Showing papers in "Journal of Clinical Microbiology in 1999"

The Calgary Biofilm Device: New Technology for Rapid Determination of Antibiotic Susceptibilities of Bacterial Biofilms

Abstract: Determination of the MIC, based on the activities of antibiotics against planktonic bacteria, is the standard assay for antibiotic susceptibility testing. Adherent bacterial populations (biofilms) present with an innate lack of antibiotic susceptibility not seen in the same bacteria grown as planktonic populations. The Calgary Biofilm Device (CBD) is described as a new technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. The CBD produces 96 equivalent biofilms for the assay of antibiotic susceptibilities by the standard 96-well technology. Biofilm formation was followed by quantitative microbiology and scanning electron microscopy. Susceptibility to a standard group of antibiotics was determined for National Committee for Clinical Laboratory Standards (NCCLS) reference strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 29213. Growth curves demonstrated that biofilms of a predetermined size could be formed on the CBD at specific time points and, furthermore, that no significant difference (P > 0.1) was seen between biofilms formed on each of the 96 pegs. The antibiotic susceptibilities for planktonic populations obtained by the NCCLS method or from the CBD were similar. Minimal biofilm eradication concentrations, derived by using the CBD, demonstrated that for biofilms of the same organisms, 100 to 1,000 times the concentration of a certain antibiotic were often required for the antibiotic to be effective, while other antibiotics were found to be effective at the MICs. The CBD offers a new technology for the rational selection of antibiotics effective against microbial biofilms and for the screening of new effective antibiotic compounds.

Evaluation of Protein A Gene Polymorphic Region DNA Sequencing for Typing of Staphylococcus aureus Strains

Abstract: Three hundred and twenty isolates of Staphylococcus aureus were typed by DNA sequence analysis of the X region of the protein A gene (spa). spa typing was compared to both phenotypic and molecular techniques for the ability to differentiate and categorize S. aureus strains into groups that correlate with epidemiological information. Two previously characterized study populations were examined. A collection of 59 isolates (F. C. Tenover, R. Arbeit, G. Archer, J. Biddle, S. Byrne, R. Goering, G. Hancock, G. A. Hebert, B. Hill, R. Hollis, W. R. Jarvis, B. Kreiswirth, W. Eisner, J. Maslow, L. K. McDougal, J. M. Miller, M. Mulligan, and M. A. Pfaller, J. Clin. Microbiol. 32:407-415, 1994) from the Centers for Disease Control and Prevention (CDC) was used to test for the ability to discriminate outbreak from epidemiologically unrelated strains. A separate collection of 261 isolates form a multicenter study (R. B. Roberts, A. de Lencastre, W. Eisner, E. P. Severina, B. Shopsin, B. N. Kreiswirth, and A. Tomasz, J. Infect. Dis. 178:164-171, 1998) of methicillin-resistant S. aureus in New York City (NYC) was used to compare the ability of spa typing to group strains along clonal lines to that of the combination of pulsed-field gel electrophoresis and Southern hybridization. In the 320 isolates studied, spa typing identified 24 distinct repeat types and 33 different strain types. spa typing distinguished 27 of 29 related strains and did not provide a unique fingerprint for 4 unrelated strains from the four outbreaks of the CDC collection. In the NYC collection, spa typing provided a clonal assignment for 185 of 195 strains within the five major groups previously described. spa sequencing appears to be a highly effective rapid typing tool for S. aureus that, despite some expense of specificity, has significant advantages in terms of speed, ease of use, ease of interpretation, and standardization among laboratories.

Principles and Applications of Methods for DNA-Based Typing of Microbial Organisms

Abstract: Outbreaks of infectious disease often result from exposure to a common source of the etiologic agent. Generally, the etiologic agent causing an outbreak of infection is derived from a single cell whose progeny are genetically identical or closely related to the source organism. In epidemiological terms, the organisms involved in the outbreak are clonally related; that is, they have a common origin. Clonally related organisms are members of the same species that share virulence factors, biochemical traits, and genomic characteristics. However, there is sufficient diversity at the species level that organisms isolated from different sources at different times and in different geographical regions may be differentiated or classified into subtypes or strains.

Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans.

Abstract: Associations between known or putative virulence factors of Shiga toxin-producing Escherichia coli and disease in humans were investigated. Univariate analysis and multivariate logistic regression analysis of a set of 237 isolates from 118 serotypes showed significant associations between the presence of genes for intimin (eae) and Shiga toxin 2 (stx2) and isolates from serotypes reported in humans. Similar associations were found with isolates from serotypes reported in hemorrhagic colitis and hemolytic-uremic syndrome. The enterohemorrhagic E. coli (EHEC) hemolysin gene was significantly associated with isolates from serotypes found in severe diseases in univariate analysis but not in multivariate logistic regression models. A strong association between the intimin and EHEC-hemolysin genes may explain the lack of statistical significance of EHEC hemolysin in these multivariate models, but a true lack of biological significance of the hemolysin in humans or in disease cannot be excluded. This result warrants further investigations of this topic. Multivariate analysis revealed an interaction between the eae and stx2 genes, thus supporting the hypothesis of the synergism between the adhesin intimin and Shiga toxin 2. A strong statistical association was observed between the stx2 gene and severity of disease for a set of 112 human isolates from eight major serotypes. A comparison of 77 isolates of bovine origin and 91 human isolates belonging to six major serotypes showed significant associations of the genes for Shiga toxin 1 and EspP protease with bovine isolates and an increased adherence on HEp-2 cell cultures for human isolates, particularly from diarrheic patients and healthy persons.

Detection of Antibody to Avian Influenza A (H5N1) Virus in Human Serum by Using a Combination of Serologic Assays

Abstract: From May to December 1997, 18 cases of mild to severe respiratory illness caused by avian influenza A (H5N1) viruses were identified in Hong Kong. The emergence of an avian virus in the human population prompted an epidemiological investigation to determine the extent of human-to-human transmission of the virus and risk factors associated with infection. The hemagglutination inhibition (HI) assay, the standard method for serologic detection of influenza virus infection in humans, has been shown to be less sensitive for the detection of antibodies induced by avian influenza viruses. Therefore, we developed a more sensitive microneutralization assay to detect antibodies to avian influenza in humans. Direct comparison of an HI assay and the microneutralization assay demonstrated that the latter was substantially more sensitive in detecting human antibodies to H5N1 virus in infected individuals. An H5-specific indirect enzyme-linked immunosorbent assay (ELISA) was also established to test children’s sera. The sensitivity and specificity of the microneutralization assay were compared with those of an H5-specific indirect ELISA. When combined with a confirmatory H5-specific Western blot test, the specificities of both assays were improved. Maximum sensitivity (80%) and specificity (96%) for the detection of anti-H5 antibody in adults aged 18 to 59 years were achieved by using the microneutralization assay combined with Western blotting. Maximum sensitivity (100%) and specificity (100%) in detecting anti-H5 antibody in sera obtained from children less than 15 years of age were achieved by using ELISA combined with Western blotting. This new test algorithm is being used for the seroepidemiologic investigations of the avian H5N1 influenza outbreak.

Development and Clinical Evaluation of a Highly Sensitive PCR-Reverse Hybridization Line Probe Assay for Detection and Identification of Anogenital Human Papillomavirus

Abstract: Human papillomavirus (HPV) can be detected by amplification of viral DNA. A novel PCR primer set generating a short PCR fragment (SPF PCR) was used for amplification of a fragment of only 65 bp from the L1 region and permitted ultrasensitive detection of a broad spectrum of HPV genotypes. The intra- and intertypic sequence variations of the 22-bp interprimer region of this amplimer were studied. Among 238 HPV sequences from GenBank and clinical specimens, HPV genotypes were correctly identified based on the 22-bp sequence in 232 cases (97.2%). Genotype-specific probes for HPV genotypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, 51 to 54, 56, 58, 59, 66, 68, 70, and 74 were selected, and a reverse hybridization line probe assay (LiPA) (the INNO-LiPA HPV prototype research assay) was developed. This LiPA permits the use of amplimers generated by the SPF as well as the MY 09/11 primers. The assay was evaluated with a total of 1,354 clinical specimens, comprising cervical scrapes (classifications ranging from normal cytology to severe dyskaryosis) and formalin-fixed, paraffin-embedded cervical carcinoma samples. LiPA results were highly concordant with sequence analysis of the SPF amplimer, genotype-specific PCR, and sequence analysis of amplimers generated by MY 09/11 primers. The sensitivity of the SPF primers was higher than that of the GP5+/6+ primers over a broad range of HPV types, especially when multiple HPV genotypes were present. In conclusion, the SPF LiPA method allows extremely sensitive detection of HPV DNA as well as reliable identification of HPV genotypes in both cervical smears and paraffin-embedded materials.

Comparison of Methods Based on Different Molecular Epidemiological Markers for Typing of Mycobacterium tuberculosis Complex Strains: Interlaboratory Study of Discriminatory Power and Reproducibility

Abstract: In this study, the currently known typing methods for Mycobacterium tuberculosis isolates were evaluated with regard to reproducibility, discrimination, and specificity Therefore, 90 M tuberculosis complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays In all methods, one or more repetitive DNA elements were targeted The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method To examine intralaboratory reproducibility, blinded duplicate samples were included The specificities of the various methods were tested by inclusion of 10 non-M tuberculosis complex strains All five RFLP typing methods were highly reproducible The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping In contrast, the double repetitive element PCR (DRE-PCR), IS6110 inverse PCR, IS6110 ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible The 90 strains were best discriminated by IS6110 RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns) We conclude that for epidemiological investigations, strain differentiation by IS6110 RFLP or mixed-linker PCR are the methods of choice A strong association was found between the results of different genetic markers, indicating a clonal population structure of M tuberculosis strains Several separate genotype families within the M tuberculosis complex could be recognized on the basis of the genetic markers used

Quantitative Analysis of Epstein-Barr Virus Load by Using a Real-Time PCR Assay

Abstract: To measure the virus load in patients with symptomatic Epstein-Barr virus (EBV) infections, we used a real-time PCR assay to quantify the amount of EBV DNA in blood. The real-time PCR assay could detect from 2 to over 10(7) copies of EBV DNA with a wide linear range. We estimated the virus load in peripheral blood mononuclear cells (PBMNC) from patients with symptomatic EBV infections. The mean EBV-DNA copy number in the PBMNC was 10(3.7) copies/microg of DNA in patients with EBV-related lymphoproliferative disorders, 10(4.1) copies/microg of DNA in patients with chronic active EBV infections, and 10(2.2) copies/microg of DNA in patients with infectious mononucleosis. These numbers were significantly larger than those in either posttransplant patients or immunocompetent control patients without EBV-related diseases. In a patient with infectious mononucleosis, the virus load decreased as the symptoms resolved. The copy number of EBV DNA in PBMNC from symptomatic EBV infections was correlated with the EBV-positive cell number determined by the in situ hybridization assay (r = 0.842; P < 0.0001). These results indicate that the real-time PCR assay is useful for diagnosing symptomatic EBV infection and for monitoring the virus load.

Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization.

Abstract: A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species of Theileria and Babesia simultaneously. We included Theileria annulata, T. parva, T. mutans, T. taurotragi, and T. velifera in the assay, as well as parasites belonging to the T. sergenti-T. buffeli-T. orientalis group. The Babesia species included were Babesia bovis, B. bigemina, and B. divergens. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all Theileria and Babesia species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences from Bos taurus or other hemoparasites (Trypanosoma species, Cowdria ruminantium, Anaplasma marginale, and Ehrlichia species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain. T. annulata, T. orientalis, and B. bigemina were identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species.

PCR Targeted to the 16S-23S rRNA Gene Intergenic Spacer Region of Clostridium difficile and Construction of a Library Consisting of 116 Different PCR Ribotypes

Abstract: A reference library of types of Clostridium difficile has been constructed by PCR ribotyping isolates (n = 2,030) from environmental (n = 89), hospital (n = 1,386), community practitioner (n = 395), veterinary (n = 27), and reference (n = 133) sources. The library consists of 116 distinct types identified on the basis of differences in profiles generated with PCR primers designed to amplify the 16S-23S rRNA gene intergenic spacer region. Isolates from 55% of infections in hospitals in the United Kingdom belonged to one ribotype (type 1), but this type was responsible for only 7.5% of community infections.

Typing of Human Enteroviruses by Partial Sequencing of VP1

Abstract: Human enteroviruses (family Picornaviridae) are the major cause of aseptic meningitis and also cause a wide range of other acute illnesses, including neonatal sepsis-like disease, acute flaccid paralysis, and acute hemorrhagic conjunctivitis. The neutralization assay is usually used for enterovirus typing, but it is labor-intensive and time-consuming and standardized antisera are in limited supply. We have developed a molecular typing system based on reverse transcription-PCR and nucleotide sequencing of the 3′ half of the genomic region encoding VP1. The standard PCR primers amplify approximately 450 bp of VP1 for most known human enterovirus serotypes. The serotype of an “unknown” may be inferred by comparison of the partial VP1 sequence to those in a database containing VP1 sequences for the prototype strains of all 66 human enterovirus serotypes. Fifty-one clinical isolates of known serotypes from the years 1991 to 1998 were amplified and sequenced, and the antigenic and molecular typing results agreed for all isolates. With one exception, the nucleotide sequences of homologous strains were at least 75% identical to one another (>88% amino acid identity). Strains with homologous serotypes were easily discriminated from those with heterologous serotypes by using these criteria for identification. This method can greatly reduce the time required to type an enterovirus isolate and can be used to type isolates that are difficult or impossible to type with standard immunological reagents. The technique may also be useful for the rapid determination of whether viruses isolated during an outbreak are epidemiologically related.

Amplified-Fragment Length Polymorphism Analysis: the State of an Art

Abstract: In the past decade, various methods have been developed for the identification and typing of prokaryotic and eukaryotic organisms at the DNA level. These methods differ in their taxonomic range, discriminatory power, reproducibility, and ease of interpretation and standardization ([62][1], [67][2

Identification of Vibrio parahaemolyticus Strains at the Species Level by PCR Targeted to the toxR Gene

Abstract: The DNA colony hybridization test with the polynucleotide probe for Vibrio parahaemolyticus toxR gene was performed. All 373 strains of V. parahaemolyticus gave positive results, and the strains belonging to four other Vibrio species including Vibrio alginolyticus gave weakly positive results, suggesting that toxR sequence variation may reflect the phylogenetic relationships of Vibrio species. We then established a toxR-targeted PCR protocol for the specific detection of V. parahaemolyticus.

Relationship between Helicobacter pylori iceA, cagA, and vacA Status and Clinical Outcome: Studies in Four Different Countries

Abstract: There is continuing interest in identifying Helicobacter pylori virulence factors that might predict the risk for symptomatic clinical outcomes. It has been proposed that iceA and cagA genes are such markers and can identify patients with peptic ulcers. We compared H. pylori isolates from four countries, looking at the cagA and vacA genotypes, iceA alleles, and presentation of the infection. We used PCR to examine iceA, vacA, and cagA status of 424 H. pylori isolates obtained from patients with different clinical presentations (peptic ulcer, gastric cancer, and atrophic gastritis). The H. pylori isolates examined included 107 strains from Bogota, Colombia, 70 from Houston, Tex., 135 from Seoul, Korea, and 112 from Kyoto, Japan. The predominant genotype differed among countries: the cagA-positive iceA1 vacA s1c-m1 genotype was predominant in Japan and Korea, the cagA-positive iceA2 vacA s1b-m1 genotype was predominant in the United States, and the cagA-positive iceA2 vacA s1a-m1 genotype was predominant in Colombia. There was no association between the iceA, vacA, or cagA status and clinical outcome in patients in the countries studied. iceA status shows considerable geographic differences, and neither iceA nor combinations of iceA, vacA, and cagA were helpful in predicting the clinical presentation of an H. pylori infection.

Detection of Prosthetic Hip Infection at Revision Arthroplasty by Immunofluorescence Microscopy and PCR Amplification of the Bacterial 16S rRNA Gene

Abstract: In this study the detection rates of bacterial infection of hip prostheses by culture and nonculture methods were compared for 120 patients with total hip revision surgery. By use of strict anaerobic bacteriological practice during the processing of samples and without enrichment, the incidence of infection by culture of material dislodged from retrieved prostheses after ultrasonication (sonicate) was 22%. Bacteria were observed by immunofluorescence microscopy in 63% of sonicate samples with a monoclonal antibody specific for Propionibacterium acnes and polyclonal antiserum specific for Staphylococcus spp. The bacteria were present either as single cells or in aggregates of up to 300 bacterial cells. These aggregates were not observed without sonication to dislodge the biofilm. Bacteria were observed in all of the culture-positive samples, and in some cases in which only one type of bacterium was identified by culture, both coccoid and coryneform bacteria were observed by immunofluorescence microscopy. Bacteria from skin-flake contamination were readily distinguishable from infecting bacteria by immunofluorescence microscopy. Examination of skin scrapings did not reveal large aggregates of bacteria but did reveal skin cells. These were not observed in the sonicates. Bacterial DNA was detected in 72% of sonicate samples by PCR amplification of a region of the bacterial 16S rRNA gene with universal primers. All of the culture-positive samples were also positive for bacterial DNA. Evidence of high-level infiltration either of neutrophils or of lymphocytes or macrophages into associated tissue was observed in 73% of patients. Our results indicate that the incidence of prosthetic joint infection is grossly underestimated by current culture detection methods. It is therefore imperative that current clinical practice with regard to the detection and subsequent treatment of prosthetic joint infection be reassessed in the light of these results.

Detection and identification of Ehrlichia, Borrelia burgdorferi sensu lato, and Bartonella species in Dutch Ixodes ricinus ticks.

Abstract: A sensitive and specific PCR hybridization assay was developed for the simultaneous detection and identification of Ehrlichia and Borrelia burgdorferi sensu lato. In separate assays the 16S rRNA gene of Ehrlichia species and the 23S-5S rRNA spacer region of B. burgdorferi sensu lato were amplified and labeled by PCR. These PCR products were used in a reverse line blot hybridization assay in which oligonucleotide probes are covalently linked to a membrane in parallel lines. Hybridization of the samples with the oligonucleotide probes on this membrane enabled the simultaneous detection and identification of Ehrlichia, B. burgdorferi, and Bartonella species in 40 different samples. The application of the assay to DNA extracts from 121 Ixodes ricinus ticks collected from roe deer demonstrated that 45% of these ticks carried Ehrlichia DNA. More than half of these positive ticks carried species with 16S rRNA gene sequences closely related to those of E. phagocytophila and the human granulocytic ehrlichiosis agent. The majority of the other positive ticks were infected with a newly identified Ehrlichia-like species. In addition, 13% of the ticks were infected with one or more B. burgdorferi genospecies. In more than 70% of the ticks 16S rRNA gene sequences for Bartonella species or other species closely related to Bartonella were found. In five of the ticks both Ehrlichia and B. burgdorferi species were detected.

Development of a PCR Assay for Rapid Detection of Enterococci

Abstract: Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR-based assay is capable of detecting all clinically important enterococci and has potential for use in clinical microbiology laboratories.

Identification of Mycobacterial Species by Comparative Sequence Analysis of the RNA Polymerase Gene (rpoB)

Abstract: The genus Mycobacterium comprises a wide range of organisms, including obligate parasites causing serious human and animal diseases, opportunistic pathogens, and saprophytic species found in nature. Human infections are caused mainly by slowly growing mycobacteria that need more than 7 days to form visible colonies on solid media. Traditionally, the definitive diagnosis of mycobacterial infections has been dependent on the isolation and identification of causative agents and requires a series of specialized physiological and biochemical tests. The procedures for these tests are complex, laborious, and usually impeded by the slow growth of mycobacteria in clinical laboratories. In particular, Mycobacterium leprae has not been cultivated in vitro. There have been increasing numbers of reports of infections caused by mycobacteria other than M. tuberculosis (MOTT), especially in association with human immunodeficiency virus infection. These are rarely disease associated, previously unknown or newly recognized mycobacteria that are not easy to identify; moreover, due to their phenotypic similarity to certain species, they cannot be easily characterized by the conventional methods of identification. However, mycobacterial systematics may help in the differentiation and identification of these phenotypically similar mycobacterial species. Descriptive taxonomic analyses have been used to classify mycobacterial species. For a clearer definition of species boundaries, macromolecular comparisons, particularly of 16S rRNA, which is highly conserved throughout organisms, have been used to determine phylogenetic relationships. Mycobacterial phylogenetic analysis based on sequences of 16S rRNA (25, 31) or the 16S rRNA gene (16S rDNA) (27) has helped to define mycobacterial species. This analysis demonstrated the usefulness of genotypic studies, especially when conventional procedures are inapplicable, particularly for the differentiation and identification of novel and uncultivable mycobacteria (19). It has been suggested, however, that for the delineation of species boundaries, 16S rRNA-based phylogenetic analysis has its limitations (11). Ambiguous results due to the presence of two different 16S rRNA genes in an organism would also limit the use of 16S rDNA sequencing in the identification of mycobacterial species (23, 26). Doubts about its usefulness were raised because M. kansasii, a pathogenic mycobacterium, could not be distinguished from nonpathogenic M. gastri by this means (35). A similar result was observed in 23S rRNA sequence analysis; the sequence for M. kansasii was identical to that for M. celatum (32). rpoB encodes the β subunit of RNA polymerase. The rpoB nucleotide sequences of three mycobacterial species were previously known (15, 16, 22). Missense mutations within rpoB’s limited region are known to be related to rifampin resistance in M. tuberculosis (34). Recently, the rpoB gene was used as an alternative tool to identify mycobacteria (14). However, only a limited number of reference species (five slowly growing and five rapidly growing species) in the genus Mycobacterium were used. In the present study, rpoB DNAs (342 bp) comprising a highly conserved region throughout the eubacteria (5) were amplified from the 44 reference strains of mycobacteria. Their nucleotide sequences (306 bp) were directly determined and compared to study their phylogenetic relationships. To demonstrate the feasibility of using this method in which rpoB sequences are compared and a phylogenetic tree with reference species is inferred, this procedure was applied to clinical isolates. We suggest that this procedure is a useful identification method that can be completed within two working days.

Cryptococcus neoformans var.grubii: Separate Varietal Status for Cryptococcus neoformans Serotype A Isolates

Abstract: Cryptococcus neoformans var. neoformans presently includes isolates which have been determined by the immunologic reactivity of their capsular polysaccharides to be serotype A and those which have been determined to be serotype D. However, recent analyses of the URA5 sequences and DNA fingerprinting patterns suggest significant genetic differences between the two serotypes. Therefore, we propose to recognize these genotypic distinctions, as well as previously reported phenotypic differences, by restricting C. neoformans var. neoformans to isolates which are serotype D and describing a new variety, C. neoformans var. grubii, for serotype A isolates.

Autopsy-Controlled Prospective Evaluation of Serial Screening for Circulating Galactomannan by a Sandwich Enzyme-Linked Immunosorbent Assay for Hematological Patients at Risk for Invasive Aspergillosis

Abstract: Efforts to improve the diagnosis of invasive aspergillosis (IA) have been directed towards the detection of fungal antigens, including galactomannan (GM). However, previous evaluations of GM detection have been hampered by a lack of proven cases of IA and by a nonserial study design. This prospective study assessed the diagnostic value of serial screening for circulating GM by using a recently developed sandwich enzyme-linked immunosorbent assay (ELISA) for prolonged-neutropenic and/or steroid-treated patients with hematological disorders. Serum GM levels were monitored twice weekly for 186 consecutive patients at increased risk for IA. The patients were stratified according to the likelihood of IA (proven, probable, possible, and no evidence of IA) by using stringent criteria. Proven IA was defined by characteristic histopathological findings together with a positive culture for Aspergillus species. Autopsy and culture from autopsy specimens was used to verify both positive and negative test results. A total of 2,172 serum samples were tested from 243 episodes (mean, 9 samples/episode). Based on the analysis of 71 patients with confirmed disease status (culture and histology), the sensitivity and specificity of serial GM monitoring were 92.6 and 95.4%, respectively. The positive predictive value was almost 93%, the negative predictive value was 95%, and the efficacy was 94%. False-positive reactions occurred at a rate of nearly 8%, although this figure might have been overestimated. Less than 1% of all tested sera were considered inconclusive. In more than half of the cases, antigenemia was detected before clinical suspicion of IA (median, 6 days before). Serial determination of serum GM by the sandwich ELISA technique is a sensitive tool for the diagnosis of IA in hematological patients at risk. This approach may substantially influence clinical management with regard to preemptive and empirical antifungal therapy.

Use of Multiplex PCR To Detect Classical and Newly Described Pyrogenic Toxin Genes in Staphylococcal Isolates

Abstract: Staphylococcus aureus may contain one or more genes that encode a variety of immunomodulatory pyrogenic toxins (PTs), including the staphylococcal enterotoxins and toxic shock syndrome toxin (TSST). The PTs interact with several cellular targets to produce disease, such as food poisoning and toxic shock syndrome. At present, nine serologically distinct enterotoxins and one immunoreactive form of TSST have been identified and characterized. As isolates of S. aureus are further assessed, it is anticipated that this number will increase. To facilitate screening, a multiplex PCR was designed to simultaneously determine which of these 10 currently known PT genes an individual S. aureus isolate possesses. We show here, using S. aureus isolates with characterized PT phenotypes, that this novel PCR technique reliably detects each of the known PTs in a single reaction.

Rapid Identification of Fungi by Using the ITS2 Genetic Region and an Automated Fluorescent Capillary Electrophoresis System

Abstract: Invasive fungal disease often plays an important role in the morbidity and mortality of immunocompromised patients. The poor sensitivity of current fungal blood culture and histological practices has led to the development of highly sensitive and specific molecular techniques, such as the PCR. Sequence variability of the internal transcribed spacer 2 (ITS2) region of fungi is potentially useful in rapid and accurate diagnosis of clinical fungal isolates. PCR with fungus-specific primers targeted toward conserved sequences of the 5.8S and 28S ribosomal DNA (rDNA) results in amplification of the species-specific ITS2 regions, which are variable in amplicon length. We have made use of the ABI PRISM 310 genetic analyzer and the ABI PRISM 310 GeneScan analysis software for the determination of variable size differences of the ITS2 region of clinically important fungi, including Candida and non-Candida yeasts, Aspergillus species, and a variety of dermatophytes. No cross-reaction occurred when samples were tested against human and bacterial genomic DNA. We have found that most clinically significant fungal isolates can be differentiated by this method, and it therefore serves to be a promising tool for the rapid (<7 h) diagnosis of fungemia and other invasive fungal infections.

Mycoplasma hyopneumoniae Potentiation of Porcine Reproductive and Respiratory Syndrome Virus-Induced Pneumonia

Abstract: An experimental model that demonstrates a mycoplasma species acting to potentiate a viral pneumonia was developed. Mycoplasma hyopneumoniae, which produces a chronic, lymphohistiocytic bronchopneumonia in pigs, was found to potentiate the severity and the duration of a virus-induced pneumonia in pigs. Pigs were inoculated with M. hyopneumoniae 21 days prior to, simultaneously with, or 10 days after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV), which induces an acute interstitial pneumonia in pigs. PRRSV-induced clinical respiratory disease and macroscopic and microscopic pneumonic lesions were more severe and persistent in M. hyopneumoniae-infected pigs. At 28 or 38 days after PRRSV inoculation, M. hyopneumoniae-infected pigs still exhibited lesions typical of PRRSV-induced pneumonia, whereas the lungs of pigs which had received only PRRSV were essentially normal. On the basis of macroscopic lung lesions, it appears that PRRSV infection did not influence the severity of M. hyopneumoniae infection, although microscopic lesions typical of M. hyopneumoniae were more severe in PRRSV-infected pigs. These results indicate that M. hyopneumoniae infection potentiates PRRSV-induced disease and lesions. Most importantly, M. hyopneumoniae-infected pigs with minimal to nondetectable mycoplasmal pneumonia lesions manifested significantly increased PRRSV-induced pneumonia lesions compared to pigs infected with PRRSV only. This discovery is important with respect to the control of respiratory disease in pigs and has implications in elucidating the potential contribution of mycoplasmas in the pathogenesis of viral infections of other species, including humans.

Clinical and Financial Benefits of Rapid Bacterial Identification and Antimicrobial Susceptibility Testing

Abstract: To assess the expected clinical and financial benefits of rapid reporting of microbiology results, we compared patients whose cultured samples were processed in the normal manner to patients whose samples were processed more rapidly due to a minor change in work flow. For the samples tested in the rapid-reporting time period, the vast majority of bacterial identification and antimicrobial susceptibility testing (AST) results were verified with the Vitek system on the same day that they were available. This time period was called rapid AST (RAST). For RAST, a technologist on the evening shift verified the data that became available during that shift. For the control time period, cultures were processed in the normal manner (normal AST [NAST]), which did not include evening-shift verification. For NAST, the results for approximately half of the cultures were verified on the first day that the result was available. The average turnaround time for the reporting of AST results was 39.2 h for RAST and 44.4 h for NAST (5.2 h faster for RAST [P = 0.001]). Subsequently, physicians were able to initiate appropriate antimicrobial therapy sooner for patients whose samples were tested as part of RAST (P = 0.006). The mortality rates were 7.9 and 9.6% for patients whose samples were tested as part of RAST and NAST, respectively (P = 0.45). The average length of stay was 10.7 days per patient for RAST and 12.6 days for NAST, a difference of 2.0 days less for RAST (P = 0.006). The average variable cost was $4,927 per patient for RAST and $6,677 for NAST, a difference of $1,750 less per patient for RAST (P = 0.001). This results in over $4 million in savings in variable costs per year in our hospital.

Case-Control Study of Enteropathogens Associated with Childhood Diarrhea in Dhaka, Bangladesh

Abstract: The International Centre for Diarrhoeal Disease Research, Bangladesh, is a major center for research into diarrheal diseases. The center treats more than 100,000 patients a year. To obtain useful information representative of all patients, a surveillance system in which a 4% systematic sample of all patients is studied in detail, including etiological agents of diarrhea, was installed in October 1979. The first paper on etiology for the surveillance patients was published in 1982, which identified a potential enteric pathogen in 66% of patients. In subsequent years, several new agents of diarrhea have been identified. To assess the importance of a broader spectrum of diarrheal agents including the ones identified relatively recently, we studied 814 children with diarrhea. The children were up to 5 years of age and were part of the surveillance system. They were matched with an equal number of community controls without diarrhea. The study was conducted from February 1993 to June 1994. A potential enteric pathogen was isolated from 74.8% of diarrheal children and 43.9% of control children (P = 0.0001). Even though the first study was not a case-control study, it identified rotavirus, Campylobacter jejuni, enterotoxigenic Escherichia coli, Shigella spp. , and Vibrio cholerae O1 as major pathogens. The present study identified these pathogens as being significantly associated with diarrhea. In addition, the study also identified six additional agents, including enteropathogenic E. coli, Aeromonas spp., V. cholerae O139, enterotoxigenic Bacteroides fragilis, Clostridium difficile, and Cryptosporidium parvum, as being significantly associated with diarrhea. Plesiomonas shigelloides, Salmonella spp., diffusely adherent E. coli, enteroaggregative E. coli, Entamoeba histolytica, and Giardia lamblia were not significantly associated with diarrhea. Enteroinvasive E. coli, enterohemorrhagic E. coli, and Cyclospora cayetanensis were not detected in any of the children. The major burden of diseases due to most pathogens occurred in the first year of life. As in the previous study, seasonal patterns were seen for diarrhea associated with rotavirus, V. cholerae, and enterotoxigenic E. coli, and infections with multiple pathogens were common. With a few exceptions, these findings are in agreement with those from other developing countries. This knowledge of a broader spectrum of etiological agents of diarrhea in the surveillance patients will help us plan studies into various aspects of diarrheal diseases in this population.

Adenoviruses from human immunodeficiency virus-infected individuals, including two strains that represent new candidate serotypes Ad50 and Ad51 of species B1 and D, respectively.

Abstract: Adenovirus (Ad) isolates from a large number of human immunodeficiency virus (HIV)-infected individuals were compared serologically and genetically with Ad isolates from immunocompetent patients. Between 1982 and 1994, stool and urine samples from 137 subjects with AIDS hospitalized in The Netherlands yielded 143 Ad strains. Forty additional Ad strains were obtained from 35 HIV-positive patients in Manchester, United Kingdom, in 1992 and 1993. Of these 183 HIV-associated Ad strains, 84% belonged to species D and 3% belonged to species C. These strains were compared with 2,301 Ad strains collected during general diagnostic examinations in The Netherlands from 1973 to 1992. Of the latter strains, 5% belonged to species D and 49% belonged to species C. Two of the Ads isolated from fecal specimens of AIDS patients represent new serotypes: candidate Ad serotype 50 (prototype strain, Wan) of subspecies B1 and candidate Ad serotype 51 (prototype strain, Bom) of species D. The DNA restriction enzyme patterns of strains Wan and Bom differed from the patterns of all established prototypes.

Occurrence of Leishmania infantum Parasitemia in Asymptomatic Blood Donors Living in an Area of Endemicity in Southern France

Abstract: Visceral leishmaniosis (VL) due to Leishmania infantum (L. chagasi) is a lethal disease if untreated, but asymptomatic L. infantum infections have been reported previously. A better understanding of parasite transmission, dissemination, and survival in the human host is needed. The purpose of this study was to assess whether L. infantum circulated in peripheral blood of subjects with no history of VL. Sera from 565 blood donors were screened by Western blotting to detect Leishmania-specific antibodies and identify individuals with probable past exposure to Leishmania. Seropositivity was found in 76 donors whose buffy coats were examined by PCR and direct culture. The parasite minicircle kinetoplast DNA was amplified from blood samples of nine donors. Promastigotes were detected by culture in blood samples from nine donors. Only two donors were PCR and culture positive. These results indicate that L. infantum circulates intermittently and at low density in the blood of healthy seropositive individuals, who thus appear to be asymptomatic carriers. Implications for the safety of blood transfusion are discussed.

Epidemiological Evidence for Dormant Cryptococcus neoformans Infection

Abstract: To date, the time of acquisition of a Cryptococcus neoformans infectious strain has never been studied. We selected a primer, (GACA)(4), and a probe, CNRE-1, that by randomly amplified polymorphic DNA (RAPD) analysis and restriction fragment length polymorphism (RFLP), respectively, regrouped strains from control samples of C. neoformans var. grubii environmental isolates according to their geographical origins. The two typing techniques were then used to analyze 103 isolates from 29 patients diagnosed with cryptococcosis in France. Nine of the 29 patients lived in Africa a median of 110 months prior to moving to France; 17 of the patients originated from Europe. Results showed a statistically significant clustering of isolate subtypes from patients originating from Africa compared to those from Europe. We conclude that the patients had acquired the C. neoformans infectious strain long before their clinical diagnoses were made.

Coinfection with Multiple Tick-Borne Pathogens in a Walker Hound Kennel in North Carolina

Abstract: Both dogs and humans can be coinfected with various Ehrlichia, Bartonella, Rickettsia, and Babesia species. We investigated a kennel of sick Walker Hounds and their owners in southeastern North Carolina for evidence of tick-borne infections and associated risk factors. A high degree of coinfection was documented in the dog population. Of the 27 dogs, 26 were seroreactive to an Ehrlichia sp., 16 to Babesia canis, and 25 to Bartonella vinsonii, and 22 seroconverted to Rickettsia rickettsii antigens. According to PCR results, 15 dogs were infected with Ehrlichia canis, 9 with Ehrlichia chaffeensis, 8 with Ehrlichia ewingii, 3 with Ehrlichia equi, 9 with Ehrlichia platys, 20 with a Rickettsia species, 16 with a Bartonella species, and 7 with B. canis. The detection of DNA from any Ehrlichia species was associated with clinical illness and with concurrent B. canis infection (by PCR). Both E. canis and an uncharacterized Rickettsia species appeared to result in chronic or recurrent infection. Death in the dog population was associated with living in a dirt lot rather than the concrete kennel. Of 23 people on whom serologic testing was conducted, eight were seroreactive to Bartonella henselae, one to E. chaffeensis, and one to R. rickettsii antigen; however, none had clinical or hematologic abnormalities consistent with illness caused by these organisms. We conclude that kennel dogs with heavy tick exposure can be infected at a high rate with multiple, potentially zoonotic, tick-borne pathogens. In addition, our findings further illustrate the utility of PCR for documenting coinfection with tick-transmitted pathogens.

Mycobacterium Species Identification and Rifampin Resistance Testing with High-Density DNA Probe Arrays

Abstract: Species identification within the genus Mycobacterium and subsequent antibiotic susceptibility testing still rely on time-consuming, culture-based methods. Despite the recent development of DNA probes, which greatly reduce assay time, there is a need for a single platform assay capable of answering the multitude of diagnostic questions associated with this genus. We describe the use of a DNA probe array based on two sequence databases: one for the species identification of mycobacteria (82 unique 16S rRNA sequences corresponding to 54 phenotypical species) and the other for detecting Mycobacterium tuberculosis rifampin resistance (rpoB alleles). Species identification or rifampin resistance was determined by hybridizing fluorescently labeled, amplified genetic material generated from bacterial colonies to the array. Seventy mycobacterial isolates from 27 different species and 15 rifampin-resistant M. tuberculosis strains were tested. A total of 26 of 27 species were correctly identified as well as all of the rpoB mutants. This parallel testing format opens new perspectives in terms of patient management for bacterial diseases by allowing a number of genetic tests to be simultaneously run.