Evaluation of an eight marker-panel including long mononucleotide repeat markers to detect microsatellite instability in colorectal, gastric, and endometrial cancers.

BACKGROUND: Accurate determination of microsatellite instability (MSI) status is critical for optimal treatment in cancer patients. Conventional MSI markers can sometimes display subtle shifts that are difficult to interpret, especially in non-colorectal cases. We evaluated an experimental eight marker-panel including long mononucleotide repeat (LMR) markers for detection of MSI. METHODS: The eight marker-panel was comprised of five conventional markers (BAT-25, BAT-26, NR-21, NR-24, and NR-27) and three LMR markers (BAT-52, BAT-59 and BAT-62). MSI testing was performed against 300 specimens of colorectal, gastric, and endometrial cancers through PCR followed by capillary electrophoresis length analysis. RESULTS: The MSI testing with eight marker-panel showed 99.3% (295/297) concordance with IHC analysis excluding 3 MMR-focal deficient cases. The sensitivity of BAT-59 and BAT-62 was higher than or comparable to that of conventional markers in gastric and endometrial cancer. The mean shift size was larger in LMR markers compared to conventional markers for gastric and endometrial cancers. CONCLUSIONS: The MSI testing with eight maker-panel showed comparable performance with IHC analysis. The LMR markers, especially BAT-59 and BAT-62, showed high sensitivity and large shifts which can contribute to increased confidence in MSI classification, especially in gastric and endometrial cancers. Further study is needed with large number of samples for the validation of these LMR markers.

Development of diagnostic tests for pathogen identification and detection of antimicrobial resistance on WHO global priority pathogens using modular real-time nucleic acid amplification test.

BACKGROUND: Concerns regarding antimicrobial resistance (AMR) have resulted in the World Health Organization (WHO) designating so-called global priority pathogens (GPPs). However, little discussion has focused on the diagnosis of GPPs. To enable the simultaneous identification of pathogens and AMR, we developed a modular real-time nucleic acid amplification test (MRT-NAAT). METHODS: Sequence-specific primers for each modular unit for MRT-NAAT pathogen identification and AMR sets were designed. The composition of the reaction mixture and the real-time PCR program were unified irrespective of primer type so to give MRT-NAAT modularity. Standard strains and clinical isolates were used to evaluate the performance of MRT-NAAT by real-time PCR and melting curve analysis. Probit analysis for the MRT-NAAT pathogen identification set was used to assess the limit of detection (LoD). RESULTS: The MRT-NAAT pathogen identification set was made up of 15 modular units 109-199 bp in product size and with a Tms of 75.5-87.5 °C. The LoD was < 15.548 fg/µL, and nine modular units successfully detected the target pathogens. The MRT-NAAT AMR set included 24 modular units 65-785 bp in product size with a Tms of 75.5-87.5 °C; it showed high performance for detecting GPP target genes and variants. CONCLUSIONS: MRT-NAAT enables pathogen identification and AMR gene detection and is time-effective. By unifying the reaction settings of each modular unit, the modularity where combinations of primers can be used according to need could be achieved. This would greatly help in reflecting the researcher's need and the AMR status of a certain region while successfully detecting pathogens and AMR genes.

Incidence and seasonality of respiratory viruses causing acute respiratory infections in the Northern United Arab Emirates.

BACKGROUND: The data on the seasonality of respiratory viruses helps to ensure the optimal vaccination period and to monitor the possible outbreaks of variant type. OBJECTIVES: This study was designed to describe the molecular epidemiology and seasonality of acute respiratory infection (ARI)-related respiratory viruses in the United Arab Emirates (UAE). METHODS: Both upper and lower respiratory specimens were collected for the analysis from all the patients who visited the Sheikh Khalifa Specialty Hospital (SKSH) with ARI for over 2 years. The multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) test was used to detect respiratory viruses, which include human adenovirus, influenza virus (FLU) A and B, respiratory syncytial virus, parainfluenza viruses, human rhinovirus (HRV), human metapneumovirus, human enterovirus, human coronavirus, and human bocavirus. RESULTS: A total of 1,362 respiratory samples were collected from 733 (53.8%) male and 629 (46.2%) female patients with ARI who visited the SKSH between November 2015 and February 2018. The rRT-PCR test revealed an overall positivity rate of 37.2% (507/1362). The positive rate increased during winter; it was highest in December and lowest in September. FLU was the most frequently detected virus (273/1362 [20.0%]), followed by human rhinovirus (146/1362 [10.7%]). The FLU positivity rate showed two peaks, which occurred in August and December. The peak-to-low ratio for FLU was 2.26 (95% confidence interval: 1.52-3.35). CONCLUSIONS: The pattern of FLU in the UAE parallels to that of temperate countries. The trend of the small peak of FLU in the summer suggests a possibility of semi-seasonal pattern in the UAE.

Verification of a method using magnetic bead enrichment and nucleic acid extraction to improve the molecular detection of bacterial contamination in blood components.

Bacterial contamination of blood products poses a significant risk in transfusion medicine. Platelets are particularly vulnerable to bacterial growth because they must be stored at room temperature with constant agitation for >5 days. The limitations of bacterial detection using conventional methods, such as blood cultures and lateral flow assays, include the long detection times, low sensitivity, and the requirement for substantial volumes of blood components. To address these limitations, we assessed the performance of a bacterial enrichment technique using antibiotic-conjugated magnetic nanobeads (AcMNBs) and real-time PCR for the detection of bacterial contamination in plasma. AcMNBs successfully captured >80% of four bacterial strains, including Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Klebsiella pneumoniae, in both plasma and phosphate-buffered saline. After 24-h incubation with bacterial enrichment, S. aureus and B. cereus were each detected at 101 CFU/mL in all trials (5/5), E. coli at 101 CFU/mL in 1/5 trials, and K. pneumoniae at 10² CFU/mL in 4/5 trials. Additionally, without incubation, the improvement was also achieved in samples with bacterial enrichment, S. aureus at 10² CFU/mL and B. cereus at 101 CFU/mL in 1/5 trials each, E. coli at 10³ CFU/mL in 3/5 trials, and K. pneumoniae at 10¹ CFU/mL in 2/5 trials. Overall, the findings from this study strongly support the superiority of bacterial enrichment in detecting low-level bacterial contamination in plasma when employing AcMNBs and PCR.IMPORTANCEThe study presents a breakthrough approach to detect bacterial contamination in plasma, a critical concern in transfusion medicine. Traditional methods, such as blood cultures and lateral flow assays, are hampered by slow detection times, low sensitivity, and the need for large blood sample volumes. Our research introduces a novel technique using antibiotic-conjugated magnetic nanobeads combined with real-time PCR, enhancing the detection of bacteria in blood products, especially platelets. This method has shown exceptional efficiency in identifying even low levels of four different species of bacteria in plasma. The ability to detect bacterial contamination rapidly and accurately is vital for ensuring the safety of blood transfusions and can significantly reduce the risk of infections transmitted through blood products. This advancement is a pivotal step in improving patient outcomes and elevating the standards of care in transfusion medicine.

Relationship between CES2 genetic variations and rifampicin metabolism.

OBJECTIVES: Rifampicin is known to be deacetylated in vivo, resulting in its metabolite 25-desacetyl rifampicin, but the enzyme metabolizing rifampicin and the association of this process with any genetic variation have not yet been elucidated. In this study, genetic variations of a surrogate enzyme, carboxylesterase 2 (CES2), and their association with the metabolism of this drug, were investigated. METHODS: Plasma concentrations of rifampicin and 25-desacetyl rifampicin were measured in 35 patients with tuberculosis receiving a first-line antituberculosis treatment. Direct PCR-based sequencing of the CES2 gene, covering all 12 exons, the 5'-untranslated region (UTR), the 3'-UTR and intronic and promoter regions, was performed. A dual luciferase reporter assay was carried out to assess whether variations in the promoter region affected the transcription of this gene. RESULTS: Ten variations were detected, of which two were in the candidate promoter region, five in introns and three in the 3'-UTR. One of the variations in the 3'-UTR was a novel variation. Genotypes at three closely linked variations (c.-2263A > G, c.269-965A > G and c.1612 + 136G > A) and c.1872*302_304delGAA were associated with significantly different plasma rifampicin concentrations. The mean plasma rifampicin concentration significantly increased with the number of risk alleles at the three closely linked variations, while the plasma concentration decreased along with an increase in the number of risk alleles at c.1872*302_304delGAA. When HepG2 cells were transfected with a luciferase reporter construct bearing the c.-2263G allele, luciferase activities were consistently decreased (by 5%-10%) compared with those harbouring the c.-2263A sequence. CONCLUSIONS: Variations in CES2, especially c.-2263A > G in the promoter region, may alter rifampicin metabolism by affecting expression of the gene.

Factors influencing oral microbiome analysis: from saliva sampling methods to next-generation sequencing platforms.

The exploration of oral microbiome has been increasing due to its relatedness with various systemic diseases, but standardization of saliva sampling for microbiome analysis has not been established, contributing to the lack of data comparability. Here, we evaluated the factors that influence the microbiome data. Saliva samples were collected by the two collection methods (passive drooling and mouthwash) using three saliva-preservation methods (OMNIgene, DNA/RNA shield, and simple collection). A total of 18 samples were sequenced by both Illumina short-read and Nanopore long-read next-generation sequencing (NGS). The component of the oral microbiome in each sample was compared with alpha and beta diversity and the taxonomic abundances, to find out the effects of factors on oral microbiome data. The alpha diversity indices of the mouthwash sample were significantly higher than that of the drooling group with both short-read and long-read NGS, while no significant differences in microbial diversities were found between the three saliva-preservation methods. Our study shows mouthwash and simple collection are not inferior to other sample collection and saliva-preservation methods, respectively. This result is promising since the convenience and cost-effectiveness of mouthwash and simple collection can simplify the saliva sample preparation, which would greatly help clinical operators and lab workers.

Comparative Analysis of Short- and Long-Read Sequencing of Vancomycin-Resistant Enterococci for Application to Molecular Epidemiology.

Vancomycin-resistant enterococci (VRE) are nosocomial pathogens with genetic plasticity and widespread antimicrobial resistance (AMR). To prevent the spread of VRE in the hospital setting, molecular epidemiological approaches such as pulsed-field gel electrophoresis and multilocus sequence typing have been implemented for pathogen outbreak surveillance. However, due to the insufficient discriminatory power of these methods, whole-genome sequencing (WGS), which enables high-resolution analysis of entire genomic sequences, is being used increasingly. Herein, we performed WGS of VRE using both short-read next-generation sequencing (SR-NGS) and long-read next-generation sequencing (LR-NGS). Since standardized workflows and pipelines for WGS-based bacterial epidemiology are lacking, we established three-step pipelines for SR- and LR-NGS, as a standardized WGS-based approach for strain typing and AMR profiling. For strain typing, we analyzed single-nucleotide polymorphisms (SNPs) of VRE isolates and constructed SNP-based maximum-likelihood phylogenies. The phylogenetic trees constructed using short and long reads showed good correspondence. Still, SR-NGS exhibited higher sensitivity for detecting nucleotide substitutions of bacterial sequences. During AMR profiling, we examined AMR genes and resistance-conferring mutations. We also assessed the concordance between genotypic and phenotypic resistance, which was generally better for LR-NGS than SR-NGS. Further validation of our pipelines based on outbreak cases is necessary to ensure the overall performance of pipelines.

Zika Virus Infection During Research Vaccine Development: Investigation of the Laboratory-Acquired Infection via Nanopore Whole-Genome Sequencing.

Zika virus (ZIKV) emerged as a serious public health problem since the first major outbreak in 2007. Current ZIKV diagnostic methods can successfully identify known ZIKV but are impossible to track the origin of viruses and pathogens other than known ZIKV strains. We planned to determine the ability of Whole Genome Sequencing (WGS) in clinical epidemiology by evaluating whether it can successfully detect the origin of ZIKV in a suspected case of laboratory-acquired infection (LAI). ZIKV found in the patient sample was sequenced with nanopore sequencing technology, followed by the production of the phylogenetic tree, based on the alignment of 38 known ZIKV strains with the consensus sequence. The closest viral strain with the consensus sequence was the strain used in the laboratory, with a percent identity of 99.27%. We think WGS showed its time-effectiveness and ability to detect the difference between strains to the level of a single base. Additionally, to determine the global number of LAIs, a literature review of articles published in the last 10 years was performed, and 53 reports of 338 LAIs were found. The lack of a universal reporting system was worrisome, as in the majority of cases (81.1%), the exposure route was unknown.

Molecular detection of bacterial contamination in plasma using magnetic-based enrichment.

Bacterial contamination of blood products is a major problem in transfusion medicine, in terms of both morbidity and mortality. Platelets (PLTs) are stored at room temperature (under constant agitation) for more than 5 days, and bacteria can thus grow significantly from a low level to high titers. However, conventional methods like blood culture and lateral flow assay have disadvantages such as long detection time, low sensitivity, and the need for a large volume of blood components. We used real-time polymerase chain reaction (PCR) assays with antibiotic-conjugated magnetic nanobeads (MNBs) to detect enriched Gram-positive and -negative bacteria. The MNBs were coated with polyethylene glycol (PEG) to prevent aggregation by blood components. Over 80% of all bacteria were captured by the MNBs, and the levels of detection were 101 colony forming unit [CFU]/mL and 102 CFU/mL for Gram-positive and -negative bacteria, respectively. The detection time is < 3 h using only small volumes of blood components. Thus, compared to conventional methods, real-time PCR using MNBs allows for rapid detection with high sensitivity using only a small volume of blood components.

Factors associated with the risk of colorectal neoplasia in young adults under age 40.

BACKGROUND/AIMS: Recent epidemiologic studies have shown a continued increase in colorectal cancer incidence among younger adults. Little is known about the factors that contribute to the development of young-onset colorectal neoplasia (CRN). METHODS: A cross-sectional analysis was performed for individuals younger than 40 years who underwent colonoscopy in Seoul St. Mary's Hospital and its affiliated health screening center. High-risk CRN was defined as adenoma or sessile serrated lesion ≥ 10 mm, with three or more adenomas, villous histology, high grade dysplasia, or carcinoma. RESULTS: Of these 13,621 included participants, 2,023 (14.9%) had one and more CRN. Young patients with CRN tended to be elderly, male, obese, smoker, having a habit of drinking, and having comorbidities such as hypertension, dyslipidemia, diabetes, and chronic kidney disease. In a multivariate analysis adjusted for age, sex, obesity, smoking status, and alcohol intake, old age (odds ratio [OR], 1.086; 95% confidence interval [CI], 1.054 to 1.119), male sex (OR, 1.748; 95% CI, 1.247 to 2.451), obesity (OR, 1.439; 95% CI, 1.133 to 1.828), and smoking (OR, 1.654; 95% CI, 1.287 to 2.127) were independent risk factors for overall CRN. Obesity and smoking as two modifiable factors increased the risk for high-risk CRN even more than for overall CRN (OR, 1.734; 95% CI, 1.168 to 2.575 and OR, 1.797; 95% CI, 1.172 to 2.753, respectively). CONCLUSION: Obesity and smoking were modifiable risk factors for CRN in young adults. They increased the risk for highrisk CRN even more than for overall CRN. A colonoscopy might be beneficial for young individuals with these factors.

Novel multiplex PCR using dual-priming oligonucleotides for detection and discrimination of the Mycobacterium tuberculosis complex and M. bovis BCG.

We developed a novel multiplex PCR assay using dual-priming oligonucleotide primers targeting the RD1 gene for simultaneous identification of the Mycobacterium tuberculosis complex and M. bovis bacillus Calmette-Guérin (BCG). This assay would be useful both for detection of the M. tuberculosis complex and for differentiation of M. bovis BCG from pathogenic M. tuberculosis complex species.

Properties of subviral particles of hepatitis B virus.

In the sera of patients infected with hepatitis B virus (HBV), in addition to infectious particles, there is an excess (typically 1,000- to 100,000-fold) of empty subviral particles (SVP) composed solely of HBV envelope proteins in the form of relatively smaller spheres and filaments of variable length. Hepatitis delta virus (HDV) assembly also uses the envelope proteins of HBV to produce an infectious particle. Rate-zonal sedimentation was used to study the particles released from liver cell lines that produced SVP only, HDV plus SVP, and HBV plus SVP. The SVP made in the absence of HBV or HDV were further examined by electron microscopy. They bound efficiently to heparin columns, consistent with an ability to bind cell surface glycosaminoglycans. However, unlike soluble forms of HBV envelope protein that were potent inhibitors, the SVP did not inhibit the ability of HBV and HDV to infect primary human hepatocytes.

Transcription of hepatitis delta virus RNA by RNA polymerase II.

Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.

Human bloodstream infection caused by Staphylococcus pettenkoferi.

Staphylococcus pettenkoferi is a recently isolated human pathogen with only a few reported cases of infection. We report a case of bloodstream infection caused by S. pettenkoferi in a patient with pulmonary tuberculosis.

A novel strategy for interpreting the T-SPOT.TB test results read by an ELISPOT plate imager.

BACKGROUND: The T-SPOT.TB can be read by an ELISPOT plate imager as an alternative to a labor-intensive and time-consuming manual reading, but its accuracy has not been sufficiently discussed to date. METHODS: 1,423 test results obtained from manual reading using a microscope and an ELISPOT plate imager were compared. The agreement of qualitative test results was assessed using Cohen's kappa coefficient. The relationship of spot counts was studied using Bland-Altman analysis. RESULTS: The overall percent agreement of the qualitative test results was 95.43% with a kappa coefficient of 0.91. Positive test results with the maximum net spot count of 8 and borderline test results showed relatively high discordance. The agreement of spot counts in panel A, panel B, and nil control was good, and variability did not increase with higher spot counts. On the basis of study findings, a novel strategy for interpreting the test results by an ELISPOT plate imager was proposed. CONCLUSIONS: To increase diagnostic accuracy, positive test results with the maximum net spot count of 8 and borderline test results should be manually confirmed. Our strategy could be a practical guide for laboratories to build their own strategies for interpreting the test results by an ELISPOT plate imager.

Genotyping of 19 red cell antigens, including RHD, using liquid bead arrays.

BACKGROUND: Traditionally, the determination of blood group has been performed using serological techniques. Due to the drawbacks of using antisera, molecular typing of blood groups has been introduced. However, the commonly used genotyping assays in blood banks have limitations because the panels for these were based on genetic variations in Caucasians. METHODS: We developed a multiplex human erythrocyte antigen genotyping assay using allele-specific primer extensions and hybridization with bead microarrays. Single nucleotide polymorphisms (SNP) were genotyped for the 18 red cell antigens and wild-type RHD, DEL, and complete deletion were examined for RHD. The cut-off of median fluorescence intensity (MFI) for each allele was optimized. In the evaluation stage, the results of 87 samples were compared with those from other genotyping methods, and 26 serologic results were also compared. RESULTS: The cut-off values were determined using -1 and -2 standard deviation (SD) of the minimum adjusted MFI for SNP detection. Complete deletion was determined with raw MFI+2SD. In comparison with the other methods, the kappa values were 0.984 overall. Compared with serologic methods, our assay showed discrepancy in 2 S/s, 3 C/c, and 3 Dia/Dib antigens. CONCLUSIONS: Our method reliably predicts the presence or the absence of the 19 antigens.

Performance characteristics of nested polymerase chain reaction vs real-time polymerase chain reaction methods for detecting Mycobacterium tuberculosis complex in paraffin-embedded human tissues.

OBJECTIVES: Nucleic acid amplification tests on formalin-fixed, paraffin-embedded (FFPE) tissue specimens enable Mycobacterium tuberculosis complex (MTB) detection and rapid tuberculosis diagnosis in the absence of microbiologic culture tests. We aimed to evaluate the efficacy of different polymerase chain reaction (PCR) methods for detecting Mycobacterium species in FFPE tissues. METHODS: We examined 110 FFPE specimens (56 nonmycobacterial cases, 32 MTB, and 22 nontuberculous mycobacteria [NTM] determined by acid-fast bacilli [AFB] culture) to assess five PCR methods: nested PCR (N-PCR) (Seeplex MTB Nested ACE Detection; Seegene, Seoul, South Korea), an in-house real-time PCR (RT-PCR) method, and three commercial RT-PCR methods (AccuPower MTB RT-PCR [Bioneer, Seoul, Korea], artus M tuberculosis TM PCR [Qiagen, Hilden, Germany], and AdvanSure tuberculosis/NTM RT-PCR [LG Life Sciences, Seoul, Korea]). RESULTS: The results of N-PCR, in-house RT-PCR, and AdvanSure RT-PCR correlated well with AFB culture results (concordance rates, 94.3%, 87.5%, and 89.5%, respectively). The sensitivity of N-PCR (87.5%) was higher than that of the RT-PCR methods, although these differences were not statistically significant between N-PCR and the in-house and AdvanSure RT-PCR methods (68.8% and 80.0%, respectively). All the PCR methods had high specificities, ranging from 98.2% to 100%. Only two NTM cases were detected by AdvanSure RT-PCR, implying a very low sensitivity. CONCLUSIONS: Well-designed RT-PCR and N-PCR can effectively identify MTB in FFPE specimens.

A review of haptoglobin typing methods for disease association study and preventing anaphylactic transfusion reaction.

Haptoglobin, the product of the Hp gene, is a glycoprotein involved in the scavenging of free hemoglobin. Haptoglobin levels increase or decrease in response to various acquired conditions, and they are also influenced by genetic predisposition. There were 2 major alleles, Hp (1) and Hp (2), and 1 minor allele, Hp (del) . Many researchers have attempted to study the haptoglobin types and their association with disease; however, no definitive conclusions have been reached yet. It is reported that patients who are genetically deficient in haptoglobin are at risk of anaphylaxis against blood components containing haptoglobin. Haptoglobin genotypes also affect the reference intervals of haptoglobin levels. Many studies have attempted to establish simple and accurate typing methods. In this paper, we have broadly reviewed several methods for haptoglobin typing-phenotyping, Southern blotting, conventional PCR, real-time PCR, and loop-mediated isothermal amplification. We discuss their characteristics, clinical applications, and limitations. The phenotyping methods are time consuming and labor intensive and not designed to detect patients harboring Hp (del) . The rapid and robust haptoglobin genotyping may help in preventing fatal anaphylactic reactions and in establishing the relationships between the haptoglobin phenotypes and diseases.

Human platelet antigen genotyping and expression of CD109 (human platelet antigen 15) mRNA in various human cell types.

CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA) 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC), two peripheral bloods (PB), 12 granulocyte products, natural killer (NK)-92, B-lymphocyte (CO88BV59-1), K-562 leukemia cell line, human embryonic stem cell (hESC), and human fibroblasts (HF). HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34- PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs.