Development of Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Azumiobodo hoyamushi (Kinetoplastea)

Ascidian soft tunic syndrome (AsSTS) caused by Azumiobodo hoyamushi (A. hoyamushi) is a serious aquaculture problem that results in mass mortality of ascidians. Accordingly, the early and accurate detection of A. hoyamushi would contribute substantially to disease management and prevention of transmission. Recently, the loop-mediated isothermal amplification (LAMP) method was adopted for clinical diagnosis of a range of infectious diseases. Here, the authors describe a rapid and efficient LAMP-based method targeting the 18S rDNA gene for detection of A. hoyamushi using ascidian DNA for the diagnosis of AsSTS. A. hoyamushi LAMP assay amplified the DNA of 0.01 parasites per reaction and detected A. hoyamushi in 10 ng of ascidian DNA. To validate A. hoyamushi 18S rDNA LAMP assays, AsSTS-suspected and non-diseased ascidians were examined by microscopy, PCR, and by using the LAMP assay. When PCR was used as a gold standard, the LAMP assay showed good agreement in terms of sensitivity, positive predictive value (PPV), and negative predictive value (NPV). In the present study, a LAMP assay based on directly heat-treated samples was found to be as efficient as DNA extraction using a commercial kit for detecting A. hoyamushi. Taken together, this study shows the devised A. hoyamushi LAMP assay could be used to diagnose AsSTS in a straightforward, sensitive, and specific manner, that it could be used for forecasting, surveillance, and quarantine of AsSTS.

Biceps Femoris Tendon and Lateral Collateral Ligament: Analysis of Insertion Pattern Using MRI

PURPOSE: The biceps femoris tendon (BFT) and lateral collateral ligament (LCL) in the knee were formerly known to form a conjoined tendon at the fibular attachment site. However, the BFT and LCL are attached into the fibular head in various patterns. We classified insertion patterns of the BFT and LCL using MR imaging, and analyzed whether the LCL attaches to the fibular head or not. MATERIALS AND METHODS: A total of 494 consecutive knee MRIs of 470 patients taken between July 2012 and December 2012 were retrospectively reviewed. There were 224 males and 246 females, and patient age varied from 10 to 88 (mean, 48.6). The exclusion criteria were previous surgery and poor image quality. Using 3T fat-suppressed proton density-weighted axial images, the fibular insertion patterns of the BFT and LCL were classified into following types: type I (the LCL passes between the anterior arm and direct arm of the BFT's long head), type II (the LCL joins with anterior arm of the long head of the BFT), type III (the BFT and LCL join to form a conjoined tendon), type IV (the LCL passes laterally around the anterior margin of the BFT), and type V (the LCL passes posteriorly to the direct arm of the BFT's long head). RESULTS: Among the 494 cases of the knee MRI, there were 433 (87.65%) type I cases, 21 (4.25%) type II cases, 2 (0.4%) type III cases, 16 (3.23%) type IV cases, and 22 (4.45%) type V cases. There were 26 cases (5.26%) in which the LCL and BFT were not attached into the fibular head. CONCLUSION: The fibular attachment pattern of the BFT and LCL shows diverse types in MR imaging. The LCL does not adhere to the head in some patients.