Fragment Length Polymorphism Typing Research Articles

Comparison of the characteristics of Mycobacterium tuberculosis isolates from sputum and lung lesions in chronic tuberculosis patients.

Mycobacterium tuberculosis (Mtb) in sputum originates from lung cavities in tuberculosis (TB) patients. But drug susceptibility testing (DST) of sputum Mtb can not be conducted the same as in the lung because mutagenesis of bacilli may be happening in the lung during treatment and result in the possibility of the presence of heterogeneous drug-resistant subpopulations in the different lung lesions. This could be one of the reasons for low cure rates for multi-drug resistant (MDR)-TB. We studied the resected lungs of nine surgery patients with chronic TB. The isolates isolated from the sputum and different lung lesions of each patient were tested for phenotypic DST and genotyped using restriction fragment length polymorphism (RFLP) typing method. Genetic analysis to resistance to first and second line drugs was also performed. Five of nine patients were MDR-TB and three XDR-TB. DST results for ten anti-TB drugs were in accordance among different lung lesions in eight patients. However, only three of these eight patients showed the concordance of DST with sputum. Even though the isolates were heteroresistant, genotyping them by RFLP showed the clonal population in each individual patient. Six of eight followed-up patients achieved successful cure. In conclusion, the heteroresistance between sputum and lung lesions and a clonal population without mixed infection might provide useful information in establishing treatment regimen and surgery decision for MDR- and XDR-TB.

The role of Azeri patients in transmission of tuberculosis to East Azerbaijan, Iran

Introduction: Human migration can transmit infectious disease like tuberculosis (TB) to recipient countries. The IS6110 is an insertion sequence used for strain differentiation of mycobacterium tuberculosis (MTB), epidemiological studies, and recent transmission. In this study, our aim was to investigate transmission TB from the Republic of Azerbaijan to Iran people. Methods: Totally 119 isolates of MTB were collected from 14 patients from Republic of Azerbaijan and 105 patients from East Azerbaijan, Iran who referred to TB centers of the province. Isolates were analyzed by IS6110 restriction fragment length polymorphism (RFLP) typing. Isolates with identical RFLP patterns were considered a cluster. Results: 93 distinct IS6110-RFLP patterns were revealed. 81 of these patterns were unique, and 12 were shared by 2-8 strains. 38 strains (31.9%) belonged to one of 12 clusters that were found among the total of 119 strains. One of these 12 clusters contained two Iranian patients with one Azeri patient. Conclusion: Finding of the present study indicates that Azeri TB patients refer to TB centers of the province for treatment can transmit TB to East Azerbaijan people. Therefore, it needs to design a suitable program for TB control and decrease transmission of TB between these two countries.

Detection of Cronobacter on gluB Gene and Differentiation of Four Cronobacter Species by Polymerase Chain Reaction‐Restriction Fragment Length Polymorphism Typing

AbstractCronobacter species are foodborne pathogens associated with neonatal meningitis, sepsis and necrotizing enterocolitis through consumption of contaminated powdered milk. However, there are no specific targets used for differentiating Cronobacter species. The DNA‐based assay and polyphasic analysis have facilitated the detection and typing of Cronobacter species in foodborne outbreaks. In this study, polymerase chain reaction (PCR) and dot hybridization for detecting Cronobacter spp. was developed targeting gluB gene, then one PCR‐restriction fragment length polymorphism (RFLP) typing assay with TaqI digestion was applied for four species differentiation within Cronobacter. Results indicated that the gluB gene is a specific target for detecting Cronobacter and the PCR‐RFLP typing assay could conveniently and rapidly differentiate Cronobacter malonaticus, C. dublinensis, C. sakazakii and C. muytjensii.Practical ApplicationsIn this study, polymerase chain reaction (PCR) and dot hybridization targeting gluB for detecting Cronobacter was developed and a PCR‐restriction fragment length polymorphism (RFLP) typing assay with TaqI was successfully applied for differentiating four Cronobacter species. Results indicated that gluB is a specific marker for detection of Cronobacter and PCR‐RFLP targeting gluB gene product digested with TaqI could conveniently differentiate four Cronobacter species.

Sequence based typing of Legionella pneumophila sg 1 isolated in nosocomial acquired infections in Apulia, Southern Italy.

The present report aims to molecularly characterize seven clinical L. pneumophila (L. pn.) sg 1 isolated from nosocomial acquired infections in Apulia region, using the European Working Group on Legionella Infections (EWGLI), sequence-based typing (SBT) and amplified fragment length polymorphism (AFLP) protocols and to compare the identified sequence types (STs) with those available in the EWGLI database. In the period, January 2000-December 2012, 151 is cases (136 of community and 15 of nosocomial origin) of Legionnaires' disease were notified to the Regional Center for Epidemiology. With regard to nosocomial cases, 8 were confirmed by the isolation of Legionella spp. from respiratory secretions. These clinical isolates were characterized by amplified fragment length polymorphism (AFLP) and sequence-based typing (SBT), using the EWGLI standardized protocol. The clinical isolates belong to ST42, ST23 and ST1. The AFLP confirms the SBT results. Comparing the STs herein detected with those already in the EWGLI SBT database, the 3 STs are frequent in other European countries. The molecular analysis demonstrates that the 3 STs are the most frequent in Italy and in Europe, supporting the hypothesis that some specific L. pn. sg 1 clones have gained widespread dissemination probably due to a common ecological niche. Further researches are required to investigate the potential changing incidence of STs and the fitness of emerging strains or clonal groups in environmental strains.

Phylogenetic analyses of bacteria in sea ice at Cape Hallett, Antarctica

Every year, an area of sea ice forms around the continent of Antarctica that is inhabited by communities of microbes. Although the microbial component of this habitat has been postulated to play an ecologically important role in the Southern Ocean, our understanding of the diversity of Antarctic sea-ice bacteria is limited. The aim of this study was to identify bacterial components present in sea ice at Cape Hallett. Total community DNA was isolated from sea-ice cores and the 16S ribosomal RNA genes were amplified by polymerase chain reaction (PCR) using a range of universal and targeted primer sets. PCR products were de-replicated using restriction fragment length polymorphism (RFLP) and distinct RFLP types were sequenced. The major bacterial classes identified consisted of α-proteobacteria, γ-proteobacteria, Cytophaga–Flavobacteria–Bacteroides group and a small number of Gram-positive species. Our findings indicate that the bacterial component of sea ice at Cape Hallett is primarily heterotrophic. Cyanobacteria and Archaea were not detected using the methods and primer sets employed in this study. However, bacteria whose 16S rDNA sequences cluster with species that encode phototrophic genes were detected in our sea-ice samples. This raised the possibility that bacterial phototrophy may occur in Antarctic sea ice.

Serotyping of DutchStaphylococcus aureusstrains from carriage and infection

International epidemiological studies have shown that clinical isolates of Staphylococcus aureus are usually capsulated with either type 5 or 8 capsular polysaccharides (CPs). Because all noncapsulated strains were found to be cross-reactive with polysaccharide 336 (336PS) antibodies, the noncapsulated strains were denoted as type 336PS. The capsular types of 162 Dutch methicillin-susceptible S. aureus strains derived from individuals living in the Rotterdam area were determined. The serotype distribution was 28.4% serotype 5, 53.7% type 8, and 17.9% type 336PS. Serotyping was in agreement with genotyping by amplified fragment length polymorphism (AFLP) and multi locus sequence typing (MLST). Among 49 nasal carriage isolates from healthy children 24.5% belonged to serotype 5, 67.3% were type 8 and 8.2% were type 336PS. For 28 adult patients on chronic ambulatory peritoneal dialysis (CAPD) the serotype incidences among carriage isolates obtained from the nose, catheter exit-site, and abdominal skin were 45.1%, 41.2% and 13.7%, respectively. Among S. aureus strains deriving from blood cultures, the serotype incidences were 17.7% serotype 5, 53.2% type 8, and 29.0% type 336PS. Apparently, type 336PS strains are more prevalent (P=0.017) among bacteraemia isolates as compared with the nasal carriage isolates obtained from healthy children and CAPD patients. In conclusion, all Dutch S. aureus isolates belonged to types 5, 8, or 336PS, which is in agreement with data from other countries. Thus, addition of the 336PS conjugate to a type 5- and type 8-CP protein conjugate vaccine would significantly extend the vaccine coverage.

Efficient Differentiation of Mycobacterium tuberculosis Strains of the W-Beijing Family from Russia using Highly Polymorphic VNTR Loci

The W-Beijing family is a widespread Mycobacterium tuberculosis clonal lineage that frequently causes epidemic outbreaks. This family is genetically homogeneous and conserved, so ETR-VNTR (exact tandem repeat-variable number of tandem repeats) typing is insufficient for strain differentiation, due to a common ETR-A to E profile (42435). This leads to the false clustering in molecular epidemiological studies, especially in the regions of predominance of the W-Beijing family. In this study, we searched for VNTR loci with a high evolutionary rate of polymorphism in the W-Beijing genome. Here we further evaluated VNTR typing on a set of 99 Mycobacterium tuberculosis clinical isolates and reference strains. These isolates were characterized and classified into several genotype families based on three ETR loci (A, C, E) and eight additional loci [previously described as QUB (Queen's University Belfast) or MIRU (Mycobacterial Interspersed Repetitive Units) or Mtubs]. Ninety-nine strains were divided into 74 VNTR-types, 51 isolates of the W-Beijing family identified by IS6110 RFLP-typing (the restriction fragment length polymorphism-typing) and/or spoligotyping were subdivided into 30 VNTR-types. HGDI (the Hunter-Gaston discriminatory index) for all studied loci was close to that of IS6110 RFLP typing, a "gold standard" method for subtyping M. tuberculosis complex strains. The QUB 26 and QUB 18 loci located in the PPE genes were highly polymorphic and more discriminative than other loci (HGDI is 0.8). Statistically significant increase of tandem repeats number in loci ETR-A, -E, QUB 26, QUB 18, QUB 11B, Mtub21 was revealed in the W-Beijing group compared to genetically divergent non-W-Beijing strains. Thirty-six isolates were subjected to IS6110 RFLP typing. The congruence between results of the IS6110 RFLP typing and 11-loci VNTR typing was estimated on 23 isolates of the W-Beijing family. These isolates were subdivided into 9 IS6110-RFLP types and 13 VNTR types. The poor profiles correlation (0.767) reflects the differences in the rate and type of evolution between genome regions targeted by IS6110-RFLP and VNTR typing. VNTR typing in proposed format is powerful tool for discrimination of M. tuberculosis strains with different level of genetic relationship.

Differentiation of Mycoplasma gallisepticum Strains Using Amplified Fragment Length Polymorphism and Other DNA-Based Typing Methods

Amplified fragment length polymorphism (AFLP) was used to type 34 strains of Mycoplasma gallisepticum (MG) including vaccine strains ts-11, 6/85, and F. Using AFLP, a total of 10 groups, with 30 distinguishable AFLP typing profiles, were generated in the analysis. The AFLP method was able to identify and differentiate both MG field strains from recent outbreaks and those that were epidemiologically related. The AFLP procedure will provide assistance in identifying the sources of mycoplasma infections. Vaccine strains were also differentiated from other field strains, which will be useful in the evaluation of vaccination programs. The AFLP discrimination potential was compared to other molecular typing techniques such as gene-targeted typing by DNA sequence analysis of the MG cytadhesin-like protein encoding gene, mgc2, and random amplified polymorphic DNA assay on the same MG isolates. The three assays correlated well with one another, with AFLP analysis having a much higher discriminatory power and reproducibility.

Vibrio vulnificus, a Notably Lethal Human Pathogen Derived from Seafood: A Review of Its Pathogenicity, Subspecies Characterization, and Molecular Methods of Detection

Vibrio vulnificus is presently considered the most infectious and lethal of all human pathogenic vibrios. The organism requires at least 0.5 NaCl for growth, is naturally ubiquitous to marine coastal waters and shellfish, and is sensitive to refrigeration temperatures. Septisemic infections by the organism usually result from the consumption of raw shellfish. Three biotypes are presently recognized and distinguished on the basis of biochemical characteristics, serology, and molecular typing. Cirrhosis of the liver due to chronic alcoholism is considered a high risk factor for infection by this organism, presumably due to increased levels of serum iron released by damaged hepatocytes. The organism produces an unusually large number of extracellular virulence factors. A number of selective agar media have been developed for isolation of the organism incorporating various levels of the antibiotics colistin, polymyxin-B, bile salts, and K tellurite. The molecular techniques applied to detecting, identifying, and characterizing the organism include: the polymerase chain reaction (PCR), real-time PCR, random amplified polymorphic DNA analysis (RAPD), multiplex PCR, ribotyping, multilocus enzyme electrophoresis (MLEEE), PCR-restriction fragment length polymorphism (PCR-RFLP) and gene sequence typing. In addition, seminested reverse transcription-PCR (RT-PCR) has been used to detect viable but non-culturable (VBNC) cells of V. vulnificus.

Habitat and host associations of Craterellus tubaeformis in northwestern Oregon

Knowledge of the habitat and host associations of Craterellus tubaeformis (winter chanterelle) is the key to understanding the ecological characteristics needed for its conservation. In this study, a survey of forest types in northwestern Oregon for mycorrhizal associates is performed and the hypotheses that stand age and the volume of well-decayed, coarse, woody debris (CWD) are significant to the standing crop biomass and the probability of C. tubaeformis occurrence are tested. Host associations were identified with polymerase chain reaction (PCR) amplification and restriction fragment-length polymorphism (RFLP) typing. Habitat associations were tested by measurements on 64 plots in the Coast and Cascade Ranges of northwestern Oregon. Data analysis found that stand age and well-decayed, coarse, woody debris were related significantly to the probability of C. tubaeformis occurrence but not to standing crop biomass. Results indicated the volume of well-decayed CWD is particularly important to the probability of C. tubaeformis occurrence in stands less than 100 yr of age. Well-decayed CWD was the substratum for 88% of C. tubaeformis sporocarps across all stands, despite the fact that ground area coverage of CWD ranged only from 3 to 26%. Slope, elevation and aspect were not related to the probability of C. tubaeformis occurrence or standing crop biomass. The occurrence of C. tubaeformis in northwestern Oregon is highly correlated to the presence of western hemlock (Tsuga heterophylla), and their mycorrhizal association was confirmed. Craterellus tubaeformis also can form mycorrhizae with Douglasfir (Pseudotsuga menziesii) and Sitka spruce (Picea sitchensis) but is encountered only rarely in stands without a hemlock component. In northwestern Oregon, the presence of Hydnum spp. in a stand is a good indicator of the presence of C. tubaeformis. Differences in genetic sequences between C. tubaeformis populations in western North America, eastern North America and Europe suggest the likelihood of several distinct species.

Association between polymorphisms of the angiotensin-converting enzyme and angiotensinogen genes and allergic rhinitis in a Korean population.

Angiotensin-converting enzyme (ACE) inactivates bradykinin, substance P, and neurokinin A, which are thought to play important roles in the pathogenesis of inflammatory diseases. Expression of angiotensinogen, a precursor of angiotensin, is enhanced by augmented secretion of proinflammatory cytokines (eg, interleukin-1) in the site of inflammation. Insertion or deletion (I/D) ACE and M235T angiotensinogen gene polymorphisms were reported to be associated with atopy in a Czech population. Using polymerase chain reaction restriction fragment length polymorphism and SNaPshot typing analysis, we investigated the frequencies of the genotypes and alleles of the ACE gene in 137 patients with allergic rhinitis, of the M235T angiotensinogen gene in 186 patients with allergic rhinitis, and of both in 219 healthy control subjects. There was no difference in the frequency of the DD genotype of the ACE gene in the controls and patients (odds ratio, 1.32 [0.66-2.60]; p > .05). The D allele was more frequent in patients, but the difference was not statistically significant (odds ratio, 1.21 [0.89-1.64]; p > .05). There was no difference in the frequency of the TT genotype of the angiotensinogen gene in the controls and patients (odds ratio, 1.01 [0.38-2.69]; p > .05). The T allele was more frequent in patients, but the difference was not statistically significant (odds ratio, 1.10 [0.78-1.55]; p > .05). Our results indicate that polymorphisms in the genes for ACE and angiotensinogen may not be related to the development of allergic rhinitis in the Korean population.

Molecular epidemiology of Enterococcus faecalis in liver transplant patients at University Hospital Groningen

We report the molecular epidemiology of Enterococcus faecalis in liver transplant patients transplanted at the University Hospital Groningen (The Netherlands) as determined by amplified fragment length polymorphism (AFLP) typing. A total of 133 E. faecalis isolates were cultured from the faeces and throat (95 isolates) or clinical sites (35 isolates) of 43 liver transplant patients. Among these 133 isolates, 15 different AFLP types could be identified with 90% AFLP similarity. Of these 15 groups, nine contained isolates from more than one patient, which may indicate transmission of E. faecalis isolates between patients. In five of these groups transmission could be explained by the fact that patients carrying identical strains were staying in the same ward at the same time. One of these epidemic isolates (AFLP type K) distinguished itself by colonizing 23 liver transplant patients during 15 months. Antimicrobial susceptibility testing did not reveal any multi-resistant isolates. This study showed that transmission of susceptible E. faecalis isolates occurs frequently on the liver transplant wards. Detection of this transmission and understanding of the mechanism is important, as it might also be an indicator of possible transmission of enterococci resistant to antibiotics.

A RT-PCR assay combined with RFLP analysis for detection and differentiation of isolates of Pepino mosaic virus (PepMV) from tomato

The partial nucleotide sequence of the RNA polymerase gene from one isolate of Pepino mosaic virus (PepMV) was determined. Phylogenetic and distance analysis indicated that this isolate was related to other isolates of PepMV previously reported. To develop a method for detecting PepMV by reverse transcriptase–polymerase chain reaction (RT–PCR), a pair of primers was designated from RNA polymerase sequences. RT–PCR with RNA from a large number of tomato samples with PepMV symptoms, positive controls of PepMV, weed samples containing PepMV, and other potexviruses as negative controls confirmed the specificity of the primers. Restriction endonuclease digestion of the RT–PCR products distinguished three restriction fragment length polymorphism (RFLP) types. The majority of isolates were included within type P1, which correspond with the PepMV isolates found in Europe. This type and type P2, which corresponded with the original PepMV isolated from Solanum muricatum, appear more closely related to each other than to type P3. Type P3 had completely different RFLPs from the other two types studied. It may represent a further line within the PepMV virus. The RT–PCR–RFLP assay is proposed as a rapid and easy method to detect and identify new isolates of the PepMV virus.

Characterization of Pinus ectomycorrhizas from mixed conifer and pygmy forests using morphotyping and molecular methods

We used morphotyping and molecular methods to characterize ectomycorrhizas of bishop pine (Pinus muricata D. Don) and Bolander pine (Pinus contorta ssp. bolanderi (Parl.) Critchf.) from mixed conifer and hydric pygmy forests on the northern California coast. Sixteen ectomycorrhizal morphotypes were described, producing 15 internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) types, and 12 were identified via ITS sequencing. From a given site, all root tips of a specific morphotype produced identical ITS-RFLP patterns. However, sometimes two morphotypes produced the same ITS-RFLP type, and sometimes samples of the same morphotype from two different sites produced two different ITS-RFLP types. These results indicate that surveys of ectomycorrhizal fungi based on morphology alone are not sufficient, and that grouping morphotypes prior to molecular analysis can expedite the process. Ectomycorrhizas from mixed conifer included Russuloid sp., Tomentella sublilacina (Ellis & Holw.) Wakef., Tuber sp., and two Thelephoroid species. Ectomycorrhizas from hydric pygmy included two Dermocybe spp., a Cortinarius sp., two Thelephoroid spp., and Suillus tomentosus (Kauffman) Singer. Both plant communities contained Cenococcum geophilum Fr.:Fr. The hydric pygmy sites were more similar to each other than to the mixed conifer site (Jaccard similarity). The presence of ectomycorrhizal taxa in one plant community type may reflect biotic (host specificity) or abiotic (soil fertility or hydrology) adaptation.Key words: ectomycorrhiza, bishop pine, Pinus muricata, Bolander pine, Pinus contorta ssp. bolanderi, morphotyping, ITS-RFLP.

Practical evaluation of molecular subtyping and phage typing in outbreaks of infection due to salmonella enterica serotype typhimurium

Identification and control of food-poisoning outbreaks due to salmonellosis depend on prompt microbiological diagnosis and subtyping to identify the causative strain. In Australia, Salmonella enterica subspecies enterica serotype typhimurium (S. typhimurium) is responsible for 40-70% of cases of human salmonellosis. Phage typing is the usual method of subtyping S. typhimurium, but on its own, has limitations. We compared it with three molecular subtyping methods using 100 isolates of S. typhimurium, representing four different phage types (PT 1, 9, 126 and 135) and comprising 74 isolates from three presumed outbreaks, 25 isolates from sporadic cases of salmonellosis and S. typhimurium ATCC 10428 (phage type 126). The isolates were divided into 11 subtypes by IS200 restriction fragment length polymorphism (RFLP) typing, four each by ribotyping and pulsed-field gel electrophoresis (PFGE) and 17 distinct strains using a combination of phage and molecular typing. Isolates from two presumed outbreaks were resolved into multiple strains, possibly explaining the failure to identify a common source for either during the original investigations. IS200 RFLP analysis was the most discriminatory and reproducible typing method. Several strains were identifiable within and shared between phage types 1, 9 and 126. Phage and IS200 RFLP typing together, would provide improved definition of S. typhimurium outbreaks.

Variable number of pig MHC class I genes in different serologically defined haplotypes identified by a 3'-untranslated region probe.

To estimate the number of porcine class I major histocompatibility genes, a short class I cDNA probe from the 3'-untranslated region was developed to be used in restriction fragment length polymorphism analysis. Six clones isolated from a pig spleen cDNA library were sequenced from their 3'-untranslated region. Three different transcripts were identified, one probably derived from the class I PD7 locus and two showing highest homology to the PD1 and the PD14 genes, respectively. Class I typing was performed both by restriction fragment length polymorphism and serology. Segregation of class I haplotypes was followed in one three-generation family (European Wild Boar x Large White: Swedish Yorkshire) and in six two-generation families (Duroc, Yorkshire and Chester White), for a total of 266 pigs. Twenty different class I haplotypes were identified either with restriction fragment length polymorphism and/or serological typing. Furthermore, previously unpublished serological haplotypes H62, H67 and H68 were identified. Two to seven polymorphic and three monomorphic fragments were detected in different restriction fragment length polymorphism haplotypes indicating that the number of class I genes in the investigated haplotypes varies.

Tuberculosis among urban health care workers: a study using restriction fragment length polymorphism typing.

Cases of tuberculosis identified during 1992-1994 through an active tuberculosis surveillance network among six hospitals that serve New York City (the TBNetwork) were analyzed according to the occupational status of the patients. Clinical data were obtained by review of medical records, and restriction fragment length polymorphism (RFLP) typing of Mycobacterium tuberculosis isolates was performed. No known nosocomial outbreaks of tuberculosis occurred at these hospitals in the study period. Occupational status was known for 142 of 201 patients whose isolates were available for strain typing. Patients infected by organisms with a clustered strain typing pattern, as determined by RFLP analysis, were presumed to have recently acquired disease. RFLP typing revealed that isolates from 13 (65%) of 20 health care workers and 50 (41%) of 122 non-health care workers had a clustered RFLP pattern. The strains infecting eight (89%) of nine health care workers seropositive for human immunodeficiency virus (HIV) had a clustered RFLP pattern. Multivariate analysis of 75 patients with known HIV and occupational status revealed that HIV status (P = .03) and health care worker status (P = .02; RR = 2.77) were independent risk factors for a clustered RFLP strain. These findings suggest that many of the apparently sporadic cases of tuberculosis among health care workers may be due to unrecognized occupational transmission.

Nosocomial Tuberculosis: An Outbreak of a Strain Resistant to Seven Drugs

AbstractObjective:To evaluate nosocomial transmission of multidrug-resistant (MDR) tuberculosis (TB).Design:Outbreak investigation: review of infection control practices and skin test results of healthcare workers (HCWs); medical records of hospitalized TB patients and mycobacteriology reports; submission of specimens for restriction fragment length polymorphism (RFLP) typing; and an assessment of the air-handling system.Setting:A teaching hospital in upstate New York.Results:Skin-test conversions occurred among 46 (6.6%) of 696 HCWs tested from August through October 1991. Rates were highest on two units (29% and 20%); HCWs primarily assigned to these units had a higher risk for conversion compared with HCWs tested following previous incidents of exposure to TB (relative risk [RR] = 53.4, 95% confidence interval [CI95] =6.9 to 411.1; and RR=37.4, CI95= 5.0 to 277.3, respectively). The likely source patient was the only TB patient hospitalized on both units during the probable exposure period. This patient appeared clinically infectious, was associated with a higher risk of conversion among HCWs providing direct care (RR = 2.37; CI95 = 1.05 to 5.34), and was a prison inmate with TB resistant to seven antituberculosis agents. The MDR-TB strain isolated from this patient also was isolated from other inmate and noninmate patients, and a prison correctional officer exposed in the hospital. Mycobacterium tuberculosis isolates from all of these patients had matching RFLP patterns. Infection control practices closely followed established guidelines; however, several rooms housing TB patients had marginal negative pressure with variable numbers of air changes per hour, and directional airflow was disrupted easily.Conclusions:These data strongly suggest nosocomial transmission of MDR-TB to HCWs, patients, and a prison correctional officer working in the hospital. Factors contributing to transmission apparently included prolonged infectiousness of the likely source patient and inadequate environmental controls. Continued urgent attention to TB infection control is needed.

HLA class II DNA-RFLP typing in 102 individuals from Northern Greece.

The serological identification of HLA class II alleles is often doubtful. Since accurate HLA typing is essential for the matching of donor-recipient pairs in allogeneic transplantation, an effort was made to establish DNA restriction fragment length polymorphism (RFLP) typing and to assess the correlation between the serological and RFLP techniques in the population of Northern Greece. One hundred and two healthy individuals (204 HLA-DR alleles) from Northern Greece were HLA-DR, DQ typed with both the microcytotoxicity and the Taq I RFLP method, using three exon-specific probes. DNA-RFLP typing revealed (1) concordant results with serology in 69.9% (142/204) of the alleles and (2) at least one HLA-DR allele discrepant to serology in 30.4% (62/204) of the alleles. Incorrect serological DR types (weak reactions or inability to distinguish between two alleles with a common epitope) were identified in 54 alleles (26.5%), while 3.9% (8/204) of serological "blank" alleles turned out to be definable alleles by RFPL. Of the individuals tested, 10.8% (11/102) were DR-homozygous by RFLP. This comparison of results obtained by serology and RFLP demonstrated the necessity of the clinical application of DNA typing, especially for organ transplantation where accurate HLA typing has an important influence on graft survival.

Localization of the Br Polymorphism on a 144 bp Exon of the GPIa Gene and Its Application in Platelet DNA Typing

Alloimmunization against the human platelet alloantigen system Br (HPA-5) is the second most common cause of neonatal alloimmune thrombocytopenia (NAIT) in Caucasian populations. We have recently shown that a single base polymorphism at position 1648 on platelet mRNA coding for GPIa results in an aminoacid substitution at position 505 on the mature GPIa which is associated with the two serological defined Br phenotypes. Since DNA-typing of platelet alloantigens offers possibilities for useful clinical applications, we designed genomic DNA-based restriction fragment length polymorphism (RFLP) typing for Br alloantigens. To establish this technique we analyzed the genomic organization of GPIa adjacent to the polymorphic base. Using the polymerase chain reaction (PCR) of blood cell DNA we have identified two introns (approximately 1.7 and 1.9 kb) flanking a 144 bp coding sequence of the GPIa gene encompassing the polymorphic base 1648. Based on the intron sequence, a PCR primer was constructed to amplify a 274 bp fragment which was used for allele-specific RFLP to determine the Br genotypes. The results of RFLP analysis using MnlI endonuclease obtained from 15 donors (2 Bra/a, 2 Bra/b and 11 Brb/b) correlate perfectly with serological typing by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay.