bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Detection and Epidemiology of Sapporo-Like Viruses
Authors: Vinjé Jan , Deijl Hanneke , van der Heide Reina , Lewis David , Hedlund Kjell-Olof , Svensson Lennart , Koopmans Marion P. G. 
Published: December 2019

ABSTRACT
Sapporo-like viruses (SLVs) are associated with acute gastroenteritis in humans. Due to a limited supply of available reagents for diagnosis, little is known about the incidence and pathogenicity of these viruses. We have developed a first-generation generic reverse transcriptase (RT) PCR assay based on a single primer pair targeting the RNA polymerase gene. With this assay, 55 (93%) of the 59 stool specimens collected in a 10-year period of time (1988 to 1998) and containing typical caliciviruses by electron microscopy tested positive and could be confirmed by Southern hybridization. By phylogenetic analysis, most SLV strains could be classified into one of the three recently described genotypes. However, three samples clustered separately, forming a potential new genotype. We sequenced the complete capsid gene of one of the strains in this cluster: Hu/SLV/Stockholm/97/SE. Alignment of the capsid sequences showed 40 to 74% amino acid identity among strains of the different clusters. Phylogenetic analysis of the aligned sequences confirmed the placing of Hu/SLV/Stockholm/97/SE into a new distinct genetic cluster. This is the first report on the development of a broadly reactive RT-PCR assay for the detection of SLVs.
Screening for Hepatitis C Virus in Human Immunodeficiency Virus-Infected Individuals
Authors: Thio Chloe L. , Nolt Karen R. , Astemborski Jacquie , Vlahov David , Nelson Kenrad E. , Thomas David L. 
Published: December 2019

ABSTRACT
Immunosuppression from human immunodeficiency virus (HIV) may impair antibody formation, and false-negative hepatitis C virus antibody (anti-HCV) tests have been reported in individuals coinfected with HIV and HCV. It is unknown if the frequency of false-negative tests is sufficiently high to change screening recommendations in this setting. Thus, the prevalence of false-negative results for anti-HCV by third-generation tests was determined with samples from HIV-infected individuals. Sera from 559 HIV-infected and 944 HIV-negative prospectively followed injection drug users were tested for anti-HCV by a third-generation enzyme immunoassay and for HCV RNA by using a branched DNA assay and the HCV COBAS AMPLICOR system. Of 559 HIV-infected participants, 547 (97.8%) were anti-HCV positive. One of the remaining 12 anti-HCV-negative participants was HCV RNA positive, and she later developed detectable anti-HCV. Of the 944 HIV-negative participants, 825 (87.4%) were anti-HCV positive. One of the remaining 119 anti-HCV-negative participants was HCV RNA positive, and she also developed detectable anti-HCV at a later visit. These data indicate that HIV infection does not alter the approach to hepatitis C virus screening, which should be performed with third-generation assays for anti-HCV unless acute infection is suspected.
Evaluation of a Specific Nested PCR Targeting Domain III of the 23S rRNA Gene of “
Tropheryma whippelii
” and Proposal of a Classification System for Its Molecular Variants
Authors: Hinrikson Hans Peter , Dutly Fabrizio , Altwegg Martin 
Published: December 2019

ABSTRACT

“
Tropheryma whippelii
”-associated infections are usually confirmed histopathologically by using light microscopy. PCR assays targeting the 16S rRNA gene (16S rDNA) of “
T. whippelii
” are increasingly being applied for this purpose. Compared to microscopic analysis, PCR seems to be more sensitive, as indicated by the fact that several cases of Whipple's disease with negative histopathological findings but positive PCR results have been reported. Considering the lack of pathognomonic clinical features for this disease and the fact that “
T. whippelii
” DNA has repeatedly been found in patients without clinical Whipple's disease, such PCR results should be confirmed by additional tests. We have, therefore, evaluated a “
T. whippelii
”-specific nested PCR targeting domain III of the 23S rDNA with 41 clinical specimens known to contain “
T. whippelii
” 16S rDNA. All of these specimens were also positive for “
T. whippelii
” 23S rDNA. The specificity of the test was shown by sequencing of the amplicons and by the absence of amplicons in 38 negative controls. We consider this PCR test to be a suitable tool for confirming the presence of “
T. whippelii
” DNA in specimens with inconclusive histopathological findings. The information derived from sequencing of the partial “
T. whippelii
” 23S rDNA was then combined with our recent data of the 16S-23S rDNA spacer region of this organism. Overall, four different rDNA types are recognized in our proposed classification system for molecular variants of “
T. whippelii
.” This preliminary scheme may provide a basis for further epidemiological and clinical studies with “
T. whippelii
” and associated diseases.
Serology of Culture-Confirmed Cases of Human Granulocytic Ehrlichiosis
Authors: Aguero-Rosenfeld Maria E. , Kalantarpour Fatemeh , Baluch Mehdi , Horowitz Harold W. , McKenna Donna F. , Raffalli John T. , Hsieh Tze-chen , Wu Joseph , Dumler J. Stephen , Wormser Gary P. 
Published: December 2019

ABSTRACT

We evaluated the antibody responses in the sera of 24 patients with culture-confirmed human granulocytic ehrlichiosis (HGE). Antibody titers were measured by an indirect immunofluorescent-antibody assay (IFA) by using a local human isolate as the source of antigen. All patients received appropriate antimicrobial treatment. One hundred five serum specimens collected at baseline and at periodic intervals for up to 14 months were included in the study. Seroconversion was observed in 21 of 23 patients (91.3%) from whom convalescent-phase sera were obtained. Antibodies were first detected at an average of 11.5 days after onset of symptoms. Peak titers (≥2,560 for 71.4% of patients and ≥640 for 95.2% of patients) were obtained an average of 14.7 days after onset of symptoms. Eleven of 13 patients (84.6%) from whom sera were collected between 6 and 10 months after onset of symptoms were still seropositive, and sera from 5 of 10 (50%) patients tested positive between 11 and 14 months after onset of symptoms. For a subset of 71 serum specimens from 17 patients with culture-confirmed HGE also tested by IFA by using either a human isolate from Wisconsin or an
Ehrlichia equi
isolate from a horse, there was qualitative agreement for 62 serum specimens (87.3%). Peak titers were higher, however, with the local human HGE isolate, but the difference was not statistically significant. In summary, most patients with culture-confirmed HGE develop antibodies within 2 weeks of onset of symptoms. Antibodies reach high titers during the first month and remain detectable in about one-half of patients at 1 year after onset of symptoms.
Prevalence of GB Virus C/Hepatitis G Virus Infection among Various Populations in Surabaya, Indonesia, and Identification of Novel Groups of Sequence Variants
Authors: Handajani Retno , Soetjipto  , Lusida Maria Inge , Suryohudoyo Purnomo , Adi Pangestu , Setiawan Poernomo Boedi , Nidom Chairul Anwar , Soemarto R. , Katayama Yuko , Fujii Motoko , Hotta Hak 
Published: December 2019

ABSTRACT

A molecular epidemiological study was performed to investigate the prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) infection among various populations in Surabaya, Indonesia. The prevalence of GBV-C/HGV RNA, determined by reverse transcription-PCR for a portion of the NS3 region of the viral genome, was 2.7% (4 of 150) among randomly collected blood donor sera, which were all negative for both hepatitis B virus surface antigen and antibodies against hepatitis C virus (HCV). On the other hand, the prevalence among anti-HCV-positive blood donors was 17.8% (13 of 73), with the ratio being significantly higher than that observed with the anti-HCV-negative blood donors (
P
< 0.001). A high prevalence of GBV-C/HGV infection was also observed among patients with chronic liver disease, such as chronic hepatitis (5.7%), liver cirrhosis (11.5%), and hepatocellular carcinoma (7.0%), and patients on maintenance hemodialysis (29.0%). No correlation was observed between GBV-C/HGV viremia and serum alanine aminotransferase levels in the populations tested, suggesting the possibility that GBV-C/HGV does not cause apparent liver injury. Phylogenetic analysis of sequences of a portion of the 5′ untranslated region and the E1 region of the viral genome identified, in addition to a previously reported then novel group of GBV-C/HGV variants (group 4), another novel group of variants (group 5). This result suggests that GBV-C/HGV can be classified into at least five genetic groups. GBV-C/HGV isolates of group 4 and group 5 were each shown to comprise approximately 40% of the total Indonesian isolates.
A Novel and Rapid PCR-Based Method for Genotyping Human Papillomaviruses in Clinical Samples
Authors: Nelson Joshua H. , Hawkins Gregory A. , Edlund Karin , Evander Magnus , Kjellberg Lennart , Wadell Göran , Dillner Joakim , Gerasimova Tsilya , Coker Ann L. , Pirisi Lucia , Petereit Daniel , Lambert Paul F. 
Published: December 2019

ABSTRACT

Many human papillomavirus (HPV) genotypes are associated with cervical carcinoma. We demonstrate the utility of an innovative technique for genotyping of HPV in cervical tissue samples. This method provides an accurate means of identification of the specific HPV genotypes present in clinical specimens. By using the MY09-MY11 and the GP5
+
-GP6
+
consensus primer pairs, HPV sequences were amplified by nested PCR from DNA isolated from cervical smear samples. This led to the production of an approximately 140-bp PCR product from the L1 (major capsid) gene of any of the HPVs present in the sample. PCR was performed with a deoxynucleoside triphosphate mixture which resulted in the incorporation of deoxyuridine into the amplified DNA product at positions where deoxythymidine would normally be incorporated at a frequency of about once or twice per strand. Following the PCR, the product was treated with an enzyme mix that contains uracil
N
-glycosylase (UNG) and endonuclease IV. UNG removes the uracil base from the nucleotide, and endonuclease IV cleaves the phosphodiester bond at this newly formed abasic site, producing fragments of various sizes. By having end labeled one of the amplification primers, a DNA ladder which is analogous to a “T-sequencing ladder” was produced upon electrophoresis of the products. By comparing this T-sequencing ladder to the known sequences of HPVs, the genotypes of unknown HPV isolates in samples were assigned. Data showing the utility of this technique for the rapid analysis of clinical samples are presented.
Use of PCR To Detect
Leishmania
(
Viannia
) spp. in Dog Blood and Bone Marrow
Authors: Reithinger Richard , Lambson Bronwen E. , Barker Douglas C. , Davies Clive R. 
Published: December 2019

ABSTRACT

A PCR-based protocol for the detection of
Leishmania
(
Viannia
) parasites in canine blood, buffy coat, and bone marrow was developed and was then tested with field samples taken from a random sample of 545 dogs from villages in Peru where
Leishmania
(
Viannia
)
braziliensis
and
Leishmania
(
Viannia
)
peruviana
are endemic. Comparative tests with cultured parasites mixed with dog blood showed that the PCR assay's sensitivity was significantly dependent on the DNA extraction protocol and the PCR primers used. Mass screening of field samples by the preferred PCR protocol detected American cutaneous leishmaniasis (ACL) in 44 of 545 (8.1%) dogs; 31 of 402 (7.7%), 20 of 223 (9.0%), and 8 of 46 (17.4%) were PCR positive when whole blood, buffy coat, and bone marrow aspirates, respectively, were tested. The high prevalence of
Leishmania
in both asymptomatic (7.6%) and symptomatic (18.0%) dogs provides further circumstantial evidence for their suspected role as reservoir hosts of ACL and indicates that hematogenous dissemination of parasites may be a more common pathological phenomenon than has previously been acknowledged. However, unlike for zoonotic visceral leishmaniasis, the comparatively low prevalence of
Leishmania
(
Viannia
) in the blood of symptomatic dogs indicates that PCR with blood cannot be the “gold standard” for the (mass) screening of samples in epidemiological studies.
Comparison of PCR, Antigenemia Assay, and Rapid Blood Culture for Detection and Prevention of Cytomegalovirus Disease after Lung Transplantation
Authors: Weinberg Adriana , Hodges Tony N. , Li Shaobing , Cai Guanyung , Zamora Martin R. 
Published: December 2019

ABSTRACT
The goal of this study was to evaluate serial cytomegalovirus (CMV) blood culture, antigenemia testing, and qualitative and quantitative plasma CMV PCR for their ability to predict CMV disease and thus to direct preemptive therapy after lung transplantation. Forty-one patients provided 414 samples for blood culture, 290 samples for antigenemia testing, and 432 samples for PCR. Seven patients developed 11 episodes of CMV disease. CMV PCR had sensitivity, specificity, and positive predictive and negative predictive values of 79, 99, 84, and 99%, respectively, compared with 48, 99, 85, and 98%, respectively, for antigenemia testing, and 8, 100, 100, and 97%, respectively, for culture. Only quantitative CMV PCR correlated with disease stage: asymptomatic patients had a mean of 1,500 CMV DNA copies/ml, whereas patients who developed CMV disease had 5,087 copies/ml 12 to 4 weeks before symptoms and 32,000 copies/ml at diagnosis. Furthermore, CMV PCR-measured DNA increased 5- to 10-fold immediately preceding symptoms. PCR and antigenemia test values decreased with anti-CMV therapy. CMV DNA (as detected by PCR), but not antigenemia, persisted in patients who later developed recurrent CMV disease. The data indicate that lung transplant recipients will benefit from monitoring of CMV disease by plasma CMV PCR.
Analysis of Genetic Variability within the Immunodominant Epitopes of Envelope gp41 from Human Immunodeficiency Virus Type 1 (HIV-1) Group M and Its Impact on HIV-1 Antibody Detection
Authors: Dorn Jonathan , Masciotra Silvina , Yang Chunfu , Downing Robert , Biryahwaho Benon , Mastro Timothy D. , Nkengasong John , Pieniazek Danuta , Rayfield Mark A. , Hu Dale J. , Lal Renu B. 
Published: December 2019

ABSTRACTThe serodiagnosis of human immunodeficiency virus type 1 (HIV-1) infection primarily relies on the detection of antibodies, most of which are directed against the immunodominant regions (IDR) of HIV-1 structural proteins. Among these, the N-terminal region of gp41 contains cluster I (amino acids [aa] 580 to 623), comprising the cytotoxic T-lymphocyte epitope (AVERYLKDQQLL) and the cysteine loop (CSGKLIC), and cluster II (aa 646 to 682), comprising an ectodomain region (ELDKWA). To delineate the epitope diversity within clusters I and II and to determine whether the diversity affects serologic detection by U.S. Food and Drug Administration (FDA)-licensed enzyme immunoassay (EIA) kits, gp41 Env sequences from 247 seropositive persons infected with HIV-1 group M, subtypes A (n= 42), B (n= 62), B′ (n= 13), C (n= 38), D (n= 41), E (n= 18), F (n= 27), and G (n= 6), and 6 HIV-1-infected but persistently seronegative (HIPS) persons were analyzed. While all IDR were highly conserved among both seropositive and HIPS persons, minor amino acid substitutions (<20% for any one residue, mostly conservative) were observed for all subtypes, except for B′, in comparison with the consensus sequence for each subtype. Most importantly, none of the observed substitutions among the group M plasma specimens affected antibody detection, since all specimens (n= 152) tested positive with all five FDA-licensed EIA kits. Furthermore, all specimens reacted with a group M consensus gp41 peptide (WGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASW), and high degrees of cross-reactivity (>80%) were observed with an HIV-1 group N peptide, an HIV-1 group O peptide, and a peptide derived from the homologous region of gp41 from simian immunodeficiency virus from chimpanzee (SIVcpz). Taken together, these data indicate that the minor substitutions observed within the IDR of gp41 of HIV-1 group M subtypes do not affect antibody recognition and that all HIV-1-seropositive specimens containing the observed substitutions react with the FDA-licensed EIA kits regardless of viral genotype and geographic origin.
Rapid Diagnosis of Bacteremia by Universal Amplification of 23S Ribosomal DNA Followed by Hybridization to an Oligonucleotide Array
Authors: Anthony R. M. , Brown T. J. , French G. L. 
Published: December 2019

ABSTRACTThe rapid identification of bacteria in blood cultures and other clinical specimens is important for patient management and antimicrobial therapy. We describe a rapid (<4 h) detection and identification system that uses universal PCR primers to amplify a variable region of bacterial 23S ribosomal DNA, followed by reverse hybridization of the products to a panel of oligonucleotides. This procedure was successful in discriminating a range of bacteria in pure cultures. When this procedure was applied directly to 158 unselected positive blood culture broths on the day when growth was detected, 125 (79.7%) were correctly identified, including 4 with mixed cultures. Nine (7.2%) yielded bacteria for which no oligonucleotide targets were present in the oligonucleotide panel, and 16 culture-positive broths (10.3%) produced no PCR product. In seven of the remaining eight broths, streptococci were identified but not subsequently grown, and one isolate ofStaphylococcus aureuswas misidentified as a coagulase-negative staphylococcus. The accuracy, range, and discriminatory power of the assay can be continually extended by adding further oligonucleotides to the panel without significantly increasing complexity or cost.
Specificity and Sensitivity of High Levels of Immunoglobulin G Antibodies against Pertussis Toxin in a Single Serum Sample for Diagnosis of Infection with
Bordetella pertussis
Authors: de Melker H. E. , Versteegh F. G. A. , Conyn-van Spaendonck M. A. E. , Elvers L. H. , Berbers G. A. M. , van der Zee A. , Schellekens J. F. P. 
Published: December 2019

ABSTRACT

Laboratory confirmation of pertussis by culture, PCR, or detection of antibody increase in paired sera is hampered by low sensitivity in the later stages of the disease. Therefore, we investigated whether, and at which level, concentrations of immunoglobulin G (IgG) antibodies against pertussis toxin (PT), IgG-PT, in a single serum sample are indicative of active or recent pertussis. IgG-PT, measured by enzyme-linked immunosorbent assay in units per milliliter, was analyzed in 7,756 sera collected in a population-based study in The Netherlands, in the sera of 3,491 patients with at least a fourfold increase of IgG-PT, in paired sera of 89 patients with positive cultures and/or PCR results, and in the sera of 57 patients with clinically documented pertussis with a median follow-up of 1.4 years. We conclude that, independently of age, IgG-PT levels of at least 100 U/ml are diagnostic of recent or active infection with
Bordetella pertussis
. Such levels are present in less than 1% of the population and are reached in most pertussis patients within 4 weeks after disease onset and persist only temporarily.
Serological Confirmation of Chagas' Disease by a Recombinant and Peptide Antigen Line Immunoassay: INNO-LIA Chagas
Authors: Saez-Alquézar Amadeo , Sabino Ester C. , Salles Nanci , Chamone Dalton F. , Hulstaert Frank , Pottel Hans , Stoops Erik , Zrein Maan 
Published: December 2019

ABSTRACT

Although screening for
Trypanosoma cruzi
antibodies is mandatory in most South American countries, current tests are insensitive and have poor specificity. A recently optimized line immunoassay (the INNO-LIA Chagas assay) for the serological confirmation of Chagas' disease was evaluated at a large blood bank in São Paulo, Brazil. Sera from blood donors who reacted in at least one of three serological screening assays (
n
= 1,604) and who returned for a follow-up were retested, and the donors were interviewed to assess their epidemiological risk. The results obtained by the confirmatory assay evaluated in this study were compared to those obtained by the three different screening assays. Upon consideration of the consensus results obtained by the three different screening assays as a “gold standard,” the INNO-LIA Chagas assay showed a sensitivity of 99.4% (95% confidence interval [CI], 98.3 to 99.9) and a specificity of 98.1% (95% CI, 96.6 to 99.0) for positive (
n
= 503) and negative (
n
= 577) sera. The INNO-LIA Chagas assay confirmed the results for significantly larger numbers of positive samples of at-risk individuals independent of the number of positive screening tests (
P
= 0.017, Mantel-Haenszel test). In conclusion, the INNO-LIA Chagas assay reliably confirmed the presence of antibodies to
T. cruzi
and can be implemented as a confirmatory assay for Chagas' disease serology.
Reliability of the MB/BacT System for Testing Susceptibility of
Mycobacterium tuberculosis
Complex Isolates to Antituberculous Drugs
Authors: Brunello Francesca , Fontana Roberta 
Published: December 2019

ABSTRACT

The susceptibility of 115
Mycobacterium tuberculosis
complex clinical isolates to isoniazid, streptomycin, ethambutol, and rifampin was assessed by the MB/BacT and BACTEC 460TB systems. The correlation between the two tests was 98.3% for isoniazid, 100% for streptomycin and rifampin, and 95.8% for ethambutol. Turnaround times for antimicrobial susceptibility testing ranged from 5 to 11 days (median, 8.5 days) for MB/BacT and from 4 to 8 days (median, 6 days) for BACTEC 460TB.
Hepatitis C Virus Quantitation: Optimization of Strategies for Detecting Low-Level Viremia
Authors: Hofgärtner Wolfgang T. , Kant Jeffrey A. , Weck Karen E. 
Published: December 2019

ABSTRACT
A long-term assessment of quantitative hepatitis C virus (HCV) testing was performed at the University of Pittsburgh Medical Center. The Quantiplex HCV RNA 2.0 branched-chain DNA (bDNA) assay (Bayer Diagnostics) for hepatitis C viral load determination was used to test 3,471 specimens. bDNA-negative samples were also tested by an in-house qualitative reverse transcriptase (RT)-PCR assay with a measured sensitivity of fewer than 100 HCV genome equivalents per milliliter. Of 1,239 bDNA-negative specimens, 74.1% were negative and 25.9% were positive by RT-PCR, indicating the presence of viremia in a significant proportion of bDNA-negative samples. We discuss the medical and economic implications of these results and propose two alternatives for clinical laboratories to consider in approaching quantitative HCV testing. For laboratories able to perform a sensitive RT-PCR assay for ≤40% of the bDNA test cost, prescreening bDNA requests by RT-PCR may be the most cost-effective approach.
Epidemic Typhoid in Vietnam: Molecular Typing of Multiple-Antibiotic-Resistant
Salmonella enterica
Serotype Typhi from Four Outbreaks
Authors: Connerton Phillippa , Wain John , Hien Tran T. , Ali Tahir , Parry Christopher , Chinh Nguyen T. , Vinh Ha , Ho Vo A. , Diep To S. , Day Nicholas P. J. , White Nicholas J. , Dougan Gordon , Farrar Jeremy J. 
Published: December 2019

ABSTRACT

Multidrug-resistant
Salmonella enterica
serotype Typhi isolates from four outbreaks of typhoid fever in southern Vietnam between 1993 and 1997 were compared. Pulsed-field gel electrophoresis, bacteriophage and plasmid typing, and antibiotic susceptibilities showed that independent outbreaks of multidrug-resistant typhoid fever in southern Vietnam are caused by single bacterial strains. However, different outbreaks do not derive from the clonal expansion of a single multidrug-resistant serotype Typhi strain.