bluuebunny/crossref_metadata_2025 huggingface dataset:
Helicobacter canis isolated from a dog liver with multifocal necrotizing hepatitis
Authors: Fox J G , Drolet R , Higgins R , Messier S , Yan L , Coleman B E , Paster B J , Dewhirst F E
Published: January 2020

On the basis of biochemical, phenotypic, and 16S rRNA analysis, a novel gram-negative bacterium, isolated from normal and diarrheic dogs as well as humans with gastroenteritis, has been recently named Helicobacter canis. A 2-month-old female crossbred puppy was submitted to necropsy with a history of weakness and vomiting for several hours prior to death. The liver had multiple and slightly irregular yellowish foci up to 1.5 cm in diameter. Histologically, the liver parenchyma contained randomly distributed, occasionally coalescing hepatocellular necrosis, often accompanied by large numbers of mononuclear cells and neutrophils. Sections of liver stained by the Warthin-Starry silver impregnation technique revealed spiral- to curve-shaped bacteria predominantly located in bile canaliculi and occasionally in bile ducts. Aerobic culture of liver was negative, whereas small colonies were noted on Campylobacter selective media after 5 days of microaerobic incubation. The bacteria were gram negative and oxidase positive but catalase, urease, and indoxyl acetate negative; nitrate was not reduced to nitrite, and the organism did not hydrolyze hippurate. The bacteria were also resistant to 1.5% bile. Electron microscopy revealed spiral-shaped bacteria with bipolar sheathed flagella. By 16S rRNA analysis, the organism was determined to be H. canis. This is the first observation of H. canis in active hepatitis in a dog and correlates with recent findings of Helicobacter hepaticus- and Helicobacter bilis-related hepatic disease in mice. Further studies are clearly warranted to ascertain whether H. canis-associated hepatitis is more widespread in canines as well as a cause of previously classified idiopathic liver disease in humans.
Vaginal microbiology of women with acute recurrent vulvovaginal candidiasis
Authors: Sobel J D , Chaim W
Published: January 2020

Alterations in the autochthonous vaginal microflora can predispose women to recurring attacks of Candida vaginitis. Quantitative aerobic and anaerobic cultures were obtained from 24 premenopausal women with acute recurrent vulvovaginal candidiasis and from 21 healthy asymptomatic premenopausal women. Lactobacillus species constituted the predominant flora in both groups, with a mean log10 CFU/ml of 8.7, a total isolation rate of 96%, and a mean of 1.6 Lactobacillus species isolated per patient compared with a mean log10 CFU/ml of 8.9, a total isolation rate of 90%, and a mean of 1.2 Lactobacillus species isolated per patient in the vaginitis and control groups, respectively. The results of this small study failed to provide evidence of an altered or abnormal vaginal bacterial flora in women with non-antibiotic-induced recurrent vulvovaginal candidiasis suffering from acute Candida vaginitis.
M proteins of group C streptococci isolated from patients with acute pharyngitis
Authors: Bisno A L , Collins C M , Turner J C
Published: January 2020

We studied 15 strains of group C (Streptococcus equi subsp. equisimilis) [corrected] isolated from the throats of college students with acute pharyngitis and 5 strains isolated from patients with noninfectious problems. Nineteen of the 20 strains resisted phagocytic killing during incubation in normal human blood, suggesting that they might express M proteins. Genomic DNA from all 20 strains hybridized with a probe corresponding to the carboxyterminal one-third of the group A M-protein gene emm24, a region that is highly conserved among M proteins of group A and group G streptococci. The DNA sequences of the N-terminal (variable) regions of the M-protein-encoding genes from two disease-associated group C isolates and one control isolate were determined. The predicted amino acid sequences of the two pharyngitis strains were identical and were 88% homologous to the amino acid sequence of a group G M-protein gene. The predicted terminal amino acid sequence of the control strain does not correspond to any such sequences in the GenBank database. All three strains studied possess the conserved region domain common to class I group A M-protein types epidemiologically associated with rheumatic fever. These studies demonstrate the presence of M proteins in strains of S. equi subsp. equisimilis [corrected] isolated in cases of endemically occurring acute pharyngitis. Certain of these proteins are similar to those of group G streptococci, while others may represent new M types. The similarity in structure and function between M proteins of nonrheumatogenic serogroups and those of rheumatogenic group A streptococci suggests that factors other than or in addition to M protein per se are likely involved in the pathogenesis of rheumatic fever.
Temporal shifts in traits of Vibrio cholerae strains isolated from hospitalized patients in Calcutta: a 3-year (1993 to 1995) analysis
Authors: Mukhopadhyay A K , Garg S , Mitra R , Basu A , Rajendran K , Dutta D , Bhattacharya S K , Shimada T , Takeda T , Takeda Y , Nair G B
Published: January 2020

This study presents results of a surveillance on cholera conducted with hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, from January 1993 to December 1995. The O139 serogroup of Vibrio cholerae dominated in 1993 but was replaced by O1 as the dominant serogroup in 1994 and 1995. The isolation rate of V. cholerae non-O1 non-O139 did not exceed 4.9% throughout the study period, while the isolation rate of the O139 serogroup in 1994 and 1995 was below 9%. No temporal clustering of any non-O1 non-O139 serogroup was observed. With the exception of 1 strain, none of the 64 strains belonging to the non-O1 non-O139 serogroup hybridized with ctx, zot, and ace gene probes, while 97.3 and 97.7% of the O139 and O1 strains, respectively, hybridized with all the three probes. Multiplex PCR studies revealed that all the O1 strains belonged to the EIT or biotype. There was a progressive increase in the cytotoxic response on CHO and HeLa cells evoked by culture supernatants of strains of V. cholerae non-O1 non-O139 isolated during 1994 and 1995 compared with the response evoked by those isolated in 1993. Dramatic shifts in patterns of resistance to antibiotics between strains of V. cholerae belonging to different serogroups and within strains of a serogroup isolated during different time periods were observed. There was a discernible increase in the incidence of multidrug-resistant strains of V. cholerae O1 isolated in 1994 and 1995 compared with that in 1993. On the basis of the results of this study, we predict the possibility of newer variants of V. cholerae emerging in the future.
Diagnosis of cytomegalovirus infection in pediatric Menetrier's disease by in situ hybridization
Authors: Hochman J A , Witte D P , Cohen M B
Published: January 2020

A previously healthy 7-year-old boy presented with a protein-losing enteropathy secondary to a hypertrophic gastropathy. The diagnosis of cytomegalovirus (CMV) infection was established by detection of CMV inclusion bodies in gastric biopsy samples and by hybridization with a CMV probe. This report further strengthens the association between CMV and pediatric Menetrier's disease.
Diff-Quik stain as a simplified alternative to Papanicolaou stain for determination of quality of endocervical specimens submitted for PCR detection of Chlamydia trachomatis
Authors: Kellogg J A , Seiple J W , Klinedinst J L , Stroll E
Published: January 2020

The simple, rapid, two-step Diff-Quik stain procedure (Baxter Diagnostics) was compared with the Papanicolaou stain for microscopic determination of endocervical specimen quality. Results from 230 (98.7%) of 233 specimens stained by both methods indicated agreement between the two staining methods for detection of the endocervical cells or erythrocytes indicating specimen adequacy. By using the Amplicor Chlamydia trachomatis Test (Roche Diagnostic Systems) to detect C.trachomatis and the Diff-Quik stain to assess specimen adequacy, PCR-positive results were obtained from 147 (9.1%) of 1,615 microscopically adequate specimens but from only 13 (2.2%) of the 583 inadequate specimens (P < 0.001).
Acidification of methyl-alpha-D-glucopyranoside: a useful test to differentiate Enterococcus casseliflavus and Enterococcus gallinarum from Enterococcus faecium species group and from Enterococcus faecalis
Authors: Devriese L A , Pot B , Kersters K , Lauwers S , Haesebrouck F
Published: January 2020

Enterococcus gallinarum and E. casseliflavus are difficult to differentiate from other enterococci, particularly E.faecium. The former two species were found to produce acid from methyl-alpha-D-glucopyranoside in phenol red broth, while E.faecalis strains and strains of the E.faecium species group, including E.faecium, E.durans, E. hirae, and E.mundtii, failed to produce acid from this substrate.
Genotyping of hepatitis C virus isolates from Lebanese hemodialysis patients by reverse transcription-PCR and restriction fragment length polymorphism analysis of 5' noncoding region
Authors: Matar G M , Sharara H M , Abdelnour G E , Abdelnoor A M
Published: January 2020

We have genotyped the 5' noncoding region of hepatitis C virus in Lebanese hemodialysis patients by reverse transcription-PCR and restriction fragment length polymorphism analysis. Of 50 patients, 15 had the expected 268-bp amplicon by reverse transcription-PCR. Specificity of the amplicons was confirmed by Southern hybridization. Restriction analysis of the amplicons showed the pattern for genotype 4 (common in the Middle East).
Modified thioglycolate medium: a simple and reliable means for detection of Trichomonas vaginalis
Authors: Poch F , Levin D , Levin S , Dan M
Published: January 2020

Despite the declining rate of sexually transmitted diseases in developed countries, trichomoniasis is still one of the most common venereal infections. While diagnosis of this condition is commonly based on the microscopic wet-mount method, culture remains the most accurate single procedure for detecting the presence of Trichomonas vaginalis in clinical samples. In the present study, the efficacy of a modified formula of the commonly available thioglycolate medium was compared with that of the standard Diamond's medium for detection of T. vaginalis in samples from 176 women with vaginal symptoms. Thioglycolate medium supplemented with yeast extract, horse serum, and antimicrobial agents was as reliable as Diamond's medium for detection of T. vaginalis in vaginal fluid samples. Modified thioglycolate medium may be used as a readily available, low-cost substitute for the standard medium for culturing T. vaginalis.
Phenotypic and genotypic characterization of spotted fever group Rickettsiae isolated from Catalan Rhipicephalus sanguineus ticks
Authors: Beati L , Roux V , Ortuño A , Castella J , Porta F S , Raoult D
Published: January 2020

Eighty-nine Rhipicephalus sanguineus ticks and 21 Rhipicephalus bursa ticks collected in Catalonia were tested by the hemolymph test to establish their infection rate with spotted fever group rickettsiae. By Giménez staining, 11.2% of the R. sanguineus isolates and 0% of the R. bursa isolates were found to contain rickettsia-like organisms. Six spotted fever group rickettsial strains (Bar29, Bar31, Gir4, Tar1, Tar2, and Tar3) were isolated from these ticks and were characterized by phenotypic and genotypic analyses. PCR followed by restriction fragment length polymorphism analysis showed that the six strains were identical and were characterized by the same restriction profiles as a strain, Mtu5, previously isolated from Rhipicephalus turanicus ticks in the South of France. Microimmunofluorescence serotyping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified organisms, and Western blot (immunoblot) assay with mouse polyclonal sera confirmed this observation. Pulsed field gel electrophoresis of the whole genome of three of the strains showed that, although closely related, the profile of Tar1 was slightly different from that of the Bar strains. Phylogenetic analysis showed that this new rickettsial sero- and genotype, which will be named the "Catalan strain," is closely related to Rickettsia massiliae. This strain shows an unexpected resistance to rifampin. The epidemiological implications of these findings are considered.
Evaluation of a quality assurance program for quantitation of human immunodeficiency virus type 1 RNA in plasma by the AIDS Clinical Trials Group virology laboratories
Authors: Yen-Lieberman B , Brambilla D , Jackson B , Bremer J , Coombs R , Cronin M , Herman S , Katzenstein D , Leung S , Lin H J , Palumbo P , Rasheed S , Todd J , Vahey M , Reichelderfer P
Published: January 2020

A number of quantitative assays have been developed by using amplification techniques to measure human immunodeficiency virus type 1 RNA in the plasma of infected individuals. The Virology Committee of the AIDS Clinical Trials Group in the Division of AIDS, National Institute of Allergy and Infectious Diseases, has established a quality assurance program (QAP) for quantitative assays of HIV-1 RNA levels in plasma. The primary objective of the QAP was to ascertain that a laboratory could maintain the precision required to have a 90% power to detect a fivefold difference in RNA copy number between two samples in the same batch. To achieve this goal, the QAP required an intra-assay standard deviation of no greater than 0.15 log10 RNA copies per ml. Panels for proficiency testing consisted of coded replicate samples and a common set of standards. To date, 41 laboratories have participated in the program and have used both commercial and in-house assays. We demonstrated that 65% of the laboratories were capable of attaining the necessary level of intra-assay precision. The fitted regressions indicated that the differences among laboratories that used the same kit were generally greater than the differences among population-average regressions for the kits themselves. The use of an external QAP and a common set of standards reduced differences both among laboratories that used the same kit and among laboratories that used different kits. Thus, use of a common set of standards across clinical trial protocols would allow for cross-protocol comparisons.
Comparison of a competitive combined reverse transcription-PCR assay with a branched-DNA assay for hepatitis C virus RNA quantitation
Authors: Mayerat C , Bürgisser P , Lavanchy D , Mantegani A , Frei P C
Published: January 2020

We have developed a sensitive and reproducible one-step competitive reverse transcriptase (RT) PCR assay, which allows hepatitis C virus (HCV) RNA quantitation in plasma over a broad range of values. The RNA samples and a constant amount of an internal standard were reverse transcribed and coamplified with the same primers in the same tube. A standard curve was obtained from an additional series of tubes containing both the internal standard and known amounts of a wild-type HCV RNA transcript, thus eliminating the need for titrating samples with the competitor. Eighty-eight anti-HCV-positive samples were tested by RT-PCR and a branched-DNA (bDNA) assay which has a detection limit of 3.5 x 10(5) copies per ml. Fifty-five samples were quantifiable by both methods (correlation coefficient, 0.72), the ranges of values found by the RT-PCR and bDNA assays being, respectively, 0.127 x 10(6) to 18.4 x 10(6) and 0.44 x10(6) to 38 x 10(6) copies per ml. Six samples that had indeterminate values by the bDNA assay had RT-PCR values between 0.37 x 10(5) and 9.6 x 10(5) copies per ml. Twenty-two samples that had values below the cutoff value by the bDNA assay had RT-PCR values between 2.5 x 10(3) and 10.4 x 10(5) (18 less than and 4 more than the limit of 3.5 x 10(5) copies per ml). The remaining five samples were negative by both assays. The level of RT-PCR interassay reproducibility was high (correlation coefficient between duplicate values, 0.94). Our method, with a detection limit of 2,500 copies per ml, was markedly more sensitive than the bDNA assay. This method is convenient for following up patients with low viremia, a common situation with alpha interferon treatment.
Evaluation of 16S rRNA gene PCR with primers Hp1 and Hp2 for detection of Helicobacter pylori
Authors: Chong S K , Lou Q , Fitzgerald J F , Lee C H
Published: January 2020

The PCR primer set Hp1-Hp2, which amplifies a 109-bp fragment of the 16S rRNA gene of Helicobacter pylori, has been widely used for the detection of H. pylori in clinical specimens. We have examined 34 stool samples and 50 human tissue samples from H. pylori-infected and uninfected patients, five human leukocyte samples, and one human cell line by this PCR method. All of these specimens produced a 109-bp PCR product. When Escherichia coli DNA was used as the template, several nonspecific bands, but not the 109-bp band, were observed. No PCR products were generated when DNA samples from five different fungi were used as templates. These results indicate that this 109-bp PCR product was amplified from the human genome. The 109-bp PCR product generated from various clinical specimens also hybridized with the probe pHp, corresponding to a region internal to the PCR product of Hp1-Hp2. We conclude that the 16S rRNA gene PCR with the primer set Hp1-Hp2 is not specific and cannot be used to detect H. pylori in clinical specimens.
Spacer oligonucleotide typing of Mycobacterium bovis strains from cattle and other animals: a tool for studying epidemiology of tuberculosis
Authors: Aranaz A , Liébana E , Mateos A , Dominguez L , Vidal D , Domingo M , Gonzolez O , Rodriguez-Ferri E F , Bunschoten A E , Van Embden J D , Cousins D
Published: January 2020

The spacer oligonucleotide typing (spoligotyping) method was evaluated for its ability to differentiate Mycobacterium bovis strains. This method detects the presence or absence of spacers of the direct repeat locus of the M. bovis genome. The spacers in the direct repeat locus are amplified by PCR and are detected by hybridization of the biotin-labelled PCR product with a membrane containing oligonucleotides derived from spacer sequences that have previously been bound to a membrane. One hundred eighty-two M. bovis isolates from domestic animals (cattle, goat, sheep, and cats) and wild animals (deer and wild boar) were spoligotyped, and the results were compared with those obtained by IS6110 restriction fragment length polymorphism analysis. Two rather homogeneous clusters of isolates containing 20 and 4 types, respectively, were identified by spoligotyping. The first cluster included isolates from cattle, cats, and feral animals. By spoligotyping, isolates from the Spanish wild boar and deer had the same pattern as some bovine isolates, suggesting transmission between these animals and cattle and highlighting the importance of the study of these reservoirs. The second cluster included all the caprine and ovine isolates. Within each cluster, the patterns of the different strains differed only slightly, suggesting that the spoligotypes may be characteristic of strains from particular animal species. Spoligotyping proved to be useful for studying the epidemiology of bovine M. bovis isolates, especially of those isolates containing only a single copy of IS6110. In view of our results, we suggest fingerprinting all M. bovis strains by the spoligotyping method initially and then by IS6110 restriction fragment length polymorphism typing of the strains belonging to the most common spoligotypes.
Monitoring Babesia bovis infections in cattle by using PCR-based tests
Authors: Calder J A , Reddy G R , Chieves L , Courtney C H , Littell R , Livengood J R , Norval R A , Smith C , Dame J B
Published: January 2020

The sensitivity and specificity of PCR tests based on the small-subunit rRNA gene sequence of Babesia bovis were compared in a blind study of experimentally infected cattle with the corresponding parameters of the complement fixation (CF) test currently used in the United States to screen for bovine babesiosis. Cattle were experimentally infected with a single inoculum of a cloned laboratory strain of B. bovis. Blood samples were collected and tested over a period covering from the day of infection to 10 months postinfection. The level of parasitemia (percent infected erythrocytes) present in each sample was estimated from test results and was plotted as a function of time postinfection. These data are the first describing the course of infection by methods capable of detecting parasitemias in the range of 10(-7)%, which frequently occur in the carrier state. Parasitemias in the samples tested strongly influenced the sensitivity and negative predictive value of the PCR-based tests which varied with time postinfection. The average sensitivities of the three PCR-based tests for B. bovis ranged from 58 to 70% for a single determination, while the sensitivity of the CF test was only 6%. Both PCR-based and CF tests for B. bovis had high specificity values ranging from 96 to 100%.
Comparative evaluation of PCR and commercial DNA probes for detection and identification to species level of Mycobacterium avium and Mycobacterium intracellulare
Authors: Devallois A , Picardeau M , Goh K S , Sola C , Vincent V , Rastogi N
Published: January 2020

Selective amplification of a 187-bp fragment within the DT6 sequence using the AV6 and AV7 primers for Mycobacterium avium and of a 666-bp fragment within the DT1 sequence of Mycobacterium intracellulare using the IN38 and IN41 primers was performed for 69 clinical isolates identified as M. avium complex by conventional methods. The results were compared in parallel with results with commercial M. avium and M. intracellulare probes. A positive response to either of the two PCRs or M. avium-M. intracellulare AccuProbes constituted positive detection as M. avium complex; this cumulative detection limit was 94.2% for PCR, compared with 90% for AccuProbe. Concordance, on the other hand, was considered an identical species identification using either DT1 PCR and the M. intracellulare probe or DT6 and DT1 PCRs are inexpensive and at least equally sensitive, in-house options to the AccuProbe system for species identification of M. avium and M. intracellulare.
Monoclonal antibodies to surface antigens of Mycobacterium tuberculosis and their use in a modified enzyme-linked immunosorbent spot assay for detection of mycobacteria
Authors: Glatman-Freedman A , Martin J M , Riska P F , Bloom B R , Casadevall A
Published: January 2020

Three monoclonal antibodies (MAbs) were generated from splenocytes of a BALB/c mouse immunized with heat-killed Mycobacterium tuberculosis. All three MAbs bound to surface epitopes of M. tuberculosis as shown by whole-cell enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and immunoelectron microscopy. One immunoglobulin M (IgM) MAb bound to lipoarabinomannan, the second IgM MAb bound to mycolyl-arabinogalactan-peptidoglycan complex, and the third MAb, an IgG3, bound to a surface epitope of an uncertain nature. The MAbs demonstrated different cross-reactivity patterns with other mycobacteria. Two of the MAbs were used to develop a modified ELISA spot assay for the detection of mycobacteria.
Myositis associated with a newly described microsporidian, Trachipleistophora hominis, in a patient with AIDS
Authors: Field A S , Marriott D J , Milliken S T , Brew B J , Canning E U , Kench J G , Darveniza P , Harkness J L
Published: January 2020

Microsporidia are zoonotic protozoa which were rare human pathogens prior to 1985, when Enterocytozoon bieneusi was described in human immunodeficiency virus-infected patients with chronic diarrhea. Another species, Encephalitozoon (Septata) intestinalis, is associated with diarrhea and chronic sinusitis, and approximately 25 cases have been reported in the literature. However, other microsporidial infections in human immunodeficiency virus-infected patients remain extremely rare. We report the first case of a Pleistophora sp.-like microsporidian infection presenting as a progressive severe myosotis associated with fever and weight loss. The organism was demonstrated by light microscopy and electron microscopy in corneal scrapings, skeletal muscle, and nasal discharge. Electron microscopy showed an electron-dense surface coat with "sunflare"-like projections surrounding all stages of development of meronts (two to four nuclei, dividing by binary fission), sporonts, and sporoblasts. Division of sporonts, in which sporonts separate from the thick outer coat, creating a sporophorous vesicle, is by binary fission, differentiating this organism from Pleistophora sp. The spore measures 4.0 by 2.5 microns and has a rugose exospore. A new genus and species, Trachipleistophora hominis, has been established for this parasite. The patient was treated with albendazole, sulfadiazine, and pyrimethamine, and the clinical symptoms resolved.
Molecular typing of Salmonella enterica serovar typhi
Authors: Navarro F , Llovet T , Echeita M A , Coll P , Aladueña A , Usera M A , Prats G
Published: January 2020

The efficiencies of different tests for epidemiological markers--phage typing, ribotyping, IS200 typing, and pulsed-field gel electrophoresis (PFGE)--were evaluated for strains from sporadic cases of typhoid fever and a well-defined outbreak. Ribotyping and PFGE proved to be the most discriminating. Both detected two different patterns among outbreak-associated strains.
PCR detection of metallo-beta-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum beta-lactams
Authors: Senda K , Arakawa Y , Ichiyama S , Nakashima K , Ito H , Ohsuka S , Shimokata K , Kato N , Ohta M
Published: January 2020

We applied PCR to the rapid detection of the metallo-beta-lactamase gene, blaIMP, in clinically isolated gram-negative rods. A total of 54 high-level ceftazidime-resistant strains (MICs, > 128 micrograms/ml) were subjected to PCR analyses with the blaIMP-specific primers, since the blaIMP-bearing clinical isolates tested in our previous study always demonstrated high-level resistance to ceftazidime. Twenty-two blaIMP-positive strains including 9 Pseudomonas aeruginosa, 9 Serratia marcescens, 2 Alcaligenes xylosoxidans, 1 Pseudomonas putida, and 1 Klebsiella pneumoniae strains were newly identified from 18 different hospitals in Japan. These strains were mostly isolated from urine samples and showed high-level resistance to almost every cephem, while their levels of resistance to carbapenems were diverse. The PCR analyses with novel integrase gene-specific (intI3) and acc(6')-Ib gene-specific primers suggested that the integron structure found in a large plasmid harbored by S. marcescens AK9373 was also well conserved among blaIMP-positive strains. These results imply that the blaIMP gene cassettes have been dispersing into various gram-negative rods with the help of the newly identified integron element. Thus, the PCR-aided rapid detection will be helpful for the early recognition of emerging blaIMP-positive clinical isolates which demonstrate consistent resistance to beta-lactams.
Analysis of PCR as a tool for detection of JC virus DNA in cerebrospinal fluid for diagnosis of progressive multifocal leukoencephalopathy
Authors: Hammarin A L , Bogdanovic G , Svedhem V , Pirskanen R , Morfeldt L , Grandien M
Published: January 2020

Two polyomaviruses, JC virus (JCV) and BK virus (BKV), affect humans. JCV is the causative agent of progressive multifocal leukoencephalopathy (PML), and detection of JCV in the central nervous system (CNS) is a prerequisite for confirmation of the disease. BKV is generally not associated with neurological disease, but involvement of BKV in patients with CNS disorders has been reported. In the present study polyomavirus DNA was detected by a nested PCR at a sensitivity corresponding to the detection of 10 copies of JCV DNA in 10 microliters of cerebrospinal fluid (CSF). CSF samples from 212 patients with neurological symptoms and immunodeficiencies were investigated for the presence of polyomavirus DNA. Of 128 human immunodeficiency virus (HIV)-infected patients, 14 (11%) had JCV DNA in their CSF, and all 14 patients had clinical PML. BKV DNA was detected in one HIV-infected patient with neurological symptoms not compatible with PML. Among 84 HIV-negative patients, 6 (7%) had detectable JCV DNA in their CSF, confirming PML in patients with clinical conditions of T-cell lymphoma, chronic lymphatic leukemia, status postliver transplantation, congenital immunodeficiency, sarcoidosis, and immunodeficiency of unknown origin. The specificity of the PCR was confirmed by a clinical follow-up study which showed full agreement between the detection of JCV DNA in CSF and clinically manifest PML. The described PCR is a rapid, reproducible, and easily performed assay.
Detection of extended-spectrum-beta-lactamase-producing members of the family Enterobacteriaceae with Vitek ESBL test
Authors: Sanders C C , Barry A L , Washington J A , Shubert C , Moland E S , Traczewski M M , Knapp C , Mulder R
Published: January 2020

A three-phase analysis of the Vitek ESBL test and a double-disk (2 disk) test was performed to assess their ability to detect extended-spectrum beta-lactamases (ESBLs) in members of the family Enterobacteriaceae. In the first two phases involving detection of ESBLs in 157 stains processing well-characterized beta-lactamases, sensitivity and specificity were found to be 99.5 and 100%, respectively, for the Vitek ESBl test and 98.1 and 99.4%, respectively, for the 2-disk test. In the third phase, in which the ability of each test to detect ESBLs in 295 clinical isolates was assessed, there was only one false positive (Vitek ESBL test). Across all three phases, the Vitek ESBL test was found to be much easier to perform than the 2-disk test. The latter also involved subjective interpretation of results. There were a total of 176 Escherichia coli and 157 Klebsiella pneumoniae isolates and less than 40 isolates of each of 14 other species evaluated. In a supplemental study of Klebsiella oxytoca, an organism possessing a chromosomal beta-lactamase similar to an ESBL, the Vitek ESBL test was found to be capable of detecting hyperproduction of this enzyme in strains of this species as well. These data indicate that the Vitek ESBL test is reliable for the detection of ESBLs in E. coli and K. pneumoniae, the two species in which ESBLs are most common, and of hyperproduction of the K. oxytoca beta-lactamase, a situation which engenders a level of resistance to this species similar to that seen with ESBLs.
Surveillance of community-acquired viral infections due to respiratory viruses in Rhone-Alpes (France) during winter 1994 to 1995
Authors: Lina B , Valette M , Foray S , Luciani J , Stagnara J , See D M , Aymard M
Published: January 2020

Nasal swab from patients with acute flu-like illness were evaluated for the presence of respiratory viruses in the Rhone-Alpes region of France from 1 October 1994 through 2 May 1995. The relative frequencies and seasonal distributions of the specific viruses were assessed. In addition, virus type was correlated with specific clinical signs and symptoms. During the study, 962 samples were collected by 75 medical practitioners participating in the Groupe Regional d'Observation de la Grippe surveillance network. One or more viruses were detected from 348 samples (36.1%), including 108 respiratory syncytial virus (RSV), 64 influenza virus A type H3N2, 47 influenza virus B, 64 coronavirus, 35 rhinovirus, 22 adenovirus, 5 enterovirus, and 3 parainfluenza-fluenza strains. There were 16 mixed infections. RSV infections peaked in the early winter, and influenza viruses A and B infections peaked during the late winter and early spring. There were two peaks of coronavirus infections (late fall and late winter). Other viruses were detected at lower levels throughout the study period. Patients from whom adenovirus was isolated were significantly more likely to have a fever of > 39.5 degrees C than were patients with other detectable viruses (P < 0.001). Furthermore, there was a significant correlation between influenza and cough (P < 0.01) and RSV and bronchiolitis (P < .001). Thus, the current study defined the overall and relative frequencies of respiratory virus detection from nasal swab specimens in patients with an acute flu-like illness in the Rhone-Alpes region of France during a 7-month period. Correlation with clinical signs and symptoms and provisional conclusions regarding seasonality were also determined.
Direct PCR analysis for toxigenic Pasteurella multocida
Authors: Lichtensteiger C A , Steenbergen S M , Lee R M , Polson D D , Vimr E R
Published: January 2020

A more rapid, accurate method to detect toxigenic Pasteurella multocida is needed for improved clinical diagnosis, farm biosecurity, and epidemiological studies. Toxigenic and nontoxigenic P. multocida isolates cannot be differentiated by morphology or standard biochemical reactions. The feasibility of using PCR for accurate, rapid detection of toxigenic P. multocida from swabs was investigated. A PCR protocol which results in amplification of an 846-nucleotide segment of the toxA gene was developed. The PCR amplification protocol is specific for toxigenic P. multocida and can detect fewer than 100 bacteria. There was concordance of PCR results with (i) detection of toxA gene with colony blot hybridization, (ii) detection of ToxA protein with colony immunoblot analysis, and (iii) lethal toxicity of sonicate in mice in a test set of 40 swine diagnostic isolates. Results of an enzyme-linked immunosorbent assay for ToxA agreed with the other assays except for a negative reaction in one of the 19 isolates that the other assays identified as toxigenic. In addition to accuracy, as required for a rapid direct specimen assay, toxigenic P. multocida was recovered efficiently from inoculated swabs without inhibition of the PCR. The results show that PCR detection of toxigenic P. multocida directly from clinical swab specimens should be feasible.
Development of two species-specific fingerprinting probes for broad computer-assisted epidemiological studies of Candida tropicalis
Authors: Joly S , Pujol C , Schröppel K , Soll D R
Published: January 2020

Candida tropicalis has emerged as the second most frequent colonizing Candida species, and it has been documented in nosocomial infections. To develop an effective fingerprinting system for this species, which is amenable to computer-assisted analyses and which provides information on the genetic relatedness of independent isolates, two DNA fragments, Ct3 (18,000 bp) and Ct14 (20,000 bp), were cloned from a genomic library of Sau3AI partial digestion products. Both probes generate complex Southern blot patterns containing 8 to 20 bands, when hybridized to EcoRI- or EcoRI-HaeIII-digested DNA of independent C. tropicalis isolates. The two probes show no cross-hybridization and are both species specific for C. tropicalis. A comparison of the capacity of the two probes to identify the same strain in different isolates, and differentiate unrelated strains, using computer-assisted computation of similarity coefficients and the genesis of dendrograms, suggests that while Ct14 is more effective in grouping independent isolates, Ct3 is more effective in discriminating small differences in the patterns of highly related isolates and is therefore a more effective probe for determining microevolution within a clonal population and substrain shuffling in recurrent infections. Because of their alternative attributes, it is suggested that both probes be used in fingerprinting studies of C. tropicalis.
Prevalence and varieties of Helicobacter species in dogs from random sources and pet dogs: animal and public health implications
Authors: Eaton K A , Dewhirst F E , Paster B J , Tzellas N , Coleman B E , Paola J , Sherding R
Published: January 2020

Gastric bacteria of a variety of ultrastructural morphologies have been identified in or isolated from domestic carnivores, but their prevalence in different populations of animals and their clinical significance are still unknown. The purposes of this study were (i) to evaluate the prevalence and morphologic types of gastric bacterial in three different populations of dogs; (ii) to determine which of the organisms were culturable, and if the cultured organisms were morphologically similar to the organisms seen in situ; (iii) to identify the isolated organisms; and (iv) to determine if gastric bacteria were associated with gastritis. Three groups of dogs were examined: healthy laboratory dogs, healthy dogs from an animal shelter, and pet dogs with various nongastric illnesses. Of these, 100% of laboratory and shelter dogs and 67% of pet dogs were colonized by large, tightly coiled gastric spiral bacteria morphologically similar to Gastrospirillum hominis or Helicobacter felis (referred to as gastrospirilla). Regardless of the presence or density of gastric bacteria, all of the dogs in the study except one had mild to moderate gastritis. Helicobacter spp. were isolated from only 6 of 39 stomachs cultured, and only three of the organisms isolated were morphologically similar to the bacteria seen in situ. Five helicobacters were identified by 16S rDNA (genes coding for rRNA) sequence analysis. Three were strains of H. felis, one was H. bilis, and one was a novel helicobacter morphologically similar to "Flexispira rappini." Gastrospirilla are almost universal in the stomachs of domestic dogs, and in most infected dogs, they do not appear to be associated with clinical signs or histologic lesions compared with uninfected dogs. Nongastrospirillum helicobacters are rare in dogs and are not histologically detectable. Helicobacter pylori was not isolated from domestic dogs.
Comparative typing of Pseudomonas aeruginosa by random amplification of polymorphic DNA or pulsed-field gel electrophoresis of DNA macrorestriction fragments
Authors: Renders N , Römling Y , Verbrugh H , van Belkum A
Published: January 2020

Eighty-seven strains of Pseudomonas aeruginosa were typed by random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments. Stains were clustered on the basis of interpretative criteria as presented previously for the PFGE analysis. Clusters of strains were also defined on the basis of epidemiological data and subsequently reanalyzed by RAPD. It was found that in an RAPD assay employing the enterobacterial repetitive intergenic consensus sequence ERIC2 as a primer, single band differences can be ignored; in this case, clonally related strains could be grouped as effectively and reliably as with PFGE. These data could be corroborated by the use of other primer species. However, some primers either showed reduced resolution or, in contrast, identified DNA polymorphisms beyond epidemiologically and PFGE-defined limits. Apparently, different primers define different windows of genetic variation. It is suggested that criteria for interpretation of the ERIC2 PCR fingerprints can be simple and straightforward: when single band differences are ignored, RAPD-determined grouping of P. aeruginosa is congruent with that obtained by PFGE. Consequently, this implies that RAPD can be used with trust as a first screen in epidemiological characterization of P. aeruginosa. The ability to measure the rate of molecular evolution of the P. aeruginosa genome clearly depends on the choice of restriction enzyme or primer when RAPD or PFGE, respectively, is applied for the detection of DNA polymorphisms.
Presence of human herpesvirus 6 variants A and B in saliva and peripheral blood mononuclear cells of healthy adults
Authors: Aberle S W , Mandl C W , Kunz C , Popow-Kraupp T
Published: January 2020

Saliva and peripheral blood mononuclear cells (PBMCs) from 44 healthy young adults were tested for human herpesvirus 6 variants A and B (HHV-6A and -6B) DNA by a sensitive nested PCR. HHV-6B infection was ascertained in 98% of the subjects, and 95% were found to excrete variant B in their saliva. HHV-6A was found in the PBMCs of 16%, but was not detected in saliva samples.
Comparison of PCR detection of mecA with standard susceptibility testing methods to determine methicillin resistance in coagulase-negative staphylococci
Authors: York M K , Gibbs L , Chehab F , Brooks G F
Published: January 2020

Approximately 75% of coagulase-negative staphylococci are resistant to methicillin, but it is suspected that even more resistance exists that is not detected by standard susceptibility assays. To determine the most accurate assay for measuring resistance, we compared the detection of mecA by PCR with detection by National Committee for Clinical Laboratory Standards methods using oxacillin as the class drug. Strains from 11 species of coagulase-negative staphylococci were selected such that 84% were susceptible by the broth microdilution method. Of 45 mecA-positive strains, 1 strain was unable to express the mecA gene product after induction and was not included in further analyses. For microdilution with 2% NaCl, the disk test without salt, and agar screen containing 4% NaCl plus-6 micrograms of oxacillin per ml, the sensitivities in detecting the 44 mecA-positive strains were 50, 84, and 70%, respectively, at 24 h and 77, 82, and 100%, respectively, at 48 h. The specificities of microdilution, disk, and agar screen in detecting the 97 strains lacking mecA were 100, 89, and 100%, respectively, at 24 h. Only the disk test proved to be less specific at 48 h (81%). Furthermore, for 10 of the mecA-positive strains plus an additional 8 strains subsequently added to the analyses, the MICs were 2 micrograms/ml at 24 h by the broth microdilution method; all 18 strains were positive for mecA by PCR. Thus, an oxacillin MIC of > or = 2 micrograms/ml indicated resistance and is probably a more appropriate breakpoint than the current National Committee for Clinical Laboratory Standards breakpoint of 4 micrograms/ml for coagulase-negative staphylococci. Strains for which MICs are < 2 micrograms/ml may be methicillin resistant and should be verified as susceptible by oxacillin agar screening with incubation for 48 h.
Predominance of two Bartonella henselae variants among cat-scratch disease patients in the Netherlands
Authors: Bergmans A M , Schellekens J F , van Embden J D , Schouls L M
Published: January 2020

Restriction endonuclease analysis of the PCR-amplified 16S-23S rRNA gene spacer region was used to investigate the prevalence of Bartonella henselae variants in samples from cat-scratch disease (CSD) patients. Analysis of spacer PCR fragments from 27 Bartonella DNA-positive samples from Dutch patients with CSD with AluI revealed two restriction fragment length polymorphism (RFLP) patterns, patterns A and B. Twenty samples yielded B. henselae pattern A, and 7 samples yielded B. henselae pattern B. Three samples from North American patients with CSD were shown to contain B. henselae with RFLP pattern B. To be able to detect and differentiate Bartonella DNA in clinical material more sensitively and faster, two B. henselae PCRs which amplify part of the 16S rRNA gene and which can discriminate between two B. henselae variants were developed. Thirty-two of 41 Bartonella DNA-positive samples from Dutch patients with CSD contained type I B. henselae, 7 samples contained type II B. henselae, and two samples were negative in both type-specific PCRs. Two samples from North American patients with CSD both contained type II B. henselae. A 100% correlation was found between the AluI spacer RFLP pattern and the 16S rRNA PCR type. We have shown that Dutch patients with CSD contain a limited number of B. henselae variants, suggesting that, in contrast to systemic bartonellosis, CSD in immunocompetent patients is caused by a limited number of B. henselae variants.
Evaluation of Anaplasma marginale major surface protein 3 (MSP3) as a diagnostic test antigen
Authors: Alleman A R , Barbet A F
Published: January 2020

An immunodominant surface protein, major surface protein 3 (MSP3), has been proposed as an antigen suitable for use in the diagnosis of bovine anaplasmosis. We further characterized MSP3 to examine its potential as a test antigen for the serological diagnosis of carrier cattle. The specificity of this antigen in detecting infected cattle as well as the conservation of MSP3 between strains of Anaplasma marginale was evaluated by using immunoblots of A. marginale proteins separated by one- and two-dimensional polyacrylamide gel electrophoreses. Immune sera from animals infected with Anaplasma ovis, Ehrlichia risticii, and Ehrlichia ewingii reacted with the MSP3 antigen of A. marginale. One-dimensional gel electrophoresis of A. marginale proteins demonstrated size polymorphism of MSP3 between different geographic isolates. Two-dimensional gel electrophoresis revealed at least three different antigens migrating at the 86-kDa molecular size, and sera from animals infected with different strains of A. marginale reacted with different 86-kDa antigens. These results indicate that MSP3 may share cross-reactive epitopes with antigens found in A. ovis and some Ehrlichia spp. In addition, MSP3 is not conserved between different isolates of A. marginale, and at least in the isolate from Florida, what was previously identified as MSP3 is actually a group of three or more 86-kDa antigens with different isoelectric points. The cross-reactivity of MSP3 with some Ehrlichia spp., the variability of MSP3 between isolates, and the multiple 86-kDa antigens recognized by various sera suggest that MSP3 is not a suitable candidate for use as a recombinant test antigen.
Analysis of HL and O serotypes of Campylobacter strains by the flagellin gene typing system
Authors: Nachamkin I , Ung H , Patton C M
Published: January 2020

We recently developed a molecular typing system for Campylobacter jejuni and Campylobacter coli based on restriction fragment length polymorphism analysis of the flagellin gene,flaA (I.Nachamkin, K. Bohachick, and C.M. Patton, J. Clin. Microbiol. 31:1531-1536, 1993). We extended the typing system to 83 flagellin types (designated flaA-1,flaA-2, etc.) on the basis of analysis of 404 isolates of C. jejuni and C. coli including common serotypes isolated in the United States, a selection of less common serotypes, and serotype reference strains. Of the 295 strains previously shown to belong to common HL and O serotypes (C. M. Patton, M.A. Nicholson, S.M. Ostroff, A.A. Ries, I.K. Wachsmuth, and R.V. Tauxe, J. Clin. Microbiol. 31:1525-1530, 1993), six flaA types accounted for 53.6% of strains as follows: flaA-1, 21.7%; flaA-7, 14.9%; flaA-27, 5.1%; flaA-49, 4.4%; flaA-13, 3.7%; and flaA-21, 3.7%. Seventy-five percent of the strains were within 15 flaA types, 90% were within 30 flaA types, and all 295 strains were contained within 52 flaA types. Within each HL or O serotype, there usually were multiple flaA types. For 12 common HL serotypes and 7 common O serotypes, more than 50% of these isolates were a single flaA type. A database was developed by using commercially available restriction fragment length polymorphism analysis software (ProRFLP; DNA ProScan, Inc., Nashville, Tenn.) that should allow other investigators to perform typing with this system.