bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative evaluation of molecular typing of strains from a national epidemic due to Salmonella brandenburg by rRNA gene and IS200 probes and pulsed-field gel electrophoresis
Authors: Baquar N , Burnens A , Stanley J
Published: January 2020

In Switzerland in 1992 there was a prolonged series of outbreaks of human salmonellosis caused by a previously rare serotype, Salmonella brandenburg. In order to examine the genotypic basis of the epidemic, molecular typing was applied to representative strains of this serovar isolated between 1983 and 1992. These included sporadic human isolates up to 1985, isolates from unrelated geographical areas, and Swiss isolates from humans, animals, and meat products isolated after 1991. Plasmid profiling was not found to be applicable to S. brandenburg, but chromosomal typing was accomplished by analyzing restriction fragment length polymorphisms with DNA probes for three marker loci; the 16S and 23S rRNA genes and sites of insertion of the mobile DNA element IS200. The macrorestriction profiles of the whole genome were examined by pulsed-field gel electrophoresis, which proved to be the most discriminatory of the typing methods. The study demonstrated the comparative value and complementary relationship between these typing methods for epidemiological purposes. All approaches concurred in identifying the 1992 isolates as a single genotypic clone, which was present in multiple (food) vehicles of infection. They were distinct from sporadic isolates of this serovar and from strains of S. brandenburg isolated in other countries.
Automated quantitative determination of hepatitis C virus viremia by reverse transcription-PCR
Authors: Besnard N C , Andre P M
Published: January 2020

An automated reverse transcription-PCR was developed for the quantitative detection of hepatitis C virus. The quantitation is based on the coamplification and labelling with digoxigenin-dUTP during PCR of two similar templates, the viral genome and a modified RNA which acts as a mimic target. Known amounts of the mimic RNA sequence were introduced into the clinical samples. The automated quantitation of the two coamplified and labelled products depends on the use of two biotinylated caputre probes which are complementary, respectively, to a deleted sequence and to an inserted sequence introduced by site-directed mutagenesis in a wild viral cloned cDNA. This method proved to be simple, reproducible, and useful for quantitate hepatitis C virus viremia in chronically infected patients. This easy-to-perform, automated assay could also be used for the accurate determination of human immunodeficiency virus viremia or other RNA molecules.
Differential susceptibilities of enterococcal species to elfamycin antibiotics
Authors: Miele A , Goldstein B P , Bandera M , Jarvis C , Resconi A , Williams R J
Published: January 2020

The elfamycins are a class of naturally occurring antibiotics not currently used in the therapy of human disease. Enterococcus faecium and closely related species (Enterococcus durans and Enterococcus hirae) are susceptible to these antibiotics, while isolates of Enterococcus faecalis and other enterococcal species are highly resistant. Among enterococci, susceptibility or resistance to elfamycins appears to be determined by the bacterial protein synthesis elongation factor EF-Tu. Elfamycin susceptibility may be a useful adjunct for rapidly distinguishing E. faecalis and E. faecium in the clinical microbiology laboratory and/or as a supplementary test for use in determining the species of enterococci.
Evaluation of RapiDEC Staph for identification of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus
Authors: Janda W M , Ristow K , Novak D
Published: January 2020

RapiDEC Staph is a test for presumptive identification of the principal human staphylococcal species, Staphylococcus aureus, S. epidermidis, and S. saprophyticus. The test includes control and test cupules for fluorogenic detection of coagulase and chromogenic substrates for alkaline phosphatase and beta-galactosidase. These tests identify S. aureus, S. epidermidis, and S. saprophyticus, respectively. Positive results with both chromogenic substrates provide a presumptive identification of S. xylosus or S. intermedius (S. xylosus-S. intermedius). Test cupules are inoculated with an organism suspension, and reactions are read after a 2-h incubation. RapiDEC-Staph was evaluated with 303 clinical and stock staphylococcal strains. Identifications were compared with those obtained by the tube coagulase test, a latex slide coagulase test (StaphAUREX), another commercial identification system (Staph-TRAC), and additional conventional tests. RapiDEC-Staph correctly identified 100% of 130 S. aureus strains, 70.3% of 74 S. epidermidis strains, and 81.3% of 32 S. saprophyticus strains. Four of five S. xylosus isolates were called S. xylosus-S. intermedius. Unidentified S. epidermidis and S. saprophyticus strains were called "Staphylococcus spp." Among the 62 other coagulase-negative staphylococci, 4 were misidentified as S. epidermidis and 7 were misidentified as S. saprophyticus. While the sensitivity and specificity of the fluorogenic coagulase test for S. aureus were 100%, failure to detect alkaline phosphatase activity in several S. epidermidis isolates resulted in fewer correct identifications by the RapiDEC-Staph test for this species.
Analysis of sera indeterminate by Ortho-HCV RIBA-2 by using three confirmatory assays for anti-hepatitis C virus antibody
Authors: Dussaix E , Charnaux N , Laurent-Puig P , Chopineau S , Laurian Y , Buffet C
Published: January 2020

The diagnostic performances of three commercially available recombinant immunoblot assays (RIBAs) for anti-hepatitis C virus antibody were evaluated on 50 ORTHO-HCV RIBA-2 (RIBA-2)-indeterminate serum samples. Concordant interpretations were obtained with the three tests in 60% of the samples, with 56% positive, 2% indeterminate, and 2% negative results. Considering test performance in regard to the number of remaining indeterminate results, analyzing sera by RIBA-3, INNO-LIA HCV Ab III, and DECISCAN HCV reduced the number of samples reacting indeterminately to 40, 6, and 8%, respectively. The three serum samples classified as indeterminate in the INNO-LIA HCV Ab III as well as three of four serum samples interpreted as indeterminate in the DECISCAN HCV and 16 of 20 samples classified as indeterminate in the RIBA-3 were hepatitis C virus RNA positive by PCR. This study clearly shows the good performance of the three tests as confirmatory assays compared with that of the RIBA-2. However, according to the manufacturers' criteria of positivity, the INNO-LIA HCV Ab III and DECISCAN HCV appeared to be more suitable than the RIBA-3 for interpreting serum samples found indeterminate in the RIBA-2.
Immunoaffinity purification of baculovirus-expressed rubella virus E1 for diagnostic purposes
Authors: Lindqvist C , Schmidt M , Heinola J , Jaatinen R , Osterblad M , Salmi A , Keränen S , Akerman K , Oker-Blom C
Published: January 2020

Three monoclonal antibodies, termed 4E10, 1E11:10, and 2D9:1, were generated against rubella virus. Immunoblot analysis with purified authentic rubella virus or recombinant baculovirus-expressed rubella virus structural proteins E1, E2, and C demonstrated that they were directed against the E1 envelope glycoprotein of the rubella virus particle. By using the yeast Ty virus-like particle system, it was possible to map the binding site of 1E11:10 within amino acids 236 to 286 of the E1 protein and the binding sites of 2D9:1 and 4E10 outside this region. Immunoaffinity purification with these monoclonal antibodies made it evident that they are useful for obtaining large quantities of pure baculovirus-expressed rubella virus envelope protein E1. The diagnostic potential of this immunoaffinity-purified recombinant rubella virus E1 protein compared with that of authentic rubella virus is demonstrated.
Confirmation of human Campylobacter concisus isolates misidentified as Campylobacter mucosalis and suggestions for improved differentiation between the two species
Authors: On S L
Published: January 2020

A strain from human diarrhea originally identified as Campylobacter mucosalis (NCTC 12408) was examined by using 64 phenotypic characters. The similarity of this strain to 297 isolates of Campylobacter, Helicobacter, Arcobacter, and related taxa was then determined with a computer-supported data analysis program, MVSP. NCTC 12408 showed closest similarity to 20 type, reference, and field isolates of Campylobacter concisus. These strains were clearly separated from those of C. mucosalis in the numerical analysis of phenotypic tests; a table was constructed from the data used to aid in differentiating these two species in the clinical laboratory. The identity of NCTC 12408 was confirmed as C. concisus by visual comparison of its sodium dodecyl sulfate-polyacrylamide gel whole-cell protein electrophoregram with those of type strains of C. concisus and C. mucosalis. These data suggest that genuine human infection with C. mucosalis has not yet been reported.
Demonstration of Borrelia burgdorferi DNA in urine samples from healthy humans whose sera contain B. burgdorferi-specific antibodies
Authors: Karch H , Huppertz H I , Böhme M , Schmidt H , Wiebecke D , Schwarzkopf A
Published: January 2020

Since the possibility of asymptomatic infection with Borrelia burgdorferi has been suggested by a positive serology found in healthy subjects, we hypothesized that these subjects might excrete borrelial DNA sequences in urine as happens in patients with Lyme borreliosis. We found borrelial sequences by nested PCR in the urine samples from 3 of 13 healthy B. burgdorferi antibody-positive adults but not in urine samples from 79 antibody-negative healthy controls. After therapy with doxycycline, the urine samples were repeatedly negative for B. burgdorferi DNA. We conclude that urinary excretion of borrelial DNA sequences may occur in seropositive healthy subjects during asymptomatic infection. Demonstration of such sequences in urine must be interpreted cautiously and may not necessarily prove a borrelial cause of disease.
DNA hybridization and contour-clamped homogeneous electric field electrophoresis for identification of enterococci to the species level
Authors: Donabedian S , Chow J W , Shlaes D M , Green M , Zervos M J
Published: January 2020

In this study, 113 Enterococcus faecium, 37 Enterococcus faecalis, 24 Enterococcus gallinarum, 15 Enterococcus raffinosus, and 13 Enterococcus casseliflavus clinical isolates and American Type Culture Collection (ATCC) strains were evaluated by contour-clamped homogeneous electric field electrophoresis. Thirty-one of the E. faecium, 22 of the E. faecalis, 24 of the E. gallinarum, 15 of the E. raffinosus, and 13 of the E. casseliflavus isolates were also evaluated by DNA-DNA hybridization. Genomic DNAs from type strains E. faecalis ATCC 19433, E. faecium ATCC 19434, E. gallinarum ATCC 49573, E. raffinosus ATCC 49427, and E. casseliflavus ATCC 25788 were labeled with biotin for use as probes. E. faecalis differed from all other species in always having a largest fragment of > 400 kb. E. gallinarum was different from all other species in having all SmaI fragments of < 200 kb. Biotin-labeled probes showed a high degree of hybridization with genomic DNA from the same species and a low degree of hybridization when hybridized to genomic DNA from different species for all isolates tested except for four isolates identified as E. faecium by conventional biochemical methods. The DNA from these four isolates hybridized strongly to DNA from E. gallinarum ATCC 49573 and weakly to E. faecium ATCC 19434 DNA and had all SmaI fragments of < 200 kb in size. These data suggest that these isolates are nonmotile E. gallinarum. DNA from each ATCC type strain hybridized strongly with itself and had only a low degree of hybridization with DNA from other ATCC type strains tested. These results suggest that contour-clamped homogeneous electric field electrophoresis patterns and DNA-DNA hybridization with biotin-labeled probes may be of use for species differentiation of some enterococci.
Comparison of molecular and microscopic techniques for detection of Treponema pallidum in genital ulcers
Authors: Jethwa H S , Schmitz J L , Dallabetta G , Behets F , Hoffman I , Hamilton H , Lule G , Cohen M , Folds J D
Published: January 2020

We compared the ability of direct immunofluorescent staining (DFA) and the PCR to detect Treponema pallidum in specimens from patients with genital ulcer disease. Touch preparations from 156 patients with genital lesions were fixed in acetone and stained with a fluorescein-labeled monoclonal antibody specific for the 37-kDa antigen of T. pallidum. After microscopic examination, the smear was removed from the slide with a swab. DNA was extracted with phenol-chloroform and precipitated with isopropanol. Ten microliters of the extracted DNA was amplified by PCR using primers for the gene encoding the 47-kDa protein of T. pallidum and hybridized to an internal probe. Twenty-two of 156 specimens were positive for T. pallidum by DFA and PCR, while 127 were negative by both methods, yielding a concordance of 95.5% (kappa = 0.84). Four specimens were positive by PCR and negative by DFA, while three specimens were negative by PCR and positive by DFA. The DFA-negative, PCR-positive specimens may have resulted from the presence of large numbers of leukocytes on the slides, obscuring visualization of treponemes. The DFA-positive, PCR-negative results were not due to inhibition of the PCR since purified T. pallidum DNA was amplified when added to aliquots of these specimens. Negative results in these specimens were most likely due to inefficient recovery of their DNA. These data suggest that DFA and PCR are equivalent methods for detection of T. pallidum on touch preparations of genital lesions. Further refinements of the PCR assay are necessary for it to significantly improve the detection of T. pallidum in genital lesions.
Pasteurella multocida septicemia and subsequent Pasteurella dagmatis septicemia in a diabetic patient
Authors: Fajfar-Whetstone C J , Coleman L , Biggs D R , Fox B C
Published: January 2020

Pasteurella species may cause zoonotic infections of humans. Serious systemic infections with these organisms are unusual, but they may occur in individuals with predisposing underlying illnesses. Occurrences of bacteremia due to P. multocida are infrequent, and P. dagmatis bacteremia is even rarer. We report independent occurrences of P. multocida and P. dagmatis septicemia in the same diabetic patient after contact with two pet dogs. We review the history of Pasteurella species and discuss the biochemical and clinical features of its association with zoonosis.
Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR
Authors: Cebula T A , Payne W L , Feng P
Published: January 2020

Mismatch amplification mutation assay primers, specific for a unique base substitution in uidA of Escherichia coli O157:H7, was coupled with primers for the Shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay. Analysis of 108 bacteria showed that all Escherichia coli serotype O157:H7 strains were identified simultaneously with the SLT types encoded by these strains.
Effect of delayed specimen processing on cytomegalovirus antigenemia test results
Authors: Landry M L , Ferguson D , Cohen S , Huber K , Wetherill P
Published: January 2020

Blood samples held at either 4 degrees C or room temperature for 1 day had similar mean decreases in number of cytomegalovirus antigenemia-positive cells (52 to 55%) and similar false-negative test results (13 to 14%). After 2 days, samples held at 4 degrees C showed no further decline, whereas samples held at room temperature had a mean 81% decrease in positive cells, a 32% false-negative rate, and a more marked deterioration in cell morphology.
Characterization of Neisseria elongata subsp. glycolytica isolates obtained from human wound specimens and blood cultures
Authors: Andersen B M , Weyant R S , Steigerwalt A G , Moss C W , Hollis D G , Weaver R E , Ashford D , Brenner D J
Published: January 2020

Four slightly yellow-pigmented, alpha-hemolytic, gram-negative coccobacilli, three from wound specimens and one from multiple blood cultures of a patient with endocarditis, were identified as Neisseria elongata subsp. glycolytica on the basis of their overall biochemical and genetic similarities to this subspecies. These strains resembled N. elongata in their guanine-plus-cytosine contents (55.6 to 57.1 mol%) and in their overall cellular fatty acid profiles, which are characterized by large amounts of 16:0, 16:1 omega 7c, and 18:1 omega 7c fatty acids. Their identities were confirmed by species-level DNA relatedness (hydroxyapatite method) to the type strains of all three N. elongata subspecies. The biochemical profiles and cultural characteristics of these strains resembled those of the type strain of N. elongata subsp. glycolytica except for the production of a weak yellow growth pigment and alpha-hemolysis on sheep blood agar. They differed from N elongata subsp. elongata by the production of catalase, by the production of alpha-hemolysis on sheep blood agar, and by acid production from D-glucose. They differed from N. elongata subsp. nitroreducens by the production of catalase and an inability to reduce nitrate. These studies suggest a pathogenic potential for N. elongata subsp. glycolytica, usually considered to be a transient colonizer in humans.
Phenotypic and genotypic analysis of variability in Aspergillus fumigatus
Authors: Rinyu E , Varga J , Ferenczy L
Published: January 2020

Sixty-one isolates and collection strains of Aspergillus fumigatus were compared for their phenotypic (morphological features and isoenzyme profiles) and genotypic (restriction enzyme-generated mitochondrial DNA and ribosomal DNA profiles and random amplified polymorphic DNA patterns) features. The examined strains exhibited highly variable colony morphologies and growth rates at different temperatures, but their micromorphologies and conidial diameters were characteristic of the species. Of the isoenzymes studied, the beta-arylesterase and phosphatase patterns were the most divergent, and the 61 strains could be classified into seven groups. The glucose 6-phosphate dehydrogenase and catalase isoenzyme patterns displayed only a limited variability, while the profiles of superoxide dismutase, lactate dehydrogenase, and glutamate dehydrogenase were highly conserved. The HaeIII-generated mitochondrial DNA patterns and SmaI-digested repetitive DNA and ribosomal DNA hybridization patterns of almost all strains were also invariable. The level of variation was much higher when random amplified polymorphic DNA analysis was applied. Although the patterns of the strains were very similar with most of the primers, the application of some primers made it possible to cluster the A. fumigatus isolates into several groups. The results indicate that the random amplified polymorphic DNA technique could be used more efficiently than isoenzyme analysis for typing A. fumigatus isolates. A good correlation was found between the dendrograms obtained from the isoenzyme and random amplified polymorphic DNA data, but the isoenzyme and amplified DNA patterns did not correlate with the pathogenicity, pigment production, or geographical origin of the strains. One "A. fumigatus" strain (strain FRR 1266) exhibited unique isoenzyme, mitochondrial DNA, ribosomal DNA, and random amplified polymorphic DNA patterns; it is proposed that this strain represents a new species of the section Fumigati.
Effect of three concentration techniques on viability of Cryptosporidium parvum oocysts recovered from bovine feces
Authors: Bukhari Z , Smith H V
Published: January 2020

Bovine fecal samples (1 g) negative for Cryptosporidium sp. oocysts were seeded with 7 x 10(4) Cryptosporidium parvum oocysts and purified by either water-ether concentration, sucrose density flotation, or zinc sulfate flotation to evaluate oocyst recovery. The effect of these purification techniques on the viability of recovered oocysts was also evaluated. Significantly higher numbers of seeded oocysts were recovered by water-ether concentration (recovery rate, 46 to 75%) than by sucrose density (24 to 65%) or zinc sulfate (22 to 41%) flotation methods. In addition, water-ether concentration did not exert a significant effect on the viability of the population of oocysts recovered, whereas sucrose density flotation and zinc sulfate flotation selectively concentrated viable oocysts. The water-ether concentration procedure is recommended for use in epidemiological studies in which both oocyst enumeration and viability assessment are required.
Establishment of enzyme-linked immunosorbent assay using purified recombinant 83-kilodalton antigen of Borrelia burgdorferi sensu stricto and Borrelia afzelii for serodiagnosis of Lyme disease
Authors: Rauer S , Kayser M , Neubert U , Rasiah C , Vogt A
Published: January 2020

The 83-kDa antigen of Borrelia burgdorferi was expressed as a recombinant protein in Escherichia coli and purified for use in an enzyme-linked immunosorbent assay (p83-ELISA). Antibodies to the 83-kDa antigen of both the immunoglobulin G (IgG) and IgM isotypes could be detected in all stages of Lyme disease. Sensitivity varied, depending on the clinical stage of illness. In early stages, as defined for 118 patients with erythema migrans, it was found to be 20% (24 of 118 patients: 7 with IgM, 16 with IgG, and 1 with IgM and IgG). Of the patients with late-stage Lyme arthritis and acrodermatitis chronica atrophicans, 94% (16 of 17:2 with IgM and IgG and 14 with IgG) and 86% (36 of 42:2 with IgG and IgM and 34 with IgG) revealed positive results in the p83-ELISA, respectively. p83 displays sequence heterogeneity according to the genomospecies, but when the reactions of serum specimens from acrodermatitis chronica atrophicans patients and arthritis patients with p83 derived from representative strains of B. burgdorferi sensu stricto and Borrelia afzelii in ELISAs were compared, no differences in specificity and sensitivity were seen. When 82 serum specimens from healthy controls were tested, none had IgG and only 3 (4%) had IgM antibodies, indicating a high specificity. Positive reactions with antibodies against Treponema pallidum (1 of 37 patients; IgG) and Epstein-Barr virus (1 of 44 patients; IgM) and with autoantibodies of various specificities (1 of 53 patients; IgG) were seen with < 3% of the serum samples te11111111111111111111 high speficicity for B. burgdorferi.2+ 13% for IgM antibodies, the IgM p83-ELISA provided little diagnostic information for Lyme disease, whereas the IgG p83-ELISA appears to be a suita ;e test for serodiagnosis of advanced-stage Lyme disease.
Ribotyping for use in studying molecular epidemiology of Serratia marcescens: comparison with biotyping
Authors: Chetoui H , Delhalle E , Osterrieth P , Rousseaux D
Published: January 2020

Ribotyping carried out with a nonradioactive probe (acetylaminofluorene ribosomal RNA kit I from Eurogentec, Seraing, Belgium) was performed for the characterization of 139 hospital strains of Serratia marcescens. These strains, which belonged to 11 biotypes and 1 nontypeable group, were isolated in seven hospitals in Belgium between 1986 and 1992. EcoRI and HindIII were used to obtain cleavage patterns. Analysis of the results produced 27 different patterns with EcoRI and 23 patterns with HindIII. Typeability reached 100%. Combination of the patterns obtained with each enzyme produced 38 distinct ribotypes. Percent similarity values, calculated by using the Dice coefficient and unweighted-pair group average linkage clustering, showed four main clusters and nine subclusters of ribopatterns at a similarity rate of approximately 80% or less. These groups did not coincide with those delimited by biotyping, although a rather good correlation was observed. The simultaneous use of the two methods has potential value in epidemiological studies.
Diagnostic value of an amplification method (Gen-Probe) compared with that of culture for diagnosis of tuberculosis
Authors: Vlaspolder F , Singer P , Roggeveen C
Published: January 2020

Five hundred fifty respiratory and nonrespiratory specimens from 340 patients were analyzed by comparing the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD) with conventional culture, which was the method of reference, for the detection of the Mycobacterium tuberculosis complex. After resolution of discrepant results by retesting the samples and reviewing the patients' clinical histories, a total of 60 respiratory specimens were MTD and culture positive, 347 were MTD and culture negative, 4 were MTD positive and culture negative, and 1 was MTD negative and culture positive. This results in a sensitivity of 98.4%, a specificity of 98.9%, and positive and negative predictive values of 93.8 and 99.7%, respectively. Repeatedly, clinicians asked to test specimens of nonpulmonary origin by MTD. Although, MTD is not approved for use with nonrespiratory specimens, the following results were shown. Sixty-one pleural exudate specimens showed disappointing results (sensitivity, 20%). However, MTD performed well with another 77 nonrespiratory specimens; 17 samples were positive and 57 samples were negative by both MTD and culture. No false-negative results were found by MTD. Three MTD-positive, culture-negative specimens had high sample relative light unit/cutoff relative light unit ratios, strongly suggesting true tuberculosis. Positive microscopy and positive culture with MTD-negative results occurred 12 times. Those cultures showed atypical mycobacteria 11 times and Actinomyces species once. The stability of the reagents in the MTD kit was also assessed by testing reagents, including the enzyme mixture, kept at -70 degrees C for at least 6 months. No loss of activity was seen.
Utility of direct immunofluorescence and virus culture for detection of varicella-zoster virus in skin lesions
Authors: Coffin S E , Hodinka R L
Published: January 2020

A direct immunofluorescence assay (DFA) with a monoclonal antibody from Ortho Diagnostic Systems was compared with conventional cell culture for the rapid detection of varicella-zoster virus (VZV) in 140 dermal lesions from 133 patients. A total of 79 (56%) specimens were positive for VZV: 40 (51%) by DFA alone, 2 (3%) by culture only, and 37 (47%) by both culture and DFA. After discordant analysis, the sensitivities and negative predictive values, respectively, were 97.5% (77 of 79) and 96.8% (61 of 63) for DFA and 49.4% (39 of 79) and 60.4% (61 of 101) for viral culture. Of the 39 positive viral cultures, VZV was isolated from 38 (97%) cultures in A549 cells, 23 (59%) in primary rhesus monkey kidney cells, and only 16 (41%) in MRC-5 cells. We conclude that DFA is the optimal method for rapid identification of VZV. In addition, better recovery of VZV in culture may be achieved by using A549 cells.
Evaluation and validation of a monoclonal immunofluorescent reagent for direct detection of Bordetella pertussis
Authors: McNicol P , Giercke S M , Gray M , Martin D , Brodeur B , Peppler M S , Williams T , Hammond G
Published: January 2020

An outbreak of pertussis in Manitoba, Canada, provided an opportunity to evaluate the recently developed monoclonal antibody (MAb) BL-5 for the direct detection of Bordetella pertussis. The MAb recognizes a lipooligosaccharide epitope. A total of 1,507 consecutive nasopharyngeal swabs for culture and companion smears for direct fluorescent-antibody (DFA) detection were evaluated at Cadham Provincial Laboratory between September and November 1994. The cutoff for DFA positivity was four fluorescing organisms with morphology characteristic of B. pertussis. PCR analysis for B. pertussis DNA was performed on a subset of 100 smears by eluting material from the slides after DFA examination. In comparison with culture, the sensitivity, specificity, and positive and negative predictive values of BL-5 were 65.1% (41 of 63 samples), 99.6% (1,438 of 1,444 samples), 87.2% (41 of 47 samples), and 98.5% (1,438 of 1,460 samples), respectively. The sensitivity of culture compared with PCR was 45.5% (10 of 22 samples) for the subset of 100 specimens tested by both procedures. An expanded "gold standard" of positivity by culture or PCR for these 100 specimens resulted in DFA sensitivity, specificity, and positive and negative predictive values of 32.3, 97.1, 83.3, and 76.1%, respectively. The utility of MAb BL-5 for direct detection of B. pertussis in a clinical laboratory setting has been demonstrated by this investigation.
Greater diversity of hepatitis C virus genotypes found in Hong Kong than in mainland China
Authors: Zhang Y Y , Lok A S , Chan D T , Widell A
Published: January 2020

A hepatitis C virus (HCV) genotyping PCR assay based on type-specific primers was expanded to include genotype 6a as well as genotypes 1a, 1b, 2a, and 3a. The nucleotide sequences of a 194-bp fragment in the center of the HCV core gene showed that the homologies between genotype 6a and genotypes 1a, 1b, 2a, 2b, 3a, and 5 were 81.2, 82.1, 73.8, 77.3, 81.4, and 78.9%, respectively. A high degree of homology (99.6%) was seen in the amplified core region among eight clinically unrelated genotype 6a isolates. Although the Hong Kong Chinese patients had predominantly genotype 1b (70%), it was noteworthy that genotype 6a was the second most common genotype (14%). Four other HCV genotypes--1a, 1b, 2a, and 2b--were also present. In contrast, HCV infection by mainland China was confined to genotypes 1b and 2a. Thus, we found a greater diversity of HCV genotypes in Hong Kong than in mainland China.
Development and testing of monoclonal antibody-based rapid immunodiagnostic test kits for direct detection of Vibrio cholerae O139 synonym Bengal
Authors: Hasan J A , Huq A , Nair G B , Garg S , Mukhopadhyay A K , Loomis L , Bernstein D , Colwell R R
Published: January 2020

We report on the development and testing of two monoclonal antibody-based rapid immunodiagnostic test kits, BengalScreen, a coagglutination test, and Bengal DFA, a direct fluorescent-antibody test, for direct detection of Vibrio cholerae O139 synonym Bengal in clinical and environmental specimens. The BengalScreen test requires less than 5 min to complete and can be used in the field. Bengal DFA, being more sensitive than BengalScreen, requires only one reagent and less than 20 min for detection and enumeration of V. cholerae O139 synonym Bengal. In tests for specificity, all 40 strains of V. cholerae O139 reacted with both test kits, whereas 157 strains of heterologous species examined did not, yielding 100% specificity in this study. A field trial was conducted in with both BengalScreen and Bengal DFA, and the results were compared with those obtained by conventional culture methods. BengalScreen demonstrated a sensitivity of 95%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94%. Results obtained by Bengal DFA, on the other hand, were 100% sensitive and 100% specific and yielded 100% positive and negative predictive values compared with culture methods. In a second evaluation, 93 stool specimens from Mexico that were negative for V. cholerae O139 by culture were also tested with both the BengalScreen and Bengal DFA kits. None of the 93 specimens were positive for V. cholerae O139 by both tests. A concentration method was optimized for screening of environmental water samples for V. cholerae O139 synonym Bengal with rapid test kits. BengalScreen results were unequivocally positive when water samples contained at least 2.0 x 10(3) CFU/ml, whereas Bengal DFA demonstrated an unequivocally positive reaction when the water sample contained at least 1.5 x 10(2) CFU/ml. When Bengal DFA was compared with conventional culture methods for enumeration of V. cholerae O139 synonym Bengal organisms, no difference was observed.
Optimization of detection of cytomegalovirus viremia in transplantation recipients by shell vial assay
Authors: Patel R , Klein D W , Espy M J , Harmsen W S , Ilstrup D M , Paya C V , Smith T F
Published: January 2020

Cytomegalovirus (CMV) viremia is a widely used laboratory marker of CMV disease following transplantation and is additionally used to trigger preemptive antiviral therapy. Despite this, the optimal method for diagnosing CMV viremia in transplantation recipients remains unknown. To determine the sampling frequency and blood volume required for the optimal diagnosis of viremia by shell vial assay, a prospective study of 46 viremic transplantation recipients was conducted. Blood specimens (2.5 and 5 ml) were collected twice, 3 h apart, at a median of 1.4 days (range, 1 to 3 days) after the triggering shell vial-positive blood had been collected. Considering a single 2.5-ml specimen, an average of only 40% of previously viremic patients had documented CMV in their blood: this increased to 50% when a second 2.5-ml sample of blood was collected 3 h later. The yields of two 2.5-ml versus two 5-ml samples were 50 versus 61%, respectively. Viremia as detected by shell vial assay is intermittent, and increasing the frequency and volume of blood sampling increases its diagnosis. These results have implications in diagnosis of CMV infection and its preemptive therapy.
Pathogenic, immunologic, and molecular differences between two Ehrlichia risticii strains
Authors: Vemulapalli R , Biswas B , Dutta S K
Published: January 2020

Ehrlichia risticii is the causative agent of Potomac horse fever (PHF), an acute infectious disease of horses. In the last few years, there have been several reports of PHF cases occurring even in vaccinated horses. We isolated a new strain of E. risticii (90-12 strain) from a vaccinated horse suffering from clinical PHF. The major pathogenic, immunologic, and molecular differences between the 90-12 strain and the 25-D stain, which was originally isolated during the outbreaks in 1984, were studied. The 90-12 strain was more pathogenic for mice and horses compared with the 25-D strain. In enzyme-linked immunosorbent assay and immunofluorescence assay with mouse and horse antisera of both the strains, two- to fourfold differences were observed between (immunoblot) with mouse and horse antisera and also with the recombinant clone-specific antibodies. Though several antigens were similar in both the strains, there were significant differences between them in the 110-, 85-, 70-, 51-, and 33-kDa antigens. The 85-kDa antigen was present only in the 90-12 strain but cross-reacted with a 50-kDa antigen of the 25-D strain. The 51-kDa antigens of both strains had different migration patterns, Southern blot hybridization of the genome from both the strains with DNA probes made from the 51-, 55-, and clones for both the strains, whereas the probe of the 85-kDa clone showed a completely different pattern. The 16S rRNA gene sequences from the two strains were identical. Neither strain replicated in gamma interferon-treated mouse peritoneal macrophages. In in vitro neutralization assay, sera from the 25-D strain-infected horse neutralized the homologous strain but did not neutralize the 90-12 strain, whereas sera from the 90-12 strain-infected horse neutralized both the strains. In mouse protection experiments, there was complete homologous protection. But in cross-protection, mice immunized with the 25-D strain were only partially protected against challenge with the 90-12 strain, whereas mice immunized with the 90-12 strain were completely protected against the 25-D strain challenge. These results clearly indicate that there are major differences between the 90-12 and 25-D strains which may have implications regarding the vaccine failure for PHF and the development of an efficient vaccine.
Haemophilus parainfluenzae biliary tract infection: rationale for an ascending route of infection from the gastrointestinal tract
Authors: Bottone E J , Zhang D Y
Published: January 2020

Haemophilus parainfluenzae was isolated from a bile specimen of a 56-year-old patient with elevated liver enzymes and chronic cholecystitis. Several factors contributing to nonbacteremic H. parainfluenzae infection of the biliary tract include (i) the not infrequent occurrence of H. parainfluenzae in stool specimens, (ii) the presence of an outer membrane protein similar to those found in enteric bacteria which facilitates adherence to the colonic mucosa, and (iii) an adequate supply of V factor (nicotinamide adenine dinucleotide) supplied from bile and the local flora, which is necessary for H. parainfluenzae's growth. Because H. parainfluenzae rarely occurs in the biliary tract, direct cultivation of bile specimens on chocolate agar seems unwarranted. However, bile specimens showing a gram-negative coccobacillus on a Gram-stained smear that fails to grow on routine media should be subcultured on chocolate agar.
Prevalence of Cyclospora species and other enteric pathogens among children less than 5 years of age in Nepal
Authors: Hoge C W , Echeverria P , Rajah R , Jacobs J , Malthouse S , Chapman E , Jimenez L M , Shlim D R
Published: January 2020

Stools from 124 Nepalese children aged 6 to 60 months with diarrhea were examined for organisms of the coccidian genus Cyclospora and for other enteric pathogens. Enterotoxigenic Escherichia coli, Giardia Lamblia, Campylobacter species, Cyclospora species, and Cryptosporidium species were the most common pathogens identified. Cyclospora species were detected in none of 74 children < 18 months of age compared with 6 (12%) of 50 children > or = 18 months of age (P = 0.004).
Bacillus licheniformis prosthetic aortic valve endocarditis
Authors: Santini F , Borghetti V , Amalfitano G , Mazzucco A
Published: January 2020

A 73-year old man developed an acute prosthetic aortic valve dehiscence for which emergent operation was undertaken. The intraoperative evidence of an aortic annular disruption and of a subannular abscess led to the hypothesis that an endocarditis process was involved. The aortic valve was replaced with a stentless porcine bioprosthesis. Cultures taken intraoperatively from the aortic area had a pure growth of aerobic, spore-forming, gram-positive bacilli identified as Bacillus licheniformis. The patient responded to specific antibiotic therapy with no relapse at a 20-month follow-up. The potentiality of B. licheniformis as a pathogen should be reconsidered.
Analysis of Bordetella pertussis isolates from an epidemic by pulsed-field gel electrophoresis
Authors: Beall B , Cassiday P K , Sanden G N
Published: January 2020

We examined genetic variation among 78 clinical isolates of Bordetella pertussis, including 54 strains recovered during a 1986 pertussis epidemic. A total of 16 pulsed-field gel electrophoresis (PFGE) profiles, generated with each of three different enzymes (XbaI, SpeI, and DraI), were obtained from the epidemic and sporadic isolates included in the study. Indistinguishable profiles were seen among strains unrelated temporally or geographically, as well as among strains isolated sporadically from the same geographic areas. All isolates from the epidemic had indistinguishable PFGE profiles. The PFGE pattern of the epidemic strains was shared with only 1 of 25 strains isolated independently of the outbreak. This isolate was cultured from a specimen from a laboratory scientist who had been working with the epidemic strains, further implicating the usefulness of PFGE for the epidemiologic study of clinical strains of B. pertussis. Differences in PFGE profiles for single epidemic strains occurred occasionally upon repeated passage on agar medium, suggesting that subculturing of initial isolates should be minimized before pulsed-field analysis.
Diagnosis of tuberculosis by Amplicor Mycobacterium tuberculosis test: a multicenter study
Authors: Carpentier E , Drouillard B , Dailloux M , Moinard D , Vallee E , Dutilh B , Maugein J , Bergogne-Berezin E , Carbonnelle B
Published: January 2020

The Amplicor Mycobacterium tuberculosis test is a new PCR assay for the direct detection of Mycobacterium tuberculosis from clinical samples. A multicenter study that included six laboratories was done to evaluate the Amplicor test in comparison with direct microscopy and culture (solid or radiometric media), and the culture method was used as the "gold standard." A total of 2,073 specimens, i.e., 1,749 respiratory specimens and 324 other specimens, were tested. A total of 184 cultures yielded M. tuberculosis. Of these 184 cultures, 77 (42%) were smear negative and 23 (12.5%) concerned extrapulmonary specimens. The sensitivity of the Amplicor test for all of the specimens and for extrapulmonary, smear-positive, and smear-negative specimens was 86, 83, 94.5, and 74%, respectively. The sensitivity of direct microscopy in comparison with that of culture was 58%. A total of 95% of patients with culture-proven tuberculosis were diagnosed by the Amplicor test, whereas direct microscopy detected mycobacteria in only 72% of these patients. The Amplicor test exhibited a high degree of specificity (98%). The assay was very rapid and easy to perform.
Coxiella burnetii blood cultures from acute and chronic Q-fever patients
Authors: Musso D , Raoult D
Published: January 2020

Q fever, a worldwide zoonosis caused by Coxiella burnetii, may present as either an acute or a chronic disease. We correlated the results of 844 C. burnetii blood cultures with serological, clinical, and therapeutic data. C. burnetii was isolated from 17% of untreated patients with acute Q fever and from 53% of untreated patients with chronic Q fever. C. burnetii was not isolated from patients who were receiving antibiotics active against C. burnetii. For seven culture-positive patients with acute Q fever, serology was negative when C. burnetii was isolated. One patient with acute Q fever had a positive blood culture 25 days after the discontinuation of specific antibiotic therapy, and another had a positive blood culture after the resolution of symptoms. In one case of chronic Q fever, a positive blood culture resulted from noncompliance with treatment. The culture method described in this report is suitable for all laboratories with cell culture facilities. Our findings suggest that blood samples must be collected prior to the initiation of an antibiotic regimen if C. burnetii is to be successfully isolated.
Molecular epidemiological studies of Mycobacterium bovis infections in humans and animals in Sweden
Authors: Szewzyk R , Svenson S B , Hoffner S E , Bölske G , Wahlström H , Englund L , Engvall A , Källenius G
Published: January 2020

Forty-nine isolates of Mycobacterium bovis from humans and animals in Sweden were analyzed by restriction fragment length polymorphism (RFLP) patterns probed by the insertion element IS6110. Most isolates had patterns indicating the presence of only one or two genomic copies of the IS6110 insertion element. This simple type of pattern was found in all human isolates. In contrast, isolates from M. bovis infections in five herds of farmed deer in Sweden showed a specific RFLP pattern with seven bands, indicating seven copies of the IS6110 sequence. In 1958, Sweden was declared free from M. bovis in cattle. However, in 1987, M. bovis was reintroduced with imported farmed deer, and since 1991, 11 outbreaks in deer herds, but not in other livestock or wildlife, have been diagnosed. Continued RFLP studies of the new Swedish M. bovis isolates can reveal possible transmission of this deer strain to other animals or humans.
Sensitivities of PCR, MicroTrak, ChlamydiaEIA, IDEIA, and PACE 2 for purified Chlamydia trachomatis elementary bodies in urine, peripheral blood, peripheral blood leukocytes, and synovial fluid
Authors: Kuipers J G , Scharmann K , Wollenhaupt J , Nettelnbreker E , Hopf S , Zeidler H
Published: January 2020

Routine microbiological diagnosis of Chlamydia-induced reactive arthritis is based mainly on the detection of Chlamydia trachomatis with urogenital swabs or in urine. Because chlamydial antigen, rRNA, and DNA are present in low quantities in the inflamed joint, highly sensitive assays are needed to detect C. trachomatis not only at the primary site of infection but also in peripheral blood and peripheral blood leukocytes, which are suspected carriers for dissemination, and in synovial fluid. To evaluate possible tools for this purpose, the sensitivities of PCR, MicroTrak, Chlamydia EIA, IDEIA, and PACE 2 for the detection of defined numbers of purified C. trachomatis elementary bodies (EB) in urine, peripheral blood, peripheral blood leukocytes, and synovial fluid were determined. In urine, PCR detected 2, MicroTrak and ChlamydiaEIA detected 2 x 10(3), and PACE 2 and IDEIA detected 2 x 10(4) EB per ml. In peripheral blood, only PCR and MicroTrak detected C. trachomatis, with detection limits of 100 and 2 x 10(7) EB per ml, respectively. For peripheral blood leukocytes, the detection limits were 2 EB per ml for PCR and 2 x 10(4) EB per ml for MicroTrak, ChlamydiaEIA, IDEIA, and PACE 2. In synovial fluid, PCR detected 200, MicroTrak and IDEIA detected 2 x 10(5), and PACE 2 detected 10(6) EB per ml. ChlamydiaEIA was unable to detect 2 x 10(6) EB per ml in synovial fluid. In summary, PCR was found to be the most sensitive method. The sensitivities of the other methods tested were at least 1,000 times lower than that of PCR. PCR should therefore be considered a most promising tool for routine diagnosis of Chlamydia-induced arthritis.
Comparison of Amplicor, in-house PCR, and conventional culture for detection of Mycobacterium tuberculosis in clinical samples
Authors: Schirm J , Oostendorp L A , Mulder J G
Published: January 2020

Five hundred four clinical specimens (337 sputum and 167 bronchial samples) from 340 patients were tested for the presence of M. tuberculosis complex by the Amplicor M. tuberculosis test and by an in-house PCR. The results were compared with those obtained by conventional culture and by direct microscopy. Thirty specimens (from 14 patients) were positive by in-house PCR, 25 (from 13 patients) were positive by the Amplicor M. tuberculosis test, and 24 (from 10 patients) were positive by culture. Cultures from 16 specimens were contaminated with other bacteria. Strong inhibition of in-house PCR was found with three samples. After discordancy analyses, with clinical data as supportive evidence for tuberculosis, 27 true-positive and 458 true-negative samples were defined. On the basis of these figures, the sensitivities of the Amplicor M. tuberculosis test, in-house PCR, culture, and microscopy were 70.4, 92.6, 88.9, and 52.4%, respectively. The specificities of all four tests were higher than 98%. The good performance of the in-house PCR for detection of M. tuberculosis makes it a very useful additional tool in M. tuberculosis diagnostics. In contrast, the Amplicor test needs to be improved. Twenty-three of the Amplicor-negative samples were further tested for inhibition of the Amplicor system by retesting the DNA extracts after the addition of M. tuberculosis DNA. In 15 of these samples, 5 true positives and 10 true negatives, inhibition of the Amplicor test was demonstrated. This might explain the lack of sensitivity of the Amplicor test. If the inhibition problem can be solved, the Amplicor M. tuberculosis test, which is already rapid, very user-friendly, and reasonably priced, may certainly become very useful in microbiological laboratories.