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PubMed1994

Molecular subtypes of env sequences around V3 region of human immunodeficiency virus type 1 in Taiwan.

Samples of peripheral blood mononuclear cells (PBMC) were collected during 1990-91 from seropositive healthy, male HIV-1 carriers visiting Taipei Venereal Disease Control Center, and a male AIDS patient admitted to a general hospital. The V3 and its flanking nucleotide (nt) sequences in their DNA were amplified by polymerase chain reaction (PCR) and compared with those of known HIV-1 prototypes. The nt sequences obtained from 21 individuals (e.g., TW92) clustered as Group A, which were highly homologous (95.6-99.5%) to that of HXB2 virus while those from 6 individuals (TW90, TW91, TW97, TW99, TW102 and TW104) were classified as Group B showing low similarities (73.2-84.2%) to those of HXB2 and moderate similarities (80.7-90.0%) to those of SC and Bangkok (BK) viruses. By comparison of their deduced amino acid sequences with those of consensus sequences for subtypes A-F as defined by Myers et al. (1993), both Groups A and B viruses (except TW102) together with those of HXB2, SC and BK viruses could be identified as members or variants of subtype B, and the TW102 virus as a member of subtype E viruses. Individuals with the Group A viruses included 4 homosexual and 17 heterosexual Taiwanese males, 2 of the latter having a history of i.v. drug abuse. Among individuals with Group B viruses, those with TW97, TW99, TW104 and TW91, who was an AIDS patient, were heterosexual Taiwanese males, whereas both TW90 and TW102 viruses were from individuals who were overseas heterosexual Chinese from Thailand, the former with a history of i.v. drug abuse and the latter without.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

Molecular subtyping of clinical and environmental strains of Cryptococcus neoformans variety neoformans serotype A isolated from southern Italy.

Analysis of ribosomal DNA (rDNA) restriction fragment-length polymorphism (RFLP) and random amplification of polymorphic DNA (RAPD) was used to investigate the genetic variability and biogeographic distribution of clinical and environmental strains of Cryptococcus neoformans isolated from a limited area of southern Italy, where the selection of a predominant cryptococcal genotype could be expected. All isolates belonged to the species Cr. neoformans variety neoformans serotype A. RFLP analysis of a specific rDNA fragment allowed the distinction of strains of Cr. neoformans from closely related fungal reference species, but neither intraspecies nor intravarieties polymorphism was detected. On the contrary, RAPD fingerprints produced by priming with four different primers [(GTG)5, (GACA)4, M13 core sequence and the 8-mer oligonucleotide (GCGGACGG)] were able to characterize the isolates up to the individual level, indicating the presence of marked heterogeneity among Cr. neoformans serotype A strains in southern Italy.

Cryptococcosis↗
PubMed1995

Molecular subtyping of prevalent M serotypes of Streptococcus pyogenes causing invasive disease.

Reproducible methodologies and a scheme for high-resolution genotyping of Streptococcus pyogenes were defined with respect to a study of six predominant M serotypes causing invasive group A streptococcal disease in the United Kingdom. Serotype reference strains were compared with nine clinical isolates of each serotype from patients with diseases such as pneumonia, puerperal sepsis, toxic shock-like-syndrome, cellulitis, or necrotizing fasciitis. Four enzymes were evaluated for their discriminatory power in 16S rRNA gene-specific ribotyping. Discriminatory power was greatest with EcoRI, which generated serotype-specific ribotypes, and with SacI, which could subdivide strains of the same M serotype. Twenty-five combined ribotypes were found among the 60 strains, and the indices of discriminatory power (D values) of this method varied from 0.51 within serotype M1 to 0.98 within strains of serotype M5. Macrorestriction with the rarely cutting endonuclease SmaI and pulsed-field gel electrophoresis gave D values varying from 0.37 within serotype M1 to the maximal 1.0 within serotype M5. Comparison of macrorestriction profiles revealed various degrees of genetic heterogeneity within M serotypes. Strains of M1, M3, M6, and M11 exhibited clonally related macrorestriction profiles, while those of R28 and M5 strains were consistent with polyphyletic origin.

Antigens, Bacterial↗
PubMed1996

Molecular subtyping of Neisseria meningitidis serogroup B: comparison of five methods.

In order to compare methods for subtyping Neisseria meningitidis serogroup B isolates, 96 isolates obtained from various locations in the United States and northwestern Europe were subtyped by five methods: monoclonal antibody (MAb)-based serotyping and serosubtyping, DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MEE), ribotyping, and PCR-restriction fragment length polymorphism of the internally transcribed spacer region of the rRNA operon (ITS PCR-RFLP). All N. meningitidis serogroup B isolates were typeable by PFGE, MEE, ribotyping, and ITS PCR-RFLP. Only 44.8% of the isolates were completely typeable (both serotype and serosubtype determination) by MAb-based serotyping and serosubtyping. 60.4% of the isolates could be serotyped but not serosubtyped, and 90.6% of the isolates could be either serotyped or serosubtyped. Simpson's discrimination indices of diversity for the methods were as follows: PFGE, 99.7%; MEE, 99.4%; ribotyping, 98.8%; MAb serotyping, 75.8%; MAb serotyping and/or serosubtyping 97.5%; and ITS PCR-RFLP, 84.2%. The high degree of diversity observed by PFGE, MEE, and ribotyping can be explained by the fact that isolates were collected from different geographic locations at various times. PFGE, MEE, and ribotyping showed greater discriminatory abilities than MAb-based serotyping and serosubtyping or ITS PCR-RFLP.

Antibodies, Monoclonal↗
PubMed2004

A surrogate marker profile for PML/RAR alpha expressing acute promyelocytic leukemia and the association of immunophenotypic markers with morphologic and molecular subtypes.

BACKGROUND: The availability of genotype-specific therapy for PML/RAR alpha(pos) acute promyelocytic leukemia (APL) requires that this disease be precisely diagnosed. Immunophenotypic characteristics heretofore proclaimed as reliably characterizing APL (HLA-DR(low), CD34(low), P-glycoprotein(low) myeloid phenotype) do not differentiate from APL-like immune profiles unassociated with the PML/RAR alpha fusion transcript. METHODS: To establish a surrogate marker profile for APL, we explored 19 potentially predictive markers compared with differentiated acute myeloid leukemia using the classification tree approach with recursive partitioning. RESULTS: In a test group of 58 APL patients, the most predictive immune profile was HLA-DR(low), CD11a(low) (alpha(L) subunit of the leukocyte integrin LFA-1), CD18(low) (beta(2) subunit of LFA-1). APL cells always expressed CD117 (c-kit) but lacked the progenitor antigen CD133 and the more mature myeloid antigen, CD11b (alpha(M) leukocyte integrin). This antigen pattern was validated in 90 additional APL patients. M3v APLs (n = 30) had more leukemic promyelocytes expressing the T-cell antigen, CD2 (P < 0.0001) or the stem cell marker, CD34 (P = 0.0003) and demonstrated higher fluorescence intensity for the binding of antibody to the common leukocyte antigen, CD45 (P = 0.0008) than M3 (n = 102). S-form APL (n = 45) had a higher percent of cells expressing CD2 or CD34 (P < 0.0001 for both) or the neural cell adhesion molecule CD56 (P = 0.001) than L-form APL (n = 66). CONCLUSIONS: PML/RAR alpha(pos) APL cells typically lack leukocyte integrins. HLA-DR(low), CD11a(low), CD18(low) is a reliable surrogate antigen expression profile for PML/RAR alpha(pos) APL, irrespective of morphology and transcript isoform.

Antigens, Neoplasm↗
PubMed2005

Molecular subtyping of clinical isolates of Candida albicans and identification of Candida dubliniensis Malaysia.

The genotypes of 221 recent isolates of Candida albicans from various clinical specimens of 213 patients admitted to the University Malaya Medical Centre, Malaysia was determined based on the amplification of a transposable intron region in the 25 S rRNA gene. The analyses of 178 C. albicans isolated from nonsterile clinical specimens showed that they could be classified into three genotypes: genotype A (138 isolates), genotype B (38 isolates) and genotype C (2 isolates). The genotyping of 43 clinical isolates from sterile specimens showed that they belonged to genotype A (29 isolates), genotype B (10 isolates), genotype C (2 isolates) and genotype D (2 isolates). The overall distribution of C. albicans genotypes in sterile and nonsterile specimens appeared similar, with genotype A being the most predominant type. This study reported the identification of C. dubliniensis (genotype D) in 2 HIV-negative patients with systemic candidiasis, which were missed by the routine mycological procedure. The study demonstrated the genetic diversity of clinical isolates of C. albicans in Malaysia.

Candida albicans↗
PubMed1991

The pathogenesis and epidemiology of catheter-related infection with pulmonary artery Swan-Ganz catheters: a prospective study utilizing molecular subtyping.

To delineate the pathogenesis and epidemiology of catheter-related infection with Swan-Ganz pulmonary artery (PA) catheters, a prospective clinical study of hospitalized adult medical and surgical patients was done. Role of catheter material was assessed by randomizing insertions to heparin-bonded PA catheters made of polyvinylchloride or polyurethane. Sources of infection and pathogenesis were studied by culturing skin, the introducer, the PA catheter tip, all hubs, infusate from each lumen, and the extravascular portion of the PA catheter beneath the external protective plastic sleeve. Concordance between isolates from sources and infected catheters was determined by speciation, antibiogram, and for coagulase-negative staphylococci, plasmid profile analysis. Risk factors for infection were determined by stepwise logistic regression. Overall, 65 (22%) of 297 Swan-Ganz catheters showed local infection of the introducer (58 catheters) or the intravascular portion of the PA catheter (20 catheters); only two catheters (0.7%) caused bacteremia. Eighty percent of infected Swan-Ganz catheters (the introducer or PA catheter) showed concordance with organisms cultured from skin of the insertion site, 17% with a contaminated hub and 18% with organisms contaminating the extravascular portion of the PA catheter beneath the sleeve. Isolates from infected PA catheters were most likely to show concordance with concomitantly infected introducers (71%). Cutaneous colonization of the insertion site with greater than 10(2) cfu/10 cm2 (relative risk [RR] 5.5; p less than 0.001), insertion into an internal jugular vein (RR 4.3; p less than 0.01), catheterization greater than 3 days (RR 3.1; p less than 0.01), and insertion in the operating room using less stringent barrier precautions (RR 2.1; p = 0.03) were each associated with a significantly increased risk of catheter-related infection. The risk of bacteremic infection with Swan-Ganz catheters is now low, in the range of 1%, with reasonable care. Swan-Ganz catheters are vulnerable to contamination from multiple sources, but the patient's skin is the single most important source of organisms causing invasive infection, which in most cases involves the introducer rather than the PA catheter. Heavy colonization of the insertion site, percutaneous insertion in the internal jugular vein rather than subclavian vein, catheterization longer than 3 days, and insertion with less stringent barrier precautions significantly increase the risk of catheter-related infection. These findings hold promise for application to management of Swan-Ganz catheters and research in catheter design to reduce the risk of catheter-related infection.

Adolescent↗
PubMed2026

Anatomical location defines distinct molecular subtypes of mucosal melanoma.

BACKGROUND: Mucosal melanoma (MM) is a rare and aggressive melanoma subtype that is understudied. The relationships between anatomical location, genomic alterations, stage at presentation, and survival remain incompletely characterized. METHODS: We carried out a retrospective single tertiary center study of 105 patients with histologically confirmed MM diagnosed between 1996 and 2025. Clinical and genomic data were analyzed to evaluate associations between anatomical location, mutational profile, stage at presentation, and survival outcomes, including melanoma-specific mortality. RESULTS: Lower-body tumors arising in the anus or genital areas were enriched for KIT and splicing factor 3 subunit B1 alterations, whereas NRAS mutations were distributed across anatomical regions. Among the two most common mutated genes, NRAS-mutant tumors were more likely than KIT-mutant tumors to present with metastatic disease [53% versus 19%; P = 0.046, odds ratio (OR) 4.7, 95% confidence interval (CI) 1.15-19.41]. Lower-body tumors were associated with worse overall survival (OS) than upper-body tumors (median 2.81 versus 8.40 years; OR = 0.05) and with higher melanoma-specific mortality. In multivariable analyses, upper-body location remained independently associated with improved OS (hazard ratio 0.14, 95% CI 0.05-0.36, P < 0.001). CONCLUSIONS: Anatomical location of MMs and genomic alterations define biologically and clinically distinct subtypes.

KIT mutation↗
PubMed2000

Pharmacological characterization, molecular subtyping, and autoradiographic localization of putative melatonin receptors in uterine endometrium of estrous rats.

The objective of this study was to determine the biochemical characteristics, subtypes, and localization of melatonin receptors in the rat uterus in estrous stage. Autoradiography with the melatonin ligand, 2-[125I]iodomelatonin, showed that melatonin receptors were localized in the rat uterine endometrium. Binding of 2-[125I]iodomelatonin in crude membrane preparations of rat uterine endometrium in estrous stage was stable, saturable, reversible and of high affinity. Rosenthal analysis yielded an equilibrium dissociation constant (Kd) of 28.9 +/- 3.59 pmol/l (n = 8) and a maximum number of binding sites (Bmax) of 1.6 +/- 0.15 fmol/mg protein (n = 8). The Kd value determined from kinetic analysis was 16.5 +/- 3.02 pmol/l (n = 3). Competition studies using various indoles and neurotransmitters demonstrated that 2-iodomelatonin, melatonin, 6-chloromelatonin, 6-hydroxymelatonin and N-acetylserotonin showed significant inhibition of the 2-[125I]iodomelatonin binding, while the other indole compounds tested had no significant inhibition. The expression of rat uterine endometrial melatonin receptor subtypes was studied by reverse transcription-polymerase chain reaction (RT-PCR) using mt1 and MT2 receptor gene-specific primers. mt1 receptor cDNA was amplified and confirmed by nucleotide sequencing. These findings indicate that mt1 receptors were present in the rat uterine endometrium, and suggest that melatonin plays an integral part in uterine physiology.

Animals↗
PubMed1999

Improved recognition of MRSA case clusters by the application of molecular subtyping using pulsed-field gel electrophoresis.

Methicillin-resistant Staphylococcus aureus (MRSA) is increasingly common in hospital and community populations, making the recognition of true nosocomial outbreaks more difficult. We have used pulsed-field gel electrophoresis (PFGE) with Sma I digestion to analyse retrospectively two perceived outbreaks of epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA 15) colonization. The first cluster of cases in patients and staff on a general ward (ward D) revealed three different antibiograms based on differences in ciprofloxacin and rifampicin sensitivities. All isolates typed using PFGE, which was more discriminatory than phage-typing. One PFGE banding profile labelled type 5 was predominant, but 12 isolates proved to be subtypes of type 5 and two were PFGE type 11. Four staff members carried a strain not found in patients, three carried strains found in patients and transient carriage was highlighted as a problem when screening staff. PFGE enhanced the epidemiological data and proved that the cases on this ward did not comprise one large outbreak but numerous sporadic cases and smaller clusters. In contrast, isolates from a second cluster of cases which occurred on ward F were indistinguishable using antibiograms, phage-typing and PFGE, confirming this was more likely to be a true outbreak of colonization. We conclude that PFGE usefully augments epidemiological information and allows more logical infection control decisions to be made, with better utilization of scarce resources.

Bacterial Typing Techniques↗
PubMed2000

Molecular subtypes of the HLA-DR antigens in pulmonary tuberculosis.

OBJECTIVE: The aim of this study was to analyze association between HLA-DRB1 alleles and pulmonary tuberculosis (PTB) in the Polish population. METHODS: The HLA-DRB1 typing was performed using sequence-specific amplification (polymerase chain reaction with sequence specific primer [PCR-SSP] in 31 patients and 58 healthy volunteers. The DRB1 primers were supplied by DYNAL in the standard kit DYNAL DR "low-resolution"-SSP. RESULTS: The study showed that the DRB1*16 alleles frequency was higher in patients with PTB than in the tested group of healthy controls (P < 0.01). When HLA-DR2 alleles were combined (i.e., the DRB1*15 with DRB1*16 alleles), their frequency was comparable with that in the healthy individuals. The highest relative risk (RR) of tuberculosis was associated with DRB1*16 alleles (RR = 9.7). When HLA-DR6 alleles were combined (i.e., the DRB1*13 with DRB1*14 alleles), only a trend for higher frequency in patients with PTB was found. Frequency of DRB1*13 alleles of HLA-DR6 was significantly lower in PTB than in the healthy individuals (P < 0.001; RR = 0.04). CONCLUSIONS: Results suggest that the presence of HLA-DRB1*16 alleles may increase the risk of development of PTB, whereas HLA-DRB1*13 alleles may be resistant to tuberculosis.

Adult↗
PubMed2000

Strengths and limitations of molecular subtyping in a community outbreak of Legionnaires' disease.

An epidemiological and microbiological investigation of a cluster of eight cases of Legionnaires' disease in Los Angeles County in November 1997 yielded conflicting results. The epidemiological part of the investigation implicated one of several mobile cooling towers used by a film studio in the centre of the outbreak area. However, water sampled from these cooling towers contained L. pneumophila serogroup 1 of another subtype than the strain that was recovered from case-patients in the outbreak. Samples from two cooling towers located downwind from all of the case-patients contained a Legionella strain that was indistinguishable from the outbreak strain by four subtyping techniques (AP-PCR, PFGE, MAb, and MLEE). It is unlikely that these cooling towers were the source of infection for all the case-patients, and they were not associated with risk of disease in the case-control study. The outbreak strain also was not distinguishable, by three subtyping techniques (AP-PCR, PFGE, and MAb), from a L. pneumophila strain that had caused an outbreak in Providence, RI, in 1993. Laboratory cross-contamination was unlikely because the initial subtyping was done in different laboratories. In this investigation, microbiology was helpful for distinguishing the outbreak cluster from unrelated cases of Legionnaires' disease occurring elsewhere. However, multiple subtyping techniques failed to distinguish environmental sources that were probably not associated with the outbreak. Persons investigating Legionnaires' disease outbreaks should be aware that microbiological subtyping does not always identify a source with absolute certainty.

Adult↗
PubMed2002

Molecular subtyping of Staphylococcus aureus from an outbreak associated with a food handler.

On 6 May 2000, a staphylococcal food poisoning outbreak occurred at a high school, affecting 10 of the 356 students who attended the breakfast. Twenty-seven Staphylococcus aureus isolates, producing enterotoxin A (SEA), SEB-, or non-SEA-E, were recovered from 7 patients, 2 food handlers and left-overs. To investigate the outbreak, we genotyped the isolates by using pulsed-field gel electrophoresis (PFGE) and three PCR-based techniques: inter-IS256 PCR typing, protein A gene (spa) typing, and coagulase gene restriction profile (CRP) analysis. Our results show that PFGE was the most discriminatory technique, whereas the three PCR-based techniques were insufficient in the discriminatory power to distinguish the S. aureus isolates from the outbreak. Based on the enterotoxin-producing types and the results of genotyping, three distinct types of strains (A1111, B2221 and N3221) were designated. Both the A1111 and B2221 strains were found in the specimens from the patients and a hand lesion of a food handler, suggesting that the source of contamination for the outbreak was most likely originated from a food handler.

Adolescent↗
PubMed2006

Distribution of molecular subtypes within Salmonella enterica serotype Enteritidis phage type 4 and S. Typhimurium definitive phage type 104 in nine European countries, 2000-2004: results of an international multi-centre study.

This study investigates the distribution of pulsed-field gel electrophoresis (PFGE) profiles within Salmonella enterica serotype Enteritidis phage type (PT) 4 and S. Typhimurium definitive phage type (DT) 104, from cases of human infection in nine European countries from 2000 to 2004. Isolates were subtyped using standardized methods and gel images submitted by each participating country to the coordinating centre (Health Protection Agency Centre for Infections, London, UK), where they were entered into a central database, developed within BioNumerics software, and designated using an agreed nomenclature. S. Enteritidis PT4 (n=3637) was differentiated into 38 different profiles. Simpson's index of diversity (D) of profiles ranged from 0.2 to 0.4. Profile SENTXB.0001 represented at least 80% of all profiles in each country. S. Typhimurium DT104 (n=1202) was differentiated into 28 different profile types. Simpson's D was at least 0.6 in all countries except in Austria and Italy. In both these countries over 74% of S. Typhimurium DT104 profiles were STYMXB.0013. Profile STYMXB.0061, was predominant in Denmark, Spain, Finland and England and Wales where it represented between 36% and 45% of profiles. Profile STYMXB.0001 represented nearly half of all profiles in Scotland and 23% in England and Wales. PFGE is proving useful for further discrimination within S. Enteritidis PT4 and S. Typhimurium DT104. Ascertainment of international outbreaks involving common serotypes and phage types may be increased by the timely pooling of PFGE profiles within a central database readily accessible to all participating countries.

Bacteriophage Typing↗
PubMed2006

TCL1 shows a regulated expression pattern in chronic lymphocytic leukemia that correlates with molecular subtypes and proliferative state.

Expression of the human oncogene TCL1 in transgenic mice produces B-cell tumors that resemble chronic lymphocytic leukemia (CLL) suggesting its role in B-cell tumorigenesis. To clarify the expression pattern and regulation of TCL1 in CLL, we assessed 213 primary tumors by immunohistochemistry (IHC), flow-cytometry and/or Western blot, using a new monoclonal antibody. TCL1 protein was detectable in the majority of CLL (90% by IHC) but showed marked variations across cases with virtual absence in approximately 10% of tumors. Higher TCL1 levels correlated with markers of the 'pre-germinal center' CLL subtype including unmutated VH status (P=0.005), ZAP70 expression (P=0.007), and presence of chromosome 11q22-23 deletions (P=0.04). Intratumoral heterogeneity in TCL1 levels was also prominent and explained in part by markedly lower TCL1 expression in proliferating tumor cells. In vitro exposure of CLL cells to interleukin-4 (but not other growth factors) produced progressive and irreversible decrease in TCL1 protein levels in association with the onset of proliferation. TCL1 expression patterns in CLL are complex and highly dynamic and appear to reflect both the histogenetic subtypes of the disease and the growth parameters of individual tumors. The observed regulation pattern suggests that TCL1 may exert its effects predominantly in the unmutated/ZAP70-positive tumor subset.

Animals↗
PubMed2000

Simultaneous molecular subtyping and shiga toxin gene detection in Escherichia coli using multiplex polymerase chain reaction.

A robust random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR) protocol was developed for the combined epidemiological typing and shiga toxin detection of clinical shiga toxin-producing O157 and non-O157 Escherichia coli isolates. Using shiga toxin gene-specific primers, combined with two short 10-mer primers, in a multiplex shiga toxin/RAPD-PCR the fingerprints generated allowed differentiation between epidemiologically unrelated strains and allowed identification of a band amplified from the shiga toxin gene(s). Hybridization with a digoxigenin-labelled probe specific for stx1 and stx2 confirmed its identity. The combination of primers in this way allows valuable additional information to be gained from discriminatory RAPD profiles, with further benefits of time and cost savings over tests performed individually.

Bacterial Toxins↗