Young Kee Kim

Evaluation of rapid immunocapture assays for diagnosis of Plasmodium vivax in Korea.

Abstract. The rapid immunocapture assays, OptiMal and ICT, were evaluated from 87 individuals for the diagnosis of malaria infections directly from whole blood. A total of 87 individuals was examined for malaria parasites by microscopic examination of Giemsa-stained blood smears, and 65 cases were positive for Plasmodium vivax by microscopy. Correspondingly, the OptiMal test identified malaria infection in 45 cases (69.2%) of microscopy positive cases. Of these, two cases were misinterpreted as Plasmodium falciparum, whereas ICT detected P. vivax infection in 29 (44.6%) patients. We would like to propose that rapid immunocapture assays are an easy method that can serve as a useful tool in addition to microscopy for the diagnosis of malaria, but sensitivity is not yet satisfactory for diagnosis of P. vivax in Korea.

Evaluation of a novel real-time RT-PCR using TOCE technology compared with culture and Seeplex RV15 for simultaneous detection of respiratory viruses

Abstract Background Various kinds of commercial molecular systems have been developed for fast and more accurate detection of respiratory viruses. Anyplex™ II RV16 [RV16] was designed for simultaneous detection of 16 respiratory viruses using multiplex PCR coupled with TOCE™ technology. Objectives To compare the performance of RV16 with those of culture and Seeplex® RV15 ACE [RV15] by determining their sensitivity and specificity. Study design Seven hundred and thirty respiratory samples were tested by modified shell vial culture method, RV16, and RV15. For molecular tests, automated nucleic acid extraction and liquid handling system using MICROLAB Nimbus IVD (Hamilton, USA) was adopted to maximize the workflow and accuracy. Performance of each assay was determined against a composite reference standard. Results Two hundred and one samples (28%) out of 730 samples were positive by culture, while additional 281 (39%) were positive by RV16 or RV15. Sensitivities of RV16, RV15, and culture for virus tested were as follows: 100/93/63% for influenza A, 90/80/69% for influenza B, 98/94/63% for RSV, 98/52/23% for adenovirus, and 100/75/46% for PIV. For viruses not covered by culture, sensitivities of RV16 and RV15 were as follows: 99/81% for rhinovirus, 92/100% for coronavirus OC43, 100/56% for coronavirus 229E/NL63, 92/88% for metapneumovirus, 100/62% for bocavirus, and 91/91% for enterovirus. Overall, the specificities of culture, RV16, and RV15 (Seegene) were 100/99.9/99.9%. Conclusions RV16 assay was superior to culture method and RV15 and will be a promising tool for patient management and public health epidemiology.

Human Bocavirus in Patients with Respiratory Tract Infection

Background Human bocavirus (HBoV) is a newly identified viral pathogen, and its clinical epidemiology and significance in respiratory infections have not yet been completely elucidated. We investigated the prevalence of HBoV infection and the association between viral (HBoV) load and clinical features of the infection in patients of all age-groups. Methods Nasopharyngeal aspirates from patients with symptoms of respiratory infection were tested for presence of HBoV by using real-time polymerase chain reaction. HBoV-positive patients were categorized into low- and high-viral-load groups using 1.0×106 copies/mL as the threshold value of viral load. Results Detection rate of HBoV was 4.8% (N=93) in a total of 1,926 samples with peak incidence of infection being observed in patients aged 6-12 months. HBoV infection was more frequently observed in young children, especially, in children aged less than 5 yr, and the HBoV load decreased with increase in age. HBoV was codetected with other respiratory viruses in 17 (18.3%) of the 93 HBoV-positive patients and 15 patients (88.2%) belonged to the low-viral-load group. Patients infected with HBoV alone showed a higher viral load than those patients in whom HBoV was codetected with other respiratory viruses (median load, 3.78×105 copies/mL vs. 1.94×104 copies/mL, P=0.014). Higher pulse rate (P=0.007) and respiratory rate (P=0.021) were observed in patients with a high-viral-load. Conclusions Our results suggest that HBoV may be the causative agent of respiratory infection in the high-viral-load group.

Enhanced detection of respiratory viruses using cryopreserved R-Mix ReadyCells

BACKGROUND R-Mix, which contains a fresh mixture of two cell lines, Mv1Lu (mink lung cells) and A549 cells, has shown good sensitivity and specificity for respiratory virus culture. However, it has until recently only been available in North America, in part due to the shipping constraints associated with cell aging and the difficulty in providing these cells to hard to reach regions. Recently, cryopreserved R-Mix ReadyCells for longer storage were developed. These cells, which are shipped on dry ice and have a shelf life as long as 6 months from date of manufacture, can be thawed and used as needed with minimal addition of refeeding media. OBJECTIVE Assess the potential for cryopreserved R-Mix ReadyCells to replace conventional culture. STUDY DESIGN Two hundred and twenty-three nasopharyngeal aspirates confirmed as respiratory virus-positive by conventional culture were inoculated into cryopreserved R-Mix ReadyCells and re-inoculated into conventional culture cells simultaneously. After 1 and 3 days of incubation cryopreserved R-Mix ReadyCells and conventional culture cells were screened using a respiratory virus fluorescent antibody pool for the detection of seven major respiratory viruses (influenza A and B viruses, parainfluenza 1, 2 and 3 viruses, respiratory syncytial virus and adenovirus). Positive pool results were further differentiated with specific monoclonal antibodies against the individual viruses. RESULTS After 1 day of incubation detection rates for conventional culture were 25%, 39%, 39%, 49%, and 10% for influenza A virus, influenza B virus, parainfluenza viruses, respiratory syncytial virus, and adenovirus, respectively. Corresponding detection rates for cryopreserved R-Mix ReadyCells were 78%, 91%, 72%, 81%, and 65%. Average detection rates of cryopreserved R-Mix ReadyCells for all respiratory viruses were 80% after 1 day incubation and 95% after 3 days incubation, compared to 35% and 70% by conventional culture. CONCLUSION The cryopreserved R-Mix ReadyCells system offers a highly sensitive and rapid method for detection of respiratory viruses that may allow it to replace conventional cell culture systems.

Evaluation of BACTEC Plus aerobic and anaerobic blood culture bottles and BacT/Alert FAN aerobic and anaerobic blood culture bottles for the detection of bacteremia in ICU patients

Blood culture is the most valuable laboratory test for the diagnosis of bacteremia and sepsis. The BACTEC FX and BacT/Alert 3D automated blood culture systems are commonly used in Korean health care facilities. A controlled clinical evaluation of the resin-containing BACTEC Plus aerobic (BA) and anaerobic (BN), and the charcoal-containing FAN aerobic (FA) and anaerobic (FN) bottles using blood from intensive care unit (ICU) patients was designed. The performances of these 2 systems with media containing particle absorbing antimicrobial agents were evaluated using the culture positivity rate and time to detection (TTD). TTD was collected using data management systems, either the Epicenter (BD Diagnostic Systems) or the hospital laboratory information system. A total of 1539 four-bottle sets were collected from 270 patients in medical and surgical ICUs. Blood culture samples included 1539 bottles each of BA, BN, FA, and FN, and yielded 113 (7.3%), 90 (5.8%), 104 (6.8%), and 80 (5.2%) positive bacterial or fungal isolates, respectively. There were significant differences between the resin-containing BA and BN samples in culture positivity and also between the charcoal-containing FA and FN samples, especially for Escherichia coli (25/27 versus 17/27, P < 0.05) and Acinetobacter baumannii (14/15 versus 7/15, P < 0.05). Significantly shorter recovery time was observed in BACTEC Plus aerobic bottles than in FAN aerobic bottles (17.2 and 24.7 h, respectively) (P < 0.001).

Evaluation of the AdvanSure™ real-time RT-PCR compared with culture and Seeplex RV15 for simultaneous detection of respiratory viruses

Abstract Recently, AdvanSure™ kit based on multiplex real-time PCR was developed for simultaneous detection of 14 respiratory viruses (RVs). We compared the performance of AdvanSure with those of Seeplex® RV 15 ACE and culture by determining their sensitivities and specificities against a composite reference standard. Four hundred thirty-seven respiratory samples were tested by modified shell vial culture method, RV 15 ACE, and AdvanSure. One hundred fourteen samples (26.2%) out of 437 samples were positive by culture, while additional 91 (20.8%) were positive by AdvanSure or RV15. One hundred twelve of 114 culture-positive samples were positive by AdvanSure except 2 samples (1 adenovirus, 1 respiratory syncytial virus [RSV]). Overall, the sensitivities of culture, RV15, and AdvanSure were 74.5%, 89.8%, and 95.1%, respectively. Sensitivities of culture, RV15, and AdvanSure for each virus tested were as follows: 91/100/96% for influenza A, 60/0/100% for influenza B, 63/95/97% for RSV, 69/81/89% for adenovirus, and 87/93/93% for parainfluenza virus. For viruses not covered by culture, sensitivities of RV15 and AdvanSure were as follows: 77/88% for rhinovirus, 100/100% for coronavirus OC43, 40/100% for coronavirus 229E/NL63, 13/100% for metapneumovirus, and 44/100% for bocavirus. The overall specificities of culture, RV15, and AdvanSure were 100/98.9/99.5%, respectively. Of 45 coinfected specimens, AdvanSure detected 41 specimens (91.1%) as coinfected, while RV15 detected 27 specimens (60.0%) as coinfected. AdvanSure assay demonstrated exquisite performance for the detection of RVs and will be a valuable tool for the management of RV infection.

The Genomic Heterogeneity among Mycobacterium terrae Complex Displayed by Sequencing of 16S rRNA and hsp65 Genes

The species identification within Mycobacterium terrae complex has been known to be very difficult. In this study, the genomic diversity of M. terrae complex with eighteen clinical isolates, which were initially identified as M. terrae complex by phenotypic method, was investigated, including that of three type strains (M. terrae, M. nonchromogenicum, and M. triviale). 16S rRNA and 65‐kDa heat shock protein (hsp65) gene sequences of mycobacteria were determined and aligned with eleven other references for the comparison using similarity search against the GenBank and Ribosomal Database Project II (RDP) databases. 16S rRNA and hsp65 genes of M. terrae complex showed genomic heterogeneity. Amongst the eighteen clinical isolates, nine were identified as M. nonchromogenicum, eight as M. terrae, one as M. mucogenicum with the molecular characteristic of rapid growth. M. nonchromogenicum could be subdivided into three subgroups, while M. terrae could be subdivided into two subgroups using a 5 bp criterion (>1% difference). Seven isolates in two subgroups of M. nonchromogenicum were Mycobacterium sp. strain MCRO 6, which was closely related to M. nonchromogenicum. The hsp65 gene could not differentiate one M. nonchromogenicum from M. avium or one M. terrae from M. intracellulare. The nucleotide sequence analysis of 16S rRNA and hsp65 genes was shown to be useful in identifying the M. terrae complex, but hsp65 was less discriminating than 16S rRNA.

Alteration of platelet counts and lipid profiles after treatment of acute Plasmodium vivax

During malaria infections, thrombocytopenia and low cholesterol levels are frequently observed changes. We compared these changes in patients admitted with fevers and infected with Plasmodium vivax, patients admitted with fevers with respiratory/urinary infections and afebrile normal (control) non-infected volunteers. Changes in the platelet count and lipid parameters are reported for malaria patients after treatment with hydroxychloroquine and primaquine for acute P. vivax malaria. Of a total 141 participants, 55 patients were diagnosed with malaria (positive blood smear) prior to treatment. Compared to the normal (n=52) and non-malaria fever groups (n=34), there was a significant decrease in five hematologic indices (white blood cell, red blood cell, hemoglobin, hematocrit and platelet) and three lipid parameters (total cholesterol, HDL-c and LDL-c) in the vivax malaria group at day 0 (pre-treatment). Following treatment, the platelet counts returned to normal limits (P<0.05) from 91,058/microL on day 0 to 246,833/microL by day 17 after treatment. However, changes in the lipid parameters of malaria patients showed a slow recovery to normal limits compared to the platelet counts. The HDL-c and LDL-c remained low for 1 month after treatment but increased at 3 and 6 months post-treatment. At 12 months after treatment, the levels of two lipid parameters had fully recovered to the normal limits. Thus, special attention should be applied when interpreting laboratory blood profiles of malaria patients, especially platelet and lipid based tests, until full recovery after treatment.

Cross-linking of CD4 induces cytoskeletal association of CD4 and p56lck.

A membrane glycoprotein CD4 functions as a co-receptor of a T lymphocyte. The co-receptor function has been attributed to a protein tyrosine kinase, p56lck, which is activated upon CD4 binding to MHC molecule. In this study, we present evidences that one of the pathways through which CD4 transmits its signal is cytoskeleton association of p56lck tyrosine kinase as well as CD4 itself. Cytoskeletal association of both proteins is inhibited by a tyrosine kinase inhibitor, genistein, indicating that tyrosine protein kinase activation is important for cytoskeletal association of CD4 and p56lck. Cytoskeletal association of these proteins by CD4 cross-linking is not affected by inhibitors of protein kinase C nor PI3-kinase. Taken together, these results suggest that CD4 cross-linking activates a tyrosine kinase which then induces the simultaneous association of CD4 and p56lck with cytoskeleton.

Acute myeloid leukemia (AML-M2) associated with variant t(8;21): report of three cases

Variants of the t(8;21)(q22;q22) involving chromosome 8, 21, and other chromosomes account for approximately 3% of all t(8;21)(q22;q22) found in patients with acute myeloid leukemia (AML). The clinicopathologic features of AML with the variant t(8;21) have not been well established. We report three cases of AML with variants of t(8;21) characterized, respectively, by derivative 8 with the interstitial inverted insertion of 21q and concurrent monosomy 21, t(8;18;21)(p22;q11.3;q22), and t(2;21;8)(q11.2;q22;q22). Fluorescence in situ hybridization or reverse transcriptase-polymerase chain reaction assay confirmed the presence of RUNX1-RUNX1T1 gene (previously AML1-ETO) rearrangements. Among these cases, three-way breakpoints 18p11.3 and 2q11.2 have not been previously reported. The present report deals with the results of hematologic, immunophenotypic, cytogenetic, fluorescence in situ hybridization, and molecular analyses of these variants. The possible role of the genes in this region in leukemogenesis, response to treatment, and clinical implications are discussed.