Detecting primary drug-resistant mutations in Korean HIV patients using ultradeep pyrosequencing.

HIV primary resistance, drug resistance in treatment-naïve patients, is an emerging public health issue. The prevalence of HIV primary resistance mutations down to the level of 1% minor variants was investigated using ultradeep pyrosequencing (UDPS) in HIV-positive Korean blood donors and in treatment naïve chronic patients for the comparison. The entire pol region was sequenced from 25 HIV-positive blood donors, and 18 treatment-naïve chronic HIV patients. UDPS was successful in 19 blood donors and 18 chronic patients. In total, 1,011,338 sequence reads were aligned, and 28,093 sequence reads were aligned on average per sample. The prevalence of HIV primary resistance mutations in the HIV-positive blood donors and chronic HIV patients were 63.2% and 44.4% according to UDPS, respectively. Protease inhibitor (PI) drugs demonstrated different patterns in HIV-positive blood donors and chronic HIV patients, whereas non-nucleoside reverse transcriptase inhibitors (NNRTI), nucleoside reverse transcriptase inhibitors (NRTI), and integrase inhibitor (INI) drugs showed similar patterns between the two groups. Higher level of primary resistance prevalence was observed mainly because UDPS method could detect mutations in minor variants with 1-10% frequency. The higher resistance prevalence was observed in HIV-positive blood donors than in chronic patients. Considering that treatments for HIV-infected patients were recently amended to start at an earlier stage, information about degree of drug resistance to each drug between the two groups would help to establish future policies, design additional clinical trials, assess HIV patient care in Korea.

Genotyping of 22 blood group antigen polymorphisms and establishing a national recipient registry in the Korean population.

It is often difficult for standard blood banks in Korea to supply adequate amounts of blood for patients with rare phenotype. Moreover, the definition of a blood in need is ambiguous, and much remains to be learned. In this study, we determined the prevalence of various red blood cell (RBC) antigens from a donor viewpoint and estimated the demand for specific antigen-negative blood from a patient viewpoint. Our data will aid the establishment of a Rare Blood Program in Korea (KRBP). RBC genotyping of 419 blood donors was performed using a Lifecodes RBC/RBC-R typing kit (Immucor, Norcross, GA). A national recipient registry website has been established. Each hospital-based blood bank voluntarily enters data on antibodies detected and identified and the outcomes of specific antigen testing. We calculated the availabilities of specific antigen-negative blood components based on these registry data and predicted the prevalence of RBC antigens via RBC genotyping. The prevalences of various RBC antigens in the D-negative population were determined for the first time, and the Cartwright, Scianna, Dombrock, Colton, Landsteiner-Wiener, Cromer, and Knops blood group systems were identified. The availabilities of specific antigen-negative units differed when calculations were based on serotyping or genotyping, especially in the D-negative group. Data on the prevalences of various blood antigens are essential for estimating the availabilities of blood components that are appropriate for use by patients expressing relevant antibodies. Then, blood banks would be able to efficiently supply safe blood products.

[Evaluation of the virus-elimination efficacy of nanofiltration (Viresolve NFP) for the parvovirus B19 and hepatitis A virus].

BACKGROUND: The safety of plasma derivatives has been reinforced since 1980s by variable pathogen inactivation or elimination techniques. Nucleic acid amplification test (NAT) for the source plasma has also been implemented worldwide. Recently nanofiltration has been used in some country for ensuring safety of plasma derivatives to eliminate non-enveloped viruses such as parvovirus B19 (B19V) and hepatitis A virus (HAV). We evaluated the efficacy of nanofiltration for the elimination of B19V and HAV. METHODS: To verify the efficacy of nanofiltration, we adopted a 20 nm Viresolve NFP (Millipore, USA) in the scaling down (1:1,370) model of the antithrombin III production. As virus stock solutions, we used B19V reactive plasma and porcine parvovirus (PPV) and HAV obtained from cell culture. And 50% tissue culture infectious dose was consumed as infectious dose. The methods used to evaluate the virus-elimination efficacy were reverse-transcriptase polymerase chain reaction for B19V and the cytopathic effect calculation after filtration for PPV and HAV. RESULTS: B19V was not detected by RT-PCR in the filtered antithrombin III solutions with initial viral load of 6.42 x 10(5) IU/mL and 1.42 x 10(5) IU/mL before filtration. The virus-elimination efficacy of nanofiltration for PPV and HAV were > or = (3.32) and > or = (3.31), respectively. CONCLUSIONS: Nanofiltration would be an effective method for the elimination of B19V and HAV. It may be used as a substitute for NAT screening of these viruses in source plasma to ensure safety of plasma derivatives in Korea.

Investigation of the prevalence of human parvovirus B19 DNA in Korean plasmapheresis donors.

BACKGROUND: To ensure the safety of plasma derivatives, some countries have been screening for the human parvovirus B19 (B19V) antigen or DNA in blood donors. We investigated the prevalence of B19V DNA and anti-B19V antibodies in Korean plasmapheresis donors to evaluate the necessity of B19V DNA screening test. METHODS: Plasma samples were collected between March and July 2008 from 10,032 plasmapheresis donors. The B19V DNA test was performed using the LightCycler 2.0 (Roche, Germany) with quantification kits. Anti-B19V IgM and IgG were tested in 928 randomly selected samples from the 10,032 donors using recomWell Parvovirus B19 ELISA IgM, IgG assay (Mikrogen, Germany). RecomLine Parvovirus B19 LIA IgG, IgM assay (Mikrogen, Germany) was used to analyze the epitopes of antibodies in donors showing positive results for B19V DNA and anti-B19V antibodies. DNA sequencing was performed to identify the genotypes. RESULTS: The prevalence of B19V DNA was 0.1% (10/10,032). Virus titers in B19V DNA positive donors were less than 10(5) IU/mL (range: 2.7 x 10(1)-3.2 x 10(4) IU/mL) except for 1 donor (1.33 x 10(8) IU/mL). All the isolated B19V DNAs from 6 donors were identified as genotype I. Nine out of 10 B19V DNA positive donors also possessed anti-B19V IgG only or IgG and IgM. The prevalence of anti-B19V IgG was 60.1% (558/928). CONCLUSIONS: The prevalence of B19V DNA in Korean blood donors was not high and most donors also possessed neutralizing anti-B19V antibodies. Thus, the implementation of a B19V screening test for Korean blood donors does not appear to be imperative.

Establishment of an HBsAg mixed titer performance panel and HBsAg working standard for quality control of HBsAg diagnostic kits in Korea.

BACKGROUND: International Standards or commercial panels used for performance validation of diagnostic kits might not reflect the viral characteristics common in Korea. Also, continuous use of these materials is difficult because of limited quantity and high cost. OBJECTIVES: Establishment of HBsAg reference materials to be used as National Standards for validation of HBsAg diagnostic kits. STUDY DESIGN: 568 plasma units with OD less than 2.0 on HBsAg EIA were collected. HBsAg testing with 3 EIAs and 1 CIA was performed on all units. HBsAg positive units were subjected to HBV DNA quantification, genotyping and subtyping. Candidates for the mixed titer performance panel and working standard were confirmed for HBsAg by neutralization. A collaborative study was conducted for the candidates of the mixed titer performance panel and the working standard. RESULTS: Based on the results of the collaborative study, a working standard (KFDA08/024) consisting of a series of four-fold dilutions of 2 materials, one with genotype/subtype C2/adr and the other with C1/adw, was established. A mixed titer performance panel composed of 2 negative and 16 positive samples was also established. A G1896A and a T/I126S mutant are included in the positive samples. CONCLUSIONS: An HBsAg mixed titer performance panel and a working standard reflecting HBV genotypes/subtypes prevalent in Korea have been established as National Standards. This will enable consistent supply of validation materials, improve the validation system of HBsAg diagnostic kits in Korea and lead to quality improvement of diagnostic kits.

Homozygous deletion of CDKN2A (p16, p14) and CDKN2B (p15) genes is a poor prognostic factor in adult but not in childhood B-lineage acute lymphoblastic leukemia: a comparative deletion and hypermethylation study.

The biological behavior of childhood B-lineage acute lymphoblastic leukemia (B-ALL) is different from that of adults. We performed a comprehensive analysis of the deletion and the methylation profile of CDKN2A (hereafter identified separately as p16 and p14, for the different proteins encoded) and CDKN2B (hereafter p15) in 91 newly diagnosed B-ALL patients (61 children, 30 adults). The prognostic significance of the profiles of these genes and the association between alterations in these genes and known cytogenetic prognostic factors (BCR/ABL; ETV6/RUNX1, formerly TEL/AML1; MLL rearrangement; and ploidy changes of chromosomes) were also assessed. The prevalence of homozygous deletion, hemizygous deletion, and no deletion of the 9p21 region was 11.5%, 16.4%, and 72.1%, respectively, in children and 30.0%, 20.0%, and 50.0%, respectively, in adults; the higher incidence of homozygous deletion in adults was significant (P=0.029). Homozygous deletion was associated with poor overall survival in adults (P=0.019), but not in children. The incidence of promoter methylation of p16, p14, and p15 was 34.4%, 14.8%, and 34.4%, respectively, in children and 26.7%, 10.0%, and 40.0%, respectively, in adults, with no significant difference between the two groups. No significant association was observed between deletion and methylation or with known cytogenetic prognostic factors. The difference in incidence, distribution, and prognostic effect of homozygous deletion in children and adults may explain the prognostic disparity.

Diagnosis of human hepatitis B virus in non-treated blood by the bovine IgG DNA-linked carbon nanotube biosensor.

OBJECTIVES: To identify an effective bioassay for human hepatitis B virus (HBV) using voltammetric sensor. STUDY DESIGN: Electrode was prepared with bovine IgG immobilized onto a DNA-linked carbon nanotube electrode (BIDCE). Analytical BIDCE conditions were optimized through square wave (SW) stripping and cyclic voltammetry (CV). RESULTS: Optimum parameters of 75 Hz SW frequency, 0.1 V SW amplitude, -1.3 V SW accumulation potential, 100 s accumulation time, and 3.0 mV SW increment potential were obtained. Within working ranges of 0.035-0.242 mg/mL anti-bovine IgG, the relative standard deviation of 0.2 mL HBV was observed to be 0.04 (n=4). CONCLUSIONS: Diagnostic application was performed through direct assay of HBV in non-treated human blood.

Seroprevalence of human T-lymphotropic virus type 1 and 2 in Korean blood donors.

The seroprevalence rate of human T-lymphotropic virus (HTLV) among the Korean blood donor population was studied to determine whether screening for HTLV should be implemented. A total of 15,173 serum samples collected from June to July 2006 at five Blood Centers which are located closely to Japan geographically, where the prevalence of HTLV is known to be high, were tested. Serological screening was done by a chemiluminescence method. Samples reactive repeatedly on serological screening were confirmed further by Western blot, line immunoassay, nested polymerase chain reaction and sequencing of proviral DNA. Six samples tested reactive with the serological assay showing a reactive rate of 0.004%. Among the six samples, one sample was confirmed as HTLV-1 positive, giving a confirmed reactive rate of 0.007%. Based on the results of this study, an extended study will be conducted to evaluate whether introduction of HTLV screening is necessary in Korea.

Prevalence of antibodies to the circumsporozite protein of Plasmodium vivax in five different regions of Korea.

Malaria has recently re-emerged in the Republic of Korea (ROK), but only few malaria seroprevalences were reported. We obtained 1014 serum samples from inhabitants of five regions of ROK during the high transmission season between June and August in 2001. The levels of anti-circumsporozoite protein (CSP) antibody were assessed in samples using an indirect enzyme-linked immunosorbent assay (ELISA). The highest IgG seroreactivity against Plasmodium vivax recombinant CSP antigen was found among male residents of Cheolwon gun (13.5%), then Incheon (4.7%). The IgG seroreactivity from other regions ranged from 0.0% to 2.0%. These epidemiological data of seroprevalence in five regions of Korea showed a similar pattern to the annual incidence of malaria in these respective regions. The prevalence of antibodies increased with age, suggesting that the age and area-related prevalence patterns reflected differences in the inoculation rates between age groups and geographic regions. Seroprevalence and annual incidence were positively correlated in some areas of Korea.