PCR-reverse Blot Hybridization Assay Research Articles

Clinical characteristics and the diagnostic role of molecular tests in musculoskeletal infections caused by nontuberculous Mycobacterium: a single-center experience.

We retrospectively investigated NTM musculoskeletal infections to understand the clinical characteristics as well as the diagnostic performance of molecular tests as a useful supplementary test. A retrospective cohort study was conducted from January 2016 to December 2019. Intraoperative specimens for culture and pathological analysis were obtained during surgery under sterile conditions. AdvanSure TB/NTM real-time PCR (LG Life Sciences, Seoul, Korea) and PCR-reverse blot hybridization assay (REBA, REBA Myco-ID; YD Diagnostics, Yongin, Republic of Korea) was performed directly from tissue specimens for the rapid and accurate identification of 20 mycobacterial species. A series of 24 patients was identified during the study period. Agricultural/aquatic exposure were predisposing factors in 9 patients, trauma in 4 patients, but no predisposing host factor was identified for 11 patients. Twenty-two patients presented with tenosynovitis and two with arthritis with substantial delays between initial presentation of symptoms and microbiologic diagnosis, with a median of 147days (range 5-396days). Pathologic tissue examination of musculoskeletal NTM infections was positive for chronic granulomatous inflammation in 18 cases. Fifteen were positive in the culture study, and nine were positive only in the molecular study using tissue specimens. All 6M. ulcerans/M. marinum isolates were identified only by PCR. This study is an important reminder that NTM infections should be a part of the differential diagnosis in patients with chronic musculoskeletal infections refractory to regular treatment. Although molecular test cannot replace the conventional smear and culture methods, it can be used as a useful supplementary test especially in diagnosing M. marinum infection.

PCR-reverse blot hybridization assay in respiratory specimens for rapid detection and differentiation of mycobacteria in HIV-negative population

BackgroundRapid identification of pathogenic Mycobacterium species is critical for a successful treatment. However, traditional method is time-consuming and cannot discriminate isolated non-tuberculosis mycobacteria (NTM) at species level. In the retrospective study, we evaluated the clinical applicability of PCR-reverse blot hybridization assay (PCR-REBA Myco-ID) with clinical specimens for rapid detection and differentiation of mycobacterial species.MethodsA total of 334 sputum and 362 bronchial alveolar lavage fluids (BALF) from 696 patients with mycobacterium pulmonary disease (MPD) and 210 patients with non-mycobacterium pulmonary disease used as controls were analyzed. Sputum or BALF were obtained for MGIT 960-TBc ID test and PCR-REBA Myco-ID assay. High resolution melt analysis (HRM) was used to resolve inconsistent results of MGIT 960-TBc ID test and PCR-REBA Myco-ID assay.ResultsA total of 334 sputum and 362 BALF specimens from 696 MPD patients (292 MTB and 404 NTM) were eventually analyzed. In total, 292 MTBC and 436 NTM isolates (mixed infection of two species in 32 specimens) across 10 Mycobacterium species were identified. The most frequently isolated NTM species were M. intracellulare (n = 236, 54.1%), followed by M. abscessus (n = 106, 24.3%), M. kansasii (n = 46, 10.6%), M. avium (n = 36, 8.3%). Twenty-two cases had M. intracellulare and M. abscessus mixed infection and ten cases had M. avium and M. abscessus mixed infection. A high level of agreement (n = 696; 94.5%) was found between MGIT 960-TBc ID and PCR-REBA Myco-ID (k = 0.845, P = 0.000). PCR-REBA Myco-ID assay had higher AUC for both MTBC and NTM than MGIT 960-TBc ID test.ConclusionPCR-REBA Myco-ID has the advantages of rapid, comparatively easy to perform, relatively low cost and superior accuracy in mycobacterial species identification compared with MGIT 960-TBc ID. We recommend it into workflow of mycobacterial laboratories especially in source-limited countries.

Comparison of Multiplex Real-Time PCR and PCR-Reverse Blot Hybridization Assays for the Direct and Rapid Detection of Porcine Circovirus Type 2 Genotypes

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), poses a serious economic threat for the swine industry. Currently, PCV2 is classified into five major genotypes: PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e. The aim of this study is to evaluate the performance of two commercially available methods, multiplex real-time PCR assay and PCR-reverse blot hybridization assay (REBA), for the rapid detection of PCV2 and direct identification of PCV2 genotypes from clinical samples as well as to compare the results with that of sequence analysis. Molecular diagnostic methods were used to evaluate a total of 180 samples, including tissues and blood samples from pigs that were suspected of PCVAD infection. The results of this study showed that the detection rate for positive PCV2 was 48.3% (n = 87) in both multiplex real-time PCR and PCR-REBA methods. Using sequence analysis, which is the gold standard, and multiplex real time PCR assay, the sensitivity, specificity, positive predictive value, and negative predictive value of PCV2 genotyping were found to be 97.1% (n = 67, 95% CI 0.894–0.998, p < 0.001), 100% (n = 93, 95% CI 0.966–1.000, p < 0.001), 100% (95% CI 0.953–1.000, p < 0.001), 97.9% (95% CI 0.921–0.998, p < 0.001), respectively. The results of PCR-REBA were found to be consistent with those of sequence analysis for all the samples and showed good agreement (κ = 1). The most prevalent genotypes detected in this study were PCV2d (n = 53, 60.9%), followed by PCV2a (n = 17, 19.5%), PCV2b (n = 14, 16.1%), and PCV2a/b co-infection (n = 3, 3.5%). Both the methods required ~3 h for completion. Therefore, we conclude that two molecular methods are rapid and reliable for the characterization of the causative pathogen with PCV2 genotypes.

Prediction of Y haplogroup by polymerase chain reaction-reverse blot hybridization assay.

The analysis of Y-SNPs from crime scene samples is helpful for investigators in narrowing down suspects by predicting biogeographical ancestry. In this study, a PCR-reverse blot hybridization assay (REBA) for predicting Y-chromosome haplogroups was employed to determine the major haplogroups worldwide, including AB, DE, C, C3, F, K, NO, O, O2, and O3 and evaluated. The REBA detects nine biallelic Y chromosome markers (M9, M89, M122, M145, M175, M214, M217, P31, and RPS4Y711) simultaneously using multiple probes. The REBA for Y-single nucleotide polymorphisms (SNP) genotyping was performed using 40 DNA samples from Asians-14 Koreans, 10 Indonesians, six Chineses, six Thais, and four Mongolians. 40 Asian samples were identified as haplogroup O2 (40%), O3 (32.5%), C3 (17.5%), O (7.5%) and K (2.5%). These cases were confirmed by DNA sequence analysis (κ = 1.00; P < 0.001). PCR-REBA is a rapid and reliable method that complements other SNP detection methods. Therefore, implementing REBA for Y-SNP testing may be a useful tool in predicting Y-chromosome haplogroups.

Development of a Rapid Reverse Blot Hybridization Assay for Detection of Clinically Relevant Antibiotic Resistance Genes in Blood Cultures Testing Positive for Gram-Negative Bacteria.

Rapid and accurate identification of the causative pathogens of bloodstream infections is crucial for the prompt initiation of appropriate antimicrobial therapy to decrease the related morbidity and mortality rates. The aim of this study was to evaluate the performance of a newly developed PCR-reverse blot hybridization assay (REBA) for the rapid detection of Gram-negative bacteria (GNB) and their extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, and carbapenemase resistance genes directly from the blood culture bottles. The REBA-EAC (ESBL, AmpC β-lactamase, carbapenemase) assay was performed on 327 isolates that were confirmed to have an ESBL producer phenotype, 200 positive blood culture (PBCs) specimens, and 200 negative blood culture specimens. The concordance rate between the results of REBA-EAC assay and ESBL phenotypic test was 94.2%. The sensitivity, specificity, positive predictive value, and negative predictive value of the REBA-EAC assay for GNB identification in blood culture specimens were 100% (95% CI 0.938–1.000, P < 0.001), 100% (95% CI 0.986–1.000, P < 0.001), 100% (95% CI 0.938–1.000, P < 0.001), and 100% (95% CI 0.986–1.000, P < 0.001), respectively. All 17 EAC-producing GNB isolates from the 73 PBCs were detected by the REBA-EAC assay. The REBA-EAC assay allowed easy differentiation between EAC and non-EAC genes in all isolates. Moreover, the REBA-EAC assay was a rapid and reliable method for identifying GNB and their β-lactamase resistance genes in PBCs. Thus, this assay may provide essential information for accelerating therapeutic decisions to achieve earlier appropriate antibiotic treatment during the acute phase of bloodstream infection.

Evaluation of the Punch-it™ NA-Sample kit for detecting microbial DNA in blood culture bottles using PCR-reverse blot hybridization assay

DNA extraction efficiency affects the success of PCR-based method applications. The Punch-it™ NA-Sample kit for extracting DNA by using paper chromatography is technically easy to use and requires just two reagents and only 10min to complete. The Punch-it™ NA-Sample kit could be offered as a rapid, accurate, and convenient method for extracting bacterial and fungal DNA from blood culture bottles. We compared the efficiencies of the commercial kit (Punch-it™ NA-Sample kit) and an in-house conventional boiling method with Chelex-100 resin for DNA extraction from blood culture bottles. The efficiency of the two DNA extraction methods was assessed by PCR-reverse blot hybridization assay (PCR-REBA, REBA Sepsis-ID) for detecting Gram positive (GP) bacteria, Gram negative (GN) bacteria, and Candida species with 196 positive and 200 negative blood culture bottles. The detection limits of the two DNA extraction methods were 103CFU/mL for GP bacteria, 103CFU/mL for GN bacteria, and 104CFU/mL for Candida. The sensitivity and specificity of the Punch-it™ NA-Sample kit by REBA Sepsis-ID were 95.4% (187/196) and 100% (200/200), respectively. The overall agreement of the two DNA extraction methods was 98.9% (392/396). Three of four samples showing discrepant results between the two extraction methods were more accurately matched up with the Punch-it™ NA-Sample kit based on conventional culture methods. The results indicated that the Punch-it™ NA-Sample kit extracted bacterial and fungal DNA in blood culture bottles and allowed extracted DNA to be used in molecular assay.

PCR-reverse blot hybridization assay for fast and accurate identification of causative species in superficial fungal infections.

Superficial fungal infections are a very common problem in dermatological clinics. The diagnostic method of fungal culture is time-consuming and has inconsistent sensitivity. Therefore, a practical method for rapid and accurate identification of the species causing superficial fungal infections is needed. To compare PCR-reverse blot hybridization assay (PCR-REBA) with conventional fungal diagnostic methods so as to determine the reliability of PCR-REBA for the diagnosis and species identification in superficial fungal infections. Potassium hydroxide (KOH) preparation, fungal culture, conventional real-time PCR and PCR-REBA were used to assess 83 specimens, and the results from each method were compared. Of the 83 specimens, 44 specimens that were positive by fungal culture had 62.7% agreement with PCR-REBA. Compared with real-time PCR, there was 68.7% agreement with fungal culture, but 91.6% agreement with PCR-REBA. When the comparison was made using the 55 specimens that gave positive results in both KOH preparation and fungal culture, there was 85.5% agreement with real-time PCR for fungal culture, but 94.5% agreement with PCR-REBA. Compared with KOH preparation or fungal culture, PCR-REBA has higher sensitivity and specificity. Therefore, PCR-REBA could be a useful method in clinical settings because it can identify species quickly and accurately, and can also determine the existence of pathogens.

Mycobacterium marinum Infection on the Face Diagnosed by Polymerase Chain Reaction Amplification and Direct Sequencing.

Dear Editor: A 56 years old Korean woman presented with asymptomatic multiple rice-sized erythematous papules on left cheek (Fig. 1A). The scaly papules were noted 2 months ago. She had no history of trauma or exposure to fish. She had been learning to swim for 3 months. Culture examination of the smear for bacteria and fungus revealed no growth. The nested polymerase chain reaction (PCR) for Mycobacterium tuberculosis showed negative results. A biopsy specimen taken from the lesion showed granulomatous inflammation with granulation tissue in the deep dermis. The granulomatous infiltration was composed of multiple histiocytes, mononuclear cells, and giant cells (Fig. 2A). Acid-fast bacilli (AFB) staining produced negative findings (Fig. 2B). Hence, the diagnosis was as nonspecific inflammation of the face, and the patient was treated with triamcinolone injection, three times for 6 weeks. After the injection, the lesions improved overall; however, a solitary lesion showed pus formation. Thus, in addition, mycobacterial culture including the pus was performed, which showed growth of yellow pigment-producing Mycobacterium at 32℃ to 33℃ in mycobacterium growth indicator tube (Fig. 2C). Zeihl-Neelsen staining of culture materials showed innumerable AFB (Fig. 2D), and the or ganism was identified as Mycobacterium. Hence, PCR-reverse blot hybridization assay was performed to identify atypical mycobacterial species, which were confirmed as M. marinum or M. ulcerans. Fig. 1 (A) Multiple, erythematous, various sized, scaly papules on the left cheek. (B) After 4 months, the lesions improved with postinflammatory hyperpigmentation and atrophic scar. Fig. 2 (A) Granulomatous infiltration in the deep dermis was composed of multiple histiocytes, mononuclear cells, and giant cells (H&E, ×200). (B) Negative findings (acid-fast bacillus, ×100). (C) Growth of yellow pigment producing mycobacterium ... Furthermore, PCR amplification and direct sequencing of 16SrRNA, tuf, rpoB, hsp65 genes were performed for the rapid and accurate identification of the organism. In 16SrRNA and tuf gene analysis, both M. marium and M. ulcerans showed >99% homology; thus, it was not possible to differentiate between the two species. Finally, in the rpoB and hsp65 gene analysis, M. marium showed homology of 100% and 86%, respectively, whereas M. ulcerans showed homology of 99% and 85%, respectively. On the basis of this result, the organism was identified as M. marium and the patient was treated with 200 mg minocycline and 500 mg clarithromycin for 2 months. After the treatment, the lesions improved, showing postinflammatory hyperpigmentation (Fig. 1B). M. marium is a nontuberculous photochromogenic mycobacterium1. The optimal temperature for its growth is 30℃ to 32℃, and it rarely grows at 37℃. Thus, M. marium infection rarely occurs on the face and most commonly affects the cooler extremities2. Thus far, only seven cases of infection on the face have been reported worldwide3. The conventional microbiological methods used for M. marium diagnosis are slow and solely rely on phenotypic characteristics. As delayed diagnosis is the main cause of adverse effects, rapid and accurate molecular diagnosis methods are necessary. Rapid detection of mycobacteria by using conventional broad-range PCR has previously been described in the literature4. Nowadays, M. marinum infection due to the use of pools rarely occurs because of chlorination disinfection of pool water. Thus, we report a rare and interesting case of M. marium infection showing an unusual location in a patient with a history of exposure to pool water. Hence, clinicians should consider the possibility of M. marium infection even if the history and location are atypical.

Correlation analysis of positive rate of HPV genotyping test and HPV nucleic acid loads

Objective To investigate the correlation between the positive rate of PCR-reverse dot blot genotyping test and the loads of the viral nucleic acid. Methods The fluorescent PCR assay was used to detect the high-risk HPV（HR-HPV）DNA loads in the cervical mucus samples from 1162 female patients. Patients with positive HR-HPV DNA were further analyzed by PCR-reverse dot blot hybridization assay for HPV genotyping. Results The overall positive rate of genotyping test was 68. 8% . The positive rate of genotyping test had a significant positive correlation with the Log Koc of HR-HPV DNA loads（r=0. 944, P﹤0. 01）. The quadratic curve fitting formula was Y= -1. 806＋0. 558X-0. 031X2（Y for genotyping positive rate,X for Log Koc of HR-HPV DNA loads）. There were significant differences with the positive rate of genotyping test among patients with different viral loads（P﹤0. 01）. When HR-HPV DNA loads were 10^4-10^5 copies/ ml,10^5-10^6 copies/ ml,10^6-10^7 copies/ ml and more than 10^7 copies/ ml,the positive rate of HPV genotyping test were 27. 8% ,48. 5% ,74. 0% ,97. 5% and 33. 3% ,51. 5% ,78. 0% ,97. 5% respectively by using different genotyping detection reagents. Conclusion The positive rate of PCR-reverse dot blot genotyping test was correlated with the loads of HPV nucleic acid. Key words: Human papillomavirus; Viral load; Polymerase chain reaction; Genotyping; Reverse dot blot

Evaluation of a PCR-Reverse Blot Hybridization Assay to Identify Six Dermatophytes Predominant in the Republic of Korea

Accurate and rapid diagnosis of dermatophytosis, a disease whose prevalence has been steadily increased, is important for successful treatment. Current laboratory methods for diagnosing dermatophytosis rely on KOH mount and fungal culture method. However, these methods have low sensitivity and are time-consuming (2~4 weeks to diagnosis). In our previous study, a rapid molecular diagnostic assay (PCR-reverse blot hybridization assay, REBA) was developed to identify the following 6 main species of dermatophytes: Trichophyton rubrum, T. mentagrophytes, T. tonsurans, Microsporum canis, M. gypseum, and Epidermophyton floccosum. However, the REBA required more evaluation to validate its use in clinical examinations. The aim of the present study was to evaluate and validate the ability of the PCR-REBA to successfully identify dermatophytes in clinical isolates from dermatophytosis patients. Both conventional identification methods and the PCR-REBA were used to assess the presence of species of dermatophytes in 148 clinical isolates. The results of the two approaches were compared, and discrepancies between the two approaches were resolved by fungal ITS1 sequence analysis. T. rubrum was the most prevalent dermatophyte identified by conventional identification methods (118/148, 79.7%) and the PCR-REBA (131/148, 88.4%). The overall rate of consistency between conventional identification methods and the PCR-REBA was 79.0% (117/148 samples). Fungal ITS1 sequence analysis showed that PCR-REBA results were correct for 93.5% (29/31) of the discrepant samples. The PCR-REBA is rapid, sensitive, and highly specific compared with conventional identification methods. Thus, the PCR-REBA is a potentially useful tool for identifying dermatophytes in clinical settings.

PCR-Reverse Blot Hybridization Assay for Screening and Identification of Pathogens in Sepsis

Rapid and accurate identification of the pathogens involved in bloodstream infections is crucial for the prompt initiation of appropriate therapy, as this can decrease morbidity and mortality rates. A PCR-reverse blot hybridization assay for sepsis, the reverse blot hybridization assay (REBA) Sepsis-ID test, was developed; it uses pan-probes to distinguish Gram-positive and -negative bacteria and fungi. In addition, the assay was designed to identify bacteria and fungi using six genus-specific and 13 species-specific probes; it uses additional probes for antibiotic resistance genes, i.e., the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA) and the vanA and vanB genes of vancomycin-resistant enterococci (VRE). The REBA Sepsis-ID test successfully identified clinical isolates and blood culture samples as containing Gram-positive bacteria, Gram-negative bacteria, or fungi. The results matched those obtained with conventional microbiological methods. For the REBA Sepsis-ID test, of the 115 blood culture samples tested, 47 (40.8%) and 49 (42.6%) samples were identified to the species and genus levels, respectively, and the remaining 19 samples (16.5%), which included five Gram-positive rods, were identified as Gram-positive bacteria, Gram-negative bacteria, or fungi. The antibiotic resistances of the MRSA and VRE strains were identified using both conventional microbiological methods and the REBA Sepsis-ID test. In conclusion, the REBA Sepsis-ID test developed for this study is a fast and reliable test for the identification of Gram-positive bacteria, Gram-negative bacteria, fungi, and antibiotic resistance genes (including mecA for MRSA and the vanA and vanB genes for VRE) in bloodstream infections.

Mycobacterial Infection after Intravesical Bacillus Calmette-Gu?rin Treatment for Bladder Cancer: A Case Report

Bacillus Calmette-Guërin (BCG) has been traditionally used as a vaccine against tuberculosis. Further, intravesical administration of BCG has been shown to be effective in treating bladder cancer. Although BCG contains a live attenuated strain of Mycobacterium bovis, complications such as M. bovis BCG infection caused by BCG administration are extremely rare. Here, we report a case of BCG infection occurring after intravesical BCG therapy. A 67-yr-old man presented with azotemia and weight loss. He had been diagnosed with bladder cancer 4 yr back, and had undergone transurethral resection of the bladder tumor and intravesical BCG (Tice strain) therapy at that time. An acid-fast bacterial strain was isolated from his urine sample. We did not detect Mycobacterium tuberculosis protein 64 (MPT-64) antigen in the isolates obtained from his sample, and multiplex PCR and PCR-reverse blot hybridization assay indicated that the isolate was a member of the M. tuberculosis complex, but was not M. tuberculosis. Finally, sequence analysis of 16S ribosomal RNA and DNA gyrase, subunit B (gyrB) suggested that the organism was M. bovis or M. bovis BCG. Although we could not confirm that M. bovis BCG was the causative agent, the results of the 3 molecular methods and the MPT-64 antigen assay suggest this finding. This is an important finding, especially because M. bovis BCG cannot be identified using common commercial molecular genetics tools.

Detection and identification of mycobacteria in sputum from suspected tuberculosis patients

BackgroundDetection of Tuberculosis agent like nontuberculous mycobacteria (NTM) species by culture and microscopic methods remains difficult and time consuming. A fast and reliable diagnosis of tuberculosis would greatly improve the control of the disease. The purpose of this study is to compare the conventional multiplex PCR and multiplex PCR reverse cross blot hybridization assay to culture method in terms of mycobacteria species detection.FindingsAmong the 117 positively cultured samples, nontuberculous mycobacteria (NTM) species were found in 9 samples of multiplex PCR reverse cross blot hybridization assay; compared to only 3 NTM species found in our conventional multiplex PCR, and 13 NTM species were successfully identified among 162 negatively cultured samples compared to only 5 NTM species identification in conventional multiplex PCR results.ConclusionsThe sensitivity of the multiplex PCR reverse cross blot hybridization assay comparing to culture method was 86.03%, the specificity is 35.46%, the positive predictive value was 41.94% and the negative predictive value was 82.41%. For conventional multiplex PCR these values are 81.62%, 38.65%, 41.89%, 79.51%, respectively. Furthermore, in terms of mycobacteria species detection, the conventional multiplex PCR was relatively equal compared to the multiplex PCR reverse cross blot hybridization assay, and to be particularly having no significant discrepant results on the identification of Mycobacteria tuberculosis in both methods.

Improved Molecular Tools for Detection of Babesia bigemina

Molecular detection of Babesia bigemina involves a nested PCR protocol and reverse line blot hybridization (RLBH) assay based on the 18S gene. In this study, we report the development of molecular tools for improving B. bigemina detection in bovine blood-a one-step PCR assay based on the amplification of rap-1a paralogous and a new RLBH Babesia spp. 18S probe. The one-step PCR assay is highly specific, with an estimated analytical sensitivity corresponding to 0.00002% parasitemia. The RLBH assay, with a new B. bigemina probe, allows the detection of all tested B. bigemina isolates showing no cross-hybridization with B. bovis 18S gene. By developing this highly specific and sensitive one-step PCR and upgrading the RLBH assay for B. bigemina, we have improved molecular assays which, together with serologic methods, provide valuable tools for epidemiologic studies of bovine babesiosis.