Animal DNA Typing and Its Research Progress in Forensic Medicine / 法医学杂志

With the advance of molecular biology, DNA analysis technology has been widely applied in forensic science. Non-human DNA analysis can be used in some special cases and has unique forensic value to provide investigation clues and trial basis. Animal DNA typing plays a more prominent role in the detection of all kinds of non-human DNA related cases and is the main content of forensic non-human DNA analysis. This paper reviews the development history, present situation, advantages and disadvantages of animal DNA typing according to its technology, characteristic, challenges facing forensic science application scenarios, and also its future development.

Molecular epidemiology of Pseudomonas aeruginosa isolated from lower respiratory tract of ICU patients / Epidemiologia molecular de Pseudomonas aeruginosa isolada em pacientes com infecções do trato respiratório inferior admitidos em UTI

Lower respiratory tract infections (LRTIs) caused by Pseudomonas aeruginosa are the most common infection among hospitalized patients, associated with increased levels of morbidity, mortality and attributable health care costs. Increased resistant Pseudomonas worldwide has been quite meaningful to patients, especially in intensive care unit (ICUs). Different species of Pseudomonas exhibit different genetic profile and varied drug resistance. The present study determines the molecular epidemiology through DNA fingerprinting method and drug resistance of P. aeruginosa isolated from patients with LTRIs admitted in ICU. A total of 79 P. aeruginosa isolated from patients with LRTIs admitted in ICU were characterized by Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD) and Repetitive Extrapalindromic PCR (REP-PCR). Antibiotic resistance was determined by minimum inhibitory concentration (MIC) assay while MDR genes, viz, blaTEM, blaOXA, blaVIM, blaCTX-M-15 were detected by polymerase chain reaction (PCR). Of the 137 Pseudomonas sp isolated from ICU patients, 57.7% of the isolates were reported to be P. aeruginosa. The overall prevalence of P. aeruginosa among the all included patients was 34.5%. The RAPD analysis yielded 45 different patterns with 72 clusters with 57% to 100% similarity level. The RFLP analysis yielded 8 different patterns with 14 clusters with 76% to 100% similarity level. The REP PCR analysis yielded 37 different patterns with 65 clusters with 56% to 100% similarity level. There was no correlation among the different DNA patterns observed between the three different methods. Predominant of the isolates (46.8%) were resistant to amikacin. Of the 79 isolates, 60.8% were positive for blaTEM gene and 39.2% were positive for blaOXA gene. P. aeruginosa was predominantly isolated from patients with LRTIs admitted in ICU. The difference in the similarity level observed between the three DNA fingerprinting methods indicates that there is high inter-strain variability. The high genetic variability and resistance patterns indicates that we should continuously monitor the trend in the prevalence and antibiotic resistance of P. aeruginosa especially in patients with LRTIs admitted in ICU.

Infecções do trato respiratório inferior (ITRIs) são as infecções mais comuns entre pacientes internados em unidade de terapia intensiva (UTI). Pseudomonas aeruginosa é a causa mais comum de ITRIs e está associada ao aumento da mortalidade. Diferentes espécies de Pseudomonas exibem diferentes perfis genéticos e resistência variada as drogas. O presente estudo determina a epidemiologia molecular através do método de fingerprinting de DNA e resistência as drogas de P. aeruginosa isoladas de pacientes com LTRIs internados em UTI. Um total de 79 P. aeruginosa isoladas de pacientes com ITRIs internados em UTI foram caracterizados por Polimorfismo de Comprimento de Fragmentos de Restrição (RFLP), DNA Polimórfico Amplificado ao Acaso (RAPD) e PCR Extrapalindrômico Repetitivo (REP-PCR). A resistência aos antibióticos foram determinadas pelos ensaios de concentrações inibitória mínima (MIC), enquanto os genes MDR, blaTEM, blaOXA, blaVIM, blaCTX-M-15 foram detectados pela reação em cadeia da polimerase (PCR). Das 137 Pseudomonas sp isoladas de pacientes de UTI, 57,7% dos isolados foram relatados como P. aeruginosa. A prevalência geral de P. aeruginosa entre os pacientes incluídos foram de 34,5%. A análise RAPD renderam 45 padrões diferentes com 72 clusters com nível de similaridade de 57% a 100%. A análise RFLP renderam 8 padrões diferentes com 14 clusters com 76% a 100% de similaridade. A análise de PCR do REP produziram 37 padrões diferentes com 65 clusters com nível de similaridade de 56% a 100%. Não houveram correlações entre os diferentes padrões de DNA observados entre os três diferentes métodos. Predominantes dos isolados (46,8%) eram resistentes à amicacina. Dos 79 isolados, 60,8% foram positivos para o gene blaTEM e 39,2% foram positivos para o gene blaOXA. P. aeruginosa foi predominantemente isolado de pacientes com ITRIs internados em UTI. A diferença no nível de similaridade observado entre os três métodos de fingerprinting do DNA indica que há alta variabilidade inter-strain. A alta variabilidade genética e os padrões de resistência indicam que devemos monitorar continuamente a tendência na prevalência e resistência a antibióticos de P. aeruginosa, especialmente em pacientes com ITRIs internados em UTI.

Molecular epidemiology of Pseudomonas aeruginosa isolated from lower respiratory tract of ICU patients

Abstract Lower respiratory tract infections (LRTIs) caused by Pseudomonas aeruginosa are the most common infection among hospitalized patients, associated with increased levels of morbidity, mortality and attributable health care costs. Increased resistant Pseudomonas worldwide has been quite meaningful to patients, especially in intensive care unit (ICUs). Different species of Pseudomonas exhibit different genetic profile and varied drug resistance. The present study determines the molecular epidemiology through DNA fingerprinting method and drug resistance of P. aeruginosa isolated from patients with LTRIs admitted in ICU. A total of 79 P. aeruginosa isolated from patients with LRTIs admitted in ICU were characterized by Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD) and Repetitive Extrapalindromic PCR (REP-PCR). Antibiotic resistance was determined by minimum inhibitory concentration (MIC) assay while MDR genes, viz, blaTEM, blaOXA, blaVIM, blaCTX-M-15 were detected by polymerase chain reaction (PCR). Of the 137 Pseudomonas sp isolated from ICU patients, 57.7% of the isolates were reported to be P. aeruginosa. The overall prevalence of P. aeruginosa among the all included patients was 34.5%. The RAPD analysis yielded 45 different patterns with 72 clusters with 57% to 100% similarity level. The RFLP analysis yielded 8 different patterns with 14 clusters with 76% to 100% similarity level. The REP PCR analysis yielded 37 different patterns with 65 clusters with 56% to 100% similarity level. There was no correlation among the different DNA patterns observed between the three different methods. Predominant of the isolates (46.8%) were resistant to amikacin. Of the 79 isolates, 60.8% were positive for blaTEM gene and 39.2% were positive for blaOXA gene. P. aeruginosa was predominantly isolated from patients with LRTIs admitted in ICU. The difference in the similarity level observed between the three DNA fingerprinting methods indicates that there is high inter-strain variability. The high genetic variability and resistance patterns indicates that we should continuously monitor the trend in the prevalence and antibiotic resistance of P. aeruginosa especially in patients with LRTIs admitted in ICU.

Resumo Infecções do trato respiratório inferior (ITRIs) são as infecções mais comuns entre pacientes internados em unidade de terapia intensiva (UTI). Pseudomonas aeruginosa é a causa mais comum de ITRIs e está associada ao aumento da mortalidade. Diferentes espécies de Pseudomonas exibem diferentes perfis genéticos e resistência variada as drogas. O presente estudo determina a epidemiologia molecular através do método de fingerprinting de DNA e resistência as drogas de P. aeruginosa isoladas de pacientes com LTRIs internados em UTI. Um total de 79 P. aeruginosa isoladas de pacientes com ITRIs internados em UTI foram caracterizados por Polimorfismo de Comprimento de Fragmentos de Restrição (RFLP), DNA Polimórfico Amplificado ao Acaso (RAPD) e PCR Extrapalindrômico Repetitivo (REP-PCR). A resistência aos antibióticos foram determinadas pelos ensaios de concentrações inibitória mínima (MIC), enquanto os genes MDR, blaTEM, blaOXA, blaVIM, blaCTX-M-15 foram detectados pela reação em cadeia da polimerase (PCR). Das 137 Pseudomonas sp isoladas de pacientes de UTI, 57,7% dos isolados foram relatados como P. aeruginosa. A prevalência geral de P. aeruginosa entre os pacientes incluídos foram de 34,5%. A análise RAPD renderam 45 padrões diferentes com 72 clusters com nível de similaridade de 57% a 100%. A análise RFLP renderam 8 padrões diferentes com 14 clusters com 76% a 100% de similaridade. A análise de PCR do REP produziram 37 padrões diferentes com 65 clusters com nível de similaridade de 56% a 100%. Não houveram correlações entre os diferentes padrões de DNA observados entre os três diferentes métodos. Predominantes dos isolados (46,8%) eram resistentes à amicacina. Dos 79 isolados, 60,8% foram positivos para o gene blaTEM e 39,2% foram positivos para o gene blaOXA. P. aeruginosa foi predominantemente isolado de pacientes com ITRIs internados em UTI. A diferença no nível de similaridade observado entre os três métodos de fingerprinting do DNA indica que há alta variabilidade inter-strain. A alta variabilidade genética e os padrões de resistência indicam que devemos monitorar continuamente a tendência na prevalência e resistência a antibióticos de P. aeruginosa, especialmente em pacientes com ITRIs internados em UTI.

The application of ultraifltration in old biological samples( / 中国法医学杂志

Objectve To investigate the application value of ultraifltration with millipore ifltration method test of old biological samples DNA. Methods 23 old blood samples separately were cut blood piece equally suitable size of three groups, labeled A, B and C group. DNA was extracted by magnetic bead methed and get DNA template with 80μL, 80μL, 20μL elution solution, respectively, then the DNA template in group A and C take right amount directly ampliifed, group B with milipore ultraifltration ifltration condensed amplication. PCR products were detected by ABI3130xl genetic analyzer and analyzed by GeneMapperID V3.2 software. The date were analyzed by using SPSS software. Results No samples were ampliifed for all STR loci in group A. There were 18 cases in group B samples amplified all STR loci and 11 samples in group C. Conclusion Application of millipore ultrafiltration method can signiifcantly improve the old biological samples DNA classiifcation success rate.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB)

Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

Genetic markers, genotyping methods & next generation sequencing in mycobacterium tuberculosis.

Molecular epidemiology (ME) is one of the main areas in tuberculosis research which is widely used to study the transmission epidemics and outbreaks of tubercle bacilli. It exploits the presence of various polymorphisms in the genome of the bacteria that can be widely used as genetic markers. Many DNA typing methods apply these genetic markers to differentiate various strains and to study the evolutionary relationships between them. The three widely used genotyping tools to differentiate Mycobacterium tuberculosis strains are IS6110 restriction fragment length polymorphism (RFLP), spacer oligotyping (Spoligotyping), and mycobacterial interspersed repeat units - variable number of tandem repeats (MIRU-VNTR). A new prospect towards ME was introduced with the development of whole genome sequencing (WGS) and the next generation sequencing (NGS) methods, where the entire genome is sequenced that not only helps in pointing out minute differences between the various sequences but also saves time and the cost. NGS is also found to be useful in identifying single nucleotide polymorphisms (SNPs), comparative genomics and also various aspects about transmission dynamics. These techniques enable the identification of mycobacterial strains and also facilitate the study of their phylogenetic and evolutionary traits.

Frequency of Alleles D16S539, D7S820, D13S317 in a population sample in National Capital District, Papua New Guinea

The allele and genotype frequency values of the three tetranucleotide short tandem repeat (STR) loci, D7S820, D13S317 and D16S359, were analysed in blood samples of 25 unrelated randomly selected individuals in the National Capital District, Papua New Guinea. Gene-Print Silver-STR III Multiplex kit (Promega Corp., Medison, WI, USA) was used for the PCR amplification in GeneAmp®PCR System 9700 thermal cycler (Applied iosystems). Data analysis was carried out using the PowerStatsV12.xl workbook template obtained from Promega Corporation. The three STR loci were in Hardy-Weinberg equilibrium. Five alleles (9 – 13) were identified for D16S539, five alleles (8 – 12) for D7S820 and six alleles (8, 9, 11 – 14) for D13S317. No new or microvariant alleles were observed. The most frequent genotypes for D16S539 were 11-11 and 11-12; for D7S820 were 10-11 and 12-12; for D13S317 was 8-12. Observed Heterozygosity was highest in D13S317 (0.880). The combined power of discrimination was 0.99733 and the combined power of exclusion was 0.9363. The data suggests that the three loci are useful for identity testing, forensics and for solving paternity cases among the population in National Capital District, Papua New Guinea.

Allelic and Haplotypic Diversity of HLA-A, -B, -C, and -DRB1 Genes in Koreans Defined by High-resolution DNA Typing / 대한진단검사의학회지

BACKGROUND: In this study, we used high-resolution DNA typing to investigate the distribution of HLA alleles and haplotypes in Koreans. METHODS: HLA-A, -B, -C, and -DRB1 alleles were genotyped at the allelic (4-digit) level in 474 healthy Koreans. HLA genotyping was performed in two steps. Initially, serologic typing or generic-level DNA typing was performed using the PCR-sequence-specific oligonucleotide method, and then allelic DNA typing (exons 2 and 3 for class I, and exon 2 for DRB1) was carried out using the PCR-single-strand conformation polymorphism method or sequence-based typing. HLA allele and haplotype frequencies and linkage disequilibrium values were calculated by the maximum likelihood method using a computer program developed for the 11th International Histocompatibility Workshop. RESULTS: A total of 21 HLA-A, 40 HLA-B, 22 HLA-C, and 29 HLA-DRB1 alleles were found in Koreans. The most frequent alleles in each locus with frequencies of > or =10% were, in decreasing order of frequency, as follows: A*24:02, A*02:01, A*33:03; B*51:01; C*01:02, C*03:03; and DRB1*09:01. The numbers of two- and three-locus haplotypes with frequencies of >0.5% were as follows: 44 A-C, 42 B-C, 51 A-B, 52 B-DRB1, 42 A-C-B, and 34 A-B-DRB1. Thirteen A-B-DRB1 haplotypes with frequencies of > or =1.0% comprised 26.0% of the total haplotypes. The six most common haplotypes were as follows: A*33:03-B*44:03-DRB1*13:02 (3.7%), A*33:03-B*44:03-DRB1*07:01 (3.0%), A*33:03-B*58:01-DRB1*13:02 (3.0%), A*24:02-B*07:02-DRB1*01:01 (2.8%), A*30:01-B*13:02-DRB1*07:01 (2.3%), and A*11:01-B*15:01-DRB1*04:06 (2.2%). CONCLUSIONS: The information obtained in this study can be used as basic data for Koreans in the fields of organ transplantation, disease association, and anthropologic studies.

Allele frequencies for 12 autosomal short tandem repeat loci in two bolivian populations

Two hundred and sixty unrelated subjects who asked for paternity testing at two Bolivian Laboratories in La Paz and Santa Cruz were studied. The loci D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, TH01, TPOX, and CSF1PO were typed from blood samples, amplifying DNA by polymerase chain reactions and electrophoresis. Allele frequencies were estimated by simple counting and the unbiased heterozygosity was calculated. Hardy-Weinberg equilibrium was studied and gene frequencies were compared between the two samples. All loci conformed to the Hardy-Weinberg law and allele frequencies were similar in samples from the two cities. The Bolivian gene frequencies estimated were significantly different from those described for Chile and the United States Hispanic-Americans for most of the loci.

Utility of In-House PCR for HLA-B27 Typing: Comparison of Concordance Rate between PCR Kit and In-House PCR / 대한진단검사의학회지

BACKGROUND: Commercial kits of PCR method are widely used in HLA-B27 typing; however, their cost is relatively high. In this study, we evaluated the utility of an in-house PCR method by comparing it with that of a commercial kit. METHODS: HLA-B27 typing was done in 188 patients by using two PCR methods, Absolute(TM) HLAB27 PCR kit (Biosewoom, Korea) and an in-house PCR method. The primers used in the in-house method were prepared by Bioneer (Korea). Both PCR tests were done by Gene Amp PCR System 9600 (Perkin-Elmer Centus Corp., USA). RESULTS: The commercial kit and in-house PCR showed 100% concordance rate with each other in HLA-B27 typing. Of 188 patients tested 72 (38.3%) were positive and 116 (61.7%) were negative by the both tests. Of 62 patients with ankylosing spondylitis, 50 were positive (80.7%). CONCLUSIONS: The in-house PCR is a reliable and cost-effective method and can replace or supplement commercial kits for HLA-B27 typing.

A utilização de técnicas de biologia molecular na genética forense: uma revisão / The use of molecular biology techniques in foresinc genetics: a review

Os avanços nas tecnologias de DNA surtiram um enorme impacto no campo da ciência forense. Com uma incrível sensibilidade e um alto poder de discriminação, a análise de DNA tem sido uma poderosa ferramenta para a identificação humana e investigaçõescriminais. O presente estudo fará uma revisão sobre as técnicas de Biologia Molecular mais significativas – RFLP, VNTR, PCR e STR – que foram desenvolvidas nas últimas décadas, tendo como princípio o estudo de diferentes polimorfismos de DNA para a identificação precisa de indivíduos. Por um longo tempo, estes polimorfismos foram detectados por técnicas que possuíam como base a eletroforese.Outra técnica que também será exposta, Southern Blotting, visa a identificação de uma seqüência de bases específicas do DNA, que foi por muito tempo aplicada tanto na detecção de SNPs como de VNTRs e STRs. Além disto, será descrita a reação em cadeia da polimerase (PCR), um método laboratorial capaz de copiar milhões de vezes um segmento do DNA, que se destaca perante outras técnicas por ser um procedimento relativamente simples e fácil de realizar em laboratório, gerando resultados precisos e satisfatórios, em um curto espaço de tempo. Por fim, serão descritos os métodos automatizados que, a partir de PCR, permitem a detecçãorápida de marcadores moleculares, a fim de facilitar e tornar mais precisa a identificação forense.

The advent of DNA-based technologies promoted significant impact in the field of forensic science. According to its highsensibility and powerful discrimination, the DNA analyses have been a powerful tool to human identification and criminal investigations. The present study will be taken to produce a review about the most important techniques of molecular biology developed in recent decades, such as RFLP, VNTR, PCR and STR. These techniques are based in the study of different polymorphisms in the DNAand it could be used in the precise subjects identification. For a period, these polymorphisms were detected through techniques based in electrophoresis. Besides, other procedures will be explained, like Southern Blotting that aims to identify specific DNA sequences and could be applied in the research of SNPs, VNTRs and STRs. It will be also described the Polimerase Chain Reaction (PCR), alaboratorial method able to amplify millions of a short DNA segment. This technique has some advantages through the others because it is a simple and easy procedure to be done in laboratories, and could offer accurate and satisfactory results in a short period of time. Concluding, automated techniques based in PCR will be present that permit fast detection of molecular evidences in order to facilitate and promote reliable forensic identification.

Evaluation of BioSewoom(TM) HLA-A, -B, -C PCR/SSP Kit / 대한진단검사의학회지

BACKGROUND: Human leukocyte antigen (HLA) typing based on polymerase chain reaction (PCR) is rapidly replacing the conventional serological method. This study was intended to evaluate Bio- SewoomTM HLA-A, -B, -C PCR/SSP kit (BioSewoom SSP) which had recently been developed in Korea. METHODS: A total of 158 samples from domestic (21) and international (137) HLA proficiency testing (PT) were genotyped with BioSewoom SSP, and its results were compared to consensus results. For comparison with INNO-LiPA HLA-A, -B, -C Typing Kit (INNO-LiPA, Innogenetics, Belgium), 20 samples of Koreans were genotyped with both kits for each HLA-A, -B, -C locus. RESULTS: Among the 21 samples of domestic PT, BioSewoom SSP showed ambiguities as follows: 4 samples (19.0%) in HLA-A, 6 (28.6%) in HLA-B, and 1 (4.8%) in HLA-C. The ambiguities could be resolved by considering the allele distribution of Koreans. Among the 137 samples from international PT, BioSewoom SSP also showed ambiguities as follows: 12 samples (8.8%) in HLA-A, 26 (19.0%) in HLA-B and 6 (4.4%) in HLA-C. Considering the allele distribution of Koreans, the serologic equivalents obtained from BioSewoom SSP showed a full agreement with those of INNO-LiPA in all the loci tested. Twelve (0.007%) among 1,760 PCR reactions from the 21 samples of domestic PT and 20 patient samples produced faint nonspecific bands, but it was negligible. PCR failure of internal control just barely occurred (15 PCR reactions, 0.009%), but it had no bearing on allele assignment. CONCLUSIONS: The performance of BioSewoom SSP was comparable to that of INNO-LiPA. All the ambiguities could be resolved by considering the allele distribution of Koreans. It is concluded that BioSewoom SSP has good performance to be used in routine HLA laboratories.

Bones unveiled gruesome murder.

A bundle of bone in a carton was sent to the Deptt. of Forensic Medicine, Guwahati Medical College for autopsy. Police suspected that the bones belonged to a young boy of Guwahati who was kidnapped and ransom demanded. Systematic examination revealed that the bones belonged to a single human being, sex was male and the age was ascertained to be between 16 to 18 years. The stature was calculated from long bones to be 160.4 cm ± 3.9 cm. The skull showed one ante mortem incised wound over the right parietal bone involving both the tables. The time since death was estimated to be 1 to 2 years. The skull, scapula, hip bone and one femur were sent to State Forensic Science Laboratory for Superimposition, DNA typing and Chemical analysis. Superimposition could not be carried out and report of DNA typing not received. Chemical analysis gave negative test for common poisons. Opinion regarding the cause of death was given subsequently as coma resulting from homicidal incised wound of the skull. The autopsy report, corroborative evidences and subsequent investigation by the police confirmed the suspicion of police.

Analysis of Ambiguities of HLA-B Typing using the Dynal RELI(TM) SSO HLA-B Test and Development of an`Interpretation Program for Koreans' / 대한진단검사의학회지

BACKGROUND: In recent years, DNA typing has been increasingly used in HLA-A and B typing, and commercial kits based on the PCR-SSO method are most commonly used in Korea. However, SSO typing kits show ambiguities to some extent in the generic level typing of HLA-B alleles. We analyzed the ambiguities in the Dynal RELI(TM) SSO HLA-B test (Dynal B test) with confirmatory typing results, and developed and evaluated the accuracy and efficacy of an `Interpretation Program for Koreans'. METHODS: A total of 2, 169 Korean marrow donor registry samples were typed for HLA-B alleles using the Dynal B test (56 probes) and all of the 222 cases showing ambiguities were subjected to confirmatory typing. We have developed an `Interpretation Program for Koreans' for the Dynal B test on the basis of the allele frequencies of Korean, Japanese and Asian populations. The samples showing ambiguities in the Dynal B test were interpreted using the `Interpretation Program for Koreans' and the results were compared with confirmatory typing results. RESULTS: The Dynal B test showed 10.2% (222/2, 169) of ambiguities and these ambiguities were classified into 47 different band patterns. These ambiguity patterns were interpreted using the `Interpretation Program for Koreans', which showed ambiguities in 14 band patterns and 3.4% (73/2, 169) of the total samples. Among these ambiguities, 4 band patterns (55 samples) arose from those alleles which are not found in Koreans and rarely found in Japanese or Asians (B*1522, *3521, *7802). Thus, excluding these rarities, only less than 1% (18/2, 169) of samples resulted in ambiguities, and most (16/18) of these were B55 vs. B56 ambiguities. The results from the `Interpretation Program for Koreans' were fully concordant with the confirmatory typing results. CONCLUSIONS: The Dynal B test showed around 10% ambiguities and the `Interpretation Program for Koreans' showed 3.4% of ambiguities. Excluding the ambiguities with extremely low probabilities arising from rare alleles in Japanese or Asians, actually >S99% of the samples could be typed accurately using the program without additional confirmatory tests.

HLA-A, -B, -DR Allele Frequencies and Haplotypic Associations in Koreans Defined by Generic-Level DNA Typing / 대한진단검사의학회지

BACKGROUND: HLA allele and haplotype distribution varies widely among different ethnic groups. For organ transplantation, anthropology and disease association studies, reliable data on the HLA distribution in each ethnic group is needed. In recent years, more accurate DNA typing methods are increasingly used in place of the serologic typing method. METHODS: We examined HLA-A, -B, and -DR alleles at the generic (serologic) level in 1, 600 Koreans registered for the Korea Marrow Donor Program (KMDP) using the PCR-sequence specific oligonucleotide (SSO) method (Dynal RELI(TM) kit). Allele and haplotype frequencies were estimated by the maximum likelihood method using the computer program developed for the 11th International Histocompatibility Workshop. RESULTS: HLA alleles found in Koreans were 13 in A, 31 in B, and 13 in DR locus. Most frequent alleles with frequencies > or =10% were: A2, A24, A33, A11; B62, B44, B51; DR4, DR15, DR13, and DR8 in each locus in decreasing order of frequency. Subtype frequencies of B61 and B75 serologic specificities were identified: B*4002 (51.1%), *4003 (7.6%) and *4006 (41.3%) for B61, and B*1502 (9.5%) and *1511 (90.5%) for B75. Two-locus haplotypes with frequencies> or =0.1% were presented (99 A-B, 115 B-DR), among which those with frequencies> or =1.0% showing significant positive linkage disequilibrium (P or =0.1% were identified in Koreans, among which 38 haplotypes showed frequencies> or =0.5%. We compared the results of this study with those of our previous study of serologically typed HLA-A, -B and DNA typed HLA-DR in 2, 000 Koreans. Results from the two studies were similar, but blank frequencies were decreased to 0% for HLA-A, -B, and -DR locus compared with the frequencies of 0.3-0.8% in the previous study (A, 0.3%; B, 0.8%; DR, 0.3%) and all of the serologic splits could be assigned in this study. CONCLUSIONS: In this study, we provided the allele and haplotype frequencies of HLA-A, -B, and -DR in Koreans defined by a DNA typing method, which can be used as basic data on Koreans for organ transplantation and disease association studies.

Molecular Epidemiology of Vancomycin-resistant Enterococci Isolated from Patients in a Neonatal Intensive Care Unit / 대한임상병리학회지

BACKGROUND: Recently, an acquired resistance to vancomycin in enterococci has become a serious clinical problem. For the prevention of further propagation of vancomycin-resistant enterococci (VRE), epidemiological study of the infection is essential, but studies on the VRE infection are rare in Korea. We conducted an analysis of the epidemiology of a VRE outbreak in a neonatal intensive care unit (NICU) to clear up the route of propagation of the VRE. METHODS: Vancomycin-resistant Enterococcus faecium (VREFM) strains were isolated from urine specimens of seven patients, rectal swabs from seven patients, and three skin swabs from two patients in the Kosin Medical Center neonatal intensive care unit, Pusan, Korea. Antimicrobial susceptibilities were tested by a disk diffusion method and agar dilution method. Genotypes of vancomycin-resistance were determined by PCR and SmaI-digested genomic enterococcal DNAs were analyzed by pulsed-field gel electrophoresis. RESULTS: All of the 17 strains of VREFM were resistant to ampicillin, vancomycin, and teicoplanin and they showed the same genotype (vanA). SmaI-digested genomic DNAs of seven strains isolated from urine were typed as I (1), II (1), IIIb (4), and IV (1). Three strains from skin swabs were I (2) and II (1). Six strains from rectal swabs were I (2), II (1), and IIIa (3). Genomic DNA typing of one isolate from a rectal swab failed. Each genomic DNA type of VREFM strains isolated from skin swabs of two patients were the same with those from urine specimens as I and II, respectively. CONCLUSIONS: This study suggests that VRE strains colonized in the intestines can cause infections after skin colonizing and can be transmitted/propagated to other people through skin contact. In conclusion, it is important for the prevention of the dissemination of VRE that controls for patients' skin hygiene, as well as hand washing by medical persons, be put in place.

Molecular Epidemiologic Analysis of a Cluster of Klebsiella Pneuminiae Infection in Neonatal Intensive Care Unit

PURPOSE: Klebsiella pneumoniae is a major nosocomial pathogen in neonatal intensive care unit (NICU). Sequential outbreaks of K. pneumoniae infection in NICU could occur due to antibiotic resistant strains or persistent environmental sources in NICU. When these outbreaks are happen, epidemiological analysis must be performed to discover the nosocomial sources and clarify the nature of the outbreaks strains. We conducted a molecular epidemiological study to recognize sources and natures of the repeated K. pneumoniae infections in our NICU from July to November 1997. METHODS: Fourteen clinical specimens isolated from K. pneumoniae infected newborn infants and 2 environmental K. pneumoniae strains isolated from surveillance cultures were studied. To establish the epidemiological analysis, we used field inversion gel electrophoresis (FIGE) for genomic DNA typing, and plasmid DNA typing. Also, an antibiogram was obtained from susceptibility tests of isolated K. pneumoniae. RESULTS: The results of genomic DNA typing using FIGE and antibiogram showed 4 different patterns, and plasmid DNA typing analysis showed 9 different patterns. Twelve strains out of 14 clinical isolates were almost identical with the two environmental strains, when comparing the genotypic patterns. And these 12 identical strains of FIGE profile showed similar plasmid DNA patterns. The strains could be classified into 5 different types. CONCLUSION: Plasmid DNA typing method and FIGE have the power to discriminate in epidemiological analysis of K. pneumoniae outbreaks. Our study results suggest that repeated K. pneumoniae infections in NICU can be caused by clusters of K. pneumoniae from environmental sources.

Establishment of HLA-B*15 Supplementary DNA Typing for Korean Samples / 대한임상병리학회지

BACKGROUND: HLA-B*15 alleles encode molecules belonging to several serologic subtypes, B62, B63, B71, B72, B75, B76, and B77. Using the conventional serologic typing method, assignment of B15 subtypes has often been prone to error specifically in samples exhibiting either an ambiguous or a B15 homozygous reaction pattern. The goal of this study was to establish a supplementary DNA typing method for accurate assignment of B15 subtypes in 'problematic B15 positive samples'. METHODS: B*15 specific gene amplification was performed using a pair of PCR primers that specifically annealed to B*15 and B*46 alleles. Nested PCR was applied to the amplified DNA using 14 sequence specific PCR primer sets. DNA sequencing was used to clarify the assigning of samples exhibiting discrepancies between the results obtained by B*15-specific nested PCR-SSP typing and serology. RESULTS: The B*15-specific nested PCR-SSP typing could clearly discriminate the 9 B*15 alleles expressed in the Korean population. In application of the system to 30 B15 positive serologically typed samples, 4 exhibited discrepancies between serology to PCR-SSP results. DNA sequencing results obtained from the samples were concordant with those from B*15-specific nested PCR-SSP typing. CONCLUSIONS: The established B*15-specific nested PCR-SSP method is superior to serology in accuracy and resolution. Therefore, the method will be useful as a supplementary DNA typing method to clarify HLA-B assignments of 'problematic B15 positive samples' in Koreans.

Genetic Diversity Of HLA Polymorphism And New Genes.

DNA level studies, based on SSOP hybridization of HLA class II PCR products have revealed considerable diversity in HLA among Asian Indians. High resolution typing of specific alleles such as DR2 and DR4 in the HLA class II region and their DR-DQ haplotypes have helped to detect unique haplotypes and novel alleles which have subsequently been confirmed by sequencing. Incidentally, remarkable stability is maintained in several other DRBI alleles, viz DR1, DR7, DR9 and DR10. Further characterization of new alleles will be carried out by sequencing of mRNA. The ARMS-PCR technology has been found to be particularly useful for typing difficult HLA-A, HLA-B and all HLA-Cw alleles. These technologies are remarkably superior over serological methods. Our studies have shown appreciable heterogeneity of common HLA-A and B alleles in Asian Indians. Molecular subtypes of HLA-A2 revealed that subtype A*0211 is found only in the Indian population and may be the result of a selection pressure in this population. Investigations into polymorphism in the HLA-B27 gene revealed that subtypes common both to the western Caucasoids and orientals occur in the Indian population. It is apparent that the population of the Indian subcontinent, placed as it is between the Caucasoids and Negroids on one hand and Australoids and Mongoloids on the other, provides a rich source of many HLA haplotypes. While the most frequent Caucasian haplotypes occurred with a reasonable frequency in Asian Indians, those found predominantly in other ethnic groups (e.g., Australian Aborigines and populations of Oceania, China and Japan) are also detected. Knowledge on this is most important for donor selection during organ and bone marrow transplantation and for designing MHC targeted vaccines in specific diseases. The major histocompatibility complex (MHC) encompasses two major classes of molecules: the MHC classes I and class II, both of which appear to have originated from a common ancestor gene. The major biological function of the MHC is to bind peptide fragments derived from protein antigens (viruses, peptides etc) and display them on the surface of antigen presenting cells (APCs), evoking effector responses upon recognition by the antigen specific receptors on T lymphocytes. This process requires an efficient intracellular machinery to fragment the protein antigens into smaller peptides capable of binding to a host MHC molecule. Since the number of peptides that can theoretically be generated is very large, there is need for an extensive MHC gene pool. Thus the HLA system which is the MHC of man is extremely polymorphic. Although the general rules for peptide-MHC interactions for both classes of MHC molecules are essentially similar; there are two fundamental differences: i) MHC class I ligands originate from endogenous sources, mainly from proteins of the cytosol or the nucleus and are delivered by the 'endogenous processing pathway: In contrast, the MHC class II ligands are generated by the degradation of proteins from the extracellular compartment. These distinctions are also reflected in the responding T cells: CD8 positive T cells being restricted by class I-peptide complexes, and CD4 positive T cells by class II-peptide complexes. ii) In accordance with the distribution of hydrogen bonds in the peptide binding groove of the MHC, the anchor residues are placed at the terminal ends of the class I groove. Contrarily, the binding forces are distributed throughout the class II groove and ensure bonds between the peptide's backbone and class II molecule (Madden et al, 1993; Stern et al, 1994; Stern and Wiley 1994). The specificity of the interaction is determined by pockets in the MHC groove that have a fixed spacing from each other and which also have specificity for anchoring particular side chains of the peptide's amino acids.

An Analysis of the Victim's Identification Results from Commercial Aircraft Accidents in Guam

No abstract available.