Usefulness Analysis of Urine Samples for Early Screening of Human Papilloma Virus Infection

Human papilloma virus (HPV) is known to be a major cause of cervical cancer. In Korea, although the mortality of cervical cancer has decreased, HPV infection rates are increasing rapidly in young women. One of the reasons for a high rate of human immunodeficiency virus (HIV) infection appears to be associated with a low frequency to visit gynecology clinics because of the uncomfortable sampling process for HPV testing. Therefore, it is necessary to develop a non-invasive method, such as urine testing to diagnose cervical cancer rather than use of the existing invasive method. This study aimed to test validity of HPV DNA detection in urine specimens that can be easily collected from women. Paired vaginal discharge and urine samples were collected prospectively from 203 women who visited the local hospital between January and August 2018 in Busan, Korea. By using the Virocheck® assay kit (Optipharm), we found that 17.2% (35/203) of vaginal discharge samples were HPV positive and 82.8% (168/203) were HPV negative. In urine samples, 15.8% (32/203) were HPV positive and 84.2% (171/203) were HPV negative. The co-incident rate for HPV DNA detection was 84.8% in both vaginal discharge and urine samples. These results suggest that the HPV DNA detection using urine samples might be an alternative way to diagnose HPV infection in a non-invasive way. This analytical approach can be utilized as a screening test to identify HIV-infected patients who need a follow-up process by using urine samples.

A Simple and Efficient Multiplex PCR Assay for the Identification of Mycobacterium Genus and Mycobacterium tuberculosis Complex to the Species Level

PURPOSE: The Mycobacterium tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis bacillus Calmette-Guerin (BCG) and M. africanum, and causes tuberculosis in humans and animals. Identification of Mycobacterium spp. and M. tuberculosis complex to the species level is important for practical use in microbiological laboratories, in addition to optimal treatment and public health. MATERIALS AND METHODS: A novel multiplex PCR assay targeting a conserved rpoB sequence in Mycobacteria spp., as well as regions of difference (RD) 1 and RD8, was developed and evaluated using 37 reference strains and 178 clinical isolates. RESULTS: All mycobacterial strains produced a 518-bp product (rpoB), while other bacteria produced no product. Virulent M. tuberculosis complex strains, M. tuberculosis, M. bovis and M. africanum, produced a 254-bp product (RD1), while M. bovis BCG, M. microti and nontuberculous mycobacteria produced no RD1 region product. Additionally, M. tuberculosis and M. africanum produced a 150-bp product (RD8), while M. bovis and M. bovis BCG produced a 360-bp product (deleted form of RD8). M. microti and nontuberculous mycobacteria produced no RD8 region product. This assay identified all Mycobacterium spp. and all M. tuberculosis complex strains to the species level. CONCLUSION: The multiplex PCR assay of the present study could be implemented as a routine test in microbiology laboratories, and may contribute to more effective treatment and surveillance of tuberculosis stemming from the M. tuberculosis complex.

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.

Evaluation of MolecuTech Real MTB-ID for MTB/NTM Detection Using Direct Specimens / 대한임상미생물학회지

BACKGROUND: The differentiation of Mycobacterium tuberculosis (MTB) from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. The diagnosis of diseases caused by NTM is difficult because NTM are prevalent in the environment and have fastidious properties. In this study, we evaluated the real-time PCR-based MTB/NTM detection kit for its usefulness in discrimination of MTB and NTM species. METHODS: A total of 155 sputum specimens whose AFB staining smear and culture were positive were used for this study. Among them, 59 and 96 samples had been identified as MTB and NTM, respectively. DNA obtained from sputum specimens was subjected to analysis with MolecuTech Real MTB-ID(R) (M&D, Korea) real-time PCR-based MTB/NTM detection kit. Subsequently, the results of MolecuTech Real MTB-ID(R) were compared with AFB staining smear and culture results. RESULTS: The positive rate of MolecuTech Real MTB-ID(R) to detect MTB and NTM was 98.3% (58/59) and 97.9 (94/96), respectively, using sputum specimens. CONCLUSION: For detection of MTB/NTM, the sensitivity and specificity of MolecuTech Real MTB-ID(R) were comparable to those of conventional methods. Therefore, this study suggests the usefulness of real-time PCR-based MolecuTech Real MTB-ID(R) for rapid detection of MTB/NTM from direct specimens.

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.