bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzyme-Linked Immunosorbent Assay Based on Recombinant Human Group C Rotavirus Inner Capsid Protein (VP6) To Detect Human Group C Rotaviruses in Fecal Samples
Authors: James V. L. A. , Lambden P. R. , Caul E. O. , Clarke , and I. N.
Published: December 2019

A recent study showed that 43% of a population in the United Kingdom were seropositive for group C rotavirus. The higher than expected incidence may be due to limited diagnosis of acute human group C rotavirus infections because no routine test is available. Human group C rotavirus infections are routinely diagnosed by electron microscopy (EM) and a negative group A rotavirus enzyme-linked immunosorbent assay (ELISA) result. An antigen-detection ELISA was developed with hyperimmune antibodies raised to human group C rotavirus recombinant VP6 (Bristol strain) expressed in insect cells. The assay was used to screen fecal samples to determine the prevalence of group C rotavirus infection. Samples positive by ELISA were confirmed by EM, polyacrylamide gel electrophoresis of double-stranded RNA, or detection of the VP6 gene by reverse transcription-PCR. Retrospective analysis indicated a 1 to 2% detection rate of positivity among samples from patients with acute diarrhea.
Inducible β-Lactam Resistance in
Aeromonas hydrophila
: Therapeutic Challenge for Antimicrobial Therapy
Authors: Ko Wen-Chien , Wu Hsiu-Mei , Chang Tsung-Chain , Yan Jing-Jou , Wu Jiunn-Jong
Published: December 2019

Despite the abundant amount of knowledge about inducible chromosomally mediated β-lactamases among
Aeromonas
species, extended-spectrum β-lactam-resistant
A. hydrophila
strains selected in clinical practice were rarely reported. In the present study, two strains of
A. hydrophila
, A136 and A139, with markedly different susceptibilities to extended-spectrum cephalosporins were isolated from blood and the tip segment of an arterial catheter of a burn patient. Another strain (A136m) was selected in vitro by culturing A136 in a subinhibitory concentration of cefotaxime, the β-lactam agent administered for the treatment of
Aeromonas
bacteremia in this patient. Typing studies by arbitrarily primed PCR and pulsed-field gel electrophoresis indicated a clonal relationship among strains A136, A136m, and A139. These strains were identified to be of DNA hybridization group 1. Wild-type strain A136 was resistant only to ampicillin and cephamycins, but A136m and A139 were highly resistant to the expanded- and broad-spectrum cephalosporins. The presence of increased β-lactamase activity in A139 suggests that A139 is a derepressed mutant which overexpresses β-lactamases. These results call attention to the use of β-lactam agents for the treatment of invasive
Aeromonas
infections.
Diagnosis of
Trichomonas vaginalis
Infection by PCR Using Vaginal Swab Samples
Authors: Madico Guillermo , Quinn Thomas C. , Rompalo Anne , McKee Kelly T. , Gaydos Charlotte A.
Published: December 2019

Trichomonas vaginalis
infection is the most prevalent nonviral sexually transmitted disease (STD) in the world. A PCR test using vaginal swab samples for the detection of
T. vaginalis
was developed to add
T. vaginalis
infection to the growing list of STDs that can be detected by DNA amplification techniques. A primer set, BTUB 9/2, was designed to target a well-conserved region in the beta-tubulin genes of
T. vaginalis
. All strains (15 of 15) of
T. vaginalis
tested were successfully detected by PCR giving a single predicted product of 112 bp in gel electrophoresis. No such targeted product was amplified with DNA from
Trichomonas tenax
,
Trichomonas gallinae
,
Chlamydia trachomatis
,
Neisseria gonorrhoeae
,
Giardia lamblia
,
Chilomastix sulcatus
,
Dientamoeba fragilis
, and
Entamoeba histolytica
. An optimal analytical sensitivity of one
T. vaginalis
organism per PCR was achieved. Culture, performed with the Inpouch TV culture system, was examined daily with a light microscope to identify
T. vaginalis
. Twenty-three of 350 (6.6%) vaginal swab samples from women attending an army medical clinic were culture positive for
T. vaginalis
. Of these culture positive specimens, PCR detected 22 of 23 (96%) with primer set BTUB 9/2, and wet preparation detected only 12 of 23 (52%). Seventeen specimens were BTUB 9/2-PCR positive and culture negative. Ten of these discordant specimens were determined to be as true positive by PCR using primer sets TVA 5-1/6 and/or AP65 A/B, which target different regions in the
T. vaginalis
genome, and seven were determined to be false positive. The sensitivity of BTUB 9/2-PCR was 97% and the specificity was 98%. The sensitivities of culture and wet preparation were 70 and 36%, respectively. The diagnosis of
T. vaginalis
infection by PCR is a sensitive and specific method that could be incorporated into a joint strategy for the screening of multiple STDs by using molecular amplification methods.
Comparison of PCR- and Hybrid Capture-Based Human Papillomavirus Detection Systems Using Multiple Cervical Specimen Collection Strategies
Authors: Peyton C. L. , Schiffman M. , Lörincz A. T. , Hunt W. C. , Mielzynska I. , Bratti C. , Eaton S. , Hildesheim A. , Morera L. A. , Rodriguez A. C. , Herrero R. , Sherman M. E. , Wheeler C. M.
Published: December 2019

This study compared the performances of three human papillomavirus (HPV) detection tests with specimens collected by three alternative procedures. The HPV tests included the Hybrid Capture Tube test (HCT), the microplate-based Hybrid Capture II test (HC II), and the MY09-MY11 L1 consensus primer PCR-based assay. Initial cervical specimens were collected from study subjects with a broom device, and after Papanicolaou smears were made, residual specimens were placed into PreservCyt (PC), a liquid cytology medium. A second specimen was collected from each subject and placed into Digene Specimen Transport Medium (STM). The device for collection of the second specimen alternated with consecutive subjects between a conical cytology brush and a Dacron swab. At the 1.0-pg/ml cutoff, the results of the HC II agreed well with those of the PCR. Specifically, when PCR data were restricted to the types found by the HC II (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), there was greater than 90% agreement between the HC II and PCR results with both STM and PC. At a lower cutoff (0.2 pg/ml), HC II-positive results increased further, especially when the test was applied to the PC specimens. However, false-positive HC II results were more often observed at the 0.2-pg/ml cutoff. HC II yielded the highest HPV positivity with specimens placed into PC, followed by specimens collected with a conical brush and placed into STM and, last, by those collected with a Dacron swab and placed into STM. Our results demonstrate the utility of both the STM and PC specimen collection methods and show good agreement between the HC II and PCR.
Multilocus Genotypic Analysis of
Cryptosporidium parvum
Isolates from Different Hosts and Geographical Origins
Authors: Spano Furio , Putignani Lorenza , Crisanti Andrea , Sallicandro Pina , Morgan Una M. , Le Blancq Sylvie M. , Tchack Laurie , Tzipori Saul , Widmer Giovanni
Published: December 2019

The genetic analysis of oocysts recovered from the stools of humans and animals infected with
Cryptosporidium parvum
has consistently shown the existence of two distinct genotypes. One of the genotypes is found exclusively in some human infections, whereas the other genotype is found in human as well as in animal infections. On the basis of these observations and the results of published epidemiological studies with single polymorphic markers, the existence of two separate transmission cycles has been postulated, one exclusively anthroponotic and the other involving both animals and humans. To test this hypothesis,
C. parvum
isolates of different geographic and host origins were analyzed by using unlinked genetic polymorphisms. A total of 28 isolates originating from Europe, North and South America, and Australia were examined. Isolates clustered into two groups, one comprising both human and animal isolates and the other comprising isolates only of human origin. The absence of recombinant genotypes is consistent with two reproductively isolated populations within the species
C. parvum.
Detection of Point Mutations Associated with Resistance of
Helicobacter pylori
to Clarithromycin by Hybridization in Liquid Phase
Authors: Pina Myriam , Occhialini Alessandra , Monteiro Lurdes , Doermann Henry-Pierre , Mégraud Francis
Published: December 2019

When the standard procedure for determining antibiotic susceptibility of bacteria is used, the results are delayed, especially for bacteria that grow slowly, such as
Helicobacter pylori
. Treatment for this bacterium may involve clarithromycin, a compound for which resistance has been associated with point mutations on the 23S rRNA gene. This resistance is currently found in organisms isolated from 0 to 15% of patients and jeopardizes the success of the treatment. We have designed a test involving amplification and colorimetric hybridization in the liquid phase to detect the mutation at the molecular level. First, four reference strains, including the wild type and three strains with the mutations A2143C, A2143G, and A2144G, were used to optimize the method. Amplification was carried out with primers previously published. The amplified products were added to probe-coated microtiter wells. A DNA enzyme immunoassay was used to detect the hybrids. The optimal conditions of the hybridization were defined for each probe. Nineteen
H. pylori
strains resistant to clarithromycin and 22 susceptible according to phenotypic data were submitted to restriction with
Bsa
I and
Bbs
I, and part of the 23S rRNA gene was sequenced in order to determine the mutation involved for the resistant strains. The new assay showed a complete correlation with the reference methods, except for one strain. Cross-hybridizations as well as application of the reaction to other bacteria did not lead to optical densities higher than the cutoff values chosen with the receiving operating characteristic curve. This method can be easily standardized and gives a result within a day. Its application directly to the biopsy specimens or infected gastric juice is planned in the future.
PCR Method for Detection of Adenovirus in Urine of Healthy and Human Immunodeficiency Virus-Infected Individuals
Authors: Echavarria Marcela , Forman Michael , Ticehurst John , Dumler J. Stephen , Charache Patricia
Published: December 2019

Adenoviruses (AdV) cause diseases that range from localized, self-limited illnesses to fatal infections in immunocompromised patients. Culture is assumed to be sensitive but requires viable virus and up to 3 weeks for detection, and it can be inhibited by bacterial contamination. A new PCR method amplifying a region of the hexon gene was developed in order to detect AdV in urine more rapidly and with greater sensitivity than obtainable by culture technology. All 18 serotypes tested were detected. Quantitatively, with optimized urine processing, AdV PCR detected 0.2 PFU/ml (serotype 11) and 10 DNA copies/ml (serotype 2). Serially collected urine samples from human immunodeficiency virus (HIV)-infected patients with concurrent cytomegalovirus retinitis were divided into three groups: AdV culture-positive samples, AdV culture-negative or bacterially contaminated samples from patients with a history of AdV culture-positive urines, and AdV culture-negative samples from patients without a history of AdV culture positivity. Urine samples from healthy adults were also tested by culture and PCR to screen for asymptomatic shedding. Amplification was assessed with and without prior DNA purification. AdV was detected by PCR in 90% of culture-positive urines (100% of unclotted samples, e.g., those culture positive after storage for PCR testing), 71% of culture-negative or bacterially contaminated urines from AdV-infected patients, and 28% from AdV culture-negative patients. Healthy volunteers were culture negative for AdV, and 96% were PCR negative. The new AdV PCR method is rapid and sensitive and can detect viral DNA in samples for which culturing is problematic. The role of AdV replication during HIV infection merits further investigation with sensitive tools such as PCR.
Development of a New Cytomegalovirus (CMV) Immunoglobulin M (IgM) Immunoblot for Detection of CMV-Specific IgM
Authors: Lazzarotto T. , Ripalti A. , Bergamini G. , Battista M. C. , Spezzacatena P. , Campanini F. , Pradelli P. , Varani S. , Gabrielli L. , Maine G. T. , Landini M. P.
Published: December 2019

We developed a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot to detect CMV-specific IgM in human sera. The new test contains four viral proteins (vp150, vp82, vp65, and vp28) purified from viral particles and four recombinant proteins (rp150, rp130, rp52, and rp38) purified from
Escherichia coli
. These antigens were individually loaded onto nitrocellulose strips, and the strips were then used to detect CMV-specific IgM by using a μ-specific conjugate. The new assay was evaluated in parallel with one or two IgM enzyme-linked immunosorbent assays (ELISAs) to test 592 serum samples from different groups of latently or acutely infected individuals. The sensitivity of the new assay with respect to the consensus of two ELISAs was 100%, the specificity was 98.6%, the positive predictive value was 96.9%, and the negative predictive value was 100%. We also evaluated the new test by testing sera from pregnant women and transplant recipients with a known clinical history. Our results suggest that the new test combines high sensitivity with high specificity, characteristics that are mutually exclusive with the other commercially available tests. Furthermore, a statistically significant correlation was observed between the number of IgM-reactive bands and the elevated risk of transmission from CMV-infected pregnant women to their offspring.
Application of Multiplex PCR for Detection of Non-O157 Verocytotoxin-Producing
Escherichia coli
in Bloody Stools: Identification of Serogroups O26 and O111
Authors: Louie M. , Read S. , Simor A. E. , Holland J. , Louie L. , Ziebell K. , Brunton J. , Hii J.
Published: December 2019

Primers were designed to amplify sequences of verocytotoxin genes and
eaeA
genes of
Escherichia coli
O26:H11, O111:H8, and O157:H7 in a multiplex PCR assay. This assay successfully detected
E. coli
O26:H11 in bloody stool specimens in which other enteric pathogens were not detected by culture-based methods. Rapid assays to detect non-O157:H7 verocytotoxin-producing
E. coli
is important to improve methods for the etiologic diagnosis of hemorrhagic colitis.
Evaluation of a Commercial PCR Kit for Diagnosis of Cytomegalovirus Infection of the Central Nervous System
Authors: Weinberg Adriana , Spiers Debbie , Cai Guan-Yung , Long Christopher M. , Sun Rita , Tevere Vince
Published: December 2019

We evaluated the AMPLICOR cytomegalovirus (CMV) PCR kit for the diagnosis of neurologic CMV infections on 43 positive and 112 negative archived cerebrospinal fluid specimens originally tested by an in-house PCR method. The AMPLICOR kit showed sensitivity and specificity of 95 and 100%, respectively, versus the home-grown assay, indicating its utility in this clinical setting.
Helicobacter pylori cagA
Status and s and m Alleles of
vacA
in Isolates from Individuals with a Variety of
H. pylori
-Associated Gastric Diseases
Authors: Evans Dolores G. , Queiroz Dulciene M. M. , Mendes Edilberto N. , Evans Doyle J.
Published: December 2019

The
cagA
gene was detected in 100% of 16
Helicobacter pylori
isolates from patients with gastric carcinoma versus 78% of 18 isolates from patients with duodenal ulcers (
P
= 0.344) and only 64% of 22 isolates from patients with gastritis only (
P
= 0.005) in Brazil. Also, there was a significant association between isolation of
cagA
+
s1-type
vacA H. pylori
in cases of stomach cancer and ulcers as opposed to cases of gastritis only (
P
= 0.004), but this was not true in Houston (
P
= 0.238), where 94% of all isolates were
cagA
+
.
Occurrence of Verocytotoxin-ProducingEscherichia coliO157 on Dutch Dairy Farms
Authors: Heuvelink A. E. , van den Biggelaar F. L. A. M. , Zwartkruis-Nahuis J. T. M. , Herbes R. G. , Huyben R. , Nagelkerke N. , Melchers W. J. G. , Monnens L. A. H. , de Boer E.
Published: December 2019

During the period from September 1996 through November 1996, 10 Dutch dairy farms were visited to collect fecal samples from all cattle present. The samples were examined for the presence of verocytotoxin (VT)-producingEscherichia coli(VTEC) of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. Cattle on 7 of the 10 dairy farms tested positive for O157 VTEC, with the proportion of cattle infected varying from 0.8 to 22.4%. On the seven farms positive for O157 VTEC, the excretion rate was highest in calves ages 4 to 12 months (21.2%). In a follow-up study, two O157 VTEC-positive farms and two O157 VTEC-negative farms identified in the prevalence study were revisited five times at intervals of approximately 3 months. Cattle on each farm tested positive at least once. The proportion of cattle infected varied from 0 to 61.0%. Excretion rates peaked in summer and were lowest in winter. Again, the highest prevalence was observed in calves ages 4 to 12 months (11.8%). O157 VTEC strains were also isolated from fecal samples from horses, ponies, and sheep and from milk filters and stable flies. O157 VTEC isolates were characterized by VT production and type, the presence of theE. coliattaching-and-effacing gene, phage type, and pulsed-field gel electrophoretic genotype. No overlapping strain types were identified among isolates from different farms except one. The predominance of a single type at each sampling suggests that horizontal transmission is an important factor in dissemination of O157 VTEC within a farm. The presence of more than one strain type, both simultaneously and over time, suggests that there was more than one source of O157 VTEC on the farms. Furthermore, this study demonstrated that the O157 VTEC status of a farm cannot be ascertained from a single visit testing a small number of cattle.
Use of Recombinant Nucleoproteins in Enzyme-Linked Immunosorbent Assays for Detection of Virus-Specific Immunoglobulin A (IgA) and IgG Antibodies in Influenza Virus A- or B-Infected Patients
Authors: Voeten J. T. M. , Groen J. , van Alphen D. , Claas E. C. J. , de Groot R. , Osterhaus A. D. M. E. , Rimmelzwaan G. F.
Published: December 2019

The nucleoprotein genes of influenza virus A/Netherlands/018/94 (H3N2) and influenza virus B/Harbin/7/94 were cloned into the bacterial expression vector pMalC to yield highly purified recombinant influenza virus A and B nucleoproteins. With these recombinant influenza nucleoproteins, enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of influenza virus A- and B-specific immunoglobulin A (IgA) and IgG serum antibodies. Serum samples were collected at consecutive time points after the onset of clinical symptoms from patients with confirmed influenza virus A or B infections. Nucleoprotein-specific IgA antibodies were detected in 41.2% of influenza virus A-infected patients and in 66.7% of influenza virus B-infected patients on day 6 after the onset of clinical symptoms. In serum samples taken on day 21 (influenza virus A-infected patients) or day 28 (influenza virus B-infected patients), nucleoprotein-specific IgA antibodies could be detected in 58.8 and 58.3% of influenza virus A- and B-infected patients, respectively. At the same time, IgG antibody rises were detected in 88.2% of influenza virus A-infected patients and in 95.8% of influenza virus B-infected patients. On comparison, hemagglutination inhibition assays detected antibody titer rises in 81.3 and 72.7% of patients infected with influenza viruses A and B, respectively. In contrast to the detection of nucleoprotein-specific IgG antibodies or hemagglutination-inhibiting antibodies, the detection of nucleoprotein-specific IgA antibodies does not require paired serum samples and therefore can be considered an attractive alternative for the rapid serological diagnosis of influenza.
Comparative Evaluation of the New Gen-Probe
Mycobacterium tuberculosis
Amplified Direct Test and the Semiautomated Abbott LCx
Mycobacterium tuberculosis
Assay for Direct Detection of
Mycobacterium tuberculosis
Complex in Respiratory and Extrapulmonary Specimens
Authors: Piersimoni Claudio , Callegaro Annapaola , Scarparo Claudio , Penati Valeria , Nista Domenico , Bornigia Stefano , Lacchini Carla , Scagnelli Mariuccia , Santini Gianfranco , De Sio Giuseppina
Published: December 2019

Two commercial assays that detect
Mycobacterium tuberculosis
complex (MTB) in clinical specimens by rRNA target amplification (AMTDII) and ligase chain reaction (LCx) were evaluated. The tests were applied to 457 respiratory (
n
= 273) and extrapulmonary (
n
= 184) specimens collected from 357 patients. The results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered to be the “gold standard.” Seventy specimens were from patients with pulmonary tuberculosis and 28 specimens were from patients with extrapulmonary tuberculosis. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values for respiratory specimens were 92.8, 99.4, 98.5, and 97%, respectively, for AMTDII and 75.7, 98.8, 96.4, and 90.5%, respectively, for LCx. With extrapulmonary specimens, the overall sensitivities, specificities, and positive and negative predictive values were 78.6, 99.3, 95.6, and 96.2%, respectively, for AMTDII and 53.6, 99.3, 93.7, and 92.1%, respectively, for LCx. The level of agreement between AMTDII and LCx assay results was 78.2%. We conclude that although both nucleic acid amplification methods are rapid and specific for the detection of MTB in clinical specimens, AMTDII is significantly more sensitive than LCx with both respiratory (
P
= 0.005) and extrapulmonary (
P
= 0.048) specimens.
Variation in Electrophoretic Karyotype and Antifungal Susceptibility of Clinical Isolates of
Cryptococcus neoformans
at a University-Affiliated Teaching Hospital from 1987 to 1994
Authors: Klepser Michael E. , Pfaller Michael A.
Published: December 2019

Ninety-eight isolates of
Cryptococcus neoformans
were collected from 30 patients at the University of Iowa Hospitals and Clinics from December 1987 through December 1994. The susceptibility of each isolate was determined against fluconazole, itraconazole, amphotericin B, and flucytosine. Of the 98 isolates, 53 were recovered from blood, 19 were recovered from cerebrospinal fluid (CSF), and 26 were recovered from other sources. Although the strains were isolated from the same institution, DNA typing by electrophoretic karyotype (EK) revealed wide genetic variation. Overall, 23 different EK profiles were identified by computer-aided analysis. An isolate exhibiting a single EK was isolated from 24 of 30 patients (80%), whereas multiple strains with unique EKs were isolated from 6 of 30 (20%) patients. Of the six patients who had multiple strains recovered, only one individual had two strains isolated from unique body sites, one strain from the blood and the other from the CSF. Six strains were isolated from multiple patients. Nine patients had multiple sequential isolates recovered over periods of time ranging from 3 days to 4 months. EK analysis revealed persistence of the same genotype in six of the cases. Three patients, however, appeared to have an isolate with a second distinct EK emerge during therapy. Of the patients with sequential positive cultures, an increase in the MICs for test agents was observed in only one case.
C. neoformans
isolates were collected over a period of 7 years, during which time MICs at our institution remained stable.
Heterogeneity of the PorB Protein in Serotype 22
Neisseria meningitidis
Authors: Urwin Rachel , Fox Andrew J. , Musilek Martin , Kriz Paula , Maiden Martin C. J.
Published: December 2019

The genetic diversity of
porB
genes from meningococcal isolates characterized as serotype 22 was investigated by gene sequencing. This procedure identified seven distinct
porB
sequences, demonstrating variation in the PorB protein recognized by the serotype 22 monoclonal antibody. This is consistent with the genetic heterogeneity of serotype 22 meningococci reported previously.
Misleading Manifestations of
Coccidioides immitis
In Vivo
Authors: Kaufman Leo , Valero Gustavo , Padhye Arvind A.
Published: December 2019

We describe a case of coccidioidomycosis in which several unusual morphologic forms of
Coccidioides immitis
occurred in biopsy tissue from the right lower lung of a patient. To our knowledge, this is the first case where so many diverse morphologic forms were manifested in a single patient in the absence of typical endosporulating spherules. Immature spherules demonstrating segmentation mimicked morula forms of
Prototheca
spp. Certain elements resembled budding cells of
Blastomyces dermatitidis
. These consisted of juxtaposed immature spherules without endospores, a germinating endospore, or thick-walled hyphal cells. Branched, septate hyphae and moniliform hyphae consisting of chains of thick-walled arthroconidia or immature spherules were also present. Complement fixation and immunodiffusion tests performed on the patient’s serum were negative for
C. immitis
,
B. dermatitidis
, and
Histoplasma capsulatum
antibodies. Fluorescent-antibody studies were carried out with a specific
C. immitis
conjugate. All of the diverse fungal tissue elements stained positive with a moderate to strong (2 to 3+) intensity.
Evaluation of the Auxacolor System for Biochemical Identification of Medically Important Yeasts
Authors: Sheppard Donald C. , deSouza Edwige , Hashmi Zubair , Robson Hugh G. , René Pierre
Published: December 2019

We compared the Auxacolor yeast identification system (Sanofi Diagnostics Pasteur) with the API 20C Aux (bioMerieux-Vitek) using 105 isolates of medically important yeasts. The Auxacolor system provided more rapid, accurate results and displayed less interobserver variability than the API 20C Aux.
Evaluation of Bias in Diagnostic-Test Sensitivity and Specificity Estimates Computed by Discrepant Analysis
Authors: Green Timothy A. , Black Carolyn M. , Johnson Robert E.
Published: December 2019

When a new diagnostic test is potentially more sensitive than the reference test used to classify persons as infected or uninfected, a substantial number of specimens from infected persons may be reference-test negative but new-test positive. Discrepant analysis involves the performance of one or more additional tests with these specimens, reclassification as infected those persons for whom the new-test-positive results are confirmed, and recalculation of the estimates of new-test sensitivity and specificity by using the revised classification. This approach has been criticized because of the bias introduced by the selective use of confirmation testing. Under conditions appropriate for evaluating a nucleic acid amplification (NAA) test for
Chlamydia trachomatis
infection with cell culture as the reference test, we compared the bias in estimates based on the discrepant-analysis classification of persons as infected or uninfected with that in estimates based on the culture classification. We concluded that the bias in estimates of NAA-test specificity based on discrepant analysis is small and generally less than that in estimates based on culture. However, the accuracy of discrepant-analysis-based estimates of NAA-test sensitivity depends critically on whether culture specificity is equal to or is slightly less than 100%, and it is affected by competing biases that are not fully taken into account by discrepant analysis.
Effects of Decontamination Methods and Culture Conditions on Viability of
Mycobacterium ulcerans
in the BACTEC System
Authors: Palomino J. C. , Portaels F.
Published: December 2019

We used the BACTEC system to evaluate the effects of several decontamination methods and the addition of antibiotics on the viability of
Mycobacterium ulcerans
. The effects of polyoxyethylene stearate or egg yolk as supplements were also evaluated to determine their impact on the growth of
M. ulcerans
. Strains of different geographic origins were subjected to Petroff, reversed Petroff, oxalic acid, and mild HCl treatments. After treatment, the viability of each strain was assessed in the BACTEC system. All of the decontamination methods tested adversely affected bacterial viability. Treatment with mild HCl gave the best results, allowing better growth rates with some strains and causing a delay in growth with others, depending on the geographic origin of the strain. A mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin did not significantly inhibit growth. Supplementing BACTEC medium with egg yolk markedly improved the recovery of
M. ulcerans
following the use of each of the decontamination methods. Our findings demonstrate a detrimental impact on the viability of
M. ulcerans
by all of the decontamination methods currently in common use. This explains, at least in part, the difficulty often experienced in cultivating this organism from clinical specimens. Egg yolk should be added to enhance the rate of successful primary cultivation of
M. ulcerans
in the BACTEC system.
Molecular Analysis of Tn
1546
in
Enterococcus faecium
Isolated from Animals and Humans
Authors: Jensen Lars Bogø , Ahrens Peter , Dons Lone , Jones Ronald N. , Hammerum Anette M. , Aarestrup Frank Møller
Published: December 2019

The internal areas and the position of integration of the glycopeptide resistance element Tn
1546
were characterized by using PCR fragment length polymorphism, sequencing, and DNA hybridization techniques with 38 high-level vancomycin-resistant
Enterococcus faecium
isolates of human and animal origins from Europe and the United States. Only minor variations in the coding regions within Tn
1546
were found, suggesting high genetic stability. The isolates originated from broilers (
n
= 5), a chicken (
n
= 1), a duck (
n
= 1), a turkey (
n
= 1), pigs (
n
= 8), a pony (
n
= 1), and humans (
n
= 23). A total of 13 different types were defined based on a single-nucleotide difference in the
vanX
gene, the presence of insertion sequences, and hybridization patterns. For some types more than one isolate were found. For type 1, 10 isolates of both human and animal origins were 1found. All were indistinguishable from the reference strain, BM4147. For type 2, 11 isolates of human and animal origins were found. Six human isolates from England were all of type 3. Two human isolates from the United States, indistinguishable from each other, were type 9. These results showed that vancomycin-resistant
E. faecium
of animal and human origins can contain indistinguishable genetic elements coding for vancomycin resistance, indicating either horizontal gene transfer between
E. faecium
organisms of human and animal origins or the existence of a common reservoir for glycopeptide resistance.
Value of Examining Multiple Sputum Specimens in the Diagnosis of Pulmonary Tuberculosis
Authors: Nelson Susan M. , Deike Marcia A. , Cartwright Charles P.
Published: December 2019

To objectively assess the value of examining multiple sputum specimens in maximizing the sensitivity of detection of
Mycobacterium tuberculosis
, we retrospectively reviewed the acid-fast bacillus smear and culture results of patients diagnosed with culture-proven pulmonary tuberculosis (TB) at Hennepin County Medical Center between 1986 and 1996. Two hundred and forty six persons were diagnosed with pulmonary TB in the time period analyzed. In 93% of these cases (229 of 246) the laboratory diagnosis was made by detection of
M. tuberculosis
in sputum specimens; however, only 52% (120 of 229) of these patients had at least three sputum specimens submitted to the laboratory at the time of diagnosis. Of the patients from whom at least three specimens were collected, 47% (56 of 120) had at least one smear-positive specimen; the third or later specimen submitted was the first smear-positive specimen for 13% (7 of 56) of these persons but was the first culture-positive specimen for only 7% (4 of 56). Of the 64 patients with smear-negative specimens, for only 5% (3 of 64) was the third or subsequent specimen submitted the first from which
M. tuberculosis
was recovered. This data indicates that, in our institution, the overwhelming majority of culture-proven pulmonary TB cases are diagnosed from the first or second sputum specimen submitted to the laboratory and that only rarely is a third specimen of diagnostic value.
Molecular Approaches to Diagnosis of Pulmonary Diseases Due to
Mycoplasma pneumoniae
Authors: Abele-Horn Marianne , Busch Ulrich , Nitschko Hans , Jacobs Enno , Bax Ralph , Pfaff Friederike , Schaffer Beatrix , Heesemann Jürgen
Published: December 2019

In this prospective study, the use of a culture-enhanced PCR assay for the detection of
Mycoplasma pneumoniae
, followed by hybridization with a specific probe (MP-HPCR) or without hybridization (MP-PCR), and the use of a nested PCR (MP-NPCR) were evaluated. Clinical samples (190 specimens) from 190 patients with respiratory complaints were incubated in culture broth overnight and then subjected to PCR. The results of the PCR were compared to those obtained by culture, the direct antigen test, and serologic testing by microparticle agglutination and by immunoblotting in unclear cases. The sensitivities were 19 CFU for MP-PCR, 1.9 CFU for MP-HPCR, and 0.019 CFU for MP-NPCR. PCR amplification of the β-globin gene was possible in 98% of cases: after dilution of the β-globin-negative samples, all samples were reactive. Correlation between negative MP-NPCR results and negative serology results was found in 89% of cases; a positive correlation was found with 10% of the patients. Samples from three immunocompromised patients were MP-NPCR positive but serologically negative. High respiratory colonization by
M. pneumoniae
(>10
5
CFU/ml) in patients with acute respiratory disease could be detected by culture, MP-PCR, and MP-NPCR. These results indicate that MP-PCR and MP-NPCR are reliable methods for the detection of
M. pneumoniae
in respiratory tract samples of patients with respiratory complaints.
Multicenter Evaluation of the New Automated Enzymun-Test Anti-HIV 1 + 2 + Subtyp O
Authors: Weber Bernard
Published: December 2019

An international multicenter study was performed to evaluate a new, automated human immunodeficiency virus (HIV) third-generation antibody assay. The Enzymun-Test Anti-HIV 1 + 2 + Subtyp O showed 100% sensitivity and 99.8% specificity among 11,172 samples from hospitalized patients and blood donors. For early HIV antibody detection in seroconversion panels, Enzymun-Test showed a sensitivity equivalent to that of the Abbott Recomb. HIV-1/HIV-2 3rd Gen. assay.
Quality Control Limits for Dilution and Disk Diffusion Susceptibility Tests of Trovafloxacin against Eight Quality Control Strains
Authors: Fuchs Peter C. , Barry Arthur L. , Brown Steven D.
Published: December 2019

A 10-laboratory collaborative effort was designed to generate data to propose quality control limits for susceptibility tests of trovafloxacin. Broth microdilution, agar dilution, and disk diffusion tests were evaluated with eight different control strains. All tests were reproducible, and control limits are proposed.
Rapid Cytomegalovirus pp65 Antigenemia Assay by Direct Erythrocyte Lysis and Immunofluorescence Staining
Authors: Ho Stephen K. N. , Lo Chi-Yuen , Cheng Ignatius K. P. , Chan Tak-Mao
Published: December 2019

A rapid cytomegalovirus (CMV) pp65 antigenemia assay with direct erythrocyte lysis (DL) with 0.8% NH
4
Cl, followed by indirect immunofluorescence staining (IF), was evaluated with 82 blood samples from renal transplant recipients, and the results were compared to those of the conventional antigenemia assay with dextran sedimentation and two-cycle alkaline phosphatase, anti-alkaline phosphatase staining (DS-APAAP). The DL-IF modification gave a higher leukocyte yield compared to DS-APAAP (75.4 versus 54.9%;
P
< 0.05), with similar leukocyte viability rates of >95%. The DL-IF methodology involved fewer technical steps, and the assay time was shortened from 5 h to less than 3 h. Nineteen of the 82 samples concordantly tested positive for pp65 antigenemia by both assays, and the readings showed a good correlation (
r
= 0.996;
P
< 0.01). No discordant results were observed. We conclude that the CMV pp65 antigenemia assay by this novel DL-IF modification is technically simpler, cheaper, and less time-consuming but yields results comparable to those of the conventional DS-APAAP assay. The shortened assay time and increased capacity to handle more samples confer distinct advantages in the rapid diagnosis and prompt treatment of CMV disease in immunosuppressed patients.
Reduction of Carriage of EnterohemorrhagicEscherichia coliO157:H7 in Cattle by Inoculation with Probiotic Bacteria
Authors: Zhao Tong , Doyle Michael P. , Harmon Barry G. , Brown Cathy A. , Mueller P. O. Eric , Parks Andrew H.
Published: December 2019

Bacteria inhibitory toEscherichia coliO157:H7 were isolated from cattle and evaluated for their potential for reducing carriage ofE. coliO157:H7 in calves. Eighteen of 1,200 bacterial isolates from cattle feces and intestinal tissue samples were screened and determined to inhibit the growth ofE. coliO157:H7 in vitro. Seventeen of the isolates wereE. coliand one wasProteus mirabilis. None produced Shiga toxin. Genomic DNA fingerprinting by pulsed-field gel electrophoresis revealed 13 distinguishable profiles among the 18 isolates. Two calves inoculated perorally with a mixture of all 18 isolates (1010CFU) appeared to be normal and did not develop signs of clinical disease throughout a 25- to 27-day observation period. These bacteria colonized segments of the gastrointestinal tract and were in feces at the termination of the experiment (25 and 27 days postinoculation) at levels of 50 to 200 CFU/g. Fifteen cannulated calves were studied to determine the efficiency of the probiotic bacteria in reducing or eliminating the carriage ofE. coliO157:H7. Nine calves served as controls, with each animal receiving perorally 1010CFU ofE. coliO157:H7.E. coliO157:H7 was detected intermittently in the rumen samples from all control animals throughout 3 weeks postinoculation, whereasE. coliO157:H7 was shed at various levels in feces continuously throughout the experiment (mean, 28 days).E. coliO157:H7 was isolated from the rumens and colons of eight of nine and nine of nine calves, respectively, at the termination of the study. Six calves each received perorally 1010CFU of probiotic bacteria and then 2 days later received 1010CFU ofE. coliO157:H7.E. coliO157:H7 was detected in the rumen for only 9 days postinoculation in two animals, for 16 days in one animal, for 17 days in two animals, and for 29 days in one animal.E. coliO157:H7 was detected in feces for only 11 days postinoculation in one animal, for 15 days in one animal, for 17 days in one animal, for 18 days in one animal, for 19 days in one animal, and for 29 days in one animal. At the end of the experiment (mean, 30 days),E. coliO157:H7 was not recovered from the rumen of any of the six animals treated with probiotic bacteria; however,E. coliO157:H7 was recovered from the feces of one of the animals. This animal was fasted twice postinoculation. These studies indicate that selected probiotic bacteria administered to cattle prior to exposure toE. coliO157:H7 can reduce the level of carriage ofE. coliO157:H7 in most animals.
Antigenic Diversity of Hepatitis B Virus Strains of Genotype F in Amerindians and Other Population Groups from Venezuela
Authors: Blitz Linda , Pujol Flor H. , Swenson Paul D. , Porto Leticia , Atencio Ricardo , Araujo Mary , Costa Luciana , Monsalve Diana Callejas , Torres Jaime R. , Fields Howard A. , Lambert Steve , Van Geyt Caroline , Norder Helene , Magnius Lars O. , Echevarría José M. , Stuyver Lieven
Published: December 2019

The adw4 subtype of hepatitis B virus (HBV) belongs to a unique genomic group (genotype F) representing the original HBV strains from the New World. Data regarding the prevalence of this subtype among HBV carriers in South America are, however, scarce, and those concerning HBV genotype F are based on only a few samples from Latin America. In this study, serum samples were obtained from 141 hepatitis B surface antigen (HBsAg) carriers from Amerindians and urban populations from Venezuela. The HBsAg subtype was identified with monoclonal antibodies in 105 samples, and the HBV genotype was identified by reverse-phase hybridization with DNA fragments in 58 samples. The adw4 subtype was highly prevalent in the population studied (75%); among the Amerindians, the prevalence was 97%. The adw2 subtype was also present (10%), while other subtypes (ayw3 and ayw4) were only occasionally found. The HBV subtype was associated with the expected genotype in most cases (80%), and thus genotype F was highly prevalent. Sequencing of viral strains that gave genotypes unpredicted by the HBsAg subtyping confirmed seven of them as belonging to not previously described genotype-subtype associations: namely, adw2 and ayw4 within genotype F.
Epidemiological Study of a Food-Borne Outbreak of Enterotoxigenic
Escherichia coli
O25:NM by Pulsed-Field Gel Electrophoresis and Randomly Amplified Polymorphic DNA Analysis
Authors: Mitsuda Toshihiro , Muto Tetsunori , Yamada Mikiko , Kobayashi Nobuyoshi , Toba Masanori , Aihara Yukoh , Ito Akira , Yokota Shumpei
Published: December 2019

This study investigated the applicability of molecular epidemiological techniques to the identification of the causal agent of an outbreak of diarrhea caused by ingestion of food contaminated with enterotoxigenic
Escherichia coli
(ETEC). The outbreak occurred at four elementary schools in July 1996 and affected more than 800 people. Illness was most strongly associated with eating tuna paste (relative risk, 1.79; 95% confidence interval = 1.16 to 2.79;
P
= 0.0001). To evaluate the epidemiological characteristics of the pathogen, the DNAs from numerous isolated ETEC strains were subjected to randomly amplified polymorphic DNA analysis, pulsed-field gel electrophoresis of nuclease S1-treated plasmid DNA, and analysis of genomic DNA restriction fragment length polymorphisms. All ETEC isolates were of the O25:NM (nonmotile) serotype, which carries a heat-stable enterotoxin Ib gene. Genotypic analysis demonstrated that the strains isolated from the patients at all four schools were identical. The isolates of ETEC O25:NM obtained from the tuna paste that had been served for lunch at these schools were genetically indistinguishable from those isolated from the patients. Results suggest that this outbreak was food borne. The molecular biology-based epidemiological techniques used in this study were useful in characterizing the causal agent in this food-borne epidemic.
Comparative Evaluation of Initial and New Versions of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for Direct Detection of
Mycobacterium tuberculosis
in Respiratory and Nonrespiratory Specimens
Authors: Gamboa Fredy , Fernandez Gregorio , Padilla Eduardo , Manterola José M. , Lonca Joan , Cardona Pere Joan , Matas Lurdes , Ausina Vicente
Published: December 2019

We evaluated the initial version of the Amplified Mycobacterium Tuberculosis Direct Test (Gen-Probe) (AMTDT 1) and the new version of AMTDT (AMTDT 2) for the detection of
Mycobacterium tuberculosis
directly from respiratory and nonrespiratory samples and compared the results with those of culture and staining methods. The assays were applied to 410 respiratory and 272 nonrespiratory samples collected from 515 patients. The combination of the culture results and clinical diagnosis was considered to be the “gold standard.” Ninety-five respiratory specimens were collected from 67 patients with a diagnosis of pulmonary tuberculosis (TB) and 68 nonrespiratory specimens were collected from 61 patients with a diagnosis of extrapulmonary TB. With respiratory specimens, the sensitivity, specificity, and positive and negative predictive values were 83, 100, 100, and 96%, respectively, for AMTDT 1 and 94.7, 100, 100, and 98.4%, respectively, for AMTDT 2. With nonrespiratory specimens, the sensitivity, specificity, and positive and negative predictive values were 83, 100, 100, and 94%, respectively, for AMTDT 1 and 86.8, 100, 100, and 98.4%, respectively, for AMTDT 2. The overall results of AMTDT 1 and AMTDT 2 were concordant for 97% (661 of 682) of the samples. Statistically significant differences in sensitivities were found between AMTDT 1 and AMTDT 2 with respiratory specimens. It was concluded that although both nucleic acid amplification methods are rapid, sensitive, and specific for the detection of
M. tuberculosis
complex in all types of clinical samples, AMTDT 2 appeared to be more sensitive than AMTDT 1 when applied to smear-negative specimens. In contrast AMTDT 2 is more susceptible than AMTDT 1 to inhibitory substances in the amplification reaction. The turnaround time of AMTDT 2 is shorter (3.5 h) than that for AMTDT 1 (5 h).
Immunosorbent Assay Based on Recombinant Hemagglutinin Protein Produced in a High-Efficiency Mammalian Expression System for Surveillance of Measles Immunity
Authors: Bouche Fabienne , Ammerlaan Wim , Berthet Francoise , Houard Sophie , Schneider Francois , Muller Claude P.
Published: December 2019

Recombinant hemagglutinin (H) protein of the measles virus (MV) was produced in mammalian cells with a high-yield expression system based on the Semliki Forest virus replicon. Crude membrane preparations of H protein-transfected BHK-21 cells were used to coat microtiter plates to measure specific immunoglobulin G antibodies in 228 serologically defined serum samples mainly from measles late-convalescent adults. The titers by the enzyme-linked immunosorbent assay for the H protein (H-ELISA) closely correlated with neutralization test (NT) titers (
R
2
= 0.66), hemagglutination inhibition test (HI) titers (
R
2
= 0.64), with the titers from a certified commercial ELISA based on whole MV-infected cells (MV-ELISA;
R
2
= 0.45). The correlations described above were better than those of the commercial MV-ELISA titers with the NT (
R
2
= 0.52) or HI (
R
2
= 0.48) titers. By using the 2nd International Standard for anti-measles serum, the detection level of the assay corresponds to 215 mIU/ml for undiluted serum, which corresponds to the estimated threshold for protective immunity. The specificity, accuracy, and positive predictive value were, in general, better for the H-ELISA than for a commercial MV-ELISA, independent of whether HI, NT, or HI and NT were used as “gold standards.” In contrast, the H-ELISA proved to be slightly less sensitive than the MV-ELISA (sensitivities, 98.6 versus 99.5%, respectively;
P
was not significant). The assays did not differ significantly in the number of serum samples with positive HI and NT results (
n
= 212) which measured false negative (H-ELISA, 2 of 212 [0.94%]; MV-ELISA, 1 of 212 [0.47%]), but the H-ELISA detected significantly more measles-susceptible individuals than the MV-ELISA (10 of 11 versus 3 of 11, respectively;
P
< 0.05) among the individuals whose sera had negative HI and NT results. Our data demonstrate that the H-protein preparation that we describe could be a cost-effective alternative to current whole-virus-based ELISAs for surveillance for immunity to measles and that such an assay could be more efficient in detecting susceptibility to measles. Furthermore, unlike whole MV-based antigens, H-protein would also be suitable for use in the development of a simple field test for the diagnosis of measles.