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

BACKGROUND: Rapid 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. METHODS: A 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. RESULTS: A 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. CONCLUSION: PCR-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.

Genotyping and Drug Susceptibility Patterns of M. Tuberculosis Isolated from HIV Seropositive Patients in Tehran Iran.

AIM: This study aims to investigate the prevalence and drug-resistance M. tuberculosis isolated from HIV seropositive individuals in Tehran, Iran. BACKGROUND: Human immunodeficiency virus (HIV) is one of the most important risk factors for developing active tuberculosis (TB). OBJECTIVE: The objective is to determine the rate of transmission and drug-resistant M. tuberculosis (MTB) strains isolated from HIV seropositive patients in Tehran province, Iran. METHODS: This study consecutively enrolled 217 TB/HIV coinfected patients from April 2018 to August 2019 at Emam Khomeini referral hospital and 5 other health centers in Tehran province. The isolates were genotyped using 15 loci Mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR). Minimum inhibitory concentration (MIC) was determined for 6 drugs. In addition, mutations were assessed in rpoB, katG, inhA, and ahpC genes using Reverse Blot Hybridization Assay System. RESULTS: A 20 (9.2%) patients were culture-positive for M. tuberculosis and typed by MIRU-VNTR, 13 (65%) strains formed 5 clusters, but 6 (30%) isolates had a unique pattern. The total Hunter- Gaston discrimination index (HGDI) for all 15 loci was 0.846, and the cluster size was 2 to 4 patients. The estimated proportion of recent transmission was 45%. The mutation was identified in 1 isolate, lost inhAW1 and mutation in MT1 loci, which was resistant to isoniazid (INH). Moreover, 1 (5%) and 3 (15%) isolates were resistant to INH and ethambutol (EMB), respectively, of which 1 was resistant to INH and EMB. CONCLUSION: The transmission rate of TB in HIV patients was relatively high; however, the prevalence of drug-resistant strains and TB infection in females was insignificant in this study (p < 0.05); none of the isolates was MDR strains.

Efficient Early Diagnosis of Sepsis Using Whole-Blood PCR-Reverse Blot Hybridization Assay Depending on Serum Procalcitonin Levels.

Sepsis is one of the medical emergencies, and its early detection, within the first hours of development, and proper management improve outcomes. Molecular diagnostic assays using whole blood collected from patients with suspected sepsis have been developed, but the decision making is difficult because of the possibility of false positives, due to contamination. Here, we evaluated the performance of the reverse blot hybridization assay (REBA) Sepsis-ID test for the detection of sepsis-causing microorganisms using whole-blood samples. In addition, the concentrations of C-reactive protein (CRP) and procalcitonin (PCT) were determined to evaluate whether these biomarkers can provide criteria for performing REBA Sepsis-ID in clinical settings. For this study, EDTA-anticoagulated whole blood was simultaneously collected for REBA Sepsis-ID and blood culture from 440 patients with suspected sepsis, from January to October 2015. In addition, CRP and PCT concentrations were measured in 227 patients. The overall positive rates of REBA Sepsis-ID and blood culture were 16.6% (73/440) and 13.9% (61/440), respectively. The pathogen-positive rates of REBA Sepsis-ID and blood culture were 9.8% (43/440) and 9.5% (42/440), respectively. The areas under the receiver operating characteristic (AUROC) curves of PCT and CRP for predicting pathogen-positive results of REBA Sepsis-ID were 0.72 and 0.69, respectively. The PCT concentrations in the group of patients aged ≥50 years were significantly higher than those in the group aged <50 years. After adjusting for age, the PCT AUROC value was 0.77 for predicting pathogen-positive results of REBA Sepsis-ID. The optimal cutoff values of PCT concentrations for subsequent application of REBA Sepsis-ID were 0.12 ng/mL in all patients and 0.22 ng/mL in patients aged ≥50 years. Our observations showed that REBA Sepsis-ID using whole blood was advantageous for the early detection of sepsis-causing microorganisms, and the PCT concentration could be used to determine the necessity of using REBA Sepsis-ID in clinical settings. The application of REBA Sepsis-ID using whole blood, based on the PCT concentration, may contribute to a highly efficient detection of sepsis-causing microorganisms.

Identification of Mycobacterium tuberculosis and non-tuberculous mycobacteria from cutaneous sarcoidosis lesions by reverse blot hybridization assay.

While the etiology of sarcoidosis remains uncertain, mycobacteria have been suggested as a causative infectious agent. To investigate the causal relationship between mycobacteria and sarcoidosis, we performed a reverse blot hybridization assay (REBA) to identify mycobacteria from the skin samples of nine patients with sarcoidosis. Six of the nine samples were shown to be positive for mycobacteria by REBA, including Mycobacterium tuberculosis and non-tuberculous mycobacteria. This is the first study to identify mycobacteria from the skin samples of sarcoidosis patients using REBA, and our results could strengthen the etiologic association between mycobacteria and sarcoidosis.

Clinical Characteristics and Genetic Variations in Early-Onset Atopic Dermatitis Patients.

BACKGROUND: Hereditary factors contribute to atopic dermatitis (AD) development. We developed the reverse blot hybridization assay (REBA) kit to simultaneously detect variations in skin barrier- and immune response-related genes prevalent in Korean AD patients. OBJECTIVE: To identify genetic variations and clinical characteristics that could predict early AD development. METHODS: We compared AD-related genetic variations between early-onset AD subjects and non-AD controls, and clinical characteristics and genetic variations between early- and late-onset AD subjects. We compared 28 early-onset AD subjects and 57 non-AD controls from a birth cohort and 108 early- (age ≤3 years) and 90 late-onset AD subjects and 189 non-AD controls from a university hospital. Genetic variations were detected via REBA. RESULTS: There were no differences in AD-related genetic variation between early-onset AD subjects and non-AD controls in the birth cohort. When the birth cohort and hospital populations were combined, early-onset AD subjects and non-AD controls showed different frequencies of genetic variations of KLK7, SPINK5 1156, DEFB1, IL5RA, IL12RB1a, and IL12RB1b. No differences in the frequency of genetic variations were observed between early- and late-onset AD subjects. Immunoglobulin E positivity for house dust mites was prevalent in late-onset AD subjects. A family history of atopic diseases was associated with early-onset AD. CONCLUSION: No AD-related genetic variations could predict early AD development in Koreans, even though neonates with a family history of atopic diseases are likely to develop AD at ≤3 years of age. Environmental exposure may be more important than genetic variation in determining the onset age of AD.

Comparison of Clinical Features according to FLG Mutation in Korean Atopic Dermatitis Patients / 대한피부과학회지

BACKGROUND: Mutation in the gene encoding filaggrin (FLG) is a major predisposing factor for atopic dermatitis (AD), in association with distinct features such as increased allergic sensitization, higher severity, and frequent skin infections. Genetic diversity in FLG mutations exists across ethnicities. OBJECTIVE: This study aimed to investigate the clinical features of AD according to the presence of FLG mutation in Korean individuals. METHODS: We performed reverse blot hybridization assay to detect FLG mutation in Korean patients with AD. Classifying subjects into AD with or without FLG mutation, clinical features of AD and patch test results were compared between the two groups. RESULTS: Among a total of 281 subjects, 39 (13.9%) were found to have FLG mutation. AD with FLG mutation was associated with higher risk of impetigo and eczema herpeticum but lower risk of prurigo nodularis. In the patch test, there was no difference in positive reactions of major contact allergens between the groups. CONCLUSION: In Korean patients with AD, FLG mutation was associated with more frequent skin infections but not with personal or family history of atopic diseases, allergic sensitization, contact allergy, and protracted course. It is important to consider other skin-barrier-related genes, such as KLK7 and SPINK5, and immune response-related genes in conjunction.

Clinical Usefulness of PCR-REBA for Diagnosis of Onychomycosis / 대한의진균학회지

BACKGROUND: PCR-based reverse blot hybridization assay (PCR-REBA) has high sensitivity and specificity, can be performed directly on nail samples, is relatively cheaper than other molecular biologic methods, and is useful for diagnosing onychomycosis. OBJECTIVE: This study aims to compare the diagnostic efficacy of fungal culture and REBA Fungus-ID® which is a commercial PCR-REBA-based kit used for onychomycosis diagnosis. METHODS: Fifty nail samples were collected from 50 patients diagnosed with onychomycosis via direct microscopic examination using KOH preparation, and subjected to fungal culture and REBA Fungus-ID® test. RESULTS: The sensitivity of conventional fungal culture and REBA Fungus-ID® was 56% and 100%, respectively. In REBA Fungus-ID®, 43 of 50 samples were found to be infected with Trichophyton rubrum. Four of the remaining 7 samples were identified as infected with Trichophyton spp., one with Trichophyton mentagrophytes, and two revealed a panfungal DNA sequence. In fungal culture, 28 of 50 samples showed growth, of which 18 samples were identified as T. rubrum, 3 as Rhodotorula mucilaginosa, 3 as Cladosporium spp., 1 as Cyphellophora europaea, 1 as Penicillium cvjetkovicii, 1 as Lachnum soppittii, and 1 as non-dermatophytic mold. REBA Fungus-ID® and fungal culture were identical in 20 cases (40%). The non-dermatophytic fungi identified in fungal culture were considered contaminants. CONCLUSION: Nail specimens can be used directly for REBA Fungus-ID®, which has a high sensitivity for onychomycosis diagnosis. Therefore, it can be considered useful for diagnosis and identification of the causative organism in mixed infections like onychomycosis.

Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

PCR-reverse Blot Hybridization Assay for Species Identification of Dermatophytes / 대한의진균학회지

BACKGROUND: Dermatophytes (Trichophyton, Microsporum, and Epidermophyton) cause cutaneous mycoses called dermatophytosis. Forproper anti-dermatophytosis therapy, accurate and early diagnosis of dermatophytes is important. Laboratory diagnosis of dermatophytosis for dermatophytes still relies on microscopic and macroscopic examination of in vitro cultures and some physiological tests. These methods (conventional methods) are time-consuming (2~4 weeks) and yet, still have low sensitivity and specificity. Recently, in order to overcome such limitations of conventional methods, molecular-based methods have been developed to identify dermatophytes. The polymerase chain reaction-reverse blot hybridization assay (PCR-REBA) allows sensitive and specific identification of dermatophytes species. OBJECTIVE: This study was aimed to develop a new PCR-REBA with higher sensitivity using less amount of probe concentration, so the assay can be more practical in clinical settings. METHODS: For this, PCR primers and species-specific oligonucleotide probes were designed within the internal transcribed sequences 1 region between 5.8S and 18S rRNA. The species-specific probes designed in this study was to identify 6 species (T. rubrum, T. mentagrophytes, T. tonsurans, M. canis, M. gypseum, and E. floccosum) comprised 99% of dermatophytes isolatedin Korea. RESULTS: The detection efficiency of the PCR-REBA was compared with the microscopic method, and the results showed that the sensitivity of the PCR-REBA developed in this study is 100 times higher than previously developed one. Subsequently, the results of PCR-REBA were evaluated using clinical isolates. DNAs from a total of 68 clinical isolates were analyzed by PCR-REBA, and the inconsistent results between PCR-REBA and conventional microscopic identification results were confirmed by sequence analysis. CONCLUSION: In brief, the results showed that results of sequence analysis were identical with PCR-REBA implying newly developed PCR-REBA is very useful method for accurate and rapid identification of dermatophytes and would provide higher simplicity, specificity, sensitivity than conventional method.

Human Papillomavirus Prevalence in Gangwon Province Using Reverse Blot Hybridization Assay

BACKGROUND: Human papillomavirus (HPV) plays an important role in the development of cervical carcinoma. Although there is a general agreement that high levels of HPV are related to cervical cancer, the prevalence and distribution of HPV genotypes seems to vary by geographical region. This study was designed to investigate the prevalence of HPV genotypes in Gangwon Province, Korea. METHODS: In total, 342 samples were examined by Pap smear and HPV-ID(R) reverse blot hybridization assay (REBA) (M&D, Wonju, Korea). RESULTS: Overall HPV positivity was 80.9% and 64.4% in women with abnormal and normal cytology by REBA, respectively. The five most common HPV types were: HPV 16, 53, 58, 56, and 33 in samples with abnormal cytology, and HPV 16, 53, 58, 70, and 18 in samples with normal cytology. CONCLUSIONS: The REBA can provide useful data regarding prevalence of HPV genotypes. Gangwon Province showed high prevalence of HPV infection in women. The most common HPV type in Gangwon Province was HPV16, and HPV 53, 58, 56, 70 were frequently present.