Recently, the iNtRON VRE vanA/vanB real-time PCR (iNtRON; iNtRON Biotechnology, Korea) assay, a multiplex real-time PCR method, was introduced. In this prospective study, we compared the iNtRON assay with the Seeplex VRE ACE detection kit (Seeplex; Seegene, Korea), a conventional multiplex PCR assay.
A chromogenic agar-based culture, in which pre-selected vancomycin-resistant enterococci (VRE) was grown and subsequently plated on blood agar with vancomycin disks, was regarded as the reference method. A total of 304 consecutive rectal swab specimens were tested for VRE by culture and by iNtRON and Seeplex PCR assays. For the PCR assays, specimens were enriched for 16-24 hr before PCR.
VRE were isolated from 44 (14.5%) specimens by chromogenic agar-based culture. The clinical sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the iNtRON assay were 100% (95% confidence interval: 89.8%-100%), 99.2% (96.9%-99.9%), 95.6% (83.6%-99.2%), and 100% (98.2%-100%), respectively, while those of the Seeplex assay were 97.7% (86.2%-99.9%), 99.6% (97.5%-99.9%), 97.7% (86.2%-99.9%), and 99.6% (97.5%-99.9%), respectively. The iNtRON assay had a detection limit of 3,159 copies/µL and 13,702 copies/µL for the vanA and vanB genes, respectively. No cross-reactivity was observed in 11 non-VRE bacterial culture isolates.
The overall performance of the iNtRON assay was comparable to that of a chromogenic agar-based culture method for prompt identification of VRE-colonized patients in hospitals. This assay could be an alternative or supportive method for the effective control of nosocomial VRE infection.
Real-time PCR; Performance; vanA; vanB; Vancomycin-resistant enterococci
We report a case of subcutaneous infection in a 55-yr-old Korean diabetic patient who presented with a cystic mass of the ankle. Black fungal colonies were observed after culturing on blood and Sabouraud dextrose agar. On microscopic observation, septated ellipsoidal or cylindrical conidia accumulating on an annellide were visualized after staining with lactophenol cotton blue. The organism was identified as Exophiala salmonis by sequencing of the ribosomal DNA internal transcribed spacer region. Phaeohyphomycosis is a heterogeneous group of mycotic infections caused by dematiaceous fungi and is commonly associated with immunocompromised patients. The most common clinical manifestations of subcutaneous lesions are abscesses or cystic masses. To the best of our knowledge, this is the first reported case in Korea of subcutaneous phaeohyphomycosis caused by E. salmonis that was confirmed by molecular analysis and identification of morphological characteristics. This case suggests that E. salmonis infections are no longer restricted to fish.
Phaeohyphomycosis; Exophiala; Soft tissue infection; Sequence analysis; Korea
ChromID C. difficile agar (CDIF; bioMérieux, France), a chromogenic medium, allows for the isolation and identification of Clostridium difficile strains within 24 hr regardless of pretreatment of stool specimens with heat or alcohol shock. In the present study, we designed and evaluated a simple procedure for the implementation C. difficile cultures using CDIF medium in a tertiary hospital setting.
We designed a simple protocol for untreated stool specimens using CDIF medium followed by Gram staining and PRO disc (PRO disc K1532B, Key Scientific Products, USA) testing for the identification of C. difficile in colonies produced on CDIF agar. A total of 1,402 prospectively collected stool specimens from patients with suspected C. difficile infection were tested. The protocol was evaluated by phenotypic or molecular identification of C. difficile using Vitek 2 ANC card (bioMérieux) or 16S rDNA/tpi gene sequencing, respectively.
Of 1,402 stool specimens, 650 isolates were cultured in CDIF. Overall, 235 (36.2%, 235/650) strains could be presumptively identified as C. difficile by using Gram staining and PRO disc testing. Of those, 231 (98.3%, 231/235) isolates were confirmed as true C. difficile by molecular assays.
The use of CDIF combined with Gram staining and PRO disc testing of untreated stool specimens would allow for isolation and accurate identification of C. difficile strains and would be advantageous in reducing the multistep process for C. difficile culture.
Clostridium difficile; Culture; Isolation
We evaluated the performance of two different array-based techniques, a bead-based multiplex genotyping method (LQ; digene HPV Genotyping LQ Test, QIAGEN, Germany) and a DNA chip-based method using peptide nucleic acid probes (PANArray; PANArray HPV Genotyping Chip, Panagene, Korea), for detection of human papillomavirus (HPV) and genotyping of high-risk (HR) or probable high-risk (PHR) HPVs in healthy patients who visited a health-promotion center.
We obtained 508 unselected, consecutive cervicovaginal swab specimens. All specimens were examined by using the PANArray and LQ tests. All HPV-positive samples were then analyzed by multiplex PCR and direct sequencing.
The LQ test detected 47 HPV-positive cases (9.3%) with HR or PHR genotypes and the PANArray test identified 36 cases (7.1%). When the results of LQ and PANArray were compared by using comprehensive genotyping (integrated interpretation of the results of LQ, PANArray, multiplex PCR, and direct sequencing) for the detection of HR or PHR genotypes, the kappa values were 0.44 and 0.30 for LQ and PANArray, respectively. In comparison to comprehensive genotyping, the LQ test yielded 53 (60.0%) concordant and 12 (13.5%) compatible results, and the PANArray yielded 36 (40.4%) concordant and three (3.4%) compatible results.
The results of the LQ test had higher concordance and/or greater compatibility with those of comprehensive genotyping for the detection of HR or PHR genotypes than those of the PANArray test.
Human papillomavirus; DNA test; Comparison; Genotyping
Geosmithia argillacea, an anamorph of Talaromyces eburneus, is a thermophilic filamentous fungus that has a phenotype similar to that of the Penicillium species, except for the creamy-white colonies and cylindrical conidia. Recently, a new genus called Rasamsonia has been proposed, which is to accommodate the Talaromyces and Geosmithia species. Here, we report the first Korean case of G. argillacea isolated from a patient with a fungal ball. The patient was a 44-yr-old Korean man with a history of pulmonary tuberculosis and aspergilloma. The newly developed fungal ball in his lung was removed and cultured to identify the fungus. The fungal colonies were white and slow-growing, and the filaments resembled those of Penicillium. Molecular identification was carried out by sequencing the internal transcribed spacer (ITS) region of the 28S rDNA and the β-tubulin genes. A comparative sequence analysis using the GenBank (http://blast.ncbi.nlm.nih.gov/) database was performed with the basic local alignment search tool (BLAST) algorithm. The results revealed a 97-100% similarity with the G. argillacea ITS sequence. This case should increase awareness among physicians about the pathogenic potential of G. argillacea in humans and help them accurately identify this fungus, because it can be easily confused with Penicillium and Paecilomyces species owing to their similar phenotypic and microscopic characteristics. A molecular approach should be employed to enable accurate identification of G. argillacea.
Geosmithia argillacea; Talaromyces eburneus; Rasamsonia argillacea; Penicillium; Paecilomyces; Pulmonary aspergillosis; Tuberculosis; Sequencing
Mutations in the transforming growth factor β-induced gene (TGFBI) are major causes of genetic corneal dystrophies (CDs), which can be grouped into TGFBI CDs. Although a few studies have reported the clinical and genetic features of Korean patients with TGFBI CD, no data are available regarding the frequency and spectrum of TGFBI mutations in a consecutive series of Korean patients with clinically diagnosed CDs.
Patients with any type of CD, who underwent both ophthalmologic examination and TGFBI gene analysis by Sanger sequencing at a tertiary care hospital in Seoul, Korea from 2006 to 2013, were enrolled in this study.
Among a total of 89 patients, 77 (86.5%) were diagnosed as having clinical TGFBI CD. Seventy-three out of 74 patients (98.6%) with granular CD type 2 (GCD2), had the p.R124H mutation. Of particular note, one patient with rapidly progressive CD had the p.R124H mutation as well as a novel nonsense variant with unknown clinical significance (p.A179*). In three patients with lattice CD type 1 (LCD1), one known mutation (p.R124C) and two novel variants (p.L569Q and p.T621P) in the TGFBI gene were identified.
This study provides epidemiological insight into CDs in a Korean population and reaffirms that GCD2 is the most common TGFBI CD phenotype and that p.R124H is the only mutation identified in patients with GCD2. In addition, we broaden the spectrum of TGFBI mutations by identifying two novel missense variants in patients with LCD1.
Granular corneal dystrophy type 2; Korean; Lattice corneal dystrophy type 1; Mutation; TGFBI
Waardenburg syndrome (WS) is a clinically and genetically heterogeneous hereditary auditory pigmentary disorder characterized by congenital sensorineural hearing loss and iris discoloration. Many genes have been linked to WS, including PAX3, MITF, SNAI2, EDNRB, EDN3, and SOX10, and many additional genes have been associated with disorders with phenotypic overlap with WS. To screen all possible genes associated with WS and congenital deafness simultaneously, we performed diagnostic exome sequencing (DES) in a male patient with clinical features consistent with WS. Using DES, we identified a novel missense variant (c.220C>G; p.Arg74Gly) in exon 2 of the PAX3 gene in the patient. Further analysis by Sanger sequencing of the patient and his parents revealed a de novo occurrence of the variant. Our findings show that DES can be a useful tool for the identification of pathogenic gene variants in WS patients and for differentiation between WS and similar disorders. To the best of our knowledge, this is the first report of genetically confirmed WS in Korea.
Exome; PAX3; Waardenburg syndrome
The heme oxygenase-1 gene (HMOX1) promoter polymorphisms modulate its transcription in response to oxidative stress. This study screened for HMOX1 polymorphisms and investigated the association between HMOX1 polymorphisms and coronary artery disease (CAD) in the Korean population.
The study population consisted of patients with CAD with obstructive lesions (n=110), CAD with minimal or no lesions (n=40), and controls (n=107). Thirty-nine patients with CAD with obstructive lesions underwent follow-up coronary angiography after six months for the presence of restenosis. The 5'-flanking region containing (GT)n repeats of the HMOX1 gene was analyzed by PCR.
The numbers of (GT)n repeats in the HMOX1 promoter showed a bimodal distribution. The alleles were divided into two subclasses, S25 and L25, depending on whether there were less than or equal to and more than 25 (GT)n repeats, respectively. The allele and genotype frequencies among groups were statistically not different. More subjects in the S25-carrier group had the low risk levels of high sensitivity C-reactive protein (hsCRP) for the CAD than those in the non-S25 carrier group (P=0.034). Multivariate logistic regression analysis revealed that the genotypes of (GT)n repeats were not related to CAD status. The restenosis group in the coronary angiography follow-up did not show any significant difference in HMOX1 genotype frequency.
The HMOX1 genotypes were not found to be associated with CAD, but the short allele carrier group contained more individuals with hsCRP values reflecting low risk of cardiovascular disease in the Korean population.
Coronary artery disease; Heme oxygenase-1; HMOX1 gene; Polymorphism
Gastric cancer (GC) is one of the most common cancers with high morbidity and mortality. Familial GC is seen in 10% of cases, and approximately 3% of familial GC cases arise owing to hereditary diffuse gastric cancer (HDGC). CDH1, which encodes the protein E-cadherin, is the only gene whose mutations are associated with HDGC. Screening for the familial GC-predisposing gene has been neglected in high-risk countries such as Korea, China, and Japan, where all the cases have been attributed to Helicobacter pylori or other carcinogens. Screening for the GC-causing CDH1 mutation may provide valuable information for genetic counseling, testing, and risk-reduction management for the as-yet unaffected family members. An asymptomatic 44-yr-old Korean male visited our genetic clinic for consultation owing to his family history of GC. Eventually, c.1018A>G in CDH1, a known disease-causing mutation, was found. As of the publication time, the individual is alive without the evidence of GC, and is on surveillance. To our knowledge, this is the first Korean case of presymptomatic detection of CDH1 mutation, and it highlights the importance of genetic screening for individuals with a family history of GC, especially in high-risk geographical areas.
Stomach; Neoplasms; Hereditary; CDH1; Asymptomatic
Atelosteogenesis type I (AO-I) is a rare lethal skeletal dysplastic disorder characterized by severe short-limbed dwarfism and dislocated hips, knees, and elbows. AO-I is caused by mutations in the filamin B (FLNB) gene; however, several other genes can cause AO-like lethal skeletal dysplasias.
In order to screen all possible genes associated with AO-like lethal skeletal dysplasias simultaneously, we performed whole-exome sequencing in a female newborn having clinical features of AO-I.
Exome sequencing identified a novel missense variant (c.517G>A; p.Ala173Thr) in exon 2 of the FLNB gene in the patient. Sanger sequencing validated this variant, and genetic analysis of the patient's parents suggested a de novo occurrence of the variant.
This study shows that exome sequencing can be a useful tool for the identification of causative mutations in lethal skeletal dysplasia patients.
Atelosteogenesis type I; FLNB; Mutation; Exome sequencing
Axenfeld-Rieger syndrome (ARS) is characterized by anomalies of the anterior segment of the eye and systemic abnormalities. Mutations in the FOXC1 and PITX2 genes are underlying causes of ARS, but there has been few reports on genetically confirmed ARS in Korea. We identified a novel PITX2 mutation (c.300_301delinsT) in 2 Korean patients from a family with ARS. We expand the spectrum of PITX2 mutations and, to the best of our knowledge, this is the first confirmed family of PITX2-related ARS in Korea.
Axenfeld-Rieger syndrome; Homeobox protein PITX2; FOXC1 protein
Chronic granulomatous disease (CGD) is a rare genetic disease, which is caused by defects in the NADPH oxidase complex (gp91phox, p22phox, p40phox, p47phox, and p67phox) of phagocytes. This defect results in impaired production of superoxide anions and other reactive oxygen species (ROS), which are necessary for killing bacterial and fungal microorganisms and leads to recurrent, life-threatening bacterial and fungal infections and granulomatous inflammation. The dihydrorhodamine (DHR) flow cytometry assay is a useful diagnostic tool for CGD that can detect absent or reduced NADPH oxidase activity in stimulated phagocytes. We report a patient with X-linked CGD carrying a novel mutation of the CYBB gene whose chimerism status following hematopoietic stem cell transplantation (HSCT) has been rapidly determined using the DHR assay. The level of DHR activity correlates well with short tandem repeat PCR analysis. Considering the advantages of this simple, rapid, and cost-effective procedure, serial measurement of DHR assay would facilitate the rapid determination of a patient's engraftment status, as a supplementary monitoring tool of chimerism status following HSCT.
Chronic granulomatous disease; Dihydrorhodamine assay; Chimerism
Familial juvenile hyperuricemic nephropathy (FJHN; OMIM 162000) is an autosomal dominant disorder characterized by hyperuricemia and gouty arthritis due to reduced kidney excretion of uric acid and progressive renal failure. Gradual progressive interstitial renal disease, with basement membrane thickening and glomerulosclerosis resulting from fibrosis, starts in early life. In most cases of FJHN, uromodulin gene (UMOD) is responsible for the disease; however, there has been only one report of a genetically confirmed FJHN family in Korea. Here we report another Korean family with FJHN, in which three male members. a father and 2 sons.developed gout and progressive renal insufficiency. The clinical, laboratory, and radiological findings were consistent with FJHN, and renal biopsy showed chronic parenchymal damage, which can be found in FJHN but is not specific to this disease. In order to confirm the diagnosis, sequence analysis of the UMOD was performed, and a novel heterozygous missense variant (c.187T>C; p.Cys63Arg) in exon 3 was identified. We assume that this variant is likely to be the causative mutation in this family, as the variant segregated with the disease. In addition, approximately two-thirds of the known mutations lead to a cysteine amino acid change in uromodulin, and all such variants have been shown to cause UMOD-associated kidney disease. In summary, we report a Korean FJHN family with three affected members by genetic analysis of the UMOD, and provide the first report of a novel heterozygous missense mutation.
Uromodulin; Familial juvenile hyperuricemic nephropathy; Mutation; Sequence analysis; Korea
Ataxia-telangiectasia (A-T) is a rare autosomal recessive neurodegenerative disorder. It is characterized by early-onset, progressive cerebellar ataxia, oculomotor apraxia, choreoathetosis, conjunctival telangiectasias, immunodeficiency, and an increased risk of malignancy. Although A-T is known to be the most common cause of progressive cerebellar ataxia in childhood, there have been no confirmed cases in Korea. We report the clinical and genetic findings of Korean siblings who presented with limb and truncal ataxia, oculomotor apraxia, choreoathetosis, and telangiectasias of the eyes. Sequence analysis of the ataxia-telangiectasia mutated (ATM) gene revealed a known missense mutation (c.8546G>C; p.Arg2849Pro) and a novel intronic variant of intron 17 (c.2639-19_2639-7del13). Reverse-transcription PCR and sequencing analysis revealed that the c.2639-19_2639-7del13 variant causes a splicing aberration that potentiates skipping exon 18. Because A-T is quite rare in Korea, the diagnosis of A-T in Korean patients can be delayed. We recommend that a diagnosis of A-T should be suspected in Korean patients exhibiting the clinical features
Ataxia; Ataxia telangiectasia; Ataxia telangiectasia mutated protein; Korea
Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disorder (LSD) caused by abnormalities of the enzyme α-N-acetylglucosaminidase (NAGLU) that is required for degradation of heparan sulfate. The patient in this study was a 4-yr-old boy. He presented with normal height and weight, pectus carinatum, and multiple persistent Mongolian spots on his back. He had mild dysmorphic features with prominent speech developmental delays and, to a lesser extent, motor developmental delays. The cetylpyridinium chloride precipitation test revealed excessive mucopolysacchariduria (657.2 mg glycosaminoglycan/g creatinine; reference range, <175 mg glycosaminoglycan/g creatinine). Thin layer chromatography showed urinary heparan sulfate excretion. NAGLU enzyme activity was significantly decreased in leukocytes (not detected; reference range, 0.9-1.51 nmol/hr/mg protein) as well as in plasma (0.14 nmol/hr/mg protein; reference range, 22.3-60.9 nmol/hr/mg protein). PCR and direct sequencing analysis of the NAGLU gene showed that the patient was a compound heterozygote for 2 mutations: c.200T>C (p.L67P) and c.1444C>T (p.R482W). The c.200T>C mutation was a novel finding. This is the first report of a Korean patient with MPS IIIB who was confirmed by molecular genetic analyses and biochemical investigation.
Korean; Mucopolysaccharidosis IIIB; NAGLU; Novel mutation
Mucopolysaccharidosis (MPS) III has 4 enzymatically distinct forms (A, B, C, and D), and MPS IIIC, also known as Sanfilippo C syndrome, is an autosomal recessive lysosomal storage disease caused by a deficiency of heparan acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT). Here, we report a case of MPS IIIC that was confirmed by molecular genetic analysis. The patient was a 2-yr-old girl presenting with skeletal deformity, hepatomegaly, and delayed motor development. Urinary excretion of glycosaminoglycan (GAG) was markedly elevated (984.4 mg GAG/g creatinine) compared with the age-specific reference range (<175 mg GAG/g creatinine), and a strong band of heparan sulfate was recognized on performing thin layer chromatography. HGSNAT enzyme activity in leukocytes was 0.7 nmol/17 hr/mg protein, which was significantly lower than the reference range (8.6-32 nmol/17 hr/mg protein). PCR and direct sequencing of the HGSNAT gene showed 2 mutations: c.234+1G>A (IVS2+1G>A) and c.1150C>T (p.Arg384*). To the best of our knowledge, this is the first case of MPS IIIC to be confirmed by clinical, biochemical, and molecular genetic findings in Korea.
Mucopolysaccharidosis IIIC; HGSNAT; Sanfilippo syndrome; Korea
The -D- phenotype is a rare Rh phenotype that strongly expresses D antigen without C, c, E, or e antigens. In -D- phenotype individuals, anti-Rh17 (Hro) is commonly found if there is a history of pregnancy or transfusion with red blood cells (RBCs) that express C, c, E, or e antigens. We report the first case of a -D- phenotype patient with multiple Rh antibodies including anti-Rh17 who had a history of two occasions of transfusion with eight random donor platelet concentrates two and six years ago. We found that a trivial amount of RBCs in the platelet components was able to trigger sensitization to RBC antigens, especially the highly immunogenic and clinically significant Rh antigens, including C, c, E, e or CcEe polypeptides. To avoid unnecessary sensitization and to minimize the risk of hemolytic transfusion reactions in patients with this rare Rh phenotype, a modified strategy for pretransfusion screenings needs to be discussed in the field of transfusion medicine.
Rh-Hr blood group system; Rh isoimmunization; Platelet transfusion
Congenital chloride diarrhea (CLD) is an autosomal recessive disorder with the hallmark of persistent watery Cl--rich diarrhea from birth. Mutations in the solute carrier family 26, member 3 (SLC26A3) gene, which encodes a coupled Cl-/HCO3- exchanger in the ileum and colon, are known to cause CLD. Although there are a few reports of CLD patients in Korea, none of these had been confirmed by genetic analysis. Here, we describe the case of a Korean infant with clinical features of CLD. Using direct sequencing analysis, we identified 2 sequence variants: a missense variant of unknown significance (c.525G>C; p.Arg175 Ser) and a splicing mutation (c.2063-1G>T) in the SLC26A3 gene; these had been inherited from the father and mother, respectively. Whilst CLD is rare, its main symptom, diarrhea, is very common in infants. Hence, the diagnosis of CLD can prove difficult. Mutational analysis of the SLC26A3 gene should be considered as a viable method to confirm a diagnosis of CLD in Korean infants with persistent diarrhea.
Congenital chloride diarrhea; SLC26A3 gene; Mutation
Papillary thyroid carcinoma (PTC) is the most common malignant thyroid tumor, and 36-69% of PTC cases are caused by mutations in the BRAF gene. The substitution of a valine for a glutamic acid (V600E) comprises up to 95-100% of BRAF mutations; therefore, most diagnostic methods, including allele-specific PCR and real-time PCR, are designed to detect this mutation. Nevertheless, other mutations can also comprise the genetic background of PTC. Recently, a novel and sensitive technique called mutant enrichment with 3'-modified oligonucleotides (MEMO) PCR has been introduced. When we applied allelespecific PCR and MEMO-PCR for the detection of the BRAF V600E mutation, we found an unusual 3' bp deletion mutation (c.1799_1801delTGA) only when using MEMO-PCR. This deletion results in the introduction of a glutamic acid into the B-Raf activation segment (p.V600_K601delinsE), leading to an elevated basal kinase activity of BRAF. This is the first report of a rare 3 bp BRAF deletion in a PTC patient that could not be detected by allele-specific PCR.
Papillary thyroid carcinoma; BRAF; Deletion; Mutation; Mutant enrichment with 3'-modified oligonucleotides (MEMO) PCR; Korean