bluuebunny/crossref_metadata_2025 huggingface dataset:
Diagnosis of Toxoplasma parasitemia in patients with AIDS by gene detection after amplification with polymerase chain reaction
Authors: Filice G A , Hitt J A , Mitchell C D , Blackstad M , Sorensen S W
Published: January 2020

We sought evidence of toxoplasma parasitemia among 37 people with active or dormant Toxoplasma gondii infection or no evidence of infection. DNA was extracted from erythrocyte-free portions of blood samples, and the T. gondii B1 gene was amplified by the polymerase chain reaction. Evidence of T. gondii parasitemia was found in six patients with severe immunosuppression from AIDS and clinical evidence suggestive of or compatible with toxoplasmosis. Results were negative for patients unlikely to have active toxoplasmosis. Gene detection after amplification with the polymerase chain reaction is a promising test for detection of parasitemia, and parasitemia should be tested for in patients with AIDS and unexplained fever or central nervous system abnormalities.
Identification and detection of Trypanosoma cruzi by using a DNA amplification fingerprint obtained from the ribosomal intergenic spacer
Authors: González N , Galindo I , Guevara P , Novak E , Scorza J V , Añez N , Da Silveira J F , Ramírez J L
Published: January 2020

We designed a PCR assay targeted on repeated elements of the ribosomal intergenic spacer which produces highly polymorphic DNA band patterns for different strains of Trypanosoma cruzi. By labeling the PCR products with digoxigenin and by chemiluminescence detection, we improved the assay sensitivity by three orders of magnitude to get T. cruzi strain fingerprints in feces of the trypanosome-infected triatomine bug vector. We also developed a capture assay for the digoxigenin-labeled PCR products that allowed us to detect T. cruzi in triatomine bug vector feces and in human serum samples with a solid support.
Bacterial vaginosis and vaginal microorganisms in idiopathic premature labor and association with pregnancy outcome
Authors: Holst E , Goffeng A R , Andersch B
Published: January 2020

The vaginal microflora of 49 women in idiopathic preterm labor was compared with that of 38 term controls to determine whether the presence of bacterial vaginosis (BV) and/or specific microorganisms would influence the rate of preterm delivery. Demographic factors, pregnancy outcome, and reproductive history were also studied. BV, as defined by the presence of clue cells in a vaginal wet mount and characteristic microbial findings in a stained vaginal smear and vaginal culture, was more common in women with preterm labor and delivery than in controls (P < 0.01). The condition, diagnosed in 41% of women who had both preterm labor and delivery (n = 22) and in 11% each of women who had preterm labor but term delivery (n = 27) and controls, was associated with a 2.1-fold risk (95% confidence intervals, 1.2 to 3.7) for preterm birth prior to 37 weeks of gestation. BV was associated with low birth weight. Of 49 women with preterm labor, 67% (8 of 12) of women with BV were delivered of low-birth-weight neonates (< 2,500 g) compared with 22% (8 of 37) of women without the condition (P < 0.0005). The presence of hydrogen peroxide-producing facultative Lactobacillus spp. was strongly negatively associated with both preterm delivery and BV. BV-associated microorganisms, i.e., Mobiluncus, Prevotella, and Peptostreptococcus species, Porphyromonas asaccharolytica, Fusobacterium nucleatum, Mycoplasma hominis, and high numbers of Gardnerella vaginalis were significantly associated with preterm delivery; all species also strongly associated with BV (P = 0.0001 for each comparison). Mobiluncus curtisii and Fusobacterium nucleatum were recovered exclusively from women with preterm delivery. Our study clearly indicates that BV and its associated organisms are correlated with idiopathic premature delivery.
Detection of Candida albicans and other yeasts in blood by PCR
Authors: Holmes A R , Cannon R D , Shepherd M G , Jenkinson H F
Published: January 2020

Primers complementary to the region of genes coding for rRNA in Candida albicans were used in PCRs to detect yeast DNA extracted from blood samples containing various Candida species. One fragment (105 bp) was amplified from all yeasts tested, whereas a second (684 bp) was only amplified when C. albicans DNA was present. The level of sensitivity was 15 +/- 5 (mean +/- standard error) CFU of C. albicans per ml of blood.
Comparison of direct and standardized testing of infected urine for antimicrobial susceptibilities by disk diffusion
Authors: Oakes A R , Badger R , Grove D I
Published: January 2020

A total of 14,272 urine specimens were examined over one year to determine the validity of direct antimicrobial agent susceptibility testing against ampicillin, amoxicillin-clavulanic acid, cephalothin, gentamicin, norfloxacin, and trimethoprim. A comparison between direct and standardized disk diffusion tests was made for a total of 1,106 urine specimens containing > or = 10(5) organisms per ml in pure culture. There were 5,821 individual organism-antimicrobial agent challenges compared for the two testing methods, and there was complete agreement of susceptibility category in 5,492 comparisons (94.3%). Initially, discordant results were reduced from 5.7 to 2.1% when the intermediate category was considered susceptible. Intralaboratory variation was assessed by testing another 453 organisms by the standard National Committee for Clinical Laboratory Standards (NCCLS) method on two consecutive days; there was complete agreement in 96.1% of comparisons. When results of direct and standardized testing were simply classified as susceptible or resistant, there was 1.1% discordance. When simple same-day tests were used together with predictable patterns of susceptibility and resistance, 536 (48.5%) of 1,106 isolates could be identified satisfactorily to the genus or species level. For laboratory reporting purposes, the direct method is equivalent to the standard method when the urine being tested is infected with > or = 10(5) organisms of a single type per ml. The presence or absence of preexisting antimicrobial agents in urine did not appreciably influence the results. This procedure allows the earlier reporting of susceptibility results and facilitates less expensive identification of many organisms. Costs and benefits need to be determined in each institution.
Utility of gram stain in evaluation of sputa from patients with cystic fibrosis
Authors: Sadeghi E , Matlow A , MacLusky I , Karmali M A
Published: January 2020

The utility of sputum Gram stain in assessing salivary contamination and in predicting the presence of pathogens on the basis of morphology was investigated in 287 respiratory specimens from patients with cystic fibrosis. Where acceptability for culture was defined as a leukocyte/squamous epithelial cell ratio of > 5, 76.6% (220 of 287) of respiratory specimens received in the laboratory were considered acceptable. Unacceptable specimens were more common in younger patients. The positive predictive value of the Gram stain for growth from acceptable sputum samples was 98% for Pseudomonas aeruginosa, 84.4% for Pseudomonas cepacia, 86.3% for Staphylococcus aureus, and 100% for Haemophilus influenzae. In cystic fibrosis patients, as has been reported for respiratory specimens in general, Gram stain of respiratory specimens in helpful for interpreting culture results.
Variations in fluconazole susceptibility and electrophoretic karyotype among oral isolates of Candida albicans from patients with AIDS and oral candidiasis
Authors: Pfaller M A , Rhine-Chalberg J , Redding S W , Smith J , Farinacci G , Fothergill A W , Rinaldi M G
Published: January 2020

DNA subtyping by pulsed-field gel electrophoresis and in vitro susceptibility testing were used to study strain variation and fluconazole resistance in Candida albicans isolates from patients with AIDS undergoing azole (fluconazole and clotrimazole) therapy for oropharyngeal candidiasis. A total of 29 patients suffered 71 episodes of oropharyngeal candidiasis. Overall, 121 isolates of C. albicans recovered throughout the course of treatment of each infection were available for further characterization. DNA subtyping revealed a total of 61 different DNA subtypes. In vitro susceptibility testing of the 121 isolates by using proposed standard methods of the National Committee for Clinical Laboratory Standards revealed MICs of fluconazole ranging from < or = 0.125 to > 64 micrograms/ml. The MIC for 50% of isolates tested was 0.25 microgram/ml, and the MIC for 90% of isolates tested was 8.0 micrograms/ml. MICs were > or = 64 micrograms/ml for only 7.4% of the isolates tested. The majority (62%) of the patients with oropharyngeal candidiasis and undergoing azole therapy were infected or colonized with more than one DNA subtype, and the introduction or selection of strains with a more resistant DNA subtype during the course of fluconazole therapy was not uncommon. With one exception, this did not appear to have an adverse effect on clinical outcome. In contrast, for patients with AIDS and oropharyngeal candidiasis infected with a single DNA subtype of C. albicans, an increase in fluconazole MICs for the infecting strain was rarely demonstrated over the course of therapy.
Description of Leeds Acinetobacter Medium, a new selective and differential medium for isolation of clinically important Acinetobacter spp., and comparison with Herellea agar and Holton's agar
Authors: Jawad A , Hawkey P M , Heritage J , Snelling A M
Published: January 2020

Acinetobacter spp. are responsible for an increasing number of opportunistic, nosocomial infections. They have been isolated from diverse inanimate objects in the hospital environment and are resistant to most of the commonly used antibiotics. Existing media for the isolation of Acinetobacter spp. are either nonselective, allowing the growth of unwanted bacteria, or too inhibitory, inhibiting the growth of many Acinetobacter strains. For the rapid isolation and effective control of Acinetobacter infection, a new selective and differential medium, Leeds Acinetobacter Medium (LAM), has been developed to isolate Acinetobacter spp. from clinical and environmental sources. The concentration of antibiotics and other ingredients in this medium have been determined according to the results of MIC and viable counts performed for these ingredients. LAM was compared with other selective and differential media for the isolation of Acinetobacter spp. from a local hospital environment and proved to be better in terms of recovery and selectivity.
Detection of enterotoxigenic Escherichia coli in stool samples by using nonradioactively labeled oligonucleotide DNA probes and PCR
Authors: Schultsz C , Pool G J , van Ketel R , de Wever B , Speelman P , Dankert J
Published: January 2020

The detection of heat-labile enterotoxin LT-A and heat-stable enterotoxin ST Ia and ST Ib genes from enterotoxigenic Escherichia coli (ETEC) by using oligonucleotide DNA probes and the PCR was evaluated in reconstruction experiments and by testing stool specimens from 29 healthy subjects and from 50 patients with diarrhea who had returned from the (sub)tropics. ETEC strains were detected in concentrations ranging from 10(6) to 10(8) CFU/g of feces when oligonucleotide probes were applied to colony blots from five randomly picked E. coli-like colonies from CLED (cystine lactose electrolyte deficient) agar plates inoculated with the feces. When these probes were applied to blots from whole stool cultures collected from the agar plates (sweep blot), the detection limit was 10(6) CFU/g of feces. PCR of the sweep material could detect toxin genes when the concentration of ETEC strains was 10(2) CFU/g of feces. Results obtained with stool specimens from 29 healthy control subjects were negative. Testing stool specimens from 50 patients confirmed the observation that the number of samples containing ETEC enterotoxin genes was higher when PCR of sweeps was used than when oligonucleotide DNA probe hybridization of either sweep blots or colony blots was used. Furthermore, PCR of sweeps is an easy and rapid method which does not require DNA extraction and purification from fecal specimens.
Rapid diagnosis of schistosomiasis by antigen detection in urine with a reagent strip
Authors: van Etten L , Folman C C , Eggelte T A , Kremsner P G , Deelder A M
Published: January 2020

For the rapid and simple diagnosis of schistosomiasis, a reagent strip assay for detection of circulating cathodic antigen (CCA) in urine was developed. The test was based on a previously developed sandwich enzyme-linked immunosorbent assay (ELISA) using a combination of two anti-CCA monoclonal antibodies. For the reagent strip assay, as the capture matrix, monoclonal antibody was coated onto a nitrocellulose membrane and mounted on polyvinyl chloride strips. Urine samples were then tested in an assay consisting of a combined incubation step of the urine sample and biotinylated detecting antibody followed by incubation in streptavidin peroxidase and a subsequent staining. The specificity and the sensitivity of the assay, as determined with urine samples of 61 uninfected controls and 67 Schistosoma mansoni-infected individuals, were 96.7 and 95.5%, respectively. The results of the reagent strip assay compared very well with microscopical parasitological diagnosis by the standard Kato-Katz method for the same individuals. The reagent strip test has a lower detection limit of 1 ng of CCA per ml and can be completed in 75 min. By the inclusion of two reference bands on the strips, standardized reading could be achieved. This reagent strip assay is a promising tool for qualitative diagnosis of S. mansoni infections in control programs.
Genetic variation in Staphylococcus aureus coagulase genes: potential and limits for use as epidemiological marker
Authors: Schwarzkopf A , Karch H
Published: January 2020

To perform coagulase gene typing, the repeated units encoding hypervariable regions of the Staphylococcus aureus coagulase gene were amplified by the PCR technique; this was followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP) patterns. In order to assess the discriminatory power of this typing method, 30 epidemiologically unrelated S. aureus strains which differed by their pulsed-field gel electrophoresis patterns were examined. Although 18 of the 30 strains had unique and unshared AluI RFLP patterns, there were only four observed patterns in the remaining 12 strains. This finding indicated that unrelated strains may share identical AluI RFLP patterns. To elucidate the degree of genetic variation in the C-terminus-encoding loci within the coagulase genes, the PCR products of these 12 strains were subjected to Taq polymerase-mediated sequencing. Sequence analysis confirmed the AluI recognition sites in each of the four RFLP groups and demonstrated that AluI appears to yield the highest RFLP in restriction enzyme analysis. By their DNA sequences the majority of strains sharing common AluI groups could be clearly differentiated from each other and revealed between 93.2 and 98.5% homology. When we determined the nucleotide sequences of two strains after six subcultivations no significant alterations were observed. Because the discriminatory power of the current coagulase gene typing method is not great enough to be used as the sole method to type S. aureus, additional techniques are necessary. Sequence analysis of the repeated unit-encoding region for the typing of S. aureus may be potentially useful as an alternative to other current molecular typing techniques.
Role of confirmatory PCRs in determining performance of Chlamydia Amplicor PCR with endocervical specimens from women with a low prevalence of infection
Authors: Mahony J B , Luinstra K E , Sellors J W , Pickard L , Chong S , Jang D , Chernesky M A
Published: January 2020

The role of confirmatory PCR assays for determining the performance of Chlamydia Amplicor PCR for endocervical specimens from women with a low prevalence of infection was evaluated. An endocervical swab was collected from 770 women and tested by culture or direct fluorescent antibody (DFA) staining. A second swab was tested by Chlamydia Amplicor PCR (Roche Molecular Systems, Branchburg, N.J.). Discordant results were resolved by three confirmatory PCRs: one targeting the plasmid by using different primers and two directed to the major outer membrane protein (MOMP) gene. Of the 30 swabs that were positive by culture or DFA (3.9%), 27 were positive by Amplicor PCR. An additional five swabs were positive by Amplicor PCR but negative by culture or DFA. Both plasmid and MOMP confirmatory PCRs identified the five culture-DFA negatives and the three Amplicor negatives as true positives. The three specimens originally classified as negative by Amplicor PCR were positive on repeat Amplicor testing. After resolution of the discordant results by confirmatory PCR testing, the sensitivity of the initial Amplicor PCR was 91.4% (32 of 35 specimens), changing to 100% after storage and repeat testing. The specificity of Amplicor PCR was 100% (735 of 735 specimens). Our results demonstrated that plasmid and MOMP confirmatory PCRs worked equally well in resolving false-positive and false-negative Amplicor PCR results. Some specimens may contain inhibitors of Amplicor PCR which may disappear with time.
Comparison of heparin and EDTA transport tubes for detection of cytomegalovirus in leukocytes by shell vial assay, pp65 antigenemia assay, and PCR
Authors: Storch G A , Gaudreault-Keener M , Welby P C
Published: January 2020

The anticoagulants heparin and EDTA were compared for inhibitory effects on the detection of cytomegalovirus from washed leukocytes in specimen transport tubes. Evaluation was made by the centrifugation/shell vial culture technique, the pp65 antigenemia assay, and PCR. For each assay, the results with heparin and EDTA were equivalent.
Equine rotaviruses with G14 serotype specificity circulate among venezuelan horses
Authors: Ciarlet M , Reggeti F , Piña C I , Liprandi F
Published: January 2020

Two group A rotavirus strains isolated from diarrheic foals in Venezuela were classified as belonging to G14 serotype by cross-neutralization tests and on the basis of the homology of the sequenced VP7 gene. This report confirms that rotavirus strains of G14 serotype specificity circulate among equine populations.
Evolution and replacement of Candida albicans strains during recurrent vaginitis demonstrated by DNA fingerprinting
Authors: Schröppel K , Rotman M , Galask R , Mac K , Soll D R
Published: January 2020

Southern blot hybridization with the Ca3 probe and the C fragment of the Ca3 probe was used to assess the genetic relatedness of Candida albicans strains from one patient with recurrent C. albicans infection in whom the same strain was maintained, one patient in whom the infecting strain was replaced, and their male sexual partners. In the patient in whom the infecting strain was maintained, the infecting strain exhibited a minor genetic change in each successive episode of Candida vaginitis. These genetic changes occurred in the C-fragment bands of the Ca3 hybridization pattern. In the patient in whom the infecting strain was replaced by another infecting strain, a transition infection involved a genetically mixed infecting population, and the replacement strain appeared to have originated from the oral cavity of the male partner. The results demonstrate that the infecting strains of recurrent Candida vaginitis are not genetically stable, that drug treatment can result in the selection of variants of the previously infecting strain or replacement by a genetically unrelated strain, and that the male partner can be the source of a replacement strain.
Primary identification of Aureobacterium spp. isolated from clinical specimens as "Corynebacterium aquaticum"
Authors: Funke G , von Graevenitz A , Weiss N
Published: January 2020

Over a 6-year period 11 yellow-pigmented gram-positive rods (GPRs) with an oxidative carbohydrate metabolism were isolated from clinical specimens or were received as reference cultures and tentatively identified as "Corynebacterium aquaticum" according to the guide of Hollis and Weaver for the differentiation of GPRs (D. G. Hollis and R. E. Weaver, Gram-Positive Organisms: a Guide to Identification, 1981). Because these isolates seemed to be rather heterogeneous, comparative analyses with the type strain of "C. aquaticum" as well as six type strains of species belonging to the genus Aureobacterium were performed by biochemical and chemotaxonomic methods. Only four clinical strains were found to be "C. aquaticum," whereas seven strains were found to belong to the genus Aureobacterium. Discriminative phenotypic reactions between "C. aquaticum" and Aureobacterium spp. included hydrolysis of gelatin and casein (both reactions negative for "C. aquaticum" strains but positive for most Aureobacterium strains). Moreover, peptidoglycan analysis provided a reliable means of differentiating yellow-pigmented GPRs at the genus level (diaminobutyric acid as the interpeptide bridge in "C. aquaticum" and glycine-ornithine as the interpeptide bridge in Aureobacterium spp.). Antimicrobial susceptibility testing revealed that vancomycin showed an intermediate MIC for three of the four clinical "C. aquaticum" isolates, whereas all Aureobacterium strains were susceptible to vancomycin. To our knowledge, this is the first report outlining the isolation of Aureobacterium spp. from clinical specimens. However, Aureobacterium isolates could not be identified to the species level by the tests used in the study.
Susceptibility of white-tailed deer (Odocoileus virginianus) to infection with Ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis
Authors: Dawson J E , Stallknecht D E , Howerth E W , Warner C , Biggie K , Davidson W R , Lockhart J M , Nettles V F , Olson J G , Childs J E
Published: January 2020

Although more than 320 cases of human ehrlichiosis have been diagnosed in 27 states since 1986, the reservoir host or hosts remain unknown. Since antibodies reactive to Ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis, have been found in white-tailed deer (Odocoileus virginianus), we experimentally evaluated the susceptibilities of four white-tailed deer to infection with E. chaffeensis and Ehrlichia canis, a closely related species. A fifth deer served as a negative control. Isolation and nested PCR amplification results from peripheral blood indicated that E. chaffeensis circulated for at least 2 weeks. The deer developed antibodies to E. chaffeensis by day 10 after inoculation, but there was no indication of clinical disease. Immunohistochemical staining identified E. chaffeensis within macrophage-type cells in lymph nodes. The deer inoculated with E. canis did not become infected and did not seroconvert. These results indicate that white-tailed deer can support an E. chaffeensis infection with resulting rickettsemia of at least 2 weeks. The resistance to infection and the absence of seroconversion upon exposure to E. canis indicate that antibody responses previously detected among wild deer are not E. canis cross-reactions. The role of deer as competent reservoirs in the life cycle of E. chaffeensis remains to be explored with suspected tick vectors.
Characterization of phenotypic, serological, and toxigenic traits of Vibrio cholerae O139 bengal
Authors: Nair G B , Shimada T , Kurazono H , Okuda J , Pal A , Karasawa T , Mihara T , Uesaka Y , Shirai H , Garg S
Published: January 2020

Biochemical and physiological traits of a collection of strains of Vibrio cholerae O139 Bengal isolated from India, Bangladesh, and Thailand showed that these strains formed a phenotypically homogeneous group with identical characteristics that were essentially similar to those of the O1 serogroup. Resistance to 150 micrograms of the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine) and Mukherjee's El Tor phage 5 and classical phage IV and the nonagglutinability of the strains with O1 antiserum were the only discernible differences between the O139 and O1 serogroups. Extensive serological characterization further revealed the O139 serogroup to be distinct from the existing 138 serogroups of V. cholerae. Antiserum raised against the O139 serogroup required absorption with the R reference strain CA385 and with the reference strain representing serogroup O22 to remove cross-reacting agglutinins. All of the 223 representative strains of V. cholerae O139 examined hybridized with DNA probes specific for the cholera toxin (CT) gene, zonula occludens toxin gene, and El Tor hemolysin gene but not with the probe specific for the heat-stable enterotoxin gene. The amount of CT present in stool samples of patients infected with the O139 serogroup was higher than that found in stools of patients infected with O1 El Tor, and this echoed findings that the amount of CT produced by O139 strains in vitro was higher than that produced by the O1 El Tor strains. The nucleotide sequences of the genes encoding the A and B subunits of CT of the O139 serogroup were identical to the sequences reported for the CT gene of O1 El Tor. The CT gene of O139 strains could be amplified by using primers developed for detection of the CT gene of the O1 serogroup by a PCR assay, which could also be used to detect the CT gene in stool samples of patients infected with strains of the O139 serogroup.
Intrafamilial transmission of hepatitis C virus
Authors: Ho M S , Yang C S , Chen P J , Mau Y C
Published: January 2020

Family members of hepatitis C virus (HCV)-infected hemodialysis patients had a high rate (8.1%) of HCV infection and were often infected by the same genotype of HCV as the renal patients. Renal patients who had a longer history of hemodialysis or more frequent arteriovenous shunt replacement posed a higher risk to their household contacts, especially to their primary-care providers at home.
Detection of Escherichia coli K95 strains by bacteriophages
Authors: Nimmich W
Published: January 2020

A set of three bacteriophages (phi K5, phi K20, and phi K95) was used to discriminate between Escherichia coli K5 and K95 strains isolated from patients with urinary tract infections. All the strains tested were detected primarily by phi K5 and thought to carry the capsular antigen K5. Ten O2, 33 O6, and 32 O18 strains proved to be sensitive to the three phages. Eleven of 44 O75:H- strains, all 8 O75:H5 strains, 1 O17 strain, and 1 OR strain were not lysed by phage phi K20, the only phage without activity toward the K95 test strain. It has been suggested that the phi K20-resistant strains carry K95. These findings were confirmed by countercurrent immunoelectrophoresis with K-antigen extracts and anti-K95 serum. These preliminary data indicate the usefulness of these phages to detect K95 strains among K5 isolates.
Unexpected isolation of Bordetella pertussis from a blood culture
Authors: Janda W M , Santos E , Stevens J , Celig D , Terrile L , Schreckenberger P C
Published: January 2020

Bordetella pertussis was isolated from a culture of blood from a 31-year-old man with Wegener's granulomatosis. The organism was detected with the BACTEC 9240 system after 6 days of incubation and was confirmed as B. pertussis by the Centers for Disease Control and Prevention. To our knowledge, this is the first published report of the recovery of B. pertussis from blood.
Isolation of Yokenella regensburgei ("Koserella trabulsii") from a patient with transient bacteremia and from a patient with a septic knee
Authors: Abbott S L , Janda J M
Published: January 2020

Yokenella regensburgei ("Koserella trabulsii") was isolated from a 74-year-old male with a septic knee and from a 35-year-old immunocompromised female whose transient bacteremia occurred without overt signs of sepsis. Neither strain was correctly identified by laboratories using a variety of techniques.
Increased phenotypic switching in strains of Candida albicans associated with invasive infections
Authors: Jones S , White G , Hunter P R
Published: January 2020

This study reports the rates of phenotypic switching in strains of Candida albicans isolated from superficial and invasive infections. Of 19 invasive strains, 68% showed switching activity, often at very high rates, compared with only 28% of 40 strains isolated from superficial sites (P = 0.004).
Comparison of goat and horse blood as culture medium supplements for isolation and identification of Haemophilus influenzae and Streptococcus pneumoniae from upper respiratory tract secretions
Authors: Gratten M , Battistutta D , Torzillo P , Dixon J , Manning K
Published: January 2020

The results of this study show that goat blood as a culture medium supplement is as supportive as horse blood for the isolation and identification of Haemophilus influenzae and Streptococcus pneumoniae from clinical material. Care is required in the preparation of goat blood chocolate agar to ensure that a thermolabile growth inhibitor of NAD-dependent Haemophilus species is eliminated.
Detection and sequencing of rotavirus VP7 gene from human materials (stools, sera, cerebrospinal fluids, and throat swabs) by reverse transcription and PCR
Authors: Ushijima H , Xin K Q , Nishimura S , Morikawa S , Abe T
Published: January 2020

Human rotavirus RNAs from stool samples, sera, cerebrospinal fluids, and throat swabs of 15 children with rotavirus gastroenteritis were detected and serotyped by reverse transcription and PCR. The reverse transcription-PCR method may allow us to consider rotavirus infections in other parts of the body in addition to the gastrointestinal tract. Moreover, sequence analysis of the VP7 gene was performed on seven samples (one stool, two serum, three cerebrospinal fluid, and 1 throat swab sample). There were no appreciable differences in viral sequences between samples from cerebrospinal fluids, sera, or stools.
Preliminary results of Pneumocystis carinii strain differentiation by using molecular biology
Authors: Latouche S , Roux P , Poirot J L , Lavrard I , Hermelin B , Bertrand V
Published: January 2020

The mode of Pneumocystis carinii transmission is controversial. Recent studies point to exogenous inoculation rather than reactivation, and person-to-person transmission has also been suggested. Comparison of nucleotide sequences of the large-subunit mitochondrial rRNA gene of P. carinii from human immunodeficiency virus-seropositive patients showed strain differences.
Antibiotic resistance of clinical isolates of Streptococcus pneumoniae in Greece
Authors: Kanavaki S , Karabela S , Marinis E , Legakis N J
Published: January 2020

The antibiotic susceptibilities of 1,002 Streptococcus pneumoniae clinical isolates from patients with community-acquired pneumonia were determined over an 18-month period. Resistance rates were 14% for penicillin, 20% for erythromycin, 26% for tetracycline, and 1% for chloramphenicol. Resistance to non-beta-lactam antibiotics was associated with penicillin resistance at statistically levels.
Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories
Authors: Noordhoek G T , Kolk A H , Bjune G , Catty D , Dale J W , Fine P E , Godfrey-Faussett P , Cho S N , Shinnick T , Svenson S B
Published: January 2020

PCR is, in principle, a simple and rapid test for use in the detection of Mycobacterium tuberculosis. However, virtually no data are available on the reliability and reproducibility of the method. In order to assess the validity of PCR for the detection of mycobacteria in clinical samples, seven laboratories participated in a blinded study of 200 sputum, saliva, and water samples containing either known numbers of Mycobacterium bovis BCG cells or no added organisms. Each laboratory used its own protocol for pretreatment, DNA extraction, and detection of the amplification product. Insertion sequence IS6110 was the target for DNA amplification. Several participating laboratories reported high levels of false-positive PCR results, with rates ranging from 3 to 20% and with one extreme value of 77%. The levels of sensitivity also ranged widely among the different participants. A positive PCR result was reported for 2 to 90% of the samples with 10(3) mycobacteria. Although most participants did include control tests to check the sensitivity and specificity of the PCR, the sequence of operations from sample pretreatment to purification of DNA from bacteria was not always monitored adequately. During these procedures cross-contaminating DNA was introduced and/or bacterial DNA was lost. The results of the study show that the implementation of an effective system for monitoring sensitivity and specificity is required before the PCR can be used reliably in the diagnosis of tuberculosis.
PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid
Authors: Greisen K , Loeffelholz M , Purohit A , Leong D
Published: January 2020

A set of broad-range PCR primers for the 16S rRNA gene in bacteria were tested, along with three series of oligonucleotide probes to detect the PCR product. The first series of probes is broad in range and consists of a universal bacterial probe, a gram-positive probe, a Bacteroides-Flavobacterium probe, and two probes for other gram-negative species. The second series was designed to detect PCR products from seven major bacterial species or groups frequently causing meningitis: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, Escherichia coli and other enteric bacteria, Listeria monocytogenes, and Staphylococcus aureus. The third series was designed for the detection of DNA from species or genera commonly considered potential contaminants of clinical samples, including cerebrospinal fluid (CSF): Bacillus, Corynebacterium, Propionibacterium, and coagulase-negative Staphylococcus spp. The primers amplified DNA from all 124 different species of bacteria tested. Southern hybridization testing of the broad-range probes with washes containing 3 M tetramethylammonium chloride indicated that this set of probes correctly identified all but two of the 102 bacterial species tested, the exceptions being Deinococcus radiopugnans and Gardnerella vaginalis. The gram-negative and gram-positive probes hybridized to isolates of two newly characterized bacteria, Alloiococcus otitis and Rochalimaea henselii, as predicted by Gram stain characteristics. The CSF pathogen and contaminant probe sequences were compared with available sequence information and with sequencing data for 32 different species. Testing of the CSF pathogen and contaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus probes, these probes provided the correct identification of bacterial species known to be found in CSF.
MT-2 cell tropism as prognostic marker for disease progression in human immunodeficiency virus type 1 infection
Authors: Karlsson A , Parsmyr K , Sandström E , Fenyö E M , Albert J
Published: January 2020

The ability of human immunodeficiency virus type 1 (HIV-1) isolates to replicate in MT-2 cells was investigated as a prognostic marker for disease progression and CD4+ lymphocyte depletion in 53 HIV-1-infected, asymptomatic individuals. MT-2-negative viruses were isolated from 49% of the patients both early and late during the follow-up period; 38% converted from being MT-2 negative to MT-2 positive, while 11% were MT-2 positive throughout the study. One individual showed a fluctuating virus phenotype. The loss of CD4+ lymphocytes was significantly more rapid in MT-2-positive patients. We found a broad spectrum of CD4+ lymphocyte changes in patients whose virus changed its MT-2 tropism. Our data suggest that the changes could be divided into three general patterns. A stable or slowly decreasing CD4+ lymphocyte count changed into a more rapid fall in 44% of the patients, no significant change in rate of decline could be noted in 44% of the patients, while a stable CD4+ lymphocyte level after a change in MT-2 tropism was noted in 12% of the patients. A correlation between MT-2 tropism and clinical symptoms was also noted. Half of the patients with MT-2-negative virus throughout the study were still asymptomatic after a mean follow-up time of 80 months, while only 15% of those who converted remained asymptomatic. All patients with MT-2-positive viruses at the time of inclusion in the study developed HIV-1-related symptoms, and half of them died during the study. The MT-2 status of 16 patients, could be determined at the time of AIDS diagnosis; 50% were Mt-2 positive, while 50% were MT-2 negative. No difference in AIDS-defining diagnoses or CD4+ lymphocyte counts at the time of diagnosis was noted. Knowledge of the HIV-1 phenotype may improve the early recognition of progressive disease.
Characterization, distribution, and microbiological associations of Fusobacterium spp. in clinical specimens of animal origin
Authors: Jang S S , Hirsh D C
Published: January 2020

During the years from 1984 through 1991, 1,067 specimens from canine, equine, exotic, feline, porcine, and ruminant animal sources were found to contain members of the genus Fusobacterium: The most common sites or conditions from which members of this genus were isolated were abscesses, the respiratory tract, and pleural and peritoneal cavities. Most specimens contained a single Fusobacterium species. The most commonly isolated species was Fusobacterium necrophorum. Almost all of the specimens contained other obligate anaerobes together with facultative and obligate aerobes. The identities of the other isolates depended upon the species of animal from which the sample was obtained.
Use of restriction fragment polymorphism analysis of rRNA genes to assign species to unknown clinical isolates of oral viridans streptococci
Authors: Rudney J D , Larson C J
Published: January 2020

This study evaluated restriction fragment length polymorphisms of rRNA genes (ribotyping) for genotypic identification of 53 oral isolates classified as "Streptococcus sanguis" by colony morphology. Isolates were from 8-h buccal plaque on lower first permanent molars of 20 subjects. DNA was digested with AatII and hybridized with digoxygenin-labeled cDNA of Escherichia coli 16S and 23S rRNA. Strains were ribotyped again with AlwNI or PvuII on the basis of the presence or absence of a 2,290-bp AatII band. Band patterns were compared with reference ribotypes for Streptococcus gordonii, Streptococcus sanguis, Streptococcus crista, Streptococcus oralis, Streptococcus mitis, and Streptococcus parasanguis strains. Forty-eight isolates could be assigned to a species (22 S. sanguis, 14 S. oralis, 12 S. gordonii). Multiple species were seen in 14 subjects; multiple strains of the same species occurred in 11 subjects. Our findings suggest that ribotyping can be used for genotypic identification of S. sanguis, S. oralis, and S. gordonii isolates.
Case report of spinal epidural abscess caused by Haemophilus paraphrophilus
Authors: Scerpella E G , Wu S , Oefinger P E
Published: January 2020

Haemophilus paraphrophilus was recovered in pure culture from purulent material collected at surgery from a patient presenting with a spinal epidural abscess and a severe neurological deficit. This is the first report of such an occurrence.
Characterization of Chlamydia pneumoniae species-specific proteins immunodominant in humans
Authors: Iijima Y , Miyashita N , Kishimoto T , Kanamoto Y , Soejima R , Matsumoto A
Published: January 2020

Proteins of Chlamydia pneumoniae immunodominant in humans were characterized with the sera of 13 patients who were not likely to have been exposed to C. trachomatis or C. psittaci. The serological responses among these patients were similar on a qualitative basis, but some differences were found quantitatively. However, the serological responses of the patients who were infected with C. pneumoniae differed markedly from those of two patients who were infected with C. trachomatis and two who were infected with C. psittaci and those of mice that were transtracheally infected with C. pneumoniae. Among proteins immunodominant in the patients who were infected with C. pneumoniae, a 40-kDa major outer membrane protein was genus specific and 53-, 46-, and 43-kDa proteins were species specific in their reactions with the majority of the human sera used. A few sera reacted strongly with a 73-kDa protein genus specifically. Some proteins with weak immunogenicity exhibited species specificity. An antigenic analysis with human sera and murine monoclonal antibodies against the 53-kDa protein showed that hte antigenicities were strictly conserved among the seven strains of C. pneumoniae tested. The genus-specific 73-kDa protein was solubilized with octylglucoside. All of the species-specific immunodominant proteins were solubilized with sodium dodecyl sulfate, but the genus-specific major outer membrane protein was not. These results suggest that a serological diagnosis of C. pneumoniae infection could be achieved species specifically by comparison of the serum responses to sodium dodecyl sulfate- and octylglucoside-soluble fractions.
Arbitrarily primed PCR, ribotyping, and plasmid pattern analysis applied to investigation of a nosocomial outbreak due to Enterobacter cloacae in a neonatal intensive care unit
Authors: Grattard F , Pozzetto B , Berthelot P , Rayet I , Ros A , Lauras B , Gaudin O G
Published: January 2020

In December 1992, Enterobacter cloacae was isolated from the oropharynx and respiratory tract of six ventilated neonates hospitalized in the intensive care unit (ICU) of our hospital. To establish the spread of the outbreak, 41 strains of E. cloacae were analyzed for genotypic markers by three methods: plasmid profile analysis, ribotyping with EcoRI or PvuII endonuclease, and arbitrarily primed (AP) PCR. The tested strains included 12 isolates from the 6 epidemic cases, 4 isolates from the respiratory tract of 4 children hospitalized in other wards during the same period, 13 isolates from 12 children hospitalized in pediatric units before or after the outbreak, and 12 epidemiologically unrelated isolates. Ribotyping and AP PCR demonstrated that each of the last 12 strains exhibited distinct genomic patterns, as did each of the strains isolated from neonates hospitalized before or after the epidemic peak. Conversely, two clones of strains were found among the isolates recovered in December, with concordant results being obtained by the three typing methods: the first clone included seven strains from five ventilated children in the ICU and two children from another ward; another clone was shared by one neonate in the ICU and an infant from another ward. These results indicate that ribotyping and AP PCR-the latter applied, to our knowledge, for the first time to the genotypic analysis of E. cloacae--represent very discriminatory tools for the investigation of nosocomial outbreaks caused by this species.
Comparison of phenotypic methods and DNA hybridization for detection of methicillin-resistant Staphylococcus aureus
Authors: Richard P , Meyran M , Carpentier E , Thabaut A , Drugeon H B
Published: January 2020

One hundred thirty-eight Staphylococcus aureus isolates from patients with severe staphylococcal infections were collected in 15 French hospitals. Detection of the mec gene was performed by dot blot hybridization with a specific DNA probe. Dot blot results were used to characterize the isolates as methicillin susceptible (77 isolates) or resistant (61 isolates). The isolates were screened for methicillin resistance by an agar spread method on Mueller-Hinton plates containing oxacillin (2 and 10 micrograms/ml) and were incubated at 37 degrees C, with 10(8) CFU as the inoculum. MICs of oxacillin and methicillin were determined by the agar dilution method on Mueller-Hinton plates without NaCl, by using 10(5) CFU per spot, after 24 and 48h of incubation at 30 or 37 degrees C. Moderately elevated MICs were found for 20 isolates (14.5%). The mec gene was detected in six (30%) of the isolates expressing a low level of resistance to methicillin and/or oxacillin. As determined by comparison with probe hybridization results, the spread plate method with oxacillin at 2 micrograms/ml was more sensitive (sensitivity, 100%) and specific (specificity, 100%) than agar dilution with either methicillin or oxacillin in identifying methicillin resistance or susceptibility. Determinations of methicillin and oxacillin MICs by the agar dilution method had a specificity of 99 to 100% depending on the conditions of incubation, but the sensitivity was below 85% whatever the duration or temperature of incubation.
Pseudomonas aeruginosa serotype O12 outbreak studied by arbitrary primer PCR
Authors: Elaichouni A , Verschraegen G , Claeys G , Devleeschouwer M , Godard C , Vaneechoutte M
Published: January 2020

A total of 16 colonizing and infecting ofloxacin-resistant Pseudomonas aeruginosa strains and two strains isolated from ventilation equipment fluids, all with similar colonial morphologies and with minor but distinct susceptibility differences, were suspected of belonging to a single outbreak and were studied by arbitrary primer (AP) PCR. Thirteen nonrelated strains were included to evaluate the discriminatory capacity of the technique. AP PCR fingerprinting was compared with serotyping, phage typing, and antibiotic susceptibility testing. AP PCR was performed independently with three different primers. The different AP PCR typing systems yielded almost identical patterns for the epidemic strains and enabled us to differentiate most of the nonrelated strains from each other and from the outbreak strains. The combination of AP PCR typing and the phenotyping techniques that we used enabled us to conclude that an outbreak was occurring. In general, the typeability of AP PCR was greater than those of phage typing and serotyping, while the discriminatory powers of the three methods were comparable.
A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples
Authors: Kox L F , Rhienthong D , Miranda A M , Udomsantisuk N , Ellis K , van Leeuwen J , van Heusden S , Kuijper S , Kolk A H
Published: January 2020

Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-N-glycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 10(10) times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n=145), tissue biopsy samples (n=25), cerebrospinal fluid (n=15), blood (n=14), pleural fluid (n=9), feces, (n=7), fluid from fistulae (n=2), and pus from a wound (n=1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.