avian Diseases ◽

10.1637/7070 ◽

2003 ◽

Vol 47 (4) ◽

pp. 1486-1490 ◽

Cited By ~ 14

Author(s):

Lisa A. Tell ◽

Janet Foley ◽

Martha L. Needham ◽

Richard L. Walker

Keyword(s):

Polymerase Chain Reaction ◽

Dna Extraction ◽

Conventional Polymerase Chain Reaction ◽

Extraction Techniques ◽

Chain Reaction ◽

Polymerase Chain Reaction Testing ◽

Polymerase Chain ◽

Mycobacterium Spp

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Comparison of Two DNA Extraction Methods and Two PCRs for Detection of Echinococcus multilocularis in the Stool Samples of Naturally Infected Red Foxes

Animals ◽

10.3390/ani10122381 ◽

2020 ◽

Vol 10 (12) ◽

pp. 2381

Author(s):

Katarzyna Skrzypek ◽

Jacek Karamon ◽

Małgorzata Samorek-Pieróg ◽

Joanna Dąbrowska ◽

Maciej Kochanowski ◽

...

Keyword(s):

Positive Result ◽

Multiplex Pcr ◽

Dna Extraction ◽

Echinococcus Multilocularis ◽

Nested Pcr ◽

Genetic Material ◽

Extraction Methods ◽

Red Foxes ◽

Stool Samples ◽

Positive Results

(1) Background: Due to the increasing distribution of Echinococcus multilocularis infections in final hosts, epidemiological investigations are important for recognizing the spreading pattern of this parasite and also to estimate risk infection for humans. (2) Methods: Investigations were conducted with two commercial kits dedicated for DNA extraction from feces: ZR Fecal DNA Mini Prep (Zymo Research, Freiburg, Germany) and QIAamp FAST DNA Stool Mini Kit (Qiagen, Hilden, Germany) (marked as Z and Q), together with two common PCR protocols (nested PCR and multiplex PCR). The goal was to compare their efficiency in detecting the genetic material of E. multilocularis in the samples of feces. Stool samples from red foxes were collected in a highly endemic area in Poland. Sedimentation and counting technique (SCT) was used as a reference method. (3) Results: From 48 samples, 35 were positive in SCT. Further investigations showed that 40.0% of samples (from those with SCT positive result) after Z-DNA extraction and 45.7% after Q-DNA extraction gave positive results in nested PCR. In multiplex PCR, positive results were obtained in 54.3% of samples after Z isolation and 48.6% of samples after Q. Additionally, one sample that resulted in being negative in SCT gave a positive result in PCR. The number of worms detected in the intestines had no influence on PCR results. (4) Conclusions: Both of the extraction methods showed similar efficiency in DNA isolation and dealing with inhibitors; however, they showed relatively low sensitivity. This was probably caused by degradation of genetic material in the field-collected samples.

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