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Download All Background: The genus Acanthamoeba is primarily a free-living protozoan in natural habitats, but also causative agent of human and animal disease. Acanthamoeba serves as host for a variety of pathogenic bacteria such as Mycobacterium avium. Infestation with Acanthamoeba is associated with potentially sight-threatening contact lens-related keratitis, serious infections of other organs and fatal granulomatous amoebic encephalitis. More than 20 species of Acanthamoeba are known, which can be classified into three morphologic groups (Group I, II and III) and 22 genotypes (T1-T22). Some species ((A. castellanii (T4), A. lugdunensis (T4), A. polyphaga (T4), A. rhysodes (T4), A. quina (T4), A. palestinensis (T2), A. griffinii (T3), A. lenticulata (T5), A. astronyxis (T7), A. culbertsoni (T10), A. hatchetti (T11), A. healyii (T12), A. byersi (T18), A. divionensis) have been recently associated with human disease. Genotype T4 has been considered the most important genotype in both ocular and CNS infection. According to literature, T4 genotype is the most prevalent Acanthamoeba genotype causing keratitis worldwide (approx. 86%) (Diehl et al., 2021).
Description: ParoReal® Kit Acanthamoeba T4 is a non-automated IVD test, based on real-time PCR, for the qualitative detection of DNA (18S rRNA gene) of Acanthamoeba species of genotype T4 (A. castellani, A. lugdunensis, A. mauritaniensis, A. polyphaga, A. rhysodes, A. royreba). T4 genotype is the most prevalent (approx. 86%) Acanthamoeba genotype causing keratitis worldwide. The test does not detect other Acanthamoeba genotypes (T3, T15, T11, and T5) also causing keratitis. Proper specimens are DNA extracts from human clinical specimens associated with keratitis (ocular swabs, corneal biopsies, ocular punctates, corneal scrapings) as well as contact lenses and contact lens solution. This test is suitable for patients of all ages with suspected infection with Acanthamoeba genotype T4 (causative agent of Acanthamoeba keratitis, AK) and is intended as an aid in the diagnosis of infection with this pathogen in combination with patient history and additional clinical information. The test is for professional use only and the use is limited to qualified personnel instructed in real-time PCR and in vitro diagnostic procedures. Acanthamoeba species of genotype T4 cause approx. 86% of Acanthamoeba keratitis worldwide (Diehl et al., 2021). The 18S rRNA gene of Acanthamoeba is a multicopy gene. The number of rRNA repeats In Acanthamoeba cells is 24 per haploid genome. However, because Acanthamoeba is polyploid, each cell contains approximately 600 rRNA genes (Yang et al., 1994). A probe-specific amplification-curve in the FAM channel (530 nm) indicates the amplification of Acanthamoeba specific DNA. An internal DNA positive control (DNA IPC) is detected in Cy5 channel and is used as DNA extraction as well as real-time PCR inhibition control. The target for the DNA IPC (artificial target DNA) is extracted with the sample.
| Order Number | Reactions | Channel Pathogen | Channel IPC | Target |
|---|---|---|---|---|
| DHUP00153 | 50 | FAM | Cy5 | 18S rRNA |
- Amplification and detection: 18S rRNA gene of Acanthamoeba genotype T4
- Real-time PCR with rapid hot-start Taq DNA polymerase
- ROX™ dye as passive reference
- Internal Positive Control System to exclude false-negative results
- Optimized to handle PCR inhibitors
- PCR- platforms: runs on all established standard real-time PCR- platforms
- Harmonized thermal profiles to run RNA and DNA samples simultaneously
PCR platforms: This test has been validated with the ABI® 7500 instrument (Thermo Fisher Scientific, fast cycle parameters are not supported) and was also tested with a LightCycler® 480 II (Roche Diagnostics), QuantStudio™ 7 real-time PCR system (Thermo Fisher Scientific), Mic instrument (bio molecular systems), CFX Opus Real-time PCR System (Bio-Rad), CFX Dx (Bio-Rad) and T-COR 8 (Tetracore). The test is also compatible with other real-time PCR instruments which detect and differentiate fluorescence in FAM and Cy5 channel (e.g., QuantStudio™ 7 real-time PCR system (Thermo Fisher Scientific), qTOWER3G (Analytik Jena), cobas z 480 Analyzer (Roche)). When using PCR-platforms not tested by ingenetix, an evaluation of the multiplex-PCR shall be done. Keep in mind that some PCR-platforms first have to be calibrated with the corresponding dye before performing multiplex-PCR.