A novel repeat sequence-based PCR (rep-PCR) using specific repeat sequences of Mycobacterium intracellulare as a DNA fingerprinting.

Repetitive sequence-based PCR (rep-PCR) is a potential epidemiological technique that can provide high-throughput genotype fingerprints of heterogeneous Mycobacterium strains rapidly. Previously published rep-PCR primers, which are based on nucleotide sequences of Gram-negative bacteria may have low specificity for mycobacteria. Moreover, it was difficult to ensure the continuity of the study after the commercial rep-PCR kit was discontinued. Here, we designed a novel rep-PCR for Mycobacterium intracellulare, a major cause of nontuberculous mycobacterial pulmonary disease with frequent recurrence. We screened the 7,645 repeat sequences for 200 fragments from the genome of M. intracellulare ATCC 13950 in silico, finally generating five primers with more than 90% identity for a total of 226 loci in the genome. The five primers could make different band patterns depending on the genome of three different M. intracellulare strains using an in silico test. The novel rep-PCR with the five primers was conducted using 34 bacterial samples of 7 species containing 25 M. intracellulare clinical isolates, compared with previous published rep-PCRs. This shows distinguished patterns depending on species and blotting assay for 6 species implied the sequence specificity of the five primers. The Designed rep-PCR had a 95-98% of similarity value in the reproducibility test and showed 7 groups of fingerprints in M. intracellulare strains. Designed rep-PCR had a correlation value of 0.814 with VNTR, reference epidemiological method. This study provides a promising genotype fingerprinting method for tracing the recurrence of heterogeneous M. intracellulare.

Fetuin as a potential serum biomarker to detect subclinical shedder of bovine paratuberculosis.

Paratuberculosis (PTB) is a chronic contagious granulomatous enteritis of wild and domestic ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). PTB causes considerable economic losses to the dairy industry through decreased milk production and premature culling. PTB-affected cattle undergo a subclinical stage without clinical signs and initiate fecal shedding of MAP into the environment. Current diagnostic tools have low sensitivity for the detection of subclinical PTB infection. Therefore, alternative diagnostic tools are required to improve the diagnostic sensitivity of subclinical PTB infection. In this study, we performed ELISA for three previously identified host biomarkers (fetuin, alpha-1-acid glycoprotein, and apolipoprotein) and analyzed their diagnostic performance with conventional PTB diagnostic methods. We observed that serum fetuin levels were significantly lowered in the subclinical shedder and clinical shedder groups than in the healthy control group, indicating its potential utility as a diagnostic biomarker for bovine PTB. Also, fetuin showed an excellent discriminatory power with an AUC = 0.949, a sensitivity of 92.6%, and a specificity of 94.4% for the detection of subclinical MAP infection. In conclusion, our results demonstrated that fetuin could be used as a diagnostic biomarker for enhancing the diagnostic sensitivity for the detection of subclinical MAP infections that are difficult to detect based on current diagnostic methods.

Prediction of laryngeal aspiration using voice analysis.

OBJECTIVE: To determine the diagnostic value of voice analysis to screen for patients with high risk of clinically significant aspiration. DESIGN: A total of 93 patients referred for a videofluoroscopic swallowing study were included in the study. Voice analyses were performed before and after videofluoroscopic swallowing study, and five acoustic variables were measured, including average fundamental frequency, relative average perturbation, shimmer percentage, noise-to-harmonic ratio, and voice turbulence index. The patients were divided into two groups based on the results of the videofluoroscopic swallowing study: a high-risk group with patients who had the ingested materials on or below the vocal cords and a low-risk group with patients who did not have the ingested materials on or below the vocal cords. The changes of each acoustic variable before and after the videofluoroscopic swallowing study were compared between the two groups. RESULTS: Relative average perturbation, shimmer percentage, noise-to-harmonic ratio, and voice turbulence index were significantly increased after videofluoroscopic swallowing study in the high-risk group as compared with the low-risk group (P < 0.05). The change of average fundamental frequency, however, was not significantly different between the two groups (P > 0.05). According to the receiver operating characteristics curve, the sensitivity of these acoustic variables in detecting aspiration or penetration ranged from 68.9% to 91.1% and specificity ranged from 68.8% to 97.9%. Relative average perturbation was the most accurate variable, with a sensitivity of 91.1% and a specificity of 97.9% in predicting aspiration or penetration. The combination of relative average perturbation and noise-to-harmonic ratio increased the sensitivity to 100% but reduced the specificity to 77.1%. CONCLUSION: Voice analysis is a safe, noninvasive, and reliable screening tool for patients with dysphagia and can detect patients at high risk of clinically significant aspiration, thereby augmenting clinical bedside examination.