Sensitive and specific method for rapid identification of Streptococcus pneumoniae using real-time fluorescence PCR.

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the identification of organisms of clinical and epidemiological importance. As the leading cause of community-acquired pneumonia, Streptococcus pneumoniae was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. Seventy clinical isolates of S. pneumoniae, verified by latex agglutination, were screened against 26 negative control clinical isolates employing a TaqMan assay on a thermocycler (LightCycler). The probe, constructed from the lytA gene, correctly detected all S. pneumoniae genomes without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from patient specimens.

Cloning of insertion sequence IS1485 from Enterococcus species.

We have cloned and identified an insertion sequence, IS1485, that was present in several members of the genus Enterococcus. IS1485 exists in varying copy numbers with at least 12 copies in E. durans (ATCC 11576), 3 copies in E. faecium (ATCC 19434), and one copy each in E. faecalis (ATCC 19433) and E. avium (ATCC 14024). It was also detected in clinical strains of E. gallinarum, E. casseliflavus, and E. saccharolyticus. IS1485 is 1366 bp in length, it has imperfect terminal inverted repeats with 25 of the terminal 39 residues matched, and it contains three open reading frames exceeding 50 codons, designated orfA, orfB, and orfC. The largest, orfB, was located 36 bp downstream and in the -1 reading frame relative to orfA; orfC is oriented in the opposite direction and overlaps orfA. The genetic organization of IS1485 resembles that of members of the IS3 family of transposable elements. Sequence homology exists with several members of the IS3 family especially with IS199 from Streptococcus mutans.

A PCR assay for identification of Enterococcus faecium.

Enterococcus faecium has recently emerged as a serious nosocomial pathogen. The prevalence and severity of enterococcal infections, the mortality rate from such infections, and the antibiotic resistance of enterococci are often species dependent. Since conventional biochemical methods fail to differentiate E. faecium from certain newly described enterococcal species, a PCR-based assay was developed for the rapid identification of E. faecium.

DNA fingerprinting of Candida rugosa via repetitive sequence-based PCR.

A repetitive sequence-based PCR (rep-PCR) technique was developed to characterize the genotypic relatedness among Candida rugosa isolates. Two repetitive sequences, viz., Care-2 and Com29 from Candida albicans, were used to design primers Ca-21, Ca-22, and Com-21, respectively. When used alone or in combination, these primers generated discriminatory fingerprints by amplifying the adjacent variable regions of the genome. Twenty-three isolates from burn patients, eight from other human sources, and four C. rugosa isolates pathogenic in animals were placed into nine fingerprinting groups. Different primers placed these isolates into identical groups, indicating that rep-PCR is a specific and reproducible technique for molecular characterization of C. rugosa. Moreover, these primers unequivocally discriminated among other important Candida species such as C. albicans, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. kefyr, and C. lusitaniae. These data confirm the conservation of repetitive sequences in Candida species. Because of its ease and sensitivity, rep-PCR offers a relatively rapid and discriminatory method for molecular typing of C. rugosa in outbreaks.

Development of a diagnostic polymerase chain reaction assay for detection of Mycoplasma hominis.

A polymerase chain reaction (PCR)-based assay was developed for the detection of Mycoplasma hominis. This assay generates a 152-bp PCR product which was part of an initial 471-bp M. hominis genomic DNA fragment. The 471-bp DNA fragment was shown by hybridization analysis to be unique for M. hominis. The PCR assay can amplify as few as 18 molecules of target DNA. This diagnostic assay offers potential for wide clinical application as it is rapid and can be successfully performed on crude sample preparations from a variety of media or biopsies. The use of this assay should aid in defining the aetiologic and pathologic roles played by M. hominis and thereby benefit patients.

Molecular genotyping of methicillin-resistant Staphylococcus aureus via fluorophore-enhanced repetitive-sequence PCR.

Methicillin resistance in Staphylococcus aureus is a frequent cause of nosocomial and community-acquired infections. Accurate, rapid epidemiologic typing is crucial to the identification of the source and spread of infectious disease and could provide detailed information on the generation of methicillin-resistant S. aureus (MRSA) strains. The high degree of genetic relatedness of MRSA strains has precluded the use of more conventional methods of genetic fingerprinting. A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described. The random chromosomal distribution of this repeat sequence provides an ideal target for detecting DNA fragment patterns specific to individual MRSA strains. Two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented. The repetitive element sequence-based PCR (rep-PCR) and fluorophore-enhanced rep-PCR (FERP) can identify epidemic strains among background MRSA. The combination of oligonucleotide primers labeled with different fluorescent dyes allowed simultaneous FERP fingerprinting and mecA gene detection. Eight different fingerprint patterns were observed in MRSA strains collected from different sources. These techniques provide a rapid discriminatory means of molecular epidemiologic typing of MRSA involved in nosocomial infections.

Development of a DNA probe for Ureaplasma urealyticum.

Ureaplasma urealyticum has been associated with a variety of disease conditions in humans. However, its exact etiologic role has not been well established because of the difficulties encountered in cultural diagnosis and the time needed for positive identifications. A DNA probe which is specific for a target DNA sequence unique to this suspected pathogen offers a rapid, sensitive and specific means of diagnosis. This study details the development of a polymerase chain reaction system for U. urealyticum. Using conventional hybridization techniques, a cloned genomic fragment was found to be specific for this organism. Sequencing of part of this probe DNA permitted the assignment of oligonucleotide primers which amplified a 186 bp target segment. This PCR system is specific for U. urealyticum but not for other closely related species of mycoplasma. This highly sensitive diagnostic technique will aid in determining the etiologic role, tissue tropism and dynamics of pathogenesis of this organism, and thereby result in better patient care.

A large nontypical outbreak of Norwalk virus. Gastroenteritis associated with exposing celery to nonpotable water and with Citrobacter freundii.

The US Air Force Academy experienced a point-source outbreak of gastroenteritis originally believed to be caused by Salmonella. The overall attack rate was 48% among approximately 3000 cadets and staff. Food-specific attack rates implicated chicken salad. The odds ratio for chicken salad consumption in ill cadets was 10.7 (95% confidence interval: 8.2; 13.8). The celery component had been exposed to nonpotable water. Citrobacter freundii were statistically associated with consumption of the suspected vehicle and subsequent illness. Most aspects were consistent with the epidemiology of Norwalk gastroenteritis. However, the clinical presentation was not typical of reported outbreaks. One hundred five cadets required intravenous rehydration. Serum samples implicated Norwalk virus as the most probable cause of this outbreak. The Centers for Disease Control (Atlanta, Ga) recently began national surveillance for viral gastroenteritis. All outbreaks of gastroenteritis associated with nonpotable water should be investigated for evidence of viral cause.

Non-01 Vibrio cholerae bacteremia--complication of a LeVeen shunt.

Vibrio cholerae bacteremia occurred in a patient with cirrhosis after placement of a LeVeen shunt. At the time of bacteremia, cultures of peritoneal fluid were negative and fluid dynamics did not suggest spontaneous bacterial peritonitis. Despite apparent successful treatment of the bacteremia, relapse and death occurred with culture positivity of peritoneal fluid for V. cholerae. Simultaneously, blood cultures were positive for Klebsiella pneumoniae. Agglutination studies demonstrated the V. cholerae to be a non-01 strain. Insertion of a LeVeen shunt, which bypasses the hepatic clearance mechanisms, appeared to have allowed bacteremia to occur with this bacterium that is rarely isolated from blood. In patients with LeVeen shunts, bacteremia with noninvasive pathogens may occur, and in coastal areas, Vibrios should be considered when bacteremia occurs.

Septic arthritis involving Capnocytophaga ochracea.

Septic arthritis of the knee developed in a 21-month-old child. The causative organism, isolated from two separate arthrocenteses, was identified as Capnocytophaga ochracea morphologically and by biochemical reactions. Previous human infections (bacteremias) have occurred in granulocytopenic hosts with concomitant oral pathology including periodontitis and gingivitis. No abnormalities of oral hygiene were present in this patient, and granulocyte numbers were normal or elevated. Eradication of the infection was accomplished with 8 weeks of antibiotic therapy combined with surgical drainage. Septic arthritis expands the spectrum of infections reported to be caused by Capnocytophaga spp.

Sterilization of complete dentures with sodium hypochlorite.

It is important to give the patient a denture that is clean and free from cross-contamination. This study was made to determine if Clorox could be used as a rapid, safe, and clinically effective way to sterilize complete dentures. The data obtained from this study indicate that a 5-minute immersion of dentures in undiluted Clorox accomplished sterilization against a variety of microorganisms, including a spore-forming bacteria and C. albicans.

The bacteriology of recurrent otitis media and the effect of sulfisoxazole chemoprophylaxis.

Middle ear effusion specimens were obtained from 31 children with recurrent episodes of acute otitis media. Of 75 total specimens 28 were obtained from children during chemoprophylaxis with sulfisoxazole. A single organism was isolated in 65 of 70 instances. Beta-lactamase was produced from Gram-negative organisms in 11 instances, and penicillin resistance from Streptococcus pneumoniae occurred in one instance. Haemophilus influenzae predominated during prophylaxis; S. pneumoniae predominated without it. Serotyping and biotyping were performed on 28 isolates from 8 children with consecutive episodes. In 17 instances the infecting organism was the same species but seven of these strains differed in serotype or biotype. The average number of weeks between onset of recurrence in children with homologous strains was shorter (2.6 weeks) than in the children from whom heterologous strains were found (5.7 weeks). Three media were evaluated for efficacy in 32 episodes, and direct plating resulted in the highest rate of recovery.

Isolation of oxidase-negative Pseudomonas aeruginosa from urine culture.

An isolate of Pseudomonas aeruginosa lacking characteristic indophenol oxidase was recovered from a catheterized urine specimen.

Bacteremia associated with Haemophilus influenzae type e biotype 4.

Haemophilus influenzae type e biotype 4 was isolated from a single antemortem blood culture obtained from a 60-year-old, white male with abdominal carcinomatosis.

Pneumonia due to Haemophilus influenzae (H. aegyptius) biotype 3.

Haemophilus influenzae (H. aegyptius) biotype 3 was isolated from eye, nasopharyngeal, and sputum cultures of a 23-month-old male and from sputum and transtracheal aspirate cultures of his 39-year-old mother, both with diffuse bronchopneumonia.