Periodontitis and dental scaling associated with pyogenic liver abscess: A population-based case-control study.

BACKGROUND AND OBJECTIVES: The purpose of this study was to investigate the relationship between periodontitis, dental scaling (DS) and pyogenic liver abscesses (PLAs). MATERIAL AND METHODS: A nationwide population-based case-control study was applied using data from the National Health Insurance Research Database in Taiwan. We identified and enrolled 691 PLA patients, who were individually matched by age and sex to 2764 controls. RESULTS: Conditional logistic regression was applied to estimate adjusted odds ratios (aORs) in patients with exposure to periodontitis and DS before PLA. After adjusting for other confounding factors, periodontitis remained a risk factor for PLA among patients aged 20-40 years, with an aOR of 2.31 (95% confidence interval [CI] = 1.37-3.90, P = .0018). In addition, the average aOR for PLA was significantly lower among patients with one DS (aOR = 0.76, 95% CI = 0.59-0.96) and more than one DS (aOR = 0.61, 95% CI = 0.39-0.95) within 1 year before the index date. CONCLUSION: According to these results, we concluded that adult patients with periodontitis aged <50 years old are more at risk for PLA than controls, particularly when they have no DS. Moreover, from 20 years of age, non-periodontal patients subjected to at least 2 DS per year are less at risk for PLA than controls.

Genomic collinearity and the genetic architecture of floral differences between the homoploid hybrid species Iris nelsonii and one of its progenitors, Iris hexagona.

Hybrid speciation represents a relatively rapid form of diversification. Early models of homoploid hybrid speciation suggested that reproductive isolation between the hybrid species and progenitors primarily resulted from karyotypic differences between the species. However, genic incompatibilities and ecological divergence may also be responsible for isolation. Iris nelsonii is an example of a homoploid hybrid species that is likely isolated from its progenitors primarily by strong prezygotic isolation, including habitat divergence, floral isolation and post-pollination prezygotic barriers. Here, we used linkage mapping and quantitative trait locus (QTL) mapping approaches to investigate genomic collinearity and the genetic architecture of floral differences between I. nelsonii and one of its progenitor species I. hexagona. The linkage map produced from this cross is highly collinear with another linkage map produced between I. fulva and I. brevicaulis (the two other species shown to have contributed to the genomic makeup of I. nelsonii), suggesting that karyotypic differences do not contribute substantially to isolation in this homoploid hybrid species. Similar to other studies of the genetic architecture of floral characteristics, at least one QTL was found that explained >20% variance in each color trait, while minor QTLs were detected for each morphological trait. These QTLs will serve as hypotheses for regions under selection by pollinators.

Surgical Fixation of a Comminuted Inter-Trochanteric Fracture in a Patient with Bilateral Below Knee Amputation.

Surgical fixation of hip fractures in patients with below knee amputation is challenging due to the difficulty in obtaining optimal traction for reduction of the fracture. Surgeons may face difficulty in positioning such patients on the traction table due to the absence of the foot and distal lower limb. There are several techniques described to overcome this technical difficulty. In this case report, we present a case of a 64-year old gentleman with bilateral below knee amputation presenting with a comminuted right intertrochanteric fracture. We highlight a simple and effective method of applying skin traction to obtain adequate reduction for hip fracture fixation.

Pan-drug-resistant Pseudomonas aeruginosa causing nosocomial infection at a university hospital in Taiwan.

This study evaluated the clinical and microbiological characteristics of 16 patients who were colonised or infected with 26 isolates of pan-drug-resistant Pseudomonas aeruginosa (PDRPA; intermediately-resistant or resistant to all cephalosporins, piperacillin-tazobactam, aztreonam, carbapenems, ciprofloxacin and aminoglycosides) in a university hospital during 1999-2002. All the isolates had colistin MICs < or = 4 mg/L, 19 (73%) isolates had bla(VIM-3), and 25 (96%) isolates had class I integrons (intI). Time-kill studies for two PDRPA blood isolates demonstrated synergism for cefepime-amikacin after 24 h. Pulsed-field gel electrophoresis analysis of the isolates revealed a polyclonal nature (12 pulsotypes), although clonal dissemination of PDRPA isolates among these patients was also present.

Expression of the human T cell receptor V beta repertoire.

We have used a sensitive assay, based on amplification of cDNA by the polymerase chain reaction, to determine in a variety of human tissues the relative levels of expression of the genes coding for each of the twenty families of human TcR V beta. We have determined the diversity of the expressed TcR V beta repertoire early in the development of the immune system. We have shown that the full TcR V beta repertoire is expressed early into the second trimester; the expressed repertoire is as diverse at this point, in both fetal thymus and spleen, as it is in mature thymus and peripheral blood lymphocytes. In addition the relative expression in the fetal thymus of each V beta gene is conserved to a large extent in the fetal spleen.

Skeletal tissue responses to thermal injury: an experimental study.

Skeletal changes occurring secondary to burn injuries were studied in an experimental animal model for thermal injury. One hindlimb of female Sprague-Dawley rats (200-250g) was subjected to a standardized thermal injury; the other hindlimb was left untreated. Control animals received no experimental treatment. Effects on skeletal architecture were studied at the proximal tibial metaphysis and tibial diaphysis using static histomorphometry. Bone formation dynamics were studied from a series of bone fluorochrome labels administered before the experiment began, early (days 8, 9) postburn treatment (PBT) and late PBT (days 17, 18). Animals were sacrificed on day 21 PBT. In proximal tibial metaphyses of burn-treated limbs, trabecular bone area (TBA) and trabecular number in all regions except the primary spongiosa, were significantly reduced. TBA was also decreased, but not significantly in nontreated limbs. Longitudinal growth rate, growth plate thickness and growth cartilage cell production rate are greater in burn-treated than in nonburned and control bones. Burn-treated diaphyses showed extensive woven bone formation at periosteal surfaces, and corresponding increases of bone areas and periosteal perimeters. Endocortical surfaces showed only typical occasional resorption areas. No intracortical changes were observed. Mineral appositional rate (MAR) and bone formation rate (BFR) at endocortical surfaces were markedly depressed after thermal injury, significant changes were noted in both limbs of treated animals. Among burned limbs, the early PBT label was absent from all specimens, indicating a virtual shutdown of osteoblast activity and recruitment. Similarly in nonburned limb bone, the label was absent from 50% of the specimens; in those bones in which the label was present, label lengths, appositional and bone formation rates were significantly reduced relative to the control specimens. Comparison of average bone formation dynamics for the total PBT interval indicates that MAR and BFR in burned treated tibiae were reduced to approximately 25% of control values. MAR and BFR from the nonburned side of treated animals were significantly reduced as well, to about 55% of control values. These data indicate that the principal metaphyseal effects of thermal injury are stimulation of growth cartilage proliferation, and depression of ossification and osteoblast activity. In diaphyses, thermal injury causes extensive local periosteal woven bone proliferation and a dramatic depression of endosteal bone formation. The latter effect, while more severe locally, is also evident systemically.

Effect of beta-adrenergic blockade on the results of exercise testing related to the extent of coronary artery disease.

Maximal treadmill testing was carried out in 50 patients with angiographically documented coronary artery disease (CAD) in the presence and absence of beta-adrenoceptor blockade. The results were related to the extent of CAD and interpreted relative to the clinical value of exercise testing. Maximal heart rate and systolic blood pressure were significantly lower during treatment with beta-blocking drugs. The average exercise duration was 1.3 +/- 1.9 minutes greater (+/- standard deviation), regardless of coronary anatomy. Of the 20 subjects with 3-vessel or left main CAD (severe CAD), 8 patients completed 3 stages (9 minutes) of exercise during treatment; only 4 did so without treatment. Angina was significantly more often the limiting symptom with severe CAD, and this association was abolished by beta blockade; 1 of 20 with severe CAD completed 3 stages of exercise and was not limited by angina without beta-blocking treatment, whereas 7 had these features during beta-blockade therapy. Maximal ST-segment depression was not related to the extent of CAD with or without therapy. Beta blockade suppressed the occurrence of ST depression, or delayed its appearance by an average of 2.0 +/- 2.3 minutes and reduced its severity by 0.5 +/- 0.9 mm. All tests in which ST depression was completely suppressed were associated with inadequate heart rate response, regarded as diagnostically inconclusive rather than negative. However, during beta-blocking treatment, 14 tests (28%) were inconclusive, which, in routine practice, would have necessitated repeat testing.(ABSTRACT TRUNCATED AT 250 WORDS)

Emergence of vancomycin-resistant enterococci at a university hospital in Taiwan: persistence of multiple species and multiple clones.

OBJECTIVES: To describe the epidemiology of vancomycin-resistant enterococci (VRE) in a university hospital in Taipei, Taiwan. DESIGN: Retrospective review over a 27-month period, from March 1996 to May 1998. SETTING: A tertiary-care teaching hospital in Taiwan. PARTICIPANTS: Patients with VRE isolated from any body site. METHODS: Patients were identified through hospital microbiology and infection control records. Patient charts were reviewed for clinical and epidemiology data, including age, gender, previous hospital admissions, underlying diseases, types of infection, and recent antibiotic use. VRE isolates were characterized by their typical biochemical reactions, cellular fatty acid profiles, and the presence of van genes. Antibiotypes using the E-test and randomly amplified polymorphic DNA (RAPD) patterns of these isolates were used to determine the clonality. RESULTS: Twenty-five isolates of VRE recovered from 12 patients were identified. One patient with a perianal abscess had 12 isolates of VRE (4 Enterococcus faecalis, 7 Enterococcus faecium, and 1 Enterococcus casseliflavus) recovered from perianal lesions. Among 3 patients who were hospitalized in the same room, 1 had a community-acquired cellulitis over the left leg caused by E. faecalis, and the other 2 patients both had anal colonization with 2 isolates of E. faecalis. The other 8 patients had 1 E. faecalis isolate each from various clinical specimens. All isolates possessed vanA resistance phenotype and vanA genes. Different antibiotypes and RAPD patterns of the isolates from different patients excluded the possibility of nosocomial spread at the hospital. CONCLUSIONS: Multiple species of VRE (E. faecalis, E. faecium, and E. casseliflavus) and multiple clones of E. faecium could colonize or infect hospitalized patients. In addition, clones of VRE can persist long-term in patients' lower gastrointestinal tracts. These results extend our knowledge of the coexistence and the persistence of multiple species and multiple clones of VRE in hospitalized patients.

Characterisation of p-nitrophenylglycerol-resistant Proteus mirabilis super-swarming mutants.

p-Nitrophenylglycerol (PNPG) inhibits the co-ordinately regulated activities of swarming behaviour and virulence factor expression in Proteus mirabilis. The inhibitory action of PNPG was investigated by the isolation of Tn5 insertion mutants that could swarm, albeit with much reduced ability, in the presence of PNPG. The mutants exhibited a super-swarming phenotype in the absence of PNPG; i.e., they migrated further in a given time than did the wild-type cells. Cloning and sequence analysis of the mutants indicated that Tn5 was inserted into the rsbA gene, which may encode a membrane sensor histidine kinase of the bacterial two-component signalling system. In the absence of PNPG, the mutants exhibited several swarming-related phenotypes that were different from those of the wild type; they initiated swarming earlier and had a less conspicuous consolidation phase, they differentiated earlier and maintained a differentiated state for longer, they started to express virulence factors earlier and maintained high expression levels of these factors for longer, and they had higher cell invasion ability than the wild type. These mutant phenotypes could be complemented by a plasmid-borne copy of rsbA. Together, these data suggest that RsbA may act as a repressor of swarming and virulence factor expression. In the presence of PNPG, these rsbA-mutated mutants could still swarm, differentiate and express virulence factors, whereas the wild type could not, suggesting that PNPG may target RsbA or RsbA-regulated pathways to exert its inhibitory effect. Together, these data reveal a novel mechanism through which bacteria may negatively regulate swarming differentiation and virulence factor expression and identify a potential target of PNPG action.

Inhibition of virulence factor expression and swarming differentiation in Proteus mirabilis by p-nitrophenylglycerol.

Proteus mirabilis is a common cause of upper urinary tract infections that can involve invasion of host urothelial cells. The ability to invade urothelial cells is coupled closely to swarming, a form of multicellular behaviour in which vegetative bacteria differentiate into hyperflagellate, filamentous swarming cells capable of co-ordinated and rapid population migration. Co-ordinate expression of virulence factors including urease, protease, haemolysin and flagellin during swarm-cell differentiation in P. mirabilis has been reported. To investigate the effects of p-nitrophenylglycerol (PNPG), a potent anti-swarming agent, on the various swarming-associated traits of P. mirabilis and to elucidate the relationships among them, P. mirabilis growth rate, swarming/swimming activity, cell invasion ability and the ability to express various virulence factors were monitored in the presence or absence of PNPG. It was found that PNPG could inhibit the growth rate, swarming differentiation and swarming/swimming activities of P. mirabilis. The expression of virulence factors such as protease, urease, haemolysin and flagellin in P. mirabilis was also inhibited by PNPG. The ability of P. mirabilis to invade human urothelial cells was reduced dramatically in the presence of PNPG. These results suggest that PNPG has the potential to be developed as an agent active against the effects of P. mirabilis infection.

Persistent bacteraemia caused by a single clone of Burkholderia cepacia with unusual phenotype.

We report a case of persistent bacteraemia caused by a single clone of Burkholderia cepacia with unusual characteristics. Six isolates of B. cepacia were recovered from a patient with acute myeloid leukaemia and chemotherapy-induced neutropenia within a 3-week period. All six isolates were initially incompletely identified as B. cepacia with the API 20NE system. The further use of cellular fatty acid analysis and PCR-restriction fragment length polymorphism of the 16S rDNA confirmed the identification. These isolates also displayed an identical but unusual antibiotype. The identical cellular fatty acid profiles and genomic typing generated by random amplified polymorphic DNA identified these isolates as derivatives of a single strain.

Streptococcus zooepidemicus (Lancefield group C) septicaemia in Hong Kong.

The clinical findings relating to 11 patients in Hong Kong (HK) and to 43 patients described elsewhere, all with Streptococcus zooepidemicus septicaemia, are reviewed. There was a particular association with cardiovascular disease (27%) with seven cases of endocarditis, three of abdominal aortic aneurysm and two of deep venous thrombosis. Associations not previously reported included two cases of pharyngitis and two patients with persistent post-operative fever. The overall mortality was 22%. Both human and porcine strains of S. zooepidemicus from HK did not hydrolyse aesculin in contrast to the aesculin-positive biotypes reported previously. HK strains also had very mucoid colonies and capsules of hyaluronic acid were seen in electron micrographs. Samples of chromosomal DNA, extracted by means of HindIII restriction endonuclease, of strains from human beings and pigs were identical. The MIC of penicillin for all strains was less than or equal to 0.03 mg/l but the MBC for all was greater than 32 mg/l. Penicillin alone is generally sufficient for cure but combination with an aminoglycoside may be indicated in seriously ill patients. In our locality, pigs were incriminated as a possible source of human infection whereas consumption of contaminated dairy products is important elsewhere.

Comparison of preservation mixtures for Helicobacter pylori.

Long-term preservation of 40 isolates of Helicobacter pylori was investigated with five storage media (brucella broth with 17% glycerol, brucella broth with 17% glycerol and 2% fetal calf serum [FCS], brucella broth with 17% glycerol and 10% FCS, brucella broth with 17% glycerol and 2% horse blood, and 10% mucin) at -70 degrees C for 4, 6 and 9 months. In addition to glycerol, FCS or horse blood in the storage media is necessary for survival of H. pylori. Storage of H. pylori isolates at -70 degrees C in 10% mucin is a simple and effective preservation procedure.

High protease activity of Chryseobacterium indologenes isolates associated with invasive infection.

In order to understand virulence factors of Chryseobacterium indologenes isolates associated with invasive infection, enzymatic activities and cellular fatty acid profiles of 42 isolates recovered at National Taiwan University Hospital from January 1994 to December 1996 were studied. Among them, 12 blood isolates were considered as invasive and 30 (recovered from urine, sputa, infected burn wounds, and catheter tips) were noninvasive. All isolates showed strong activities of alkaline phosphatase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and N-acetyl-beta-glucosaminidase, and had no activities for alpha-galactosidase, beta-galactosidase, beta-glucuronidase, alpha-mannosidase, and alpha-fucosidase. The activities of other enzymes were variable. Thirty-two isolates (76%) had varying degrees of protease activity. Two profiles (profiles I and II) of cellular fatty acids of the isolates were found and profile I predominated. There was no significant difference of distribution of cellular fatty acid profiles and activities of enzymes between invasive and noninvasive isolates, except protease activity which was significantly higher in invasive isolates than that in noninvasive isolates. Protease activity may play an important role in virulence on invasive infections caused by C. indologenes.

Quality assurance in clinical laboratories in Taiwan.

BACKGROUND AND PURPOSE: In 1994, the Taiwan National Health Administration assigned the execution of a quality assurance (QA) survey program to the Association of Laboratory Medicine. The purpose of this program was to investigate the quality of clinical laboratory assessments and to promote QA in the fields of clinical microscopy, hematology, chemistry, microbiology, serology, and blood banking. We report the findings of QA surveys conducted in 1998 and the effect of voluntary training on improvement of clinical laboratory testing. METHODS: A total of 1,008 clinical laboratories were included in the program in 1998. Proficiency testing (PT) was performed to evaluate various laboratory tests. Continuing education programs were conducted and experts visited laboratories that sought guidance before the PT was conducted. The full mark was set at a score of 100 for each PT scheme. The criterion for acceptability of PT results was set at a score of 80 or more. RESULTS: The rates of acceptable results were 82.4% (607/736) for hematology, 57.4% (267/465) for blood banking, 69.3% (561/810) for chemistry, and 80.1% (321/401) for microbiology. The rates of acceptable microscopy results were 90.9% (509/560) for urine chemical tests and 84.6% (610/721) for others. The rates of acceptable serology tests were 73.3% (384/524) for hepatitis and 85.6% (441/515) for syphilis. The rates of acceptable performance differed significantly among clinical laboratories with different rankings: clinical laboratories at institutions classified below the level of district hospital showed comparatively poor performance. Laboratories that received guidance showed significant improvement in performance from 1997 to 1998. CONCLUSIONS: A QA program is urgently needed in Taiwan to improve laboratory performance.

Antimicrobial activities of six new oral cephem antibiotics.

Six newly developed oral cephem antibiotics, which included 5 with an aminothiazolyl side chain, viz., cefixime, cefotiam hexetil, cefpodoxime proxetil, cefteram pivoxil, ceftibuten, and one with a conventional side chain design, BMY 28100, were tested for their antimicrobial activities. Three traditional oral beta-lactam antibiotics, ampicillin, cephalexin, and cefaclor, were used as reference antibiotics. The 5 aminothiazolyl cephems were more active than the reference drugs against clinical isolates of Escherichia coli, Klebsiella pneumoniae and Haemophilus influenzae. All 6 new drugs showed poor activity against clinical isolates of Enterobacter cloacae, Pseudomonas aeruginosa, and enterococci. They were better than cephalexin and cefaclor, but slightly poorer than or the same as ampicillin in activities against Streptococcus pyogenes and Streptococcus pneumoniae. Cefotiam hexetil, BMY 28100, and cefpodoxime proxetil were also more active than cephalexin against Staphylococcus aureus. Although the 6 tested new oral cephems demonstrated little difference in their activities against different bacteria, they all showed greater antimicrobial activity than the traditional oral antibiotics now in clinical use.

In vitro activities of 36 antimicrobial agents against clinically isolated Bacteroides fragilis.

Thirty-six antimicrobial agents were evaluated for in vitro activities against 100 clinical isolates of Bacteroides fragilis. The minimal inhibitory concentration (MIC) of each agent for each isolate was determined by the agar dilution method. Among 25 beta-lactam antibiotics, the most active agent was imipenem with an MIC90 and a geometric mean of 1 and 0.15 micrograms/ml, respectively; followed by ticarcillin-clavulanic acid, and amoxicillin-clavulanic acid. Ampicillin-sulbactam, piperacillin-tazobactam, moxalactam, and flomoxef were the next most active agents. Piperacillin, ticarcillin, ceftizoxime, cefotaxime, cefuzonam, cefoxitin, and cefmetazole were equally active with the MIC50s ranging from 4 to 16 micrograms/ml, and MIC90s ranging from 32 to greater than or equal to 256 micrograms/ml. The remaining 10 beta-lactam antibiotics, ampicillin, amoxicillin, cefazolin, cefuroxime, cefoperazone, cefmenoxime, ceftazidime, cefpirome, aztreonam, and carumonam were less active. All isolates were resistant to cefotiam at a low breakpoint. Among 6 quinolones, ciprofloxacin was the most active agent with an MIC50 and an MIC90 of 4 and 16 micrograms/ml, respectively. All isolates were resistant to nalidixic acid, pipemidic acid, cinoxacin, enoxacin, and norfloxacin. Among 5 frequently used agents, chloramphenicol, ornidazole, and metronidazole were the most effective agents which inhibited 100% of the isolates at 8, 2, and 2 micrograms/ml, respectively; while clindamycin and minocycline had less activity.

Antimicrobial susceptibility testing for Klebsiella pneumoniae isolates resistant to extended-spectrum beta-lactam antibiotics.

Detection of Klebsiella pneumoniae strains with extended-spectrum beta-lactamase (ESBL)-related resistance phenotypes is becoming important in clinical microbiology laboratories. In this study, we investigated the usefulness of three screening methods, the Etest ESBL screen, the double-disk synergy test, and the ceftazidime disk test, for identifying ESBL-producing K. pneumoniae strains. The agar dilution method was used as the standard. We also determined the in vitro activity of several new antimicrobial agents against these organisms. Strains that exhibited an increase in the minimum inhibitory concentration (MIC) to the third-generation cephalosporins or aztreonam of 2 micrograms/mL or more, but were susceptible to the three cephamycins tested, were considered to have ESBL-related resistance phenotypes. The frequency of ESBL-producing K. pneumoniae isolates (according to the disk-diffusion method) has increased markedly in recent years, from 3.4% in 1993 to 10.3% in 1997. A total of 93 preserved isolates of K. pneumoniae collected from December 1995 through March 1997 were found to be resistant to at least one of the third-generation cephalosporins (cefotaxime and ceftazidime) or aztreonam using the routine disk diffusion method. Among these isolates, 35 were classified as having an ESBL phenotype using the agar dilution method. The remaining 58 isolates were classified as cephamycin resistant, which indicated resistance to both cephamycins and third-generation cephalosporins or aztreonam. The susceptibility rates of the ESBL-producing isolates were 11% for cefotaxime, 14% for ceftazidime, and 6% for aztreonam. The susceptibility rates of these 35 isolates to imipenem, ciprofloxacin, and ofloxacin were 100%, 80%, and 86%, respectively. Both the MIC50 and MIC90 of meropenem were 0.06 microgram/mL, while the MIC50 and MIC90 of BAY 12-8039 were 0.125 and 2 micrograms/mL, respectively. Thirty-two (91%) of the 35 isolates of K. pneumoniae with the ESBL-related resistance phenotype were detected by the Etest ESBL screen, while the ceftazidime disk screen test detected 77% of these isolates, and the double-disk synergy test detected 74%. The Etest ESBL screen appears to be an acceptable, convenient, and sensitive method for the detection of ESBL-producing isolates in the clinical microbiology laboratory.

Heterogeneity of resistance elements in clinical isolates of enterococci with high-level gentamicin resistance.

High-level resistance (minimum inhibitory concentration, MIC > 1,000 micrograms/ml) to gentamicin (HLGR) in enterococci is common in Taiwan. In this study, we investigated the distribution of gentamicin resistance elements in enterococci isolated at National Taiwan University Hospital in a 1-year period, and also examined the transfer and the genetic variability of the resistance elements of different isolates. Among 109 isolates tested, 43 (39%) HLGR isolates were identified. HLGR was most common in Enterococcus faecium isolates (7/15, 47%), followed by Enterococcus faecalis (34/80, 43%), Enterococcus avium (1/5, 20%), and Enterococcus casseliflavus (1/9, 11%). To understand the mechanism of resistance transfer, four isolates of E. faecalis and five isolates of E. faecium showing HLGR were studied. Transfer of resistance markers to a plasmid-free recipient strain of E. faecalis JH2-7 was observed, with transfer frequencies ranging from 10(-2) to 10(-8). All of the transconjugants contained plasmids, with sizes ranging from 45 kb to larger than 70 kb. At least three plasmid patterns were observed on digestion with HaeIII. Hybridization with a probe specific for the aac6'aph2" gentamicin resistance gene confirmed that all of these HLGR isolates carried a Gm(r) determinant, though the hybridization patterns of the plasmids from E. faecalis and E. faecium were different. Although many similarities exist among enterococcal Gm(r) determinants, the results suggest heterogeneity may occur in the flanking regions of resistance elements.

Detection of enterovirus RNA in patients with idiopathic dilated cardiomyopathy by polymerase chain reaction.

The pathogenic role of enterovirus in patients with idiopathic dilated cardiomyopathy has been determined through a molecular biologic approach. Sensitivity in the detection of viral genomes in tissues varied between conventional nucleic acid hybridization and polymerase chain gene amplification. To improve diagnosis, we developed a strategy for reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA. We synthesized two sequence-specific oligonucleotides, primer 1 (5'dACCGACGAATACCACTGTTA3') and primer 2 (5'dCCTCCGGCCCCTGAATGCGGCTAAT3'), complementary to the 5' conserved viral genomic fragments. Viral RNA was amplified by double PCR with these two primers and hybridized with a 32-P labeled inter-primer probe (5'dATGAAACCCACAGGCACAAAG3'). Using this strategy, we detected as little as 10(-8) micrograms of coxsackievirus B3 RNA after amplification with RT-PCR, but detected none in the plasma of eight healthy adults. Among 15 patients with idiopathic dilated cardiomyopathy, viral RNA could be detected in one out of 12 plasmas (8%) and three out of four explanted heart tissues (75%). In contrast, no viral RNA could be detected in six samples of myocardial tissue from patients with other heart diseases. The only patient who had viral RNA in his plasma also had viral RNA in his myocardium. Thus, the high incidence of viral RNA in these patients suggests a possible etiologic link between them. Correct selection of specific PCR primers and the application of double PCR can improve chances of diagnosing enteroviral infection.