bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Epstein-Barr virus DNA in cerebrospinal fluid for diagnosis of AIDS-related central nervous system lymphoma
Authors: Arribas J R , Clifford D B , Fichtenbaum C J , Roberts R L , Powderly W G , Storch G A
Published: January 2020

The diagnostic utility of Epstein-Barr virus (EBV) DNA detection in cerebrospinal fluid for the diagnosis of central nervous system lymphoma was evaluated with two different PCR assays to test a collection of cerebrospinal fluid samples from 24 AIDS patients with central nervous system disorders. A PCR assay amplifying a fragment from the BamHI-W region had the highest clinical and analytic sensitivity. The BamHI-W PCR assay detected EBV DNA in cerebrospinal fluid from 83% (5 of 6) of patients with pathologically proven primary central nervous system lymphoma and 7% (1 of 16) of controls with autopsy-proven nonlymphomatous central nervous system disorders. EBV DNA was also detected in one patient with autopsy-proven systemic lymphoma involving the central nervous system and one patient with probable primary central nervous system lymphoma. EBV DNA was detected consistently when central nervous system lymphoma involved meningeal surfaces. PCR for EBV in cerebrospinal fluid appears to be useful for diagnosis of AIDS-related central nervous system lymphoma, but additional studies are required to better define the sensitivity of the assay and to understand the significance of a positive test in the absence of lymphoma.
Genotypic detection of Mycobacterium tuberculosis rifampin resistance: comparison of single-strand conformation polymorphism and dideoxy fingerprinting
Authors: Felmlee T A , Liu Q , Whelen A C , Williams D , Sommer S S , Persing D H
Published: January 2020

Detection of mutations in the rpoB gene of Mycobacterium tuberculosis can be used as an accurate predictor of rifampin resistance in the majority of strains tested. Simple but highly accurate screening methods must be developed for the detection of these mutations. Either DNA sequence analysis or single-strand conformation polymorphism (SSCP) screening can be used to detect rpoB mutations, but these techniques either are expensive or yield results that may prove difficult to interpret when used in a clinical setting. This report describes the use of dideoxy fingerprinting (ddF) as a postamplification screening method to identify rifampin-resistant genotypes. The ddF protocol was performed on the amplified rpoB fragment with no preparatory steps, thus making ddF practical for laboratories equipped for polyacrylamide gel electrophoresis. When compared with the results of SSCP analysis, ddF results were more easily interpreted and contained more sequence-dependent information that facilitated differentiation of functionally significant and silent mutations. The ddF method was used for genotypic determination of rifampin susceptibility of 20 multidrug-resistant strains of M. tuberculosis. The results of this analysis were concordant with DNA sequence analysis and conventional clinical laboratory methods.
Multicenter clinical laboratory evaluation of a beta-lactamase disk assay employing a novel chromogenic cephalosporin, S1
Authors: Doern G V , Jones R N , Gerlach E H , Washington J A , Biedenbach D J , Brueggemann A , Erwin M E , Knapp C , Raymond J
Published: January 2020

S1, a new chromogenic cephalosporin (International BioClinical, Inc., Portland, Oreg.), was used to detect beta-lactamase production among a variety of commonly encountered bacteria in a four-center collaborative study. Results of an S1 disk assay were compared with those obtained by a nitrocefin-based disk procedure (Cefinase; Becton-Dickinson Microbiology Systems, Cockeysville, Md.), with repetitive testing of five quality control organisms and with individual tests of recent clinical isolates of Neisseria gonorrhoeae (162 strains), Haemophilus influenzae (162 strains), Moraxella catarrhalis (155 strains), Staphylococcus aureus (161 strains), and Bacteroides fragilis (164 strains). The performances of the two beta-lactamase disk assays were comparable for the first three species cited above. However, the S1 assay appeared to be a more sensitive procedure than the Cefinase assay when applied to S. aureus and B. fragilis, with respect to both total numbers of positive results and length of time to a definitive positive endpoint.
Analysis of clonal relationships among isolates of Shigella sonnei by different molecular typing methods
Authors: Liu P Y , Lau Y J , Hu B S , Shyr J M , Shi Z Y , Tsai W S , Lin Y H , Tseng C Y
Published: January 2020

Shigella sonnei is a major cause of diarrheal disease in developed as well as in developing countries. Epidemiologic studies of this organism have been limited by the lack of a simple and effective method for comparing strains. In this study, we have compared different molecular typing methods, i.e., plasmid profile analysis, restriction endonuclease analysis of plasmids, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis (PFGE), and enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR (ERIC-PCR) for typing 20 clinical isolates of S. sonnei collected from six incidents of infection. PFGE and ERIC-PCR fingerprintings had the highest discriminatory power for discrimination of epidemiologically related isolates from epidemiologically unrelated strains of S. sonnei, and both gave seven distinct strain types among these isolates and the type strain of the species. Plasmid study and ribotyping produced only six and typing techniques demonstrated two distinct patterns, respectively, among these strains. All of these molecular an identical fingerprint for eight temporally related sporadic isolates. It is possible that these temporally related isolates belonged to a single bacterial clone and circulated obscurely through the community. Our results indicate that the ERIC-PCR technique represents a rapid and simple means for typing S. sonnei with a level of discrimination equivalent to that of PFGE but greater than those of plasmid profile analysis, restriction endonuclease analysis of plasmids, and ribotyping.
Clinical comparison of the isolator and BacT/Alert aerobic blood culture systems
Authors: Hellinger W C , Cawley J J , Alvarez S , Hogan S F , Harmsen W S , Ilstrup D M , Cockerill F R
Published: January 2020

The performance characteristics of the Isolator (Wampole Laboratories, Cranbury, N.J.) and the BacT/Alert (Organon Teknika Corporation, Durham, N.C.) aerobic blood culture systems were compared for 6,009 blood culture sets obtained from patients with suspected bloodstream infections. The BacT/Alert aerobic bottle [BTA(O2)] was continuously agitated while it was incubated in 5% CO2 at 36 degrees C; culture plates prepared from the Isolator tube [I(O2)] were incubated in 5% CO2 at 37 degrees C. From 394 blood cultures, 416 clinically significant isolates of bacteria and yeasts were recovered. The overall yields for BTA(O2) and I(O2) were not significantly different (319 versus 336; P = 0.20). I(O2) recovered significantly more staphylococcus (P < 0.05) and yeast isolates (P < 0.01). BTA(O2) recovered significantly more aerobic and facultatively anaerobic gram-negative bacilli (P < 0.05). In blood culture sets which produced growth of the same organisms in both the BTA(O2) and I(O2) systems, the BTA(O2) system detected growth sooner, but more rapid identification was possible with the I(O2) system by virtue of earlier isolation of colonies on solid media.
Significance of Salmonella typhi bacteriuria
Authors: Mathai E , John T J , Rani M , Mathai D , Chacko N , Nath V , Cherian A M
Published: January 2020

Bacteriuria due to Salmonella typhi usually occurs following recent typhoid fever or in chronic carrier states. Data from 18 patients with S. typhi bacteriuria, seen during 5 years, were analyzed. Fourteen patients had localized urinary tract infection due to S. typhi. Four others had bacteriuria, probably associated with typhoid fever. Localized abnormalities of the urinary tract and kidneys and also systemic diseases were found to predispose patients to S. typhi bacteriuria. Local abnormalities encountered included urolithiasis (n = 3), prostatic hypertrophy (n = 1), and tuberculosis (n = 1). One renal transplant recipient and another with lupus nephritis had S. typhi bacteriuria. One had associated strongyloidosis, and another was pregnant.
Three distinct genotypes within Candida parapsilosis from clinical sources
Authors: Lin D , Wu L C , Rinaldi M G , Lehmann P F
Published: January 2020

Three genetically distinct groups of Candida parapsilosis were detected among clinical isolates. These were distinguishable on the basis of isoenzyme profiles and DNA sequences of internally transcribed spacer (ITS) sequences flanking the 5.8S RNA gene. In an investigation of 45 strains, including 32 clinical isolates from Texas, C. parapsilosis group I composed the majority of the common clinical isolates. The type strain of C. parapsilosis was a member of this group. The 10 group II isolates were indistinguishable from group I strains when tested with the API 20C kit. The two group III isolates differed from those in groups I and II by being D-xylitol positive by the API 20C kit; however, isolates in all groups assimilated D-xylitol from broth. Isoenzyme profiles excluded the close relationship of any of these groups to Lodderomyces elongisporus, which is a teleomorphic yeast that has a physiological profile similar to that of C. parapsilosis. Although there were insignificant differences in the ITS2 rDNA sequences, comparisons of the ITS1 sequences revealed several differences. A sequence analysis of ITS1 in which missing bases were counted as mismatches showed the following similarities: group I versus group II, 87.7%; group I versus group III, 82.1%; group II versus group III, 84.5%. Also, the activity of secreted proteinase showed differences among the three groups, with many group I isolates having moderate to high activity. The degree of susceptibility to antifungal agents, amphotericin B, ketoconazole, and 5-fluorocytosine, could not be used to determine an isolate's group.
Comparison of CAPTIA syphilis G enzyme immunoassay with rapid plasma reagin test for detection of syphilis
Authors: Silletti R P
Published: January 2020

The CAPTIA Syphilis G enzyme-linked immunosorbent assay compared favorably with the rapid plasma reagin test when used to screen for syphilis in a low-risk population. The sensitivity and specificity of the CAPTIA Syphilis G test were 100 and 97.8%, respectively, for 646 routine specimens and 100 and 99.2%, respectively, for 265 specimens from obstetrics patients. Overall, for 911 specimens, the CAPTIA Syphilis G test showed a sensitivity of 100%, a specificity of 98.2%, and positive and negative predictive values of 78.9 and 100%, respectively. For the same population, the rapid plasma reagin test showed a sensitivity and a specificity of 96.4 and 97.5%, respectively, and positive and negative predictive values of 72 and 99.8%, respectively.
Determination of MICs for Mycobacterium avium-M. intracellulare complex in liquid medium by a colorimetric method
Authors: Gomez-Flores R , Gupta S , Tamez-Guerra R , Mehta R T
Published: January 2020

We investigated the potential of a rapid colorimetric microassay based on the reduction of dimethylthiazol-diphenyltetrazolium bromide (MTT) for determining the growth of Mycobacterium avium-M. intracellulare complex (MAC) and MICs of clofazimine, resorcinomycin A, and the quinolone PD 127391 against MAC. The reduction of MTT was directly proportional to the number of viable bacteria. A comparison of the MTT reduction test with the [3H]glycerol uptake assay showed the former to possess higher analytical sensitivity for detecting MAC growth in microtiter plates. The MIT reduction test avoids the use of radioisotopes and costly material and equipment; it is reliable, reproducible, and convenient for rapid routine susceptibility testing of MAC.
Direct detection of Mycobacterium tuberculosis complex in respiratory specimens by Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and Roche Amplicor Mycobacterium Tuberculosis Test
Authors: Vuorinen P , Miettinen A , Vuento R , Hällström O
Published: January 2020

Two hundred and fifty-six sputum, bronchoalveolar lavage, and bronchial and tracheal aspirate specimens from 243 patients were tested for the presence of Mycobacterium tuberculosis complex by auramine fluorochrome staining, rRNA target amplification (Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test [AMTD]), and PCR (Roche Amplicor Mycobacterium Tuberculosis Test [Amplicor PCR]. The results were compared with those of conventional Löwenstein-Jensen tube culture and BACTEC radiometric liquid culture. A total of 26 specimens from 18 patients were culture positive for M. tuberculosis. In addition, seven specimens were positive by staining and by culture for other Mycobacterium species but negative by nucleic acid amplification methods and were not included in the comparison. When compared with that for culture, the sensitivities of the techniques were as follows: for staining, 80.8%; for Gen-Probe AMTD, 84.6%; and for Roche Amplicor PCR, 84.6%. The specificities were 99.1, 98.7, and 99.1%, respectively. After resolution of discrepant results by review of the patients' clinical data, 29 specimens from 21 patients were considered positive, and the overall sensitivities, specificities, and positive and negative predictive values were 89.7, 100, 100, and 98.7% for culture; 75.9, 99.5, 95.7, and 96.9% for staining; 86.2, 100, 100, and 98.2% for Gen-Probe AMTD; and 82.8, 100, 100, and 97.9% for Roche Amplicor PCR, respectively. It is concluded that both nucleic acid amplification methods are rapid, sensitive, and specific methods for the detection of M. tuberculosis in respiratory specimens.
Comparison of three methods for culturing throat swabs from cystic fibrosis patients
Authors: Hoppe J E , Theurer-Mainka U , Stern M
Published: January 2020

In patients with cystic fibrosis who do not produce sputum, deep throat swabs are cultured for potential respiratory pathogens. Usually these swabs are directly streaked onto selective agar media. In a study of 50 pediatric cystic fibrosis patients, we compared this traditional method using rayon swabs with two methods having quantitative modifications: calcium alginate swabs eluted in Ringer's lactate and rayon swabs eluted in normal saline. The eluates were then processed quantitatively (three-step dilution series). The yield of potential pathogens was significantly higher with the two quantitative methods. Overall, the combination of alginate with Ringer's lactate was superior to the combination of rayon with saline, although only some of these differences achieved statistical significance.
Comparison of conventional stool concentration and preserved-smear methods with Merifluor Cryptosporidium/Giardia Direct Immunofluorescence Assay and ProSpecT Giardia EZ Microplate Assay for detection of Giardia lamblia
Authors: Zimmerman S K , Needham C A
Published: January 2020

Giardiasis is the most common parasitic infection in the United States. Variation in the numbers of cysts and/or trophozoites that are present along with the need for a skilled microscopist offer challenges in diagnosis. We compared the sediment wet preparation and permanent stained smear results (concentration in formalin-ethyl acetate and preparation of a smear prepared from a polyvinyl alcohol-preserved specimen) from 512 consecutive specimens with the results obtained by using the Merifluor Cryptosporidium/Giardia Direct Immunofluorescence Assay (DFA; Meridian Diagnostics, Inc., Cincinnati, Ohio) and the ProSpecT Giardia EZ Microplate Assay (EIA; Alexon, Inc., Sunnyvale, Calif.). The Merifluor DFA detected 33 of 33 positive specimens, and the ProSpecT EIA detected 32 of 33 positive specimens. The diagnostic sensitivities of the Merifluor DFA and the ProSpecT EIA were 100 and 97%, respectively. The specificities of the assays were 99.8%. The Merifluor DFA and the ProSpecT EIA appear to be equally sensitive, and both are more sensitive than conventional microscopy.
Evaluation of incubation times for urine cultures
Authors: Cavagnolo R
Published: January 2020

Nine hundred eighty-two predominantly outpatient urine cultures were evaluated for detection of uropathogens and identification and susceptibility testing after incubation for 12 to 16 h. These results were compared with results obtained after 36 and 60 h of incubation. There was 98% agreement in the detection of uropathogens and interpretation of culture results. The only consistent problem was in the detection of yeasts. Although uropathogens could be reliably detected as early as 12 h, the percentage of cultures considered too young to work on increased as incubation times decreased.
Evaluation of cleaving agents other than trypsin in direct agglutination test for further improving diagnosis of visceral leishmaniasis
Authors: el Harith A , Chowdhury S , al-Masum A , Semião-Santos S , Karim E , el-Safi S , Haque I
Published: January 2020

Trypsin treatment of Leishmania promastigote antigen has proved to be indispensible in the direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). In the present study four antigen batches were prepared with pronase (400 micrograms/ml), lipase (0.45% [wt/vol]), pancreatin (0.3% [wt/vol]), or 2-mercaptoethanol (2-ME) (1.2% [vol/vol]) at a ratio of 20:1 versus promastigote packed cell volume or a density of 10(8)/ml. Batches prepared in this way performed satisfactorily when compared with the performance of the initial trypsinated antigen. Even higher was the sensitivity and specificity of the 2-ME-processed antigen, scoring a minimum DAT titer of 1:102,400 in the VL and CVL group and a maximum of 1:400 in the negative control group. Corresponding titers ranging from 1:6,400 to 1:12,800 and 1:800 to 1:1,600 were obtained with the antigen variants processed with pronase, lipase, pancreatin, or trypsin. By combining the use of indigenous Leishmania donovani subspecies from Sudan, Bangladesh, or Morocco and incorporating 2-ME instead of trypsin in the antigen processing step, a threefold increase in titer was attained in sera from the respective areas where VL is endemic. 2-ME-processed antigen suspensions maintained stability at 4 degrees C for up to 9 months, as evidenced by the absence of autoagglutination and the reproducibility of DAT readings with standard sera. The specificity of DAT was further improved by supplementation of the sample diluent with 0.03 M urea and incubation of the test plates at 37 degrees C for 1 h. Titers ranging from 1:200 to 1:12,800 in the sera of patients and laboratory animals infected with various trypanosoma species were significantly reduce (</=1:200) or were rendered negative dilution of 1:25. Regardless of the infections caused by trypanosoma species, the sensitivity, specificity, and predictive value of a positive or negative test in DAT were 100%. Sera from patients who formerly had VL and who had been treated 6 to 36 months earlier remained reactive (>/=1:51,200) against 2-ME-processed antigen, despite the incorporation of urea into the DAT.
Identification of some clinical strains of CDC coryneform group A-3 and A-4 bacteria as Cellulomonas species and proposal of Cellulomonas hominis sp. nov. for some group A-3 strains
Authors: Funke G , Ramos C P , Collins M D
Published: January 2020

CDC coryneform group A-3 and A-4 bacteria were defined by Hollis and Weaver in 1981, but their taxonomic position is still unclear. By using biochemical and chemotaxonomical methods, four clinical strains belonging to CDC coryneform groups A-3 (n = 2) and A-4 (n = 2) were studied and could be assigned to the genus Cellulomonas, resulting in the first description of Cellulomonas strains isolated from clinical specimens. CDC coryneform group A-3 and A-4 strains were compared with the type strains of the seven species constituting the genus Cellulomonas at present as well as with the closely related species Oerskovia turbata, Oerskovia xanthineolytica, and Jonesia denitrificans, but their biochemical patterns were not compatible with the patterns of any of those species. Almost the entire sequences of the 16S rRNA genes of one representative strain of both CDC taxa were determined, and comparative sequence analysis confirmed the placement of the CDC coryneform group A-3 and A-4 strains studied in the Cellulomonas-Oerskovia subbranch of the actinomycetes. Both CDC taxa exhibited > 99% base pair homology within their 16S rDNAs. On the basis of phenotypic and molecular data, we formally propose a new species, Cellulomonas hominis sp. nov., for the CDC coryneform group A-3 bacteria examined. The type strain is DSM 9581. The precise taxonomic status of the CDC coryneform group A-4 strains studied remains to be established by quantitative DNA-DNA hybridizations.
Report of cases of and taxonomic considerations for large-colony-forming Lancefield group C streptococcal bacteremia
Authors: Carmeli Y , Ruoff K L
Published: January 2020

Traditionally, group C streptococci include four species: Streptococcus equisimilis, S. zooepidemicus, S. equi, and S. dysgalactiae, the first three of which are group C beta-hemolytic streptococci (GCBHS). However, many of the beta-hemolytic streptococci carrying Lancefield group C antigen isolated from clinical specimens are S. milleri. These organisms can be differentiated by colony size. We retrospectively collected data concerning large-colony-forming GCBHS bacteremia that occurred during a period of 8 years at the Massachusetts General Hospital. A total of 222 cases of beta-hemolytic streptococcal bacteremia were identified; data on the Lancefield grouping were available in 192 cases: 45 cases (23.6%) were group A, 96 cases (50%) were group B, 7 cases (3.6%) were group C (large colony forming), and 44 cases (22.9%) were group G. The medical records for cases of large-colony-forming GCBHS bacteremia were reviewed. In one case, the isolate was thought to be a contaminant; the other six cases are reported (five males and one female; mean age, 55 years). All patients had severe underlying conditions, and none had a history of exposure to animals. The clinical syndromes included two cases of cellulitis and one case each of endocarditis, myocardial infarction complicated by infection, pneumonia, and myofasciitis. The diagnoses for two patients with endovascular infections were delayed. Three of the six patients had fatal outcomes, and other two, after prolonged hospitalization, were transferred to a long-term rehabilitation center. We concluded that the severe outcomes reflect delay in diagnosis and treatment as well as the severity of the underlying diseases. The taxonomy of GCBHS is discussed. More reports differentiating large- and small-colony-forming GCBHS are needed.
Clinically practical seminested PCR for Burkholderia pseudomallei quantitated by enzyme immunoassay with and without solution hybridization
Authors: Kunakorn M , Markham R B
Published: January 2020

Diagnosis of melioidosis, an infectious disease caused by Burkholderia pseudomallei (formerly Pseudomonas pseudomallei), is made initially by antibody testing, which is not always sensitive or specific. We have developed two seminested PCR protocols combined with enzyme immunoassay (EIA) to detect the conserved ribosomal regulatory region of B. pseudomallei. Both PCRs used one biotinylated primer for capturing PCR products on EIA plates. One system, termed solution hybridization EIA (SHEIA), hybridized PCR products with a digoxigenin-labeled probe in solution. Another system, termed primer-labeled EIA (PLEIA), used a digoxigenin-labeled nested primer to generate products that were directly detected without hybridization. To prevent amplicon contamination, pre-PCR uracil DNA glycosylase treatment or post-PCR UV irradiation was incorporated into each system. By a rapid method of blood sample preparation for PCR, these systems had sensitivities of 75 bacteria per ml for SHEIA and 300 bacteria per ml for PLEIA. No nonspecific amplification of other bacterial DNAs was detected. This seminested PCR coupled with SHEIA or PLEIA fulfills all the requirements for a diagnostic test to be used in developing countries where B. pseudomallei is endemic.
Development of specific fluorescent-antibody test for tissue form of Penicillium marneffei
Authors: Kaufman L , Standard P G , Anderson S A , Jalbert M , Swisher B L
Published: January 2020

The diagnosis of penicilliosis marneffei can be difficult because the clinical manifestations mimic those of tuberculosis, histoplasmosis, and other mycotic infections. Furthermore, the tissue form of Penicillium marneffei can be confused with those of Histoplasma capsulatum and Cryptococcus neoformans. To facilitate the rapid detection and identification of P. marneffei in clinical materials, we sought to develop a specific indirect fluorescent-antibody (IFA) reagent for this dimorphic pathogen. Preliminary IFA studies with yeast-like cells (fission arthroconidia) of P. marneffei indicated that these cellular elements stained with antiglobulins against culture filtrate antigens and whole yeast-like cellular antigens. Both types of antiglobulins reacted with the yeast-like cells of P. marneffei and with H. capsulatum, but not with their respective mycelial forms. The antiglobulins also failed to react with the yeast and hyphal forms of a variety of other heterologous fungi. Specific antiglobulins useful in an IFA test for identifying P. marneffei yeast-like cells in culture or in clinical materials were produced by adsorptions with yeast-form cells of H. capsulatum. The yeast-like culture filtrate antigens of P. marneffei are preferred for use in the production of the specific antiglobulins because they stained P. marneffei yeast-like elements more intensely than antiglobulins produced against intact yeast-like cells.
Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae
Authors: Westerman R B , Phillips R M , Joens L A
Published: January 2020

Serpulina (Treponema) hyodysenteriae is the causative agent of swine dysentery, a contagious mucohemorrhagic disease of the colon. Diagnosis of swine dysentery is extremely difficult because of the presence of cross-reactive antibodies to the proteins of S. hyodysenteriae and Serpulina innocens, a nonpathogenic inhabitant of the porcine large intestine. Therefore, monoclonal antibodies (MAbs) against the serotype-specific lipooligosaccharide (LOS) antigens of S. hyodysenteriae were produced to rapidly differentiate S. hyodysenteriae from S. innocens. Whole-cell preparations of S. hyodysenteriae serotypes 1 through 7 were used as antigens. MAbs were characterized by an indirect enzyme-linked immunosorbent assay with whole-cell or LOS antigen and by Western blot (immunoblot) analysis with whole-cell lysates as antigen. A total of 12 LOS-specific MAbs which could identify and differentiate the seven original serotypes of S. hyodysenteriae were produced. The MAb serospecificities are as follows: MAb 9G8, serotype 1; MAb 31D9, serotype 2; MAb 7D3, serotypes 2 and 7; MAb 24B7, serotype 3; MAb 13C2, serotype 4; MAb 18E9, serotype 4; MAb 2B7, serotype 6; MAb 1D2, serotypes 2, 5, and 7; MAb 9C5, serotypes 2, 5, and 7; MAb 11C9, serotype 7; MAb 11E10, serotype 7; and MAb 6G11, serotype 7.
Genotypic analysis of pseudoepidemic due to contamination of Hanks' balanced salt solution with Candida parapsilosis
Authors: Deresinski S C , Clemons K V , Kemper C A , Roesch K , Walton B , Stevens D A
Published: January 2020

A cluster of isolates of Candida parapsilosis recovered from clinical specimens was demonstrated, by both classical and molecular epidemiological techniques, to have resulted from contamination in the laboratory. The source of the pseudoepidemic was a repeatedly utilized contaminated container of Hanks' balanced salt solution used in specimen processing. The patterns of restriction fragment length polymorphisms of DNA extracted from the clinical and environmental isolates were identical to each other but composed a newly identified unique C. parapsilosis DNA type.
Antibodies against whole sonicated Borrelia burgdorferi spirochetes, 41-kilodalton flagellin, and P39 protein in patients with PCR- or culture-proven late Lyme borreliosis
Authors: Oksi J , Uksila J , Marjamäki M , Nikoskelainen J , Viljanen M K
Published: January 2020

The sensitivities and specificities of three enzyme-linked immunosorbent assays (ELISAs) for Borrelia burgdorferi antibodies were compared for 41 patients presenting with symptoms compatible with late Lyme borreliosis (LB) and 37 healthy controls. All subjects were living in southwestern Finland, where LB is endemic. Only patients with culture- or PCR-proven disease were enrolled in the study. The antigens of the ELISAs consisted of sonicated spirochetes, 41-kDa flagellin, and recombinant P39 protein of B. burgdorferi. Fifteen patients had strongly or moderately positive results in the serological assay(s), 19 patients had only weakly positive or borderline antibody levels, and the remaining 7 patients were seronegative by ELISA. The sensitivities of the ELISAs were 78.0% with sonicate antigen, 41.5% with 41-kDa flagellin, and 14.6% with P39 protein. The specificities of the tests were 89.2, 86.5, and 94.6%, respectively. The sonicate antigen ELISA seems to be an effective screening method. These results show that antibodies to B. burgdorferi may be present in low levels or even absent in patients with culture- or PCR-proven late LB. Therefore, in addition to serological testing, the use of PCR and cultivation is recommended in the diagnosis of LB.
Characterization of feline Helicobacter pylori strains and associated gastritis in a colony of domestic cats
Authors: Handt L K , Fox J G , Stalis I H , Rufo R , Lee G , Linn J , Li X , Kleanthous H
Published: January 2020

Twenty-four young adult domestic cats from a commercial vendor were found to be infected with Helicobacter pylori. Histopathologic analyses, selected electron microscopy, and urease mapping were performed on mucosal samples collected from the cardias and fundi, bodies, and antra of these cats' stomachs. H. pylori organisms were abundant in all areas of the stomach on the basis of histologic evaluation and urease mapping. H. pylori infection was associated with a moderate to severe lymphofollicular gastritis in 21 of 24 cats (88%). The gastritis was most pronounced in the antral region and consisted mainly of multifocal lymphoplasmacytic follicular infiltrates in the deep mucosa. The severity of gastritis in the antrum corresponded to high numbers of H. pylori there on the basis of the use of the urease assay as an indicator of H. pylori colonization. Ten of 24 cats (42%) also had small to moderate numbers of eosinophils in the gastric mucosa. All 24 cats had gastric lymphoid follicles, with follicles being most prevalent in the antrum. Electron microscopy of gastric tissue revealed numerous H. pylori organisms, some of which were closely adhered to the mucosal epithelium. Human H. pylori gene-specific primers to ureA and ureB amplified products of similar sizes from H. pylori cat isolates. Digestion of the products with restriction enzymes resulted in fragments characteristic of the restriction fragment length polymorphism patterns of H. pylori isolates from humans. In the domestic cat, H. pylori infection is associated with a lymphofollicular gastritis, consisting of lymphocytic and plasmacytic infiltration into the lamina propria, and the organism appears to provide chronic antigenic stimulation resulting in the formation of gastric lymphoid follicles.
Evaluation of Rapid ATB Staph for 5-hour antimicrobial susceptibility testing of Staphylococcus aureus. Groupement pour le Dépistage, L'Etude et la Prévention des Infections Hospitalières-Groep ter Opsporing, Studie en Preventie van Infecties in de Ziekenhuizen
Authors: Struelens M J , Nonhoff C , van der Auwera P , Mertens R , Serruys E
Published: January 2020

The accuracy of Rapid ATB Staph (bioMérieux, La Balme-Les Grottes, France) for detection of oxacillin resistance and for detection susceptibility to 11 other antimicrobial agents in 553 and 519 Staphylococcus aureus isolates, respectively, was evaluated by comparing results with those produced by oxacillin agar screen and agar dilution methods, respectively. Further characterization of isolates with discrepant results for oxacillin testing was done by PCR detection of the nuc and mecA genes. By oxacillin agar screening, there were 307 oxacillin-resistant and 246 oxacillin-susceptible isolates. Rapid ATB results were obtained in 5 h for 515 (93.2%) of the isolates tested. Rapid ATB showed 97.0% sensitivity for detection of oxacillin resistance, confirmed by the presence of the mecA gene. After repeat testing of isolates flagged by the ATB software as possible errors, sensitivity increased to 99% for oxacillin-resistant isolates. Essential agreement with agar dilution testing for susceptibility to amoxicillin-clavulanic acid, gentamicin, erythromycin, clindamycin, and ciprofloxacin, as estimated by Youden's J statistic, was > 0.90. Subpopulations of isolates with significantly increased MICs of amikacin, rifampin, and minocycline, indicating borderline susceptibility, were detected by Rapid ATB and categorized as resistant. Rapid ATB Staph showed adequate accuracy for detection within 5 h of the oxacillin- and multiple-drug-resistant S. aureus isolates currently prevalent in Belgium.
Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay
Authors: van Vliet A H , van der Zeijst B A , Camus E , Mahan S M , Martinez D , Jongejan F
Published: January 2020

Currently available serological tests for cowdriosis (Cowdria ruminantium infection) in domestic ruminants are hampered by their low specificities because of cross-reactivity with Ehrlichia spp. The use of recombinant major antigenic protein (MAP1) of C. ruminantium for serodiagnosis was investigated. Overlapping fragments of the MAP1 protein were expressed in Escherichia coli and were reacted with sera from sheep infected with either C. ruminantium or Ehrlichia ovina. Two immunogenic regions on the MAP1 protein, designated MAP1-A and MAP1-B, were identified. MAP1-A was reactive with C. ruminantium antisera, E. ovina antisera, and three MAP1-specific monoclonal antibodies, whereas MAP1-B reacted only with C. ruminantium antisera. An indirect enzyme-linked immunosorbent assay (ELISA) based on MAP1-B was further developed and validated with sera from animals experimentally infected with C. ruminantium or several Ehrlichia spp. Antibodies raised in sheep, cattle, and goats against nine isolates of C. ruminantium reacted with MAP1-B. Cross-reactivity with MAP1-B was limited to Ehrlichia canis and Ehrlichia chaffeensis, two rickettsias which do not infect ruminants. Antibodies to Ehrlichia spp. which do infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not react with MAP1-B. Antibody titers to C. ruminantium in sera from experimentally infected cattle, goats, and sheep were detectable for 50 to 200 days postinfection. Further validation of the recombinant MAP1-B-based ELISA was done with sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. A total of 159 of 169 samples which were considered to be false positive by immunoblotting or indirect ELISA did not react with MAP1-B. In conclusion, recombinant MAP1-B may be a suitable antigen for a sensitive serological test for cowdriosis, with dramatically improved specificity.
Phenotypic and genotypic characterization of unusual vaginal isolates of Candida albicans from Africa
Authors: Tietz H J , Küssner A , Thanos M , De Andrade M P , Presber W , Schönian G
Published: January 2020

As expected by its global prevalence, the most frequently isolated species of yeast from vaginal swabs obtained from patients in Africa was Candida albicans, which accounted for 53 of 85 (62.4%) of the isolates from women in Madagascar and 35 of 54 (64.8%) of the culture-positive women in Angola. However, 40% of the Madagascan and 23% of the isolates from Angola, as well as two isolates obtained from one German patient, were not able to utilize the amino sugars glucosamine and N-acetylglucosamine as the sole carbon source. These isolates were able to form germ tubes but did not form chlamydospores. The correct identification as C. albicans was made possible only by using a PCR-based method of DNA fingerprinting. Only minor phenotypic and genotypic variation was observed among these strains. Whether they represent a distinct clone that is found mainly in Africa is not clear. The relevance of the amino sugar catabolic pathway in C. albicans is discussed in view of these results.
Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans
Authors: Aguero-Rosenfeld M E , Nowakowski J , Bittker S , Cooper D , Nadelman R B , Wormser G P
Published: January 2020

We investigated the appearance and evolution of immunoglobulin M (IgM) and IgG antibodies to Borrelia burgdorferi in 46 patients with culture-proven erythema migrans (EM). All patients received antimicrobial treatment and were prospectively evaluated for up to 1 year. A total of 257 serially collected serum samples were tested by commercial IgG-IgM enzyme-linked immunosorbent assay and separate IgM and IgG immunoblots (IBs). At the baseline, 33% of the patients had a positive ELISA result and 43% of the patients had a positive IgM IB result by using the criteria of the Centers for Disease Control and Prevention-Association of State and Territorial Public Health Laboratory Directors for the interpretation of IB results. Positive serology at the baseline and the rate of seroconversion correlated directly with disease duration and/or evidence of dissemination prior to treatment. At days 8 to 14 after the baseline, 91% of patients had a positive ELISA result and/or IgM IB result. Peak IgM antibody levels were seen at this time in patients with localized or disseminated disease. The most frequent IgM bands at the baseline and the peak were of 24 kDa (OspC), 41 kDa, and 37 kDa. Although 89% of the patients developed IgG antibodies as determined at a follow-up examination, only 22% were positive by the IgG IB criteria of the Centers for Disease Control and Prevention-Association of State and Territorial Public Health Laboratory Directors. The persistence of antibodies was directly related to disease duration and/or dissemination prior to treatment. Since IgM antibodies to the 24- and 41-kDa antigens remained detectable for long periods, 38% of IgM IBs were still positive at 1 year postbaseline. IgM to antigens of 39, 58, 60, 66, or 93 kDa, conversely, were most often seen in sera obtained within 1 month postbaseline. Their presence may be of assistance in confirming a recent infection with B. burgdorferi in individuals living in areas where Lyme disease is endemic.
Evaluation of commercial methods for determining antimicrobial susceptibility of Streptococcus pneumoniae
Authors: Tenover F C , Baker C N , Swenson J M
Published: January 2020

Seven commercial systems for antimicrobial susceptibility testing of Streptococcus pneumoniae were evaluated by using a challenge set of 55 pneumococcal isolates with a variety of resistance phenotypes and genotypes. Overall, the results produced by the Pasco and Etest methods were found to be acceptable for all drugs tested except for trimethoprim-sulfamethoxazole testing by the Etest. The Just One system for penicillin MIC testing was also judged to be acceptable (minor error rate, 5.5%). Although the Sensititre and MicroTech methods both produced 12.7% minor errors with penicillin, the Sensititre method classified penicillin-intermediate strains as resistant or vice versa, while four of MicroTech's errors were among intermediate strains that were classified as susceptible. The MicroMedia (minor error rate, 16.4%) and MicroScan Rapid (minor error rate, 63.6%) methods produced unacceptably high levels of errors when testing penicillin. Minor error rates for cefotaxime and ceftriaxone ranged from a low of 12.7% (Etest and Sensititre) to a high of 28% (MicroMedia). Error rates were low for erythromycin, tetracycline, and chloramphenicol by most methods with the exception of the MicroScan method, which had a high very major error rate for erythromycin (34.6%). For testing of beta-lactam drugs, the Pasco, Etest, and Just One tests for penicillin are the most accurate methods; the Sensititre method also provided acceptable results.
Genotypic and phenotypic characterization of Escherichia coli isolates from dogs manifesting attaching and effacing lesions
Authors: Beaudry M , Zhu C , Fairbrother J M , Harel J
Published: January 2020

Thirteen Escherichia coli isolates from dogs manifesting attaching and effacing lesions were characterized genetically with respect to the presence of the following virulence determinants associated with human enteropathogenic E. coli (EPEC): eaeA, encoding the outer membrane protein intimin; eaeB, which is necessary for inducing signal transduction; bfpA, encoding the bundle-forming pilus; and the EAF (stands for EPEC adherence factor) plasmid. These isolates were also analyzed phenotypically with respect to adherence to mammalian cells in vivo and in vitro. Nine of these 13 isolates were found to be eaeA positive by PCR: four of these nine were eaeB positive. The 5' end, but not the 3' end, of the eaeA gene was amplified by PCR when primers derived from the eaeA gene of EPEC were used. Six and eight of these 13 isolates were found to be bfpA positive and EAF positive, respectively. The bfpA gene and EAF locus were found on high-molecular-weight plasmids, whereas the eaeA and eaeB genes were chromosomally located when present. Only one canine E. coli isolate, 4221, which was positive for eaeA, eaeB, bfpA, and EAF, adhered to HEp-2 cells in a localized manner and was positive in the fluorescence actin staining test. The nine eaeA-positive isolates adhered to the mucosal surface of piglet ileal explants and induced some microvillus effacement. However, when tested in experimentally inoculated gnotobiotic piglets, isolate 4221 did not induce attaching and effacing lesions at any level of the intestinal tract. Our results indicate that canine E. coli isolates associated with attaching and effacing lesions share some properties with human EPEC but form a heterogeneous group.
Comparison of two leukocyte extraction methods for cytomegalovirus antigenemia assay
Authors: García A , Niubò J , Benítez M A , Viqueira M , Pérez J L
Published: January 2020

We carried out a prospective, parallel, and blind study on 113 blood samples from immunocompromised patients in order to compare two leukocyte extraction methods (6% dextran sedimentation and Polymorphprep separation) for cytomegalovirus (CMV) antigenemia assay. CMV was detected in 38 samples by antigenemia assay (34 by dextran sedimentation and 35 by Polymorphprep separation). No differences either in the number of positive specimens (P = 1) or in the mean CMV-positive cell counts (P = 0.41) were observed between the two leukocyte extraction methods. In conclusion, the two methods performed equally well for this assay.
Genomic DNA fingerprint analysis of biotype 1 Gardnerella vaginalis from patients with and without bacterial vaginosis
Authors: Wu S R , Hillier S L , Nath K
Published: January 2020

Of the 20 biotype 1 Gardnerella vaginalis isolates analyzed, 10 from patients with bacterial vaginosis and 10 from patients without bacterial vaginosis, none shared the same DNA fingerprint. However, a 1.18-kb HindIII fragment was common among 18 of the 20 biotype 1 isolates in a restriction fragment length polymorphism analysis with a 7.9-kb G. vaginalis DNA probe.
Serratia rubidaea as an invasive pathogen
Authors: Ursua P R , Unzaga M J , Melero P , Iturburu I , Ezpeleta C , Cisterna R
Published: January 2020

Serratia rubidaea biotype 1 was isolated from the bile and blood of a patient with a bile tract carcinoma obstructing the common bile duct and who underwent invasive procedures. The infection was cleared after adequate treatment with antibiotics.
New laboratory guidelines for serologic diagnosis of Lyme disease: evaluation of the two-test protocol
Authors: Ledue T B , Collins M F , Craig W Y
Published: January 2020

Recent guidelines established by the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) and the U.S. Centers for Disease Control and Prevention (CDC) recommend the use of a two-test protocol for the serologic diagnosis of Lyme disease (LD). The two-test protocol relies on a sensitive screening test, which is followed by specific immunoglobulin M (IgM) and/or IgG immunoblotting (IB), depending on the date of disease onset, of all samples with equivocal and positive screening test results. We evaluated a commercially available IgM-IgG enzyme-linked immunosorbent assay (ELISA) and separate IB tests for IgM and IgG antibodies to Borrelia burgdorferi as candidate assays for the two-test protocol. Serum samples obtained from healthy controls (n = 29), from patients with diagnoses or laboratory findings associated with serologic cross-reactivity to LD (n = 24), and from patients with well-documented early- and late-stage LD provided by the CDC and the College of American Pathologists (n = 53) were examined to determine each assay's individual sensitivity and specificity. No false-positive results were detected among the healthy controls by either ELISA or IB, whereas four false-positive ELISA results were recorded within the cross-reactive group. None of these sera, however, were positive for either IgM or IgG reactivity according to IB band criteria. With regard to the patients with LD, we determined the sensitivity and specificity of the ELISA to be 96 and 100%, respectively, compared with the reference data provided for these specimens. When we compared our IB results with data from CDC, the assay sensitivity and specificity were 80 and 96.2%, respectively, for IgM and 81.8 and 95.8%, respectively, for IgG. Pursuant to this evaluation we assessed the suitability of the two-test protocol by performing a retrospective analysis using clinical history to define samples as positive or negative for LD. We determined clinical sensitivity and specificity for all study subjects (n = 112) to be 50 and 100%, respectively. A reduction in the clinical sensitivity of the two-test protocol was associated with a lack of antibody response or seroconversion in LD patients treated with antibiotics. We conclude that the CDC-ASTPHLD guidelines provide useful criteria for test performance and interpretation aimed at standardizing the serologic diagnosis of LD.
Differentiation of Mycobacterium bovis isolates from animals by DNA typing
Authors: Skuce R A , Brittain D , Hughes M S , Neill S D
Published: January 2020

The insertion sequence IS6110 and the direct repeat (DR) specific to tuberculosis complex mycobacteria and the highly repeated DNA sequence, the polymorphic GC-rich repeat sequence (PGRS), were systematically used to identify restriction fragment length polymorphisms (RFLPs) within 210 isolates of Mycobacterium bovis. The isolates were primarily of bovine origin, but isolates from badgers, feral deer, sheep, humans, and a pig were included. The RFLP probes IS6110, DR, and PGRS individually identified 17, 18, and 18 different RFLP types, respectively, but in combination these probes identified a total of 39 different M. bovis RFLP types. The recommendations (J. D. A. van Embden, M. D. Cave, J. T. Crawford, J. W. Dale, K. D. Eisenach, B. Gicquel, P. W. M. Hermans, C. Martin, R. McAdam, T. M. Shinnick, and P. M. Small, J. Clin. Microbiol. 31:406-409, 1993) for a standardized RFLP analysis for M. tuberculosis were adapted to facilitate gel documentation, image analysis, and construction of a database of RFLP types. In the present study the same M. bovis RFLP types were evident in the various animal species included, indicating that the strains were not host restricted. Application of these techniques to defined field studies should help elucidate more accurately aspects of the epidemiology of bovine tuberculosis in different countries.