bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of Lassa Virus Antinucleoprotein Immunoglobulin G (IgG) and IgM Antibodies by a Simple Recombinant Immunoblot Assay for Field Use
Authors: ter Meulen J. , Koulemou K. , Wittekindt T. , Windisch K. , Strigl S. , Conde S. , Schmitz H. 
Published: December 2019

ABSTRACT

The nucleoprotein of Lassa virus, strain Josiah, was expressed in
Escherichia coli
as an N-terminally truncated, histidine-tagged recombinant protein. Following affinity purification the protein was completely denatured and spotted onto nitrocellulose membrane. A total of 1 μg of protein was applied for detection of Lassa virus antibodies (LVA) in a simple immunoblot assay. Specific anti-Lassa immunoglobulin M (IgM) antibodies could be detected by increasing the amount of protein to 5 μg. A panel of 913 serum specimens from regions in which Lassa virus was endemic and from regions in which Lassa virus was not endemic was used for evaluating the sensitivity and specificity of the LVA immunoblot in comparison to those of an indirect immunofluorescence (IIF) assay. The sera originated from field studies conducted in the Republic of Guinea (570 serum samples) and Liberia (99 serum samples), from inpatients of the clinical department of the Bernhard-Nocht-Institute, Hamburg, Germany (94 serum samples), and from healthy German blood donors (150 serum samples). In comparison to the IIF assay the LVA immunoblot assay had a specificity of 90.0 to 99.3%, depending on the origin of the specimens. The sensitivity was found to be highest for the Guinean samples (90.7%) and was lower for the Liberian samples (75%). Acute Lassa fever was diagnosed by PCR in 12 of 59 (20.3%) patients with fever of unknown origin (FUO) from the Republic of Guinea. On admission to the hospital, nine Lassa fever patients (75%) were reactive by the IgM immunoblot assay. One of the patients was infected with a new Lassa variant, which showed 10.4% variation on the amino acid level in comparison to the prototype strain of Lassa virus, Josiah. Seven PCR-negative patients were reactive by immunoblotting. The positive and negative predictive values of a single IgM immunoblot result for acute, PCR-confirmed Lassa fever were therefore 53.6 and 93.0%, respectively. Because of its high negative predictive value, a single IgM immunoblot result will be valuable for excluding acute Lassa fever for cases of FUO in areas where Lassa fever is endemic.
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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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

ABSTRACTDuring 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

ABSTRACT
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.