Ja Young Seo

Evaluation of PCR-based screening for vancomycin-resistant enterococci compared with a chromogenic agar-based culture method

Rapid detection of vancomycin-resistant enterococci (VRE) infection is very important for control and prevention of nosocomial spread of these bacteria. A multiplex PCR method for rapid screening of VRE has recently been developed. We performed a prospective study of VRE screening tests to compare the performance of PCR to that of a chromogenic agar-based culture method. From January to December 2009, a total of 8815 rectal swab specimens were tested simultaneously for VRE by VRE selective culture and by PCR. The specimens were inoculated onto ChromID VRE agar containing 8 µg vancomycin ml⁻¹ and examined after 24 and 48 h of incubation. Identification and antibiotic susceptibility tests were performed using the automated VITEK-2 system and a supplementary E-test and disk diffusion test. Detection of the vanA and vanB genes was performed with the Seeplex VRE detection kit. Specimens were inoculated in enterococcosel broth for 16-24 h before PCR for enrichment of VRE. VRE were isolated from 741 of the 8815 specimens by chromogenic agar-based culture (8.4 %). vanA and vanB genotypes were detected in 758 (8.6 %) and 3 (0.03 %) specimens, respectively, by multiplex PCR. Sensitivity, specificity, positive predictive value and negative predictive value of PCR for detection of VRE were 98.2 %, 99.6 %, 95.7 %, and 99.8 %. No VRE were isolated from vanB-positive specimens. The overall performance of PCR is comparable to that of a chromogenic agar-based culture method for screening of VRE, so PCR could be an alternative or supportive method for effective control of nosocomial VRE infection.

Steroid profiling for congenital adrenal hyperplasia by tandem mass spectrometry as a second-tier test reduces follow-up burdens in a tertiary care hospital: A retrospective and prospective evaluation

Abstract Background: Newborn screening for congenital adrenal hyperplasia (CAH) based on measuring 17-hydroxyprogesterone (17-OHP) by immunoassay generates a number of false-positive results, especially in preterm neonates. We applied steroid profiling by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a second-tier test in newborns with positive CAH screening and evaluated its clinical utility in a tertiary care hospital setting. Methods: By performing a 4-year retrospective data review, we were able to test 121 dried blood spots from newborns with positive CAH screening for 17-OHP, androstenedione and cortisol levels by LC-MS/MS. We prospectively evaluated the clinical utility of steroid profiling after the implementation of steroid profiling as a second-tier test in our routine clinical practice. During the 2-year prospective study period, 104 cases with positive initial screening by FIA were tested by LC-MS/MS. Clinical and laboratory follow-up were performed for at least 6 months. Results: The preterm neonates accounted for 50.7% (76/150) and 70.4% (88/125) of screening-positive cases in retrospective and prospective cohorts, respectively. By applying steroid profiling as a second-tier test for positive CAH screening, we eliminated all false-positive results and decreased the median follow-up time from 75 to 8 days. Conclusions: Our data showed that steroid profiling reduced the burden of follow-up exams by improving the positive predictive value of the CAH screening program. The use of steroid profiling as a second-tier test for positive CAH screening will improve clinical practice particularly in a tertiary care hospital setting where positive CAH screening from preterm neonates is frequently encountered.

A novel PRF1 gene mutation in a fatal neonate case with type 2 familial hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) occurs in the primary form (genetic or familial) or secondary form (acquired). The familial form of HLH (FHL) is a potentially fatal autosomal recessive disorder that occurs because of constitutional defects in cell-mediated cytotoxicity. Here, we report a fatal neonatal case of type 2 FHL (FHL2) that involved a novel frameshift mutation. Clinically, the newborn presented with severe sepsis-like features and required mechanical ventilation and continuous venovenous hemodiafiltration. Flow cytometry analysis showed marked HLH and complete absence of intracytoplasmic perforin expression in cytotoxic cells; therefore, we performed molecular genetic analyses for PRF1 mutations, which showed that the patient had a compound heterozygous mutation in PRF1, that is, c.65delC (p.Pro22Argfs*2) and c.1090_1091delCT (p.Leu364Glufs*93). Clinical and genetic assessments for FHL are required for neonates with refractory fever and progressive multiple organ failure, particularly when there is no evidence of microbiological or metabolic cause.

Comparison of platelet parameters in thrombocytopenic patients associated with acute myeloid leukemia and primary immune thrombocytopenia.

Thrombocytopenia is caused by insufficient production and excessive destruction of platelets. Recent improvement of automated blood cell analyzers has allowed measurement of several platelet parameters, providing better understanding of the underlying mechanisms of thrombocytopenia. We investigated the significance of platelet parameters in thrombocytopenic patients. Thrombocytopenic patients (platelet <100 × 103/&mgr;l) who were newly diagnosed with acute myeloid leukemia and primary immune thrombocytopenia were enrolled, and platelet, mean platelet volume, platelet distribution width, platelet crit, mean platelet component, mean platelet mass, and large platelet count were measured, and the percentages of large platelets were calculated. The parameters were also measured in the reference population. The mean values of each parameter were as follows: platelet, 259 × 103/&mgr;l; mean platelet volume, 7.9 fl; platelet distribution width, 51.3%; platelet crit, 0.20%; mean platelet component, 26.0 g/dl; mean platelet mass, 1.9 pg; large platelet, 4.7 × 103/&mgr;l; large platelet percentage, 1.7%. In comparison with acute myeloid leukemia patients, patients with primary immune thrombocytopenia showed significantly higher mean platelet volume, platelet distribution width, mean platelet component, mean platelet mass, large platelet, and large platelet % (P < 0.05). Because of increased destruction of platelets, primary immune thrombocytopenia patients have increased mean platelet volume, platelet distribution width, mean platelet component, mean platelet mass, large platelet, and large platelet percentage compared with acute myeloid leukemia patients who have ineffective platelet production. Parameters measured by automated analyzer provide better understanding of thrombopoiesis in the bone marrow and the status of the peripheral blood in the clinical field.

The First Korean Case of Mucopolysaccharidosis IIIC (Sanfilippo Syndrome Type C) Confirmed by Biochemical and Molecular Investigation

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.

Evaluation of overnight storage conditions for autologous peripheral blood stem cell products: comparison of three different conditions

Background  Overnight (ON) storage of peripheral blood stem cell (PBSC) occurs frequently in clinical settings. However, there are no standard guidelines for optimal storage conditions of freshly harvested PBSC. The aim of this study was to investigate the influence of storage temperatures on the quality of autologous PBSC and establish optimal storage conditions before cryopreservation.

A case of disseminated Penicillium marneffei infection in a liver transplant recipient.

Penicillium marneffei is the only dimorphic fungus among Penicillium spp. that can cause a fatal infection in immunocompromised patients. P. marneffei is endemic in Southeast Asia and eastern China. P. marneffei infection is an AIDS-defining illness and the third most common opportunistic infection in the endemic regions. Here, the authors report a case of disseminated P. marneffei infection in a patient who underwent liver transplantation in China. During the hospital stay, the mold form of the fungus that produced a red wine-colored pigment on the agar plate was isolated from the patients urine, transtracheal aspirate, and blood. The fungus was identified as P. marneffei by direct sequencing of the D1-D2 and ITS regions. Thermal dimorphism was also confirmed by subculturing the colony at 37 degrees C. To the best of our knowledge, this is the first Korean case of disseminated P. marneffei infection in a liver transplant recipient.

Sequence variation data of F8 and F9 genes in functionally validated control individuals: implications on the molecular diagnosis of hemophilia

Background The F8 and F9 genes encode for coagulation factor VIII (FVIII) and FIX, respectively, and mutations in these genes are the genetic basis of hemophilia A/B. To determine whether a sequence variation in F8/F9 is a disease-causing mutation, frequency data from a control population is needed. This study aimed to obtain data on sequence variation in F8/F9 in a set of functionally validated control chromosomes of Korean descent. Methods We re-sequenced F8 and F9 from DNA samples of 100 Korean male control individuals with normal PT, aPTT, and FVIII activity. PCR and direct sequencing analyses were performed using primer pairs to cover all coding regions and the flanking intronic sequences. Results Thirteen individuals (13%) were hemizygous for sequence variations in the coding region of F8. Six (6%) had c.3780C>G (p.Asp1260Glu), five (5%) had c.3864A>C (p.Ser1288=). One each individual (1%) had c.4794G>T (p.Glu1598Asp) and c.5069 A>G (p.Glu1690Gly). Asp1260Glu and Ser1288= were known SNPs (rs1800291 and rs1800292, respectively). Glu1598Asp was assigned as a missense mutation in public databases (HGMD and HAMSTeRS), and Glu1690Gly was a novel variation. Based on the normal FVIII activities in control individuals carrying these variations (109% and 148%, respectively), they were considered to be rare SNPs. No variation was observed in F9 of control individuals. Conclusion A significant proportion of control individuals carried sequence variations in F8, but not in F9. These results can be used as a reference dataset for molecular diagnosis of hemophilia A and B, particularly in Korea.

Evaluation of the iNtRON VRE vanA/vanB Real-Time PCR Assay for Detection of Vancomycin-Resistant Enterococci

Background 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. Methods 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. Results 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. Conclusions 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.

Reference Intervals for Platelet Parameters in Korean Adults Using ADVIA 2120

Analysis of thrombopoiesis is important in evaluating hemato-logic and non-hematologic diseases. Recent improvements in automated blood 4cell analyzers allow measurement of several platelet parameters, providing additional information on the un-derlying mechanisms of thrombocytosis and thrombocytopenia. The ADVIA