bluuebunny/crossref_metadata_2025 huggingface dataset:
Serotyping and Genotyping of Genital
Chlamydia trachomatis
Isolates Reveal Variants of Serovars Ba, G, and J as Confirmed by
omp1
Nucleotide Sequence Analysis
Authors: Morré Servaas A. , Ossewaarde Jacobus M. , Lan Jar , van Doornum Gerard J. J. , Walboomers Jan M. M. , MacLaren David M. , Meijer Chris J. L. M. , van den Brule Adriaan J. C.
Published: December 2019

Urogenital isolates (
n
= 93) of
Chlamydia trachomatis
were differentiated into serovars and variants by serotyping with monoclonal antibodies and genotyping by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified
omp1
gene, respectively. The types of 87 of the 93 isolates (94%) were identical, as determined by both methods. Among these 87 isolates, 3 isolates were identified as the recently described new serovariant Ga/IOL-238 by
omp1
nucleotide sequence analysis of the variable domains. Of the remaining six isolates, three isolates serotyped as both L2 and Ba but were identified as Ba/A-7 by genotyping by RFLP analysis of
omp1
. The
omp1
nucleotide sequences of variable domains VD1, VD2, and VD4 of these urogenital Ba strains were identical to the sequences of the variable domains of Ba/J160, an ocular Ba type. The three remaining isolates were serotyped as J, but the patterns obtained by RFLP analysis of
omp1
, which were identical for the three isolates, differed from that of prototype serovar J/UW36.
omp1
nucleotide sequence analysis revealed that these strains are genovariants of serovar J/UW36. Nucleotide sequence differences between serovar J/UW36 and this J genovariant, designated Jv, were found in both variable and constant domains. In conclusion, this study shows that the PCR-based genotyping of clinical
C. trachomatis
isolates by RFLP analysis of
omp1
has a higher discriminatory power and is more convenient than serotyping. Variants of
C. trachomatis
serovars Ba, G, and J were identified and characterized.
Evaluation of a New Competitive Immunoassay (BioElisa Syphilis) for Screening for
Treponema pallidum
Antibodies at Various Stages of Syphilis
Authors: Ebel Anne , Bachelart Loïc , Alonso Jean-Michel
Published: December 2019

The BioElisa Syphilis, a new competitive enzyme immunoassay (EIA) for
Treponema pallidum
whole antigen that uses specific human immunoglobulin G (IgG) antibodies as the competitor, was evaluated for potential use in screening for syphilis at various stages. The results obtained by this competitive EIA were compared with those obtained by the fluorescent treponemal antibody absorption (FTA-abs) test and the
T. pallidum
hemagglutination assay (TPHA). Serum samples from 434 patients with positive TPHA and FTA-abs test results, including patients with primary, latent, secondary, and tertiary syphilis and neurosyphilis, were investigated. Two samples tested negative by competitive EIA but were weakly reactive by the TPHA and the FTA-abs test. Sixteen serum samples from patients with clinically documented active syphilis, including several patients infected with human immunodeficiency virus, tested positive by the competitive EIA. There was a direct inverse correlation between EIA indices and titers in the TPHA and the FTA-abs test for all samples that tested positive. Specificity was assessed by testing 358 serum samples which tested negative for syphilis by TPHA and the FTA-abs test, including 100 serum samples from patients with documented infectious or autoimmune diseases. Only two serum samples gave a weakly positive EIA result. Thus, competitive EIA had a sensitivity of 99.5% and a specificity of 99.4% relative to the results of the FTA-abs test and TPHA. Our evaluation shows that BioElisa Syphilis is a sensitive, specific, and simple assay for screening for syphilis.
Rapid, Low-Technology MIC Determination with Clinical
Mycobacterium tuberculosis
Isolates by Using the Microplate Alamar Blue Assay
Authors: Franzblau Scott G. , Witzig Richard S. , McLaughlin James C. , Torres Patricia , Madico Guillermo , Hernandez Antonio , Degnan Michelle T. , Cook Mary B. , Quenzer Virginia K. , Ferguson Robert M. , Gilman Robert H.
Published: December 2019

A colorimetric, microplate-based Alamar Blue assay (MABA) method was used to determine the MICs of isoniazid (INH), rifampin, streptomycin (SM), and ethambutol (EMB) for 34 Peruvian
Mycobacterium tuberculosis
isolates (including both pansensitive and multidrug-resistant strains) and the H
37
Rv strain by using bacterial suspensions prepared directly from solid media. Results for all isolates were available within 8 days. Discordant results were observed on initial tests for 3 of 16 INH-susceptible isolates, 5 of 31 EMB-susceptible isolates, and 2 of 4 SM-resistant isolates (by the BACTEC 460 system). The overall agreements between the MICs obtained by MABA and the results obtained with the BACTEC 460 system were 87.9% for initial results and 93.6% after retesting 12 of 17 samples with discrepant results. Interpretation of MABA endpoints improved with technical experience. The MABA is a simple, rapid, low-cost, appropriate technology which does not require expensive instrumentation and which makes use of a nontoxic, temperature-stable reagent.
Detection of
Chlamydia trachomatis
by the Gen-Probe AMPLIFIED Chlamydia Trachomatis Assay (AMP CT) in Urine Specimens from Men and Women and Endocervical Specimens from Women
Authors: Crotchfelt Kimberly A. , Pare Barbara , Gaydos Charlotte , Quinn Thomas C.
Published: December 2019

Molecular biology-based amplification methods are significantly more sensitive than other methods for the detection of
Chlamydia trachomatis
. The performance characteristics of the new Gen-Probe AMPLIFIED Chlamydia Trachomatis Assay (AMP CT) with endocervical and urine specimens were compared to those of culture for patients attending two Baltimore City sexually transmitted disease clinics and a clinic for adolescents. AMP CT uses transcription-mediated amplification (TMA) and hybridization protection assay procedures to qualitatively detect
C. trachomatis
by targeting a 23S rRNA. Discrepant results between culture-negative and AMP CT-positive specimens were resolved by direct fluorescent-antibody staining of sedimented culture transport medium for elementary bodies and by TMA with 16S rRNA as a target. Following discrepant analysis, for 480 female urine specimens AMP CT had a sensitivity of 93.8% and a specificity of 100%. For 464 male urine specimens, the resolved sensitivity and specificity of AMP CT were 95.6 and 98.7%, respectively. For the 479 endocervical swab specimens the sensitivity of AMP CT was 100% and the specificity was 99.5%. Resolved culture sensitivities of AMP CT for female and male swab specimens were 52.3 and 58.9%, respectively. These results demonstrate that AMP CT is highly sensitive for the detection of
C. trachomatis
in endocervical specimens and in urine specimens from men and women.
Rapid Detection of Penicillin-Resistant
Streptococcus pneumoniae
in Cerebrospinal Fluid by a Seminested-PCR Strategy
Authors: du Plessis Mignon , Smith Anthony M. , Klugman Keith P.
Published: December 2019

A seminested-PCR assay, based on the amplification of the pneumococcal penicillin-binding protein 2B gene (
pbp2B
), was developed for the detection of penicillin-resistant and -susceptible pneumococci in cerebrospinal fluid (CSF) specimens. Species-specific primers (P5 and P6) which amplified a 682-bp conserved region of the transpeptidase-encoding region of the
pbp2B
gene were used. Four “resistance” primers were designed to bind to altered areas of the
pbp2B
gene identified in penicillin-resistant South African wild-type strains. Together with the downstream primer P6, the upstream resistance primers amplified fragments which were used to detect the presence of penicillin resistance. This system identified all 35 of the
S. pneumoniae
isolates evaluated, including strains of 11 different serotypes and a range of penicillin-resistant and -susceptible strains. The specificity of the assay was demonstrated by its inability to amplify DNA from other bacterial species which commonly cause meningitis. It was possible to detect pneumococcal DNA from culture-negative CSF inoculated with 2.5 pg of purified DNA or 18 CFU. Analysis of 285 CSF specimens showed that PCR detected the pneumococcus in 18 samples positive by culture, including the identification of four penicillin-resistant isolates. The positive predictive value and the negative predictive value of the assay were each 100%.
First Identification of Autochthonous
Cryptococcus neoformans
var.
gattii
Isolated from Goats with Predominantly Severe Pulmonary Disease in Spain
Authors: Baró Teresa , Torres-Rodríguez Josep M. , De Mendoza Miguel Hermoso , Morera Yolanda , Alía Concepción
Published: December 2019

Cryptococcus neoformans
var.
gattii
is associated with
Eucalyptus
trees growing in various tropical and subtropical regions of the world. The identification of 13 autochthonous strains of
C. neoformans
var.
gattii
in Spain is reported. These strains were isolated from lung (10 samples), liver (1 sample), and brain (2 samples) tissue specimens from six goats suffering from predominantly severe pulmonary disease that were autopsied. The animals were members of five different herds of goats grazing in rural areas of the province of Cáceres (Extremadura, Spain). Between 1990 and 1994, there were five outbreaks, in which between 2.5 and 12% of the goats were affected. Although respiratory symptoms (pneumonia) associated with cachexia were the predominant clinical picture in all outbreaks, brain and liver involvement was also documented in three of the five outbreaks. Biotyping was performed by culturing the isolates on
l
-canavanine-glycine-bromothymol blue medium and testing them for the assimilation of
d
-proline and
d
-tryptophan. Serotyping by agglutination tests confirmed the characterization of all strains as
C. neoformans
var.
gattii
serotype B. This is the first confirmation of the presence of this variety in Spain, with a peculiar ability to produce severe pulmonary and systemic disease in normal goats, particularly in the form of outbreaks of pneumonia in association with cachexia.
Development and Evaluation of a PCR-Based Assay for Detection of
Haemobartonella felis
in Cats and Differentiation of
H. felis
from Related Bacteria by Restriction Fragment Length Polymorphism Analysis
Authors: Messick Joanne B. , Berent Linda M. , Cooper Sandra K.
Published: December 2019

The 16S rRNA gene of
Haemobartonella felis
was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of
H. felis
-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of
H. felis
from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of
Eperythrozoon suis
,
Mycoplasma genitalium
, and
Bartonella bacilliformis
. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of
H. felis
organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes
Dde
I and
Mnl
I for distinguishing
H. felis
from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of
H. felis
infection in cats.
Separation among Species of
Mycobacterium terrae
Complex by Lipid Analyses: Comparison with Biochemical Tests and 16S rRNA Sequencing
Authors: Torkko Pirjo , Suutari Merja , Suomalainen Sini , Paulin Lars , Larsson Lennart , Katila Marja-Leena
Published: December 2019

Fatty acids, alcohols, and mycolic acid cleavage products were determined for 13 ATCC strains and 24 clinical isolates, which were initially identified by biochemical and growth characteristics as the
Mycobacterium terrae
complex. The clinical isolates were also analyzed by partial sequencing of the 16S rRNA gene, which divided them into five genetic entities,
M. triviale
(three strains),
M. terrae
(four strains),
M. nonchromogenicum
sensu stricto (seven strains),
Mycobacterium
sp. strain MCRO 6 (seven strains), and
Mycobacterium
sp. strain 31958 (one strain). After acidic methanolysis, secondary alcohols were a characteristic feature in all members of the
M. terrae
complex but
M. triviale
. In addition to the prominent secondary alcohols, 2-octadecanol and 2-eicosanol, two previously unidentified alcohols, 2-(8,15-dimethyl)docosenol and 2-(8,17-dimethyl)tetracosenol, were detected in
M. nonchromogenicum
,
Mycobacterium
sp. strain MCRO 6, and
Mycobacterium
sp. strain 31958. Only 2-(8,17-dimethyl)tetracosenol was detected in trace amounts in
M. terrae
. Genetic differences were associated with differences in phenotypic characteristics, including growth at 42°C and pyrazinamidase production. Based on fatty acid and alcohol composition and biochemical and genetic characteristics,
M. nonchromogenicum
and
Mycobacterium
sp. strains MCRO 6 and 31958 were found to be a closely related group, named the
M. nonchromogenicum
complex. Detected genetic variations associated with phenotypic characteristics may indicate further species separation of this complex. In conclusion, the results of gas-liquid chromatography fatty acid analysis, combined with those of a Tween 80 test, enable identification of the species of the
M. terrae
complex and their separation from other nonpigmented slowly growing mycobacteria.
Evolution of an Endemic Methicillin-Resistant
Staphylococcus aureus
Population in an Australian Hospital from 1967 to 1996
Authors: Givney Rodney , Vickery Alison , Holliday Anne , Pegler Mary , Benn Richard
Published: December 2019

The evolution over 30 years of a population of methicillin-resistant
Staphylococcus aureus
(MRSA) from a tertiary referral hospital was studied by phylogenetic analysis of
Sma
I-generated restriction fragment length polymorphisms (RFLPs). The results suggest that a new clone of MRSA appeared at the hospital in the early 1980s, which, although usually retaining its ancestral phage-type, developed four different RFLP pulsotypes in the next 16 years. This finding indicates that multiple RFLP patterns in MRSA do not necessarily represent multiple clones deriving from different
mec
gene transfer events. Such variation within a clone may be significant in the interpretation of RFLP patterns during outbreaks and emphasizes the need to use two typing methods in studies of such populations. Since the appearance of new clones of MRSA is a relatively rare event, cross-infection control is paramount in the prevention of MRSA dissemination.
Epidemiological Investigation of Vaginal
Saccharomyces cerevisiae
Isolates by a Genotypic Method
Authors: McCullough Michael J. , Clemons Karl V. , Farina Claudio , McCusker John H. , Stevens David A.
Published: December 2019

Saccharomyces cerevisiae
is a ubiquitous, ascomycetous yeast, and vaginitis caused by this organism has been reported only very rarely. The aim of the present investigation was to assess the epidemiological relatedness of a group of vaginal and commercial
S. cerevisiae
isolates by a previously reported genetic typing method, which divided the isolates into two broad groups with numerous subtypes. Nineteen
S. cerevisiae
isolates obtained from patients suffering from vaginitis and four isolates from commercial products in the same city were analyzed. The cellular DNA from each isolate was digested with the restriction endonuclease
Eco
RI, and restriction fragment length polymorphisms were generated by horizontal gel electrophoresis. The results showed that although vaginal isolates did not cluster in any particular genetic subtype, multiple patients were infected with indistinguishable strains (there were nine distinct strains among 23 isolates). For two of three patients, all three with two episodes of
S. cerevisiae
vaginitis, different strains were isolated during the recurrence of this disease. Three other patients with indistinguishable isolates were epidemiologically related in that two were practitioners in the same clinic and the third was a patient at this clinic. We also found that one commercial strain was indistinguishable from the strain isolated from three different women at the time that they were suffering from vaginitis. The findings of the present study suggest that some
S. cerevisiae
strains may possess properties permitting persistence in the human host. Furthermore, person-to-person contact and the proliferation of the use of
S. cerevisiae
as a health-food product, in home baking, and in home brewing may be a contributing factor in human colonization and infection with this organism.
Fatal Pulmonary Infection with
Mycobacterium celatum
in an Apparently Immunocompetent Patient
Authors: Bux-Gewehr Irene , Hagen Hans P. , Rüsch-Gerdes Sabine , Feurle Gerhard E.
Published: December 2019

Mycobacterium celatum
is a recently described mycobacterium isolated from patients who have suppressed cell-mediated immunity, such as AIDS. We present here, to our knowledge, the first report of a fatal pulmonary infection caused by
M. celatum
in a 73-year-old immunocompetent female patient. The mycobacterium was identified by a 16S rRNA sequence analysis.
Species-Specific and Ubiquitous-DNA-Based Assays for Rapid Identification ofStaphylococcus aureus
Authors: Martineau Francis , Picard François J. , Roy Paul H. , Ouellette Marc , Bergeron Michel G.
Published: December 2019

Staphylococcus aureusis the cause of serious infections in humans, including endocarditis, deep-seated abscesses, and bacteremia, which lead to toxic and septic shock syndromes. Rapid and direct identification of this bacterium specifically and ubiquitously directly from clinical specimens would be useful in improving the diagnosis ofS. aureusinfections in the clinical microbiology laboratory. A wide variety of kits based on biochemical characteristics efficiently identifyS. aureus, but the rapidity and the accuracy of each of these methods combined with testing of clinically relevant antibiotic resistance genes need to be improved. On the basis of hybridization assays with randomly selected clones from anS. aureusgenomic library, we have identified a chromosomal DNA fragment which is specific forS. aureusand which detected all 82S. aureusisolates tested. This 442-bp fragment was sequenced and was used to design a set of PCR amplification primers. The PCR assay was also specific and ubiquitous for the identification from bacterial cultures of 195 clinical strains ofS. aureusisolated from a variety of anatomical sites and obtained from hospitals throughout the world. The PCR assay that we have developed is simple and can be performed in about 1 h. This DNA-based test provides a novel diagnostic tool for the diagnosis ofS. aureusinfections.
Detection of Intimins α, β, γ, and δ, Four Intimin Derivatives Expressed by Attaching and Effacing Microbial Pathogens
Authors: Adu-Bobie Jeannette , Frankel Gad , Bain Christopher , Goncalves Azizedite Guedes , Trabulsi Luiz R. , Douce Gill , Knutton Stuart , Dougan Gordon
Published: December 2019

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic
Escherichia coli
(EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic
E. coli
(EHEC) isolates, a rabbit diarrheagenic
E. coli
strain (RDEC-1), and
Citrobacter rodentium
. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and
C. rodentium
. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding
eae
gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different
eae
gene derivatives. Accordingly, the different intimin types were designated α, β, δ, and γ, respectively.
Discrimination of Strains of
Salmonella enteritidis
with Differing Levels of Virulence by an In Vitro Glass Adherence Test
Authors: Solano C. , Sesma B. , Alvarez M. , Humphrey T. J. , Thorns C. J. , Gamazo C.
Published: December 2019

The objective of this study was the in vitro differentiation of isolates of
Salmonella enteritidis
whose virulences differed in a chick model. A total of 14 strains of
S. enteritidis
were isolated from either the environment, dairy products, or infected patients. The isolates could be divided into two groups on the basis of their virulence (50% lethal dose) in chickens infected intraperitoneally. When the strains were incubated in adherence test medium (Spanish patent 9700408), only the virulent strains produced aggregates and formed visible filaments attached to the glass tube. These results suggest, although for a limited number of strains, that aggregation in such a medium could be used as a diagnostic tool to discriminate virulent strains of
S. enteritidis.
Typhlitis Caused by Intestinal
Serpulina
-Like Bacteria in Domestic Guinea Pigs (
Cavia porcellus
)
Authors: Vanrobaeys Mia , De Herdt Peter , Ducatelle Richard , Devriese Luc A. , Charlier Gerard , Haesebrouck Freddy
Published: December 2019

Between January 1992 and December 1996,
Serpulina
-like bacteria were demonstrated in intestinal tract lesions from 37 of 88 guinea pigs submitted to the University of Ghent in Ghent, Belgium, for necropsy because of disease and death from different unknown causes. All infected animals had a history of sudden death with minimal introductory clinical signs. Occasionally, they produced yellow, slimy feces or showed nervous signs, but the condition always had a fatal outcome within 24 h. When larger colonies of guinea pigs were involved, the disease spread very rapidly unless treatment with ronidazole was initiated. Lesions consisted of a catarrhal or hemorrhagic inflammation of the colon and cecum (typhlitis). Electron microscopy demonstrated the presence of large numbers of
Serpulina
-like organisms adhering to the cecal mucosae of these animals. Attempts to isolate the agents failed. The organisms did not stain by an immunofluorescence technique for the detection of
Serpulina hyodysenteriae
. The present data provide evidence that intestinal
Serpulina
-like organisms can be important as a cause of disease in guinea pigs.
U.S. Case Report of Cerebral Phaeohyphomycosis Caused byRamichloridium obovoideum (R. mackenziei): Criteria for Identification, Therapy, and Review of Other Known Dematiaceous Neurotropic Taxa
Authors: Sutton Deanna A. , Slifkin Malcolm , Yakulis Robert , Rinaldi Michael G.
Published: December 2019

We report a case of cerebral phaeohyphomycosis in a 36-year-old male caused by the neurotropic fungusRamichloridium obovoideum(Matushima) de Hoog 1977 (Ramichloridium mackenzieiCampbell et Al-Hedaithy 1993). This man resided in the Middle East, where the fungus appears to be endemic and, possibly, geographically restricted, since all previous reports of brain abscesses due to this organism have been for patients indigenous to this area. As a servant of the Saudi Arabian royal family, he appeared in the United States seeking treatment for chronic weight loss, fatigue, decreased memory, and a more recent 2-week history of right-hand weakness which worsened to involve the entire right upper extremity. On the day prior to his admission, he had a focal motor seizure with rotation of the head and eyes to the right, followed by secondary generalization. A computerized tomogram showed a ring-enhancing hypodense lesion in the left parietal subcortical region with associated edema and mass effect. Diagnosis of a fungal etiology was made following a parietal craniotomy and excisional biopsy by observation of septate, dematiaceous hyphal elements 2 to 3 μm in width on hematoxylin-and-eosin-stained sections from within areas of inflammation and necrosis. Culture of the excised material grew out a dematiaceous mould which was subsequently identified asR. obovoideum. At two months postsurgery and with a regimen of 200 mg of itraconazole twice a day, the patient was doing well and returned to Saudi Arabia. His condition subsequently deteriorated, however, and following a 7-month course of itraconzole, he expired. We use this case to alert clinicians and personnel in clinical mycology laboratories of the pathogenicity of this organism and its potential occurrence in patients with central nervous system signs and symptoms who have resided in the Middle East and to review and/or compareR. obovoideumwith other neurotropic, dematiaceous taxa and similar nonneurotropic, dematiaceous species.
Practical Approach for Detection and Identification of OXA-10-Derived Ceftazidime-Hydrolyzing Extended-Spectrum β-Lactamases
Authors: Vahaboglu Haluk , Ozturk Recep , Akbal Huriye , Saribas Suat , Tansel Ozlem , Coşkunkan Figen
Published: December 2019

A practical approach to detect and identify ceftazidime-hydrolyzing extended-spectrum mutants of OXA-10 β-lactamase is presented. Large numbers of bacteria were screened by colony hybridization, a 720-bp part of
bla
OXA
was amplified by PCR from the hybridization-positive isolates, and the products were digested by
Pvu
II and
Hae
III.
Comparison of Two Enzyme-Linked Immunosorbent Assays and One Rapid Immunoblot Assay for Detection of Herpes Simplex Virus Type 2-Specific Antibodies in Serum
Authors: Groen Jan , Van Dijk Grietje , Niesters Hubert G. M. , Van Der Meijden Willem I. , Osterhaus Albert D. M. E.
Published: December 2019

The sensitivities and specificities of three immunoassays for the detection of herpes simplex virus type 2 (HSV-2)-specific immunoglobulin G antibodies in serum, including the one-strip rapid immunoblot assay (RIBA; Chiron Corporation) and two indirect enzyme immunosorbent assays (EIA; Gull Laboratories and Centocor), were compared by testing a panel of 1,250 serum samples from individuals attending an outpatient clinic for sexually transmitted diseases. A qualitative agreement among the three assays was observed with 1,080 serum samples (86.4%); 291 of the serum samples (23.3%) were positive, 789 samples (63.1%) were negative, and 170 serum samples (13.6%) gave a discordant result. Results were considered conclusive when a concordant result was obtained with two of three assays. The sensitivities and specificities of the RIBA, the Gull EIA, and the Centocor EIA proved to be 99.2, 99.7, and 89.9% and 97.1, 96.7, and 99.3%, respectively. These results indicate that the Chiron RIBA and the Gull EIA are especially useful and reliable for the detection of HSV-2-specific antibodies in serum.
Utility of Major Leukocyte Subpopulations for Monitoring Secondary Cytomegalovirus Infections in Renal-Allograft Recipients by PCR
Authors: Schäfer Peter , Tenschert Werner , Cremaschi Liana , Gutensohn Kai , Laufs Rainer
Published: December 2019

The feasibility of the major peripheral blood leukocyte (PBL) subsets for use in qualitative and quantitative PCR to monitor secondary cytomegalovirus (CMV) infection and ganciclovir therapy was assessed with 188 blood samples derived from 40 CMV immunoglobulin G-positive renal-allograft recipients. In pp65 antigen-positive patients all leukocyte fractions, but only 79.5% of plasma preparations, were PCR positive. In pp65 antigen-negative samples from patients after antiviral treatment only 7.3% of polymorphonuclear cell (PMNL) samples, but 81.8% of peripheral blood mononuclear cells (PBMC), and 10.9% of plasma samples remained PCR positive. Similarly, in patients with latent infections only 5.0% of PMNL, but 51.7% of PBMC preparations, and 8.0% of plasma samples were PCR positive. Regarding patients with active CMV infection, CMV DNA copy numbers in PMNL correlated significantly with pp65 antigen-positive cell counts before and after onset of ganciclovir therapy. Significant differences in CMV DNA copy numbers in PMNL and plasma were observed (i) between patients with symptomatic infection and those with asymptomatic infection and (ii) between patients with active infection and those with latent infection. In contrast, PBMC harbored equally low CMV DNA levels both in patients with active infection and those with latent infections, and no decline of CMV DNA load in PBMC was observed during antiviral treatment. We conclude that detection of CMV DNA in PMNL, not in PBMC, is associated with active infections and is more sensitive than detection of CMV DNA in plasma. Negative PCR results for PMNL after antiviral therapy indicate recovery, and fewer unwanted positive results occur compared to PBMC and plasma. Therefore, purified PMNL should be preferred for analysis by qualitative CMV PCR to avoid unwanted positive results. The CMV DNA load in PBMC compared with that in PMNL is negligible during active infection, so mixed PBL are sufficient for use in quantitative PCR.
Detection and Identification of
Mycobacterium tuberculosis
Directly from Sputum Sediments by Ligase Chain Reaction
Authors: Moore Douglas F. , Curry Janis I.
Published: December 2019

Sputum specimens received for the diagnosis of tuberculosis or other mycobacterial infections were tested by a ligase chain reaction (LCR)-based assay and acid-fast stain and culture techniques. Results from the LCR assay (Abbott LCx
Mycobacterium tuberculosis
[MTB] Assay) were compared to results from standard culture techniques held for 6 weeks. Four hundred ninety-three specimens from 205 patients suspected of pulmonary tuberculosis were included in the prospective study. Thirty-four (6.9%) of the specimens were culture positive for
M. tuberculosis
, and 13 (38%) of these were also fluorochrome stain positive. LCR sensitivities and specificities compared to culture were 74 and 98%, respectively. LCR sensitivity was 100% for fluorochrome stain-positive specimens and 57% for fluorochrome stain-negative specimens. Nine LCR-negative, culture-positive specimens were the result of low concentrations of
M. tuberculosis
. No inhibitors were detected in any of these specimens. Of the eight LCR-positive, culture-negative specimens, five were from patients with active tuberculosis. With these considered culture misses, final LCR sensitivity, specificity, positive predictive value, and negative predictive value were 77, 99, 91, and 98%, respectively. The same performance values for the fluorochrome acid-fast bacillus smear were 33, 98, 62, and 94%, respectively. After normal laboratory sputum processing, the Abbott LCx MTB Assay can be completed in 6 h. Thus, it is possible to have results available within 8 h of specimen submission.
Intergenic Transcribed Spacer PCR Ribotyping for Differentiation of
Saccharomyces
Species and Interspecific Hybrids
Authors: McCullough Michael J. , Clemons Karl V. , McCusker John H. , Stevens David A.
Published: December 2019

The taxonomy of the genus
Saccharomyces
has undergone significant changes recently with the use of genotypic rather than phenotypic methods for the identification of strains to the species level. The sequence of rRNA genes has been utilized for the identification of a variety of fungi to the species level. This methodology, applied to species of
Saccharomyces
, allows unknown
Saccharomyces
isolates to be assigned to the type strains. It was the aim of the present study to assess whether typing of the intergenic spacer region by using restriction fragment length polymorphisms of PCR products (intergenic transcribed spacer PCR [ITS-PCR] ribotyping) could distinguish among type strains of the 10 accepted species of
Saccharomyces
and further to assess if this method could distinguish strains that were interspecific hybrids. Cellular DNA, isolated after the lysis of protoplasts, was amplified by PCR using ITS1 and ITS4 primers, purified by liquid chromatography, and digested with restriction endonucleases. Ribotyping patterns using the restriction enzymes
Mae
I and
Hae
III could distinguish all species of
Saccharomyces
from each other, as well as from
Candida glabrata
,
Candida albicans
, and
Blastomyces dermatitidis
. The only exception to this was the inability to distinguish between
Saccharomyces bayanus
and
S. pastorianus
(
S. carlsbergensis
). Furthermore, interspecific hybrids resulting from the mating of sibling species of
Saccharomyces
were shown to share the ITS-PCR ribotyping patterns of both parental species. It should now be possible, by this simple PCR-based technique, to accurately identify these strains to the species level, thereby allowing an increase in our understanding of the characteristics required by these interspecific hybrids for their particular ecological niches.
Nested PCR Assay for Detection of Granulocytic Ehrlichiae
Authors: Massung Robert F. , Slater Kim , Owens Jessica H. , Nicholson William L. , Mather Thomas N. , Solberg Victoria B. , Olson James G.
Published: December 2019

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from
Ixodes scapularis
ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.
Multicenter Evaluation of the COBAS AMPLICOR HCV Assay, an Integrated PCR System for Rapid Detection of Hepatitis C Virus RNA in the Diagnostic Laboratory
Authors: Albadalejo J. , Alonso R. , Antinozzi R. , Bogard M. , Bourgault A.-M. , Colucci G. , Fenner T. , Petersen H. , Sala E. , Vincelette J. , Young C.
Published: December 2019

The benefits shown by the recent introduction of PCR for the in vitro diagnosis of hepatitis C virus (HCV) infection has prompted the development of standardized, ready-to-use assays that can be implemented in routine clinical laboratories. We have evaluated the clinical performance of COBAS AMPLICOR HCV (COBAS), the first instrument system that allows the automation of HCV RNA amplification and detection, to determine its performance in the routine laboratory setting. More than 2,000 specimens collected at five centers were analyzed in parallel by the COBAS and the manual AMPLICOR HCV (AMPLICOR) tests, and the results were compared with the results for biochemical and serological markers of HCV. In this study the two PCR systems showed the same accuracy, with a concordance rate of 99.8%. As expected, the correlation between serology and PCR was not absolute because the presence of anti-HCV antibodies may be associated with a latent or past infection. On the other hand, if the presence of confirmed anti-HCV antibodies and elevated alanine aminotransferase levels are taken as the “gold standard,” indicating an active, ongoing infection, the COBAS and AMPLICOR tests show high and comparable sensitivities (100%) and specificities (98%), with positive and negative predictive values of 100 and 97%, respectively. During the study no false-positive reactions were detected. The use of an internal control allowed the identification of inhibitory substances that prevented amplification for 0.3 and 0.4% of samples tested by the COBAS and AMPLICOR tests, respectively. Compared to the manual system, the COBAS system allowed a significant reduction of hands-on time and could improve the overall laboratory work flow. In conclusion, these results support the use of the COBAS and AMPLICOR tests for the molecular diagnosis of active HCV infections.
Isolation and Characterization of Verocytotoxin-Producing
Escherichia coli
O157 Strains from Dutch Cattle and Sheep
Authors: Heuvelink A. E. , van den Biggelaar F. L. A. M. , de Boer E. , Herbes R. G. , Melchers W. J. G. , Huis In ’t Veld J. H. J. , Monnens L. A. H.
Published: December 2019

In the periods from July to November 1995 and 1996, fecal samples from Dutch cattle and sheep were collected at the main slaughterhouses of The Netherlands, located at different geographic sites. The samples were examined for the presence of verocytotoxin (VT)-producing
Escherichia coli
(VTEC) of serogroup O157.
E. coli
O157 strains could be isolated from 57 (10.6%) of 540 adult cattle, 2 (0.5%) of 397 veal calves, 2 (3.8%) of 52 ewes, and 2 (4.1%) of 49 lambs. Immunomagnetic separation with O157-specific-antibody-coated beads appeared to be significantly more sensitive than conventional plating for detection of the organism in feces. With the exception of two isolates from adult cattle which appeared to be negative for VT genes, all animal isolates were positive for both VT (VT1 and/or VT2) and
E. coli
attaching-and-effacing gene sequences, and therefore, they were regarded as potential human pathogens. Although genomic typing by pulsed-field gel electrophoresis revealed a wide variety of distinct restriction patterns, comparison of the 63 animal isolates with 33 fecal O157 VTEC strains previously isolated from humans with the diarrhea-associated form of the hemolytic-uremic syndrome by their phage types and VT genotypes showed a marked similarity between animal and human isolates: 30 (90.9%) of the 33 human isolates appeared to be of
E. coli
O157 strain types also isolated from cattle and sheep. It was concluded that Dutch cattle and sheep are an important reservoir of
E. coli
O157 strains that are potentially pathogenic for humans.
Novel Recombinant-Antigen Enzyme Immunoassay for Serological Diagnosis of Syphilis
Authors: Young H. , Moyes A. , Seagar L. , McMillan A.
Published: December 2019

Enzyme immunoassay (EIA) is an ideal method for screening large numbers of patients for syphilis. We evaluated a novel immune-capture EIA (ICE Syphilis; Murex Diagnostics) that uses three recombinantTreponema pallidumantigens (TpN15, TpN17, and TpN47) and compared the results with those obtained by the nativeT. pallidumantigen EIA (Captia SelectSyph-G; Centocor) that we currently use for the serodiagnosis of syphilis. Specificity was evaluated by screening 1,184 unselected serum specimens in parallel by the ICE Syphilis and SelectSyph-G assays, while sensitivity was tested with a panel of 101 serum specimens containing antitreponemal antibodies (treated and untreated) from patients with various stages of infection. The specificity of the ICE Syphilis EIA (99.8%) on screening was significantly higher (P< 0.02) than that of the SelectSyph-G EIA (99.2%). The sensitivity of the ICE Syphilis EIA was significantly higher (P< 0.01) than that of the SelectSyph-G EIA on both initial (99 versus 91.4%) and repeat (100 versus 92.4%) testing. The ICE Syphilis EIA was also significantly more sensitive (P< 0.01) than the fluorescent treponemal antibody-abs (92.4%) but not theT. pallidumhemagglutination assay (97.1%). Sera containing antitreponemal antibodies gave a much higher antibody index (absorbance of test serum/kit cutoff) by the ICE Syphilis EIA than by the SelectSyph-G EIA. This combined with the overall high sensitivity makes the ICE Syphilis EIA an ideal test for excluding or detecting treponemal infection in human immunodeficiency virus (HIV)-infected patients. The ICE Syphilis EIA was positive with sera from all 15 HIV-infected patients in the study, whereas sera from 3 HIV-infected patients were negative by the SelectSyph-G EIA. We conclude that the high sensitivity and specificity of the ICE Syphilis EIA and its suitability for automation make it an ideal screening test.
Use of Immunoblot Assay To Define Serum Antibody Patterns Associated with
Helicobacter pylori
Infection and with
H. pylori
-Related Ulcers
Authors: Aucher P. , Petit M. L. , Mannant P. R. , Pezennec L. , Babin P. , Fauchere J. L.
Published: December 2019

Serology has been used worldwide to detect
Helicobacter pylori
infection. Using an immunoblot assay with an antigen from strain ATCC 43579, we sought to determine the antibodies which were good markers of colonization and the antibody patterns associated with ulcers or atrophy. Out of 98 dyspeptic patients, 41 were colonized by
H. pylori
, based on a positive culture or on positive results of both a urease test and direct examination. These 41 patients were seropositive by an enzyme immunoassay, and 12 of them had ulcers and 29 had evidence of atrophy. Fifty-seven of the 98 patients were noncolonized. Twenty-five of the 57 had evidence of gastric atrophy, and 10 were seropositive; 5 of these 10 had ulcers. By Western blot analysis, 12 antibodies were significantly more frequent in sera from colonized patients, and they produced immunoreactive bands at 125, 87, 74, 66, 54, 48, 46, 42, 35, 30, 16 and 14 kDa. The presence of at least one band at 54, 35, or 42 kDa was the best marker of infection (sensitivity, 95%; specificity, 82%). In the group of colonized patients, none of the antibody patterns were correlated to gastric atrophy. Conversely, the presence of a band at 125, 87, or 35 kDa was statistically associated with the presence of an ulcer. The simultaneous presence of bands at 87 and 35 kDa predicted the risk of ulcers with 83% sensitivity and 69% specificity. By using CagA-positive and VacA-positive strains and CagA-negative and VacA-negative isogenic mutants, the antigens corresponding to the bands at 125 and 87 kDa were shown to be CagA and VacA, respectively. On the other hand, the 35-kDa antigen is a novel uncharacterized component of
H. pylori
. These results may help to optimize the composition of antigenic preparations for serologic detection of
H. pylori
colonization. Immunoblot assay would be useful for screening patients at high risk of ulcers.
Comparison of PCR and Microscopy for Detection of
Cryptosporidium parvum
in Human Fecal Specimens: Clinical Trial
Authors: Morgan U. M. , Pallant L. , Dwyer B. W. , Forbes D. A. , Rich G. , Thompson R. C. A.
Published: December 2019

PCR technology offers alternatives to conventional diagnosis of
Cryptosporidium
for both clinical and environmental samples. We compared microscopic examination by a conventional acid-fast staining procedure with a recently developed PCR test that can not only detect
Cryptosporidium
but is also able to differentiate between what appear to be host-adapted genotypes of the parasite. Examinations were performed on 511 stool specimens referred for screening on the basis of diarrhea. PCR detected a total of 36 positives out of the 511 samples, while routine microscopy detected 29 positives. Additional positives detected by PCR were eventually confirmed to be positive by microscopy. A total of five samples that were positive by routine microscopy at Western Diagnostic Pathology but negative by PCR and by microscopy in our laboratory were treated as false positives. Microscopy therefore exhibited 83.7% sensitivity and 98.9% specificity compared to PCR. PCR was more sensitive and easier to interpret but required more hands-on time to perform and was more expensive than microscopy. PCR, however, was very adaptable to batch analysis, reducing the costs considerably. Bulk buying of reagents and modifications to the procedure would decrease the cost of the PCR test even more. An important advantage of the PCR test, its ability to directly differentiate between different
Cryptosporidium
genotypes, will assist in determining the source of cryptosporidial outbreaks. Sensitivity, specificity, ability to genotype, ease of use, and adaptability to batch testing make PCR a useful tool for future diagnosis and studies on the molecular epidemiology of
Cryptosporidium
infections.
Evaluation of BACTEC MYCO/F Lytic Medium for Recovery of Mycobacteria and Fungi from Blood
Authors: Waite Rebecca T. , Woods Gail L.
Published: December 2019

The reliability of MYCO/F Lytic medium in the BACTEC 9240 blood culture system was evaluated by comparing its performance to that of the Isolator system for the recovery of fungi and to that of the ESP II system for the recovery of mycobacteria. Of 717 specimens of blood cultured for fungi, 24 were positive; 12 samples were positive with both systems, 7 samples were positive with the Isolator system only, and 5 samples were positive with MYCO/F Lytic medium only. Fourteen samples grew
Histoplasma capsulatum
; both systems detected
H. capsulatum
in seven samples but the Isolator system alone detected
H. capsulatum
in seven samples. The mean times to the detection of
H. capsulatum
were 8 days (range, 4 to 13 days) for MYCO/F Lytic medium and 9 days (range, 6 to 18 days) for the Isolator system; the mean times to identification were 20 days (range, 15 to 24 days) for isolates recovered with MYCO/F Lytic medium and 11 days (range, 6 to 18 days) for those recovered with the Isolator system (
P
< 0.05).
Cryptococcus neoformans
was isolated from 10 fungal cultures; five isolates grew in both systems, and five isolates grew in MYCO/F Lytic medium only. The mean times to detection of
C. neoformans
were 4 days (range, 2 to 6 days) for MYCO/F Lytic medium and 7 days (range, 5 to 7 days) for the Isolator system (
P
< 0.05); the mean times to identification were 15 days (range, 7 to 27 days) for isolates recovered with MYCO/F Lytic medium and 8 days (range, 7 to 11 days) for those recovered with the Isolator system. Of the 687 samples of blood cultured for mycobacteria, 64 blood samples from 42 patients grew mycobacteria (58 grew
Mycobacterium avium
complex, 4 grew
Mycobacterium kansasii
, and 2 grew
Mycobacterium tuberculosis
); 42 isolates were recovered with both systems, 18 were isolated with MYCO/F medium only, and 4 were isolated with the ESP II system only alone (
P
< 0.05). The mean time to detection of mycobacteria with MYCO/F Lytic medium was 14 days, whereas it was 17 days with the ESP II system (
P
< 0.05). In summary, the combination of MYCO/F Lytic medium and the BACTEC 9240 instrument is an excellent blood culture system for the growth and detection of mycobacteria. A valid assessment of MYCO/F Lytic medium with regard to fungal isolation, however, was not possible due to the small number of isolates recovered.