PCR-reverse Hybridization Research Articles

Evaluation and clinical impact of MALDI Biotyper Mycobacteria Library v6.0 for identification of nontuberculous mycobacteria by MALDI‐TOF mass spectrometry

Major challenges in the identification of non-tuberculous mycobacteria (NTM) by MALDI-TOF MS include protein extraction protocol and updating of the NTM database. The aim of this study was to evaluate MALDI Biotyper Mycobacteria Library v6.0 (Bruker Daltonics GmbH, Bremen, Germany) for identification of clinical NTM isolates and its impact on clinical management. NTM isolates cultivated from clinical samples in 101 patients were identified simultaneously by PCR-reverse hybridization (Hain Lifescience GmbH, Nehren, Germany) as a routinely used reference molecular method and using MALDI Biotyper Microflex LT/SH after protein extraction. Each isolate was applied to eight spots, and mean scores were used in analysis. MALDI-TOF MS obtained correct identification to the species level for 95 (94.06%) NTM isolates. The majority of correctly identified isolates (92/95; 96.84%) were identified with high-confidence score of ≥1.80 and only 3.16% (3/95) with a score of <1.80. Mean value ± SD of RGM NTM isolates (2.127 ± 0.172) was statistically significant higher in comparison to SGM NTM isolates (2.027 ± 0.142) with a p value of 0.007. In comparison to PCR-reverse hybridization, discordant identification results by MALDI-TOF MS were found in six (6/101; 5.94%) NTM isolates for which clinical data were analyzed. We demonstrated a high confidence NTM identifications using Mycobacterium Library v 6.0 on routine clinical isolates. This is the first study that analyzed MALDI-TOF MS identification results of NTM isolates in the context of clinical data, and it showed that MALDI-TOF MS with its updated databases could help clarify the epidemiology, clinical characteristics, and course of infections caused by less frequent NTM species.

Prevalence of Human Papillomavirus (HPV) Genotypes in Cervicovaginal Secretions of Human Immunodeficiency Virus (HIV) Positive Indian Women and Correlation With Clinico-Virological Parameters.

Introduction and Background: Both human papillomavirus (HPV) and the human immunodeficiency virus (HIV) are sexually transmitted. High-risk (HR) HPV types are a causal factor in cervical cancer. Persistent HPV infection in this subset of immunocompromised women results in faster disease progression. The study determined the prevalence of HPV genotypes in cervicovaginal secretions of HIV seropositive women and the correlation with CD4 counts and cytology.Method: One hundred, non-pregnant, HIV-positive women of 18 years of age and above were enrolled in this cross-sectional study following approval by the institutional ethical committee. A written consent, questionnaire, followed by sample collection including a Papanicolaou (Pap) smear for cytology was undertaken. Cervicovaginal secretion samples were collected in the Digene® specimen transport medium (STM) (Qiagen Gaithersburg Inc., MD, USA). HPV genotyping was carried out with PCR amplification of a 65-base pair (bp) fragment in the L1 region of the HPV genome using the short PCR fragment (SPF10) primers followed by reverse hybridization by line probe assay (LPA) using the INNOLiPA HPV Genotyping Extra kit (Fujirebio, Belgium). Quantitation of HPV-16 and−18 viral loads (VLs) was done by real-time PCR. Results of Pap smear cytology were correlated with CD4 counts and HPV-16 and−18 VLs.Results: Mean age of the subjects was 34.9 years ± 7.2 years (median 33.0 years, range 24–60 years). HPV was detected in 62 of 93 (66.6%) samples. Twenty (32.25%) of these 62 samples harbored a single HPV genotype. Multiple genotypes (more than two) were detected in 38 (61.3%) samples. HPV-16 was the commonest genotype detected in 26 (27.9%) of all samples and 41.9% of HPV positive samples. Pap smear cytology was reported for 93 women included in the study. Women who had normal cytology were reported as negative for intraepithelial malignancy or lesion (NILM; n = 62; 71.36%), two women had a high-grade squamous intraepithelial lesion (HSIL), low-grade squamous intraepithelial lesion (LSIL; n = 11), atypical squamous cells of undetermined significance (ASCUS; n = 12). Those smears with inadequate material were reported as scant (n = 6). The median CD4 count was 363/cu.mm (range 39–787) in HPV-positive women compared to 423/cu.mm (range 141–996) in those HPV-negative women. Quantitation of HPV-16 and−18 VL was done in duplicate for samples positive by PCR reverse hybridization (INNOLiPA). Of these 20 samples (65%), 12 samples were positive by real-time PCR. The normalized HPV-16 VL ranged between 18 and 240,000 copies/cell. The normalized HPV-18 VL in cervical samples ranged between ~24 and 60,000 copies/cell.Conclusion: HIV-positive women may be infected with multiple genotypes other than HPV-16 and−18. This may have implications on the vaccines available currently which target few specific genotypes only. Studies are required to determine the predictive role of HR HPV genotypes, in significant copy numbers especially in HIV seropositive women. It would be clinically relevant if the HPV VLs, cervical cytology, and CD4 counts are considered into cervical cancer screening programs for triage and follow-up of these women.

Evaluation of MALDI Biotyper Interpretation Criteria for Accurate Identification of Nontuberculous Mycobacteria.

Identification of mycobacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) requires not only a good protein extraction protocol but also an adequate cutoff score in order to provide reliable results. The aim of this study was to assess the cutoff scores proposed by the MALDI-TOF MS system for mycobacterial identification. A total of 693 clinical isolates from a liquid medium and 760 from a solid medium were analyzed, encompassing 67 different species of nontuberculous mycobacteria (NTM). MALDI-TOF MS identified 558 (80.5%) isolates from the liquid medium and 712 (93.7%) isolates from the solid medium with scores of ≥1.60. Among these, four (0.7%) misidentifications were obtained from the liquid medium and four (0.5%) from the solid medium. With regard to species diversity, MALDI-TOF MS successfully identified 64 (95.5%) different species, while PCR-reverse hybridization (GenoType Mycobacterium CM and AS assays) identified 24 (35.8%) different species. With MALDI-TOF MS scores of ≥2, all isolates were correctly identified, and with scores in the range from 1.60 to 1.99, most isolates were correctly identified, except for Mycobacterium angelicum, M. parascrofulaceum, M. peregrinum, M. porcinum, and M. gastri In conclusion, MALDI-TOF MS is a useful method for identifying a large diversity of NTM species. A score threshold of 1.60 proved useful for identifying almost all the isolates tested; only a few species required a higher score (≥2.00) to obtain a valid definitive identification.

Genetic risk factors for venous thromboembolism among infertile men with Klinefelter syndrome

BackgroundKlinefelter syndrome (KS) is one of the commonest sex chromosome disorders. Affected males become infertile and highly susceptible to several health problems, including vascular thromboembolism (VTE). The risk of VTE may be exacerbated by an underlying genetically inherited thrombophilia. In this study, we aimed to investigate the genotype and allele frequencies of common gene polymorphisms related to hereditary thrombophilia in infertile males with KS compared to normal, fertile men. MethodsEighty-five infertile males with KS and 75 healthy control males were included in this case-control study. Genetic testing was done using an extended thrombophilia gene panel by Multiplex PCR reverse hybridization method. ResultsThere was an increased frequency of mutant alleles and heterozygous genotypes of FV Leiden, FV H 1299R, Pro G20210A, MTHFR C677T and PAI-1 4G/5G thrombophilic gene polymorphisms in KS patients compared to the control group. It was shown that 10.7% of KS patients had the A3 haplotype of the EPCR gene in comparison to 5.3% of control patients. The A3/A3 genotype was found only in KS patients (7.1%). Carriers of more than one mutant allele in KS patients exceeded the control (p < 0.001). ConclusionA high prevalence of thrombophilic gene polymorphisms and the coexistence of different mutant alleles were evident in infertile KS males. These data highlight the importance of conducting further studies to understand the role of hereditary thrombophilia in predicting venous thrombosis in patients with Klinefelter syndrome.

Molecular detection and genotyping of human papillomavirus

The development of cervical cancer is associated with persistent infection of high-risk human papillomavirus. The detection and genotyping of HPV could be used to evaluate the epidemiology of HPV infections, monitor HPV vaccine efficacy, and screen cervical cancer. There are a lot of commercially available molecular tests for HPV, based on different methodologies and detection systems. In principle, it mainly includes two categories, namely signal amplification and target amplification. Most of them are based on PCR amplification, such as fluorescent PCR, PCR-reverse hybridization, etc. The performances of detection reagents are different. In addition, HPV genotyping assays based on next-generation sequencing and quantitative HPV detection kits are developed. However, only a small number of commercial assays have been clinically verified. A large number of assays which may bring greater values in the screening of cervical cancer are needed to be clinically validated.

Mutation analysis and early pregnancy prenatal diagnosis for two families affected with non-syndromic hearing loss

Objective:The aim of this study is to identify the pathogenic genes of two non-syndromic hearing loss families, provide accurate genetic counseling and early pregnancy prenatal diagnosis services for second birth families, which determine the genotype of the fetus, early diagnosis and early intervention to prevent the birth of deaf children. Method:Two families with a severe sensorineural hearing loss were enrolled in this study. Genomic DNA was extracted from the peripheral venous blood of the proband and parents. The mutations of the four hearing loss susceptibility genes were analyzed by the hereditary hearing loss gene detection kit (PCR reverse hybridization) and Sanger sequencing. After confirming the genotype of the subject, the genotype of the fetus was examined for the fetus with 10 to 12 weeks of gestation, and the neonates were diagnosed with prenatal diagnosis. Result:In the two families, the No. 1 family proband was the compound heterozygous mutation of SLC26A4 gene c.IVS72A＞G/c.2177insCTAT, and parents were carriers. The prenatal diagnosis showed that the fetal genotype of No. 1 family was a compound heterozygous mutation, and the fetus was not born. The No. 2 family proband genotype was the compound heterozygous of GJB2 gene c.605ins46/c.512insAACG, and the parents were both carriers. The No. 2 family, the prenatal diagnosis showed the fetal genotype of No. 2 family GJB2 gene sequence was normal, the newborn passed the hearing screening. Conclusion:Genetic testing of hereditary hearing loss combined with prenatal diagnosis plays a significant role in guiding deaf children with re-fertility needs, especially prenatal diagnosis in early pregnancy, which can achieve early diagnosis, early detection and early intervention, effectively reducing the birth rate of children with deafness.

The effect of ‘cytochrome P-450 2C9’ and ‘vitamin K epoxide reductase complex 1’ genetic polymorphism upon oral anticoagulation requirements

Background Polymorphisms in the gene encoding the cytochrome P-450 2C9 enzyme (CYP2C9) are known to contribute to variability in sensitivity to Marevan. CYP2C9 is the enzyme primarily responsible for the metabolic clearance of s-enantiomer of Marevan. The vitamin K epoxide reductase complex 1 (VKORC1) is the target of coumarin anticoagulants, and its common genetic variants result in altered sensitivity to Marevan. VKORC1 polymorphisms are associated with a need for lower doses of Marevan during long-term therapy. Aim of work This study aimed to assess the allelic frequencies and to investigate the relationship between ‘CYP2C9’ and ‘VKORC1’ genotype and vitamin K antagonist anticoagulation. Patients and methods This study was conducted on 40 patients. There were 24 females and 16 males, with a male to female ratio of 2 : 3. Their ages ranged from 28 to 72 years. All the studied patients were laboratory investigated with international normalized ratio, complete blood count, and detection of VKORC1 and CYP2C9 by PCR reverse hybridization method using PGX thrombo strip assay (Vienna Lab.). Results Regarding the distribution of patients according to frequency of deep venous thrombosis (DVT) attacks, 16 (40%) patients showed single attack of DVT and 24 (60%) patients showed recurrent attacks. Patients with single attack of DVT comprised 12 (75%) females and four (25%) males, with male to female ratio of 1 : 3. As for the patients with recurrent attacks of DVT, there were 13 (54.2%) female and 11(45.8%) male patients, with a male to female ratio of 1 : 1.2. Conclusion Detection of genetic polymorphisms in CYP2C9 and VKORC1 genes before onset of warfarin therapy greatly influenced response to warfarin and shortened the time required to reach target international normalized ratio, and hence reduced the risk of recurrence of DVT.

Is an integrative laboratory algorithm more effective in detecting alpha-1-antitrypsin deficiency in patients with premature chronic obstructive pulmonary disease than AAT concentration based screening approach?

Introduction:Alpha-1-antitrypsin deficiency (AATD), genetic risk factor for premature chronic obstructive pulmonary disease (COPD), often remains undetected. The aim of our study was to analyse the effectiveness of an integrative laboratory algorithm for AATD detection in patients diagnosed with COPD by the age of 45 years, in comparison with the screening approach based on AAT concentration measurement alone.Subjects and methods:50 unrelated patients (28 males/22 females, age 52 (24–75 years) diagnosed with COPD before the age of 45 years were enrolled. Immunonephelometric assay for alpha-1-antitrypsin (AAT) and PCR-reverse hybridization for Z and S allele were first-line, and isoelectric focusing and DNA sequencing (ABI Prism BigDye) were reflex tests.Results:AATD associated genotypes were detected in 7 patients (5 ZZ, 1 ZMmalton, 1 ZQ0amersfoort), 10 were heterozygous carriers (8 MZ and 2 MS genotypes) and 33 were without AATD (MM genotype). Carriers and patients without AATD had comparable AAT concentrations (P = 0.125). In majority of participants (48) first line tests were sufficient to analyze AATD presence. In two remaining cases reflex tests identified rare alleles, Mmalton and Q0amersfoort, the later one being reported for the first time in Serbian population. Detection rate did not differ between algorithm and screening both for AATD (P = 0.500) and carriers (P = 0.063).Conclusion:There is a high prevalence of AATD affected subjects and carriers in a group of patients with premature COPD. The use of integrative laboratory algorithm does not improve the effectiveness of AATD detection in comparison with the screening based on AAT concentration alone.

Low-Colony Counts of Nontuberculous Mycobacteria: Clinical Significance Analysis

Background: Diagnosis of nontuberculous mycobacterium (NTM) is challenging, and clinical, radiological and microbiological criteria should be met. Traditionally, culture results on solid media have been reported semi-quantitatively, but no study exists regarding the clinical significance of low-colony count culture reports. The authors of the present study analyzed the clinical significance of low-colony count specimens of NTM with a greater than three-year follow-up period. Methods: A total of 341 clinical isolates were evaluated among the isolates at Seoul National University Hospital and Seoul National University Borame Hospital from October 2005 to September 2006. Colony count less than 50 was considered a low-colony count specimen. Identifications of NTM from all the isolates were performed using a DNA chip (PCR reverse hybridization, LG Life Science, Korea). Clinical significance was analyzed by reviewing the medical records of patients with greater than three years of follow-up data after NTM isolation from respiratory samples. Results: NTM lung disease was observed in 27.0% of the patients with low-colony count specimens among 167 patients with respiratory samples, and 70.4% of the patients were treated. The low-colony count patients had less NTM lung disease, longer incubation period, and less acid fast bacilli-positivity than patients with a colony count greater than 50. Conclusion: The prevalence of NTM lung disease with a low-colony count specimen was greater than 25%. In a clinical setting, NTM lung disease should not be excluded only on the basis of a low-colony count. (Korean J Clin Microbiol 2012;15:9-13)

Evaluation of a novel microplate colorimetric hybridization genotyping assay for human papillomavirus

Persistent infection with high-risk human papillomavirus (HR-HPV) has been associated with cervical cancer. Developing assays for the identification of these viral types is of great importance for monitoring patients and controlling strategies. The development of the MCHA (microplate colorimetric hybridization assay), a PCR-based method for identifying six of the most common HR-HPV types (HPV 16, 18, 31, 33, 39 and 45) is described. The MCHA combines the amplification with the GP5+/GP6+ consensus primers followed by PCR reverse hybridization with specific probes and detection through a colorimetric assay. The performance of the MCHA was evaluated using 108 DNA samples typed previously by the PapilloCheck®. The agreement between both methods was 69.4% for HPV 16; 79.1% for HPV 45; 82.4% for HPV 18; 93.6% for HPV 31; 87.9% for HPV 33, and 17.6% for HPV 39. The assay had higher sensitivity than the Papillocheck®, particularly for identifying HPV 16 and 18. The MCHA seemed to be sensitive and specific for the identification of the most prevalent HPV types in invasive cervical cancer, HPV 16, 18, 45, 33 and 31. It requires low-cost reagents and common laboratory apparatus.

Human Papillomavirus type distribution in invasive cervical cancer in Uganda

BackgroundWe conducted a study aiming to describe Human Papillomavirus (HPV) type distribution in invasive cervical carcinoma in Uganda.Methods191 archival cervical carcinoma samples diagnosed in the Department of Pathology, Makerere University in Kampala between 1968 and 1992 were analysed using a sensitive PCR-Reverse Hybridization Line Probe Assay.ResultsOut of the 186 cases of confirmed invasive cervical cancer in the study paraffin blocks, 114 were positive for HPV DNA. Specific HPV genotypes were identifiable in 109 cases: HPV 16, 18, 31, 35, 39, 44, 45, 51, 52 and 70. These occurred as single infections in 105 cases (96.3%) and as multiple infections in 4 cases (3.7%). HPV 16 or 18 accounted for 80% (84/105) of cases with single infection.ConclusionThe results of this study confirm the role of HPV 16 and 18 in cervical cancer pathogenesis in the Ugandan population. The results suggest that the currently available HPV vaccines against HPV 16 and 18 could possibly prevent the majority of invasive cervical cancers in Uganda.

Development of a Sensitive and Specific Multiplex PCR Method Combined with DNA Microarray Primer Extension To Detect Betapapillomavirus Types

Emerging lines of evidence indicate that the cutaneous human papillomavirus (HPV) types that belong to the genus Betapapillomavirus (beta HPV) are involved in the development of nonmelanoma skin cancer. Unlike the situation for mucosal HPV types, highly sensitive and reliable methods to identify characterized cutaneous HPV types in a single assay are limited. Here, we describe a novel one-shot method for the detection of all characterized beta HPV types, namely, HPV type 5 (HPV5), 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96. This assay combines two different techniques: multiplex PCR using HPV type-specific primers for amplification of each E7 gene and array primer extension (APEX) for typing. This method has been validated using clinical samples which were analyzed simultaneously for the presence of cutaneous HPV types by two additional methods, i.e., the FAP59/64 PCR protocol and a commercially available PCR-reverse hybridization assay (PM-PCR RHA). Our data show good agreement between the results obtained with the multiplex PCR/APEX assay and the PM-PCR RHA method (overall HPV positivity of 92.2% for multiplex PCR/APEX assay versus 90.6% with the PM-PCR RHA) (kappa value, 50; 95% confidence interval, 13 to 88). In addition, the multiplex PCR/APEX assay showed higher sensitivity than the PM-PCR RHA did. This favorable feature and the high-throughput potential make this assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of cutaneous types in skin cancer.

Evaluation of a Novel Highly Sensitive, Broad-Spectrum PCR-Reverse Hybridization Assay for Detection and Identification of Beta-Papillomavirus DNA

Human papillomavirus can be detected by amplification of viral DNA. A novel one-step PCR (PM-PCR) was evaluated for amplification of a 117-bp fragment from the E1 region. It permitted ultrasensitive detection of all 25 known human papillomavirus genotypes from the beta-papillomavirus genus. The intra- and intertypic sequence variations of the 77-bp interprimer region were studied. Genotype-specific probes as well as general probes were selected for the 25 established beta-papillomavirus types, and a reverse hybridization assay (RHA) was developed (PM-PCR RHA method). The analytical sensitivity of the PM-PCR RHA method was 10 to 100 viral genomes. The one-step PM-PCR turned out to be more sensitive than the previously described nested MaHa-PCR for beta-papillomavirus detection. The PM-PCR RHA method was able to detect and identify beta-papillomavirus types in frozen patient material as well as in poorly amplifiable material such as formalin-fixed, paraffin-embedded skin biopsy specimens. Inter- and intralaboratory variability experiments showed that the reproducibility of the assay was very high. In conclusion, the one-step PM-PCR together with the RHA allows extremely sensitive, specific, and reproducible detection of beta-papillomavirus DNA as well as reliable identification of beta-papillomavirus genotypes in both fresh and paraffin-embedded patient material.

Co-cutaneous infection in a dog: PCR-reverse identification of Candida tropicalis on skin biopsy

Canine leishmaniasis is a very common disease in all countries of the Mediterranean area. In these territories, Leishmania infantum MONT-1 is the species producing visceral infections in humans and visceral-cutaneous infections in dogs, the principal domestic reservoir. The alterations of the immune response during leishmaniasis could predispose the host to other infectious, parasitic or mycologic diseases. In the domain of mycology, concurrent infections by dermatophytes and Aspergillus were reported. It is reported a clinical case of Candida tropicalis and L. infantum co-infection in a dog with skin lesions; it could be the first report of a such co-infection. The animal is a 10-year-old mongrel male dog showing, since about 1 year, a diffuse dermatitis on the face, the back and the flanks, characterized by alopecia and scaling. Serological tests for ehrlichiosis and rickettsiosis gave negative result, while indirect immunofluorescence test for IgG anti- L. infantum antibodies gave a positive titre of 1:640. Due to the severity of the lesions euthanasia of the dog was decided. Histopathology and immunochemistry performed on skin biopsies confirmed the positivity for Leishmania; by mycological culture and PCR-reverse hybridization assay on skin biopsies C. tropicalis was identified as associated to the protozoan. An impaired immune function and skin lesions caused by L. infantum could have played a direct role in the development of C. tropicalis.

Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.

Human papillomavirus (HPV) can be detected by amplification of viral DNA. A novel PCR primer set generating a short PCR fragment (SPF PCR) was used for amplification of a fragment of only 65 bp from the L1 region and permitted ultrasensitive detection of a broad spectrum of HPV genotypes. The intra- and intertypic sequence variations of the 22-bp interprimer region of this amplimer were studied. Among 238 HPV sequences from GenBank and clinical specimens, HPV genotypes were correctly identified based on the 22-bp sequence in 232 cases (97.2%). Genotype-specific probes for HPV genotypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, 51 to 54, 56, 58, 59, 66, 68, 70, and 74 were selected, and a reverse hybridization line probe assay (LiPA) (the INNO-LiPA HPV prototype research assay) was developed. This LiPA permits the use of amplimers generated by the SPF as well as the MY 09/11 primers. The assay was evaluated with a total of 1, 354 clinical specimens, comprising cervical scrapes (classifications ranging from normal cytology to severe dyskaryosis) and formalin-fixed, paraffin-embedded cervical carcinoma samples. LiPA results were highly concordant with sequence analysis of the SPF amplimer, genotype-specific PCR, and sequence analysis of amplimers generated by MY 09/11 primers. The sensitivity of the SPF primers was higher than that of the GP5(+)/6(+) primers over a broad range of HPV types, especially when multiple HPV genotypes were present. In conclusion, the SPF LiPA method allows extremely sensitive detection of HPV DNA as well as reliable identification of HPV genotypes in both cervical smears and paraffin-embedded materials.