Reverse Hybridization Research Articles

HLA-DRB1∗1502 Is Associated With Anti-N-Methyl-D-aspartate Receptor Encephalitis in Thai Children

BackgroundAnti-N-methyl-d-aspartate receptor encephalitis (anti-NMDARE) is one of the most common types of autoimmune encephalitis. Most patients have no apparent immunologic triggers, which suggests a genetic predisposition. This study was conducted to identify human leukocyte antigen (HLA) class II alleles associated with anti-NMDARE in Thai children. MethodsThis case-control study enrolled patients younger than 18 years who were diagnosed with anti-NMDARE between January 2010 and December 2020. A “good outcome” was determined as a patient with a modified Rankin scale score of less than 2 at any follow-up visit. HLA genotypes were determined at four-digit alleles using reverse sequence-specific oligonucleotide probe hybridization. The HLA class II allele frequency in patients was compared with that in a database of 101 healthy control Thai children. ResultsThirty-four patients were enrolled with a mean age of 12.8 ± 5.6 years (females 85.3%). The HLA-DRB1∗1502 allele frequency was significantly higher in patients than in controls (odds ratio, 2.32; 95% confidence interval, 1.11-4.8, P = 0.023). A good outcome was noted in 14 of 14 (100%) HLA-DRB1∗1502-positive patients (median time to a good outcome, 6 months) and 14 of 17 (82.3%) HLA-DRB1∗1502-negative patients (median time to a good outcome, 3 months). Two (11.8%) HLA-DRB1∗1502-positive patients had one relapse each, and six (35.3%) HLA-DRB1∗1502-negative patients had one to three relapses. ConclusionsHLA-DRB1∗1502 was significantly associated with anti-NMDARE in our patients. Most patients had good outcomes. HLA-DRB1∗1502-positive patients tended to require a longer time to achieve a good outcome but had less frequent relapses than HLA-DRB1∗1502-negative patients.

Clinical evaluation of a new molecular method for the detection of multidrug-resistant microorganisms

IntroductionThe main objective of this work is to carry out the clinical validation of the trial with the AMR Direct Flow Chip starting from either nasal swabs, rectal swabs directly or from isolated strains to detect antibiotic resistance genes. MethodsWe developed the preclinical validation of the assay with 104 known bacterial isolates. A total of 210 nasal or rectal swab samples were analyzed. The AMR assay is based on multiplex PCR followed by reverse dot blot hybridization on DNA arrays fully automated by using the HS24 platform. ResultsBoth the sensitivity and specificity of the preclinical assay were 100%, with the 104 samples correctly identified. In the clinical validation, the sensitivity was 100% and the specificity was between 100% in nasal swabs and 97% in rectal swabs. ConclusionsThe AMR Direct Flow Chip® is a rapid and effective assay for the detection of multidrug-resistant microorganisms (MDR) from nasal and rectal swab samples.

Identification of a Novel Hb H Disease with Glucose-6-Phosphate Dehydrogenase Deficiency Using Whole Genome Sequencing

With the development of sequencing technology, more and more rare thalassemia types have been found. In this article, we found a novel Hb H disease combined with glucose-6-phosphate dehydrogenase (G6PD) deficiency through whole genome sequencing (WGS), which was verified by Sanger sequencing and polymerase chain reaction (PCR)-reverse dot-blot hybridization, respectively.

Substantial Improvement in Nontuberculous Mycobacterial Identification Using ASTA MicroIDSys Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry with an Upgraded Database.

Identifying Mycobacterium using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging. We evaluated the performance of MALDI-TOF MS in identifying nontuberculous mycobacteria (NTM) using the ASTA MicroIDSys system (ASTA Inc., Suwon, Korea) with the MycoDB v1.95s and upgraded MycoDB v2.0-beta databases. We tested 124 NTM isolates collected from Ogawa medium at Severance Hospital, Seoul, Korea, between January and April 2019. MicroIDSys scores were categorized into three groups: ≥140, reliable identification; 130–139, ambiguous identification; and <130, invalid identification. To validate the results, we used the reverse blot hybridization assay (Molecutech REBA MycoID, YD Diagnostics Corp., Korea). Initial analysis using MycoDB v1.95s resulted in 26.6% (33/124) reliable, 43.5% (54/124) ambiguous, and 29.8% (37/124) invalid identifications. Re-analysis using the upgraded MycoDB v2.0-beta database resulted in 94.4% (117/124) reliable, 4.0% (5/124) ambiguous, and 1.6% invalid (2/124) identifications. The percentage of reliable identifications that matched with the reference increased from 26.6% (33/124) with MycoDB v1.95s to 93.5% (116/124) with MycoDB v2.0-beta. The upgraded databases enable substantially improved NTM identification through deep learning in the inference algorithm and by considering more axes in the correlation analysis. MALDI-TOF MS using the upgraded database unambiguously identified most NTM species. Our study lays a foundation for applying MALDI-TOF MS for the simple and rapid identification of NTM isolated from solid media.

Hepatitis C virus genotypes and viremia in a tertiary hospital in Istanbul, Turkey.

The World Health Organization estimates that 71 million people with chronic HCV infection lived worldwide in 2015. HCV is a globally prevalent pathogen, that genotype1 is the most common. In this study, the prevalence of anti-HCV, distributions of HCV genotype, and viremia rates in patients with chronic hepatitis C were evaluated. In this retrospective single-center study, anti-HCV results of 197,081 patients were evaluated between 2017 and 2020. Quantitative HCV-RNA PCR tests were performed on the Rotor-Gene Q real-time PCR instrument. HCV genotypes determination of 546 samples was carried out with the Gen-C 2.0 Reverse Hybridization strip and HCV Genotype Plus Real-TM kit. The prevalence of anti-HCV was 0.95% and viremic HCV infection was 0.3% (610/197,081). HCV viremia rate was 33.17%. HCV viremia rate was highest in 2017 (52.36%) and the lowest in 2020 (18.3%) (p < 0.001). Genotype1 (72%) was the most common genotype, followed by genotype3 (14.1%), and genotype4 (8.8%). The most common subtypes were determined as genotype1b (56.2%) and genotype1a (13.2%). The viral load was higher in patients infected with genotype5. In this study, the rate of viremic HCV infection was found to be 0.3%. This rate was lower than the worldwide rate of HCV viremia. The distribution of HCV genotypes was like the global data. The identification of circulating genotypes and subtypes is essential for epidemiological purposes and remains important in the choice of treatment in patients with chronic HCV.

Consistency of Mycobacterium tuberculosis Complex Spoligotyping between the Membrane-Based Method and In Silico Approach.

ABSTRACTTo tackle the spread of tuberculosis (TB), epidemiological studies are undertaken worldwide to investigate TB transmission chains. Clustered regulatory interspaced short palindromic repeats (CRISPR) locus diversity, also called spoligotyping, is a widely used genotyping assay for the characterization of Mycobacterium tuberculosis complex (MTBC). We compared herein the spoligotyping of MTBC clinical isolates using a membrane-based method (following an initial PCR step) and whole-genome sequencing (WGS)-based method (i.e., in silico spoligotyping). All MTBC strains isolated at the Lyon University Hospital, France, between November 2016 and December 2020 were included (n = 597). Spoligotyping profiles were also used for species identification among the MTBC. Outputs of both methods were analyzed, and discrepant results were investigated thanks to CRISPRbuilder-TB. The overall agreement was 85.7%. Spacer discrepancies observed between the methods were due to the insertion of IS6110 within the direct repeat (DR) sequence upstream or downstream of spacers, mutated DR sequences, or truncated spacers. Discrepancies did not impact species identification. Although spoligotyping-based species identification was inconclusive for 29 isolates, SNP-based phylogeny conducted after WGS allowed the identification of 23 M. tuberculosis (Mtb), 2 M. canettii, and 4 mixed MTBC infections. WGS yielded very few discrepancies compared to membrane-based spoligotyping. Overall agreement was significantly improved (92.4%) by the CRISPR locus reconstruction using CRISPRbuilder-TB for the MTBC isolates with the shared international type 53 in silico spoligotyping. A smooth transition from the membrane-based to the in silico-based genotyping of M. tuberculosis isolates is, therefore, possible for TB diagnosis and epidemiologic survey.IMPORTANCE Whole-genome sequencing (WGS) has profoundly transformed the perspectives of tuberculosis (TB) diagnosis, providing a better discriminatory power to determine relatedness between Mycobacterium tuberculosis complex (MTBC) isolates. Previous genotyping approaches, such as spoligotyping consisting of an initial PCR step followed by reverse dot hybridization, are currently being replaced by WGS. Several pipelines have been developed to extract a spoligotype from WGS data (in silico spoligotyping) allowing for the continuity of MTBC molecular surveys before and after WGS implementation. The present study found very good overall agreement between hybridization to membrane-based spoligotyping and in silico spoligotyping, indicating the possibility of a smooth transition from the traditional to the in silico-based genotyping of MTBC isolates for TB diagnosis and epidemiological survey.

Association of high-risk human papillomavirus with ocular surface squamous neoplasia: a case-control study in Mexico.

To investigate the association of high-risk hu-man papilloma virus (HR-HPV) and other risk factors with ocular surface squamous cell neoplasia (OSSN). We obtained DNA from 22 fresh frozen OSSN tissues and 22 pterygia as controls, we used a broad-spectrum HPV DNA amplification short PCR fragment to identify HPV infection in all specimens and then genotyped HPV by a reverse hybridization line probe assay. We also obtained demographic, sun exposure, and tobacco consump-tion information. HR-HPV frequency was 40.9% in the OSSN group and 4.5% in the pterygia group (p=0.009). After covariate adjustment, OSSN was associated with HR-HPV (OR=16.3, 95%CI=1.2,218.1, p=0.03) and sunburn (OR=10.8, 95%CI=1.8,86.0, p=0.02). Ocular surface squamous cell neoplasia is a multifactorial disease. The strong association between HR-HPV and OSSN, suggests that HR-HPV could play an etiological role in OSSN development.

Trends in cocirculation of oncogenic HPV genotypes in single and multiple infections among theunvaccinated community.

Cocirculation of multiple human papillomavirus (HPV) infections with low, probably high, and high‐risk genotypes are to be associated with various grades of infections and cancer progression. The oncogenic high‐risk HPVs are distributed and cocirculated throughout the world. This study was investigated to identify HPV genotypes related to genital disorders in unvaccinated women. The subjects were referred from clinics to a molecular lab for HPV testing in Iran as a low‐coverage vaccinated country. HPVs DNAs of cervical scrapping and genital tissue specimens of 1,133 un‐vaccinated women were genotyped using an in vitro diagnostic line probe (reverse hybridization) assay. In addition, phylogenetic trees were constructed on 100 MY09/MY11 polymerase chain reaction (PCR) amplicons of common genotypes of HPV L1 gene by Sanger sequencing. The mean age of the population study was 32.7 ± 8.0 and the mean age of HPV‐positive cases was 31.6 ± 7.8. HPV DNA was detected in 57.8% (655/1133) of women subjects and 42.2% (478/1133) of cases were undetected. Among 655 HPV‐positive cases, 639 subjects (56.4%) were related to defined genotypes and 16 subjects (1.4%) were untypeable. The highest prevalence rate of HPV genotypes was identified in the 25–34 years. The top 6 dominant HPVs in single and multiple genotypes were HPV6 (284/655 [43.4%]), HPV16 (111/655 [16.9%]), HPV31 (72/655 [11%]), HPV53 (67/655 [10.2%]), HPV11 (62/655 [9.5%]), and HPV52 (62/655 [9.5%]). Moreover, single, multiple and untypeable HPV genotypes were diagnosed as follows: 1 type (318/655 [48.5%]), 2 types (162/655 [24.8%]), 3 types (83/655 [12.7%]), 4 types (42/655 [6.5%]), more than 5 types (34/655 [5.3%]), and 1.4% un‐typeable subjects. The sequenced partial L1 gene of HPV genotypes (GenBank databases under the accession numbers: MH253467‐MH253566) confirmed and determined the cocirculated HPV genotypes' origins and addressed helpful insights into the future viral epidemiology investigations. Multiple HPV infections and cocirculation of various oncogenic HPV genotypes among the normal population (women and men) with asymptomatic forms are still challenging in unvaccinated communities. The preventive and organized surveillance programs for HPV screening are needed to be considered and compiled by health policy makers of low or unvaccinated countries.

β-Thalassemia Intermedia: Interaction of α-Globin Gene Triplication With β-thalassemia Heterozygous in Spain.

ObjectivesTo verify with hematimetric data that the diagnosis and clinical grade of β-TI can be established when a triplication of alpha genes (αααanti 3.7) and heterozygous β-thalassemia coexist.Materials and MethodsRetrospective study in which 73 patients of Caucasian origin participated, who simultaneously showed a triplication or quadruplication of genes α and β-thalassemia.Screening for the most frequent α-thalassemia mutations as well as gene triplication (αααanti 3.7) was carried out by multiplex PCR followed by reverse hybridization with a commercial Alpha-Globin StripAssay kit and confirmed by MLPA (Multiplex ligation-dependent probe amplification). The molecular diagnosis of β-thalassemia was carried out by automatic sequencing according to the Sanger method.ResultsThe genotypes have been classified into three groups according to the number of α globin genes and the severity of the alteration in the β globin gene. All had a mutation in the HBB gene (β0-thalassemia, β+-thalassemia severe, and β+-thalassemia mild). Group I patients who have coherent 6 α genes and groups II and III with 5 α globin genes. In group III, the patients were carriers of mutations affecting the β and δ globin genes. The most significant hematological parameters were hemoglobin levels, MCV, RDW, and the percentage of Hb F.ConclusionsIn group I, regardless of the distribution of the 6 α globin genes, homozygous triplication (ααα/ααα) or heterozygous quadruplication (αααα/αα), the association with heterozygous β-thalassemia results in severe to moderate anemia that may or may not require transfusion therapy, is the severity of the HBB gene mutation that would determine the clinical variation. Group II patients phenotypically behaved like mild thalassemia intermedia, except for one case that presented thalassemic trait because it also presented an associated α-thalassemia (ααα/-α3.7). Finally, group III patients behaved as a thalassemic trait since all were carriers of mutations that increase the overexpression of γ genes.

Investigation of frequency and typing of human papillomavirus among genital warts using a reverse dot blot hybridization approach

BackgroundHuman papillomavirus (HPV) is the most common sexually transmitted infection worldwide, affecting about 80% of women up to the age of 50. The persistent infection of high risk-HPV types (HR-HPV) is the leading cause of cervical cancer, the fourth most common cancer of women. Therefore, we aimed to evaluate the frequency and typing of HPV in the genital lesions in the Iranian population.MethodsThis descriptive-analytic study was conducted on a population in the South-Khorasan province of Iran. All of the participants were sexually active and were checked for evident cervical warts. Biopsy samples were collected from various lesions, and all samples were tested for detection and genotyping of HPV using a reverse dot blot hybridization method (HPV direct flow CHIP).ResultsIn overall, 370 samples were evaluated; 10 cases (2.7%) were male and the rest were female. The mean age of patients was 33.3 ± 8.5 years, of which 48.1% were in the age range from 25 to 36 years. Among the samples, 345 (93.2%) were positive for HPV-DNA; the low risk HPV types (LR-HPV) and HR-HPV were identified among 80.9% and 15.5% of tissue samples, respectively. Among the LR-HPV, HPV-6, 11, 42 and 54 were the most common genotypes, and HPV-16 and 39 were prevalent HR-HPV types detected. The number of pregnancies, marriage age, and partner infection were not significantly related to the HPV types. Types 42 had a declining pattern toward aging, and HPV-11 was increasing toward aging.ConclusionThe number of samples with HR-HPV was rather high. Due to the greater frequency of infection in the age range of 25–35 years, it is advised that all individuals referred to gynecological clinics at gestational age be tested for HPV types.

Physcomitrium patens pentatricopeptide repeat protein PpPPR_32 is involved in the accumulation of psaC mRNA encoding the iron sulfur protein of photosystem I.

Pentatricopeptide repeat (PPR) proteins are involved in RNA metabolism and also play a role in posttranscriptional regulation during plant organellar gene expression. Although a hundred of PPR proteins exist in the moss Physcomitrium patens, their functions are not fully understood. Here, we report the function of P-class PPR protein PpPPR_32 in P. patens. A transient expression assay using green fluorescent protein demonstrated that the N-terminal region of PpPPR_32 functions as a chloroplast-targeting transit peptide, indicating that PpPPR_32 is localized in chloroplasts. PpPPR_32 knockout mutants grew autotrophically but with reduced protonema growth and the poor formation of photosystem I (PSI) complexes. Quantitative real-time reverse transcription-polymerase chain reaction and RNA gel blot hybridization analyses revealed a significant reduction in the transcript level of the psaC gene encoding the iron sulfur protein of PSI but no alteration to the transcript levels of other PSI genes. This suggests that PpPPR_32 is specifically involved in the expression level of the psaC gene. Our results indicate that PpPPR_32 is essential for the accumulation of psaC transcript and PSI complexes.

Association of Human Papillomavirus and Epstein-Barr Virus Infection with Tonsil Cancer in Northeastern Thailand.

Background:Human papillomavirus (HPV) and Epstein-Barr virus (EBV) are associated with head and neck cancer, including tonsil cancer (TC) in the oropharyngeal area. Increasing incidence of HPV and EBV infection in different cancer tissues of oropharynx in both epithelial and lymphoid tissues, have been reported. However, little is known about association of these tumor viruses with TC in the Thai population. Here, we investigated the prevalence of HPV and EBV infection in different histology of TC and their association with TC from Thai patients. Methods:Eighty-three exfoliated tonsil cells from non-cancer controls (NCC) and 65 formalin-fixed paraffin-embedded TC tissues (TC) that were histologically classified as tonsillar squamous-cell carcinoma (TSCC) or diffuse large B-cell lymphoma (DLBCL) were studied. Prevalence of HPV and EBV infection was determined by real-time PCR. HPV genotyping was performed by reverse line blot hybridization and HPV genome status was investigated by multiplex qPCR. Localization of EBV infection was determined by EBER in situ hybridization. Results:Infection of HPV and EBV in TC cases was 16.9% and 30.8%, whereas in exfoliated tonsil cells was 1.2% and 66.3% respectively. HPV infection was significantly higher in TSCC (30.6%) than DLBCL samples (13.8%). HPV58 was commonly detected and presented as an integrated form in TSCC, whereas only episomal form was found in DLBCL. EBV infection was significantly higher in DLBCL (44.8%) than TSCC samples (19.4%), and detected in both lower than among exfoliated tonsil cell samples (66.3%). By EBER in situ hybridization in TSCC, EBV infection localized both in epithelial cells and infiltrating lymphocytes. The co-occurrence of HPV and EBV infection was 11.11% and 13.79% of TSCC and DLBCL, respectively, was associated with well-differentiated TSCC. Conclusion:HPV and EBV infection was significantly involved in a specific TC tissue, and associated with a good clinical outcome in TSCC.

Study on Thalassemia in Han Population in Sanya of Hainan Province

To study the distribution characteristics of thalassemia genotype in Han Population in Sanya of Hainan Province. Gap PCR and reverse dot hybridization were used to detect and analyze the thalassemia gene in 572 suspected thalassemia carriers of Han Population in Sanya. Among the 572 Han Population in Sanya, 271 cases of thalassemia gene abnormality were detected, among which 161 cases were founded to be carriers of α-thalassemia gene. A total of 9 genotypes were detected, in the following order of the detection rate was －－SEA/αα，－α3.7/αα，－α4.2/αα，－－SEA/－α3.7，－－SEA/－α4.2，－α4.2/－α4.2，－α3.7/－α4.2，－α3.7/－α3.7，－－SEA/－－SEA. Among them, the deletion type (－－SEA/αα) in southeast Asia was the most common, accounting for 66 cases. 99 cases of β-thalassemia were detected, there were 7 genotypes, all of which were heterozygous. The order of the detection rate was CD41-42/βN, IVS-II-654/βN, CD17/βN, CD71-72/βN, -28/βN, －29/βN, CD27-28/βN. Among them, CD41-42/βN was the most common, accounting for 51 cases. In addition, 11 cases of combined α and β thalassemia were detected. Five kinds of genotypes were checked out, the order of detection rate was －α3.7/αα composite CD41-42/βN, －－SEA/αα composite IVS-II-654/βN, －α4.2/－α4.2 composite CD41-42/βN, －α4.2/αα composite －29/βN , －－SEA/ －α4.2 composite CD41-42/βN. Han Population in Sanya of Hainan Province is a high-risk population of thalassemia, the genotype characteristics are different from other areas with high incidence of thalassemia in China. The main type of α-thalassemia is the deficiency mutation of southeast Asia, while CD41-42 heterozygous mutation is the main type of β-thalassemia.

Human papillomavirus genotyping in high-grade squamous intraepithelial lesion.

Human papillomavirus infection is one of the most common sexually transmitted infections. Long-term exposure to the HPV leads to high-grade squamous intraepithelial lesions affecting cervical cancer. Knowledge about the distribution of HPV genotypes is crucial to guide the introduction of prophylactic vaccines. We aimed the genotype distribution in patients reporting due to abnormal Pap - smear tests. We provide a prospective observational cohort study. We obtained material from 428 women registered to Provincial Hospital in Poznan and Specialist Medical Practice in 2018-2020. In the current study, we analyze results from the first 110 inclusions with the diagnosis of HSIL from a cervical biopsy. The probe for the molecular test was collected with a combi brush and passed to an independent, standardized laboratory. HPV detection was done using PCR followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe assay. Sequence analysis was performed to characterize HPV-positive samples with unknown HPV genotypes. The molecular test detected DNA of 41 HPV genotypes. We performed statistical analyzes using the STATISTICA package 13.3. We found that 98.2% of patients received HPV-positive test results. The most frequent HPV genotype was 16, which assumed for 54.1%. In patients negative for HPV 16, the percentage decreased with increasing age. We detected that the following HPV types are next most common: HPV 31 (16.2 %), HPV 52 (11.7%), HPV 51 (9.9%), HPV 18 (9.0%), HPV 33 (9%). Moreover, thyroid diseases were the most common comorbidities and occurred in 15 patients CONCLUSIONS: To our knowledge, this study is the most extensive assessment of HPV genotypes in HSIL diagnoses in Poland.

Human papillomavirus genotyping in low-grade squamous intraepithelial lesions.

Human papillomavirus infection is one of the most common sexually transmitted infections. Histological LSIL in 70-80% of cases will regress spontaneously, while a subset is associated with residual risk for a future precancerous lesion. This study evaluates the performance of HPV genotypes for LSIL preceded by normal or mildly abnormal Pap smear. We provide a prospective observational cohort study. We obtained material from 428 women registered to Specialist Medical Practice and Provincial Hospital in Poznań in 2018-2021. In the current study, we analyze results from the first 112 inclusions with the diagnosis of LSIL from a cervical biopsy. The probe for the molecular test was collected with a combi brush and passed to the independent, standardized laboratory. HPV detection was done using PCR followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe assay. Sequence analysis was performed to characterize HPV - positive samples with unknown HPV genotypes. The molecular test detected DNA of 42 HPV genotypes. We performed statistical analyzes using the STATISTICA package 13.3. We found that 77.7% of patients received HPV-positive test results. The most frequent HPV genotype was 16, which was assumed for 22.3%. We detected that following HPV types are next most common: HPV 56 (11.6%), HPV 52 (8.9%), HPV 31 (8.0%) and HPV 51 (8.0%). Among HPV 16-negative women, the vast majority are those living in the town (p = 0.048). Moreover, thyroid diseases were the most common comorbidities. To our knowledge, this study is the most extensive assessment of HPV genotypes in LSIL diagnoses in Poland.

Investigation of Hepatitis C Virus (HCV) Genotypes by "Reverse Hybridisation Strip Assay" and DNA Sequence Analysis Methods

Hepatitis C Virus (HCV) is a virus that is estimated to infect approximately 185 million people worldwide and causes 350 000 deaths each year. The target in HCV treatment is the sustained virological response (SVR), and the most important virological factors determining SVR are genotype and baseline HCV-RNA levels. In this study, it was aimed to compare the HCV genotypes and subtypes detected by the "line probe assay (LIPA)" method with sequence analysis. HCV genotypes and subtypes were investigated by line probe assay (LIPA) (NLM analytica, Italy) and sequence analysis methods in 212 patients with chronic HCV diagnosis and with HCV RNA viral load of 10⁴ IU/ml and above. 5'UTR and core regions were studied in LIPA method and NS5B gene region was studied in the sequencing method. After amplifying 340 bp region in the NS5B gene region with the hemi-nested PCR method, sequence analysis (ABI 3500 Genetic Analyzer [Applied Biosystems, USA]) was performed. Statistical analysis of the study was determined by using TURCOSA Analytics program. In the study, HCV genotypes and subtypes that were determined in 212 patients by LIPA method were; 40 (18.8%) genotype 1a, 97 (45.75%) genotype 1b, 7 (3.3%) genotype 2, 12 (5.6%) genotype 2a/c, 2 (0.94%) genotype 2b, 19 (8.96%) genotype 3, 16 (7.55%) genotype 4, 1 (0.47%) genotype 4a, 15 (7.5%) genotype 4c/d, 1 (0.47%) genotype 4h and 2 (0.94%) genotype 5. The HCV genotypes and subtypes determined in 212 patients by sequence analysis were; 20 (9.43%) genotype 1a, 118 (55.6%) genotype 1b, 16 (7.55%) genotype 2a, 4 (1.89%) genotype 2b, 1 (0.47%) genotype 2k, 21 (9.91%) genotype 3a, 2 (0.94%) genotype 4a, 28 (13.21%) genotype 4d and 2 (0.94%) genotype 5a. The difference in results between the two methods was found to be statistically significant (p< 0.001). According to the viral load-genotype relationship, the highest viral load was detected in genotype 1a patients (p< 0.001). In conclusion, in order to determine HCV genotypes and subtypes in our study, LIPA and sequence analysis methods were compared and the genotype compatibility between the two methods were determined as 97.5% on the basis of genotype and 19% on the basis of subtype. Since the direct-acting antiviral (DEA) treatment protocol in chronic HCV patients is planned according to the genotype/subtype determination, the inconsistency of the results obtained in the LIPA method, which is routinely widely used, with the sequence analysis method is remarkable, and it was concluded that these results should be supported by studies containing more samples.

Reverse dot blot hybridization assay - An expeditious diagnostic tool for drug resistant TB.

Tuberculosis (TB) is one of the leading infectious diseases that is highly transmissible. It is reported to cause approximately 10 million infections and 1.4 million deaths globally in the year of 2019.¹ Prompt detection and treatment of TB are essential to intercept the proliferation of the disease. In the case of drug- resistant TB, failure to detect the resistance of anti TB drugs may give rise to extensively drug- resistant tuberculosis (XDR-TB). Thus, the rapid detection of drug resistance is vital;, however, the current standard drug susceptibility tests (DST) may take up to 12 weeks raising an alarming concern.² A study was done in China by Li Wan et al, on the accuracy of the reverse dot blot hybridization assay (RDBH) for rapidly detecting the resistance of four anti TB drugs (rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB)) in mycobacterium tuberculosis (MTB) isolates.³ Continuous...

Bacterial vaginosis, cervical Human Papillomavirus infection and cervical cytological abnormalities in adult women in Central Brazil: A cross-sectional study

Introduction: Bacterial vaginosis is the most common cause of vaginal discharge and occurs when there is an imbalance in the vaginal microbiota, predominantly composed of Lactobacillus spp. Human Papillomavirus is the most common sexually transmitted virus in the world. Persistent infection with high-risk Human Papillomavirus genotypes is the main cause of the development of cervical intraepithelial neoplasia and cervical cancer. Objective: To investigate the association between bacterial vaginosis and cervical Human Papillomavirus infection and between bacterial vaginosis and cervical cytological abnormalities in adult women. Methods: Cross-sectional study carried out in a gynecology outpatient clinic of the public health network. A total of 202 women were included in the study and underwent gynecological examination with cervical specimen collection. Cervical cytopathological examinations and bacterioscopy by the Nugent method were performed to identify bacterial vaginosis, and PCR and reverse hybridization were carried out for Human Papillomavirus detection and genotyping. Bivariate analysis was performed to investigate the association between bacterial vaginosis and cervical Human Papillomavirus infection, and between bacterial vaginosis and cervical cytological abnormalities. The odds ratio was calculated, with the respective 95% confidence intervals (95%CI) and 5% significance level (p≤0.05). Results: The prevalence of bacterial vaginosis was 33.2% (67/202), the prevalence of cervical Human Papillomavirus infection was 38.6% (78/202) and the prevalence of cervical cytological abnormalities was 6.0% (12/202). Bivariate analysis showed no significant association between bacterial vaginosis and cervical Human Papillomavirus infection (OR 0.69; 95% CI 0.37–1.27; p=0.23), or between bacterial vaginosis and cervical cytological abnormalities (OR 0.65; 95%CI 0.17–2.50; p=0.54). Conclusion: In this study, bacterial vaginosis did not represent a risk factor for cervical Human Papillomavirus infection or for the presence of cervical cytological abnormalities in the investigated adult women.

Clinical and genetic characteristics of hemoglobin H disease in Iran

Hemoglobin H (Hb H) disease is a subtype of α-thalassemia caused by deletional and/or non-deletional mutations in three alpha-globin genes in which the various genotypes determine the disease severity. This study was aimed to investigate the frequency of alpha gene mutations and genotypes and their correlation with hematological and clinical characteristics in Iran. Among 202 patients diagnosed with Hb H disease through a national study in Iran according to standard methods, we had access to the hematologic and clinical findings and genetic data of 101 patients in whom genetic study was performed. Genomic DNA from peripheral blood was extracted and analyzed for identification of α-globin gene mutations using Multiplex Gap Polymerase Chain Reaction, Reverse Hybridization Assay, and finally Direct DNA Sequencing method. Twenty-one different mutations and thirty genotypes were detected in 101 patients with Hb H disease. In total, 39 patients (38.6%) were deletional and 62 patients (61.4%) were non-deletional type of the disease. The -- MED mutation was highly prevalent in almost half of the patients (56.4%). Among various genotypes, –MED/-a3.7 (29.7%) and -α20 .5/-α5NT (6.9%) were the most prevalent genotypes found in the studied group. Patients with non-deletional type presented with more severe hematological and clinical findings. Hb H percentage and serum ferritin levels were significantly higher in non-deletional patients in comparison to the deletional group (p < 0.05). 12 (11.9%) and 40 (39.6%) out of 101 patients were on regular and occasional transfusions, respectively. 83% of those with regular transfusion belonged to the non-deletional group. Among transfusion-dependent patients, –MED/αCSα and α20 .5/-α5NT were the most common genotypes. In this study, two patients with -α20 .5/αCSα and –MED/α−5NT genotypes experienced thrombotic events. This study indicated that although non-deletional genotypes of Hb H disease were responsible for more clinical severity of the disease, due to the presence of severe phenotypes even in deletional types, no definite correlation was found between genotype and phenotype.

Human papillomavirus prevalence and genotype distribution in Vietnamese male patients between 2016 and 2020.

Human papillomavirus (HPV) infection in men is a serious issue because it is associated with genital warts, anogenital cancers, and HPV transmission to their sex partners. This study aimed to investigate the prevalence and genotypes of HPVs in Vietnamese male patients hospitalized with sexually transmitted infection (STI) symptoms between 2016 and 2020 by using polymerase chain reaction and reverse dot blot hybridization analysis. HPV DNA was detected in 191/941 (20.3%) penile cell samples. The HPV patient's mean age was 30.3 in the range of 16- and 69-year-old. The highest HPV prevalence (84.7%) was found in patients between 20- and 39-year-old. A total of 313 HPV genotypes were identified. The multiple-infection rate was 42.9%. The most common high-risk (HR)-HPV genotypes were HPV-16 (8.0%), HPV-51 (7.7%), HPV-52 (4.8%), HPV-56 (4.2%), and HPV-18 (3.8%). Furthermore, HPV-11 and HPV-6 genotypes were the two most common low-risk (LR)-HPV genotypes with the rate of 36.7% and 21.4%, respectively. Notably, HPV-52 was found circulating in Vietnam for the first time. In conclusion, this study results showed that HPV prevalence in Vietnamese male patients was common and diverse. In addition, regarding public health and cancer prevention, the inclusion of the HPV vaccination into the national vaccination program for both men and women is recommended.