bluuebunny/crossref_metadata_2025 huggingface dataset:
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

The 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

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

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

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

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

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.
Multilocus Sequence Typing for Characterization of Methicillin-Resistant and Methicillin-Susceptible Clones ofStaphylococcus aureus
Authors: Enright Mark C. , Day Nicholas P. J. , Davies Catrin E. , Peacock Sharon J. , Spratt Brian G.
Published: December 2019

A multilocus sequence typing (MLST) scheme has been developed forStaphylococcus aureus. The sequences of internal fragments of seven housekeeping genes were obtained for 155S. aureusisolates from patients with community-acquired and hospital-acquired invasive disease in the Oxford, United Kingdom, area. Fifty-three different allelic profiles were identified, and 17 of these were represented by at least two isolates. The MLST scheme was highly discriminatory and was validated by showing that pairs of isolates with the same allelic profile produced very similarSmaI restriction fragment patterns by pulsed-field gel electrophoresis. All 22 isolates with the most prevalent allelic profile were methicillin-resistantS. aureus(MRSA) isolates and had allelic profiles identical to that of a reference strain of the epidemic MRSA clone 16 (EMRSA-16). Four MRSA isolates that were identical in allelic profile to the other major epidemic MRSA clone prevalent in British hospitals (clone EMRSA-15) were also identified. The majority of isolates (81%) were methicillin-susceptibleS. aureus(MSSA) isolates, and seven MSSA clones included five or more isolates. Three of the MSSA clones included at least five isolates from patients with community-acquired invasive disease and may represent virulent clones with an increased ability to cause disease in otherwise healthy individuals. The most prevalent MSSA clone (17 isolates) was very closely related to EMRSA-16, and the success of the latter clone at causing disease in hospitals may be due to its emergence from a virulent MSSA clone that was already a major cause of invasive disease in both the community and hospital settings. MLST provides an unambiguous method for assigning MRSA and MSSA isolates to known clones or assigning them as novel clones via the Internet.
Comparison of Different PCR Approaches for Typing of
Francisella tularensis
Strains
Authors: de la Puente-Redondo V. A. , del Blanco N. García , Gutiérrez-Martín C. B. , García-Peña F. J. , Ferri E. F. Rodríguez
Published: December 2019

In this study, we evaluated three PCR methods for epidemiological typing of
Francisella tularensis
: repetitive extragenic palindromic element PCR (REP-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and random amplified polymorphic DNA (RAPD) assay with both M13 and T3-T7 primers. The analysis was performed with 40 strains of
F. tularensis
isolated from hares, humans, ticks, and a vole. On the basis of the combination of REP, ERIC, and RAPD fingerprints,
F. tularensis
strains were divided into 17 genetic groups (designated A to Q), and one
Francisella novicida
strain was classified in group R. The
F. novicida
strain is of special concern, since previous genetic methods have been unable to clearly distinguish between
F. tularensis
and
F. novicida
. The
F. tularensis
isolates recovered from hares were included in groups A to J, M, and P; those recovered from humans were included in groups A, D, G, J, L, O, and N; those isolated from ticks were included in groups B and Q; and that recovered from a vole was in group K. The diversities calculated for the 40
F. tularensis
isolates, according to Simpson's index, were 0.14 for REP-PCR, 0.52 for ERIC-PCR, 0.39 for RAPD assay with the M13 primer (RAPD/M13-PCR), and 0.65 for RAPD/T3-T7-PCR, and the diversity increased up to 0.90 when ERIC-PCR, RAPD/M13-PCR, and RAPD/T3-T7-PCR were combined. Our results suggest that although limited genetic heterogeneity among
F. tularensis
strains was observed, this small variation is enough to validate the PCR methods used in this study and their combinations, because they can provide safe, useful, and rapid tools for the typing of
F. tularensis
.
Failure of an Automated Blood Culture System To Detect Nonfermentative Gram-Negative Bacteria
Authors: Klaerner H.-G. , Eschenbach U. , Kamereck K. , Lehn N. , Wagner H. , Miethke T.
Published: December 2019

During a 1-year study we observed that both aerobic and anaerobic blood culture bottles from patients were negative by the BacT/Alert system during a 7-day incubation period. However, upon subcultivation of negative bottles, growth of
Pseudomonas aeruginosa
was detectable. In an attempt to explain this observation, aerobic BacT/Alert Fan bottles were seeded with a defined inoculum (0.5 McFarland standard; 1 ml) of
Escherichia coli
,
Klebsiella pneumoniae
,
Serratia marcescens
,
P. aeruginosa
,
Stenotrophomonas maltophilia
, or
Acinetobacter baumannii
. Half of the inoculated bottles were loaded into the BacT/Alert system immediately, and the remainder were preincubated for 4, 8, 16, and 24 h at 36°C. With preincubation all bottles seeded with the
Enterobacteriaceae
signaled positive during the next 1.5 h. Organisms in bottles seeded with the nonfermentative species
P. aeruginosa
and
A. baumannii
remained undetected by the BacT/Alert system for 7 days.
S. maltophilia
was detected if the preincubation time was equal or less than 8 h. Without preincubation all bottles seeded with the
Enterobacteriaceae
or nonfermentative species signaled positive. Since nonfermentative species seem to enter a state of bacteriostasis within the preincubation period, we reasoned that an unknown factor is consumed. Accordingly, a smaller inoculum should allow the detection of nonfermentative species, even after preincubation, and serial dilutions of
P. aeruginosa
were detected in preincubated bottles. In this case preincubated bottles signaled positive faster than bottles without preincubation. We conclude that all bottles from clinical settings should be subcultured prior to loading to avoid false negatives. An alternative may be preincubation at room temperature.
Simultaneous Detection of Multiplex-Amplified Human Immunodeficiency Virus Type 1 RNA, Hepatitis C Virus RNA, and Hepatitis B Virus DNA Using a Flow Cytometer Microsphere-Based Hybridization Assay
Authors: Defoort J.-P. , Martin M. , Casano B. , Prato S. , Camilla C. , Fert V.
Published: December 2019

The feasibility of performing a multiplex assay for the detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNAs and hepatitis B virus (HBV) DNA is demonstrated. This assay is based (i) on the coamplification of a 142-bp fragment from the
gag
region of the HIV-1 genome and a 142-bp HIV-1 quantitation standard fragment, a 244-bp fragment from the 5′ noncoding region of the HCV genome, and a 104-bp fragment from the pre-C and C gene regions of the HBV genome, using three sets of specific primers; (ii) on the capacity of these four biotinylated PCR products to hybridize to their specific oligonucleotide probe-coated microspheres; and (iii) on the ability of the flow cytometer to discriminate between distinct fluorescent-microsphere categories. Absence of cross-hybridization between the unrelated oligonucleotide probes and PCR products generated by the multiplex reverse transcription-PCR (RT-PCR) and the highly sensitive detection method allowed us to assess unambiguously the HIV-1 viral load and the infectious status of 35 serologically well-established clinical samples and 20 seronegative blood donor plasma samples tested. The results indicate that multiplex RT-PCR and flow cytometer microsphere-based hybridization assays, when combined, provide a rapid, sensitive, and specific method for the quantitation and detection of the major viral agents of infectious diseases in a single plasma sample.
Multicenter Evaluation of the AMPLICOR and Automated COBAS AMPLICOR CT/NG Tests for Detection of
Chlamydia trachomatis
Authors: Van Der Pol Barbara , Quinn Thomas C. , Gaydos Charlotte A. , Crotchfelt Kimberly , Schachter Julius , Moncada Jeanne , Jungkind D. , Martin David H. , Turner Buffy , Peyton Cynthia , Jones Robert B.
Published: December 2019

The fully automated COBAS AMPLICOR CT/NG and semiautomated AMPLICOR CT/NG tests were evaluated in a multicenter trial for the ability to detect
Chlamydia trachomatis
infections. Test performance compared to that of culture was evaluated for 2,236 matched endocervical swab and urine specimens obtained from women and for 1,940 matched urethral swab and urine specimens obtained from men. Culture-negative, PCR-positive specimens that tested positive in a direct fluorescent-antibody test or in a confirmatory PCR test for an alternative target sequence were resolved as true positives. The overall prevalences of chlamydia were 2.4% in women and 7.2% in men. The COBAS AMPLICOR and AMPLICOR formats yielded concordant results for 98.1% of the specimens. With the infected patient as the reference standard, the resolved sensitivities of COBAS AMPLICOR were 89.7% for endocervical swab specimens, 89.2% for female urine specimens, 88.6% for male urethral swab specimens, and 90.3% for male urine specimens. When results were analyzed as if only a single test had been performed on a single specimen type, the resolved sensitivity was always higher. The resolved specificities of PCR were 99.4% for endocervical swab specimens, 99.0% for female urine specimens, 98.7% for male urethral swab specimens, and 98.4% for male urine specimens. The internal control revealed that 2.4% of the specimens were inhibitory when initially tested. Nevertheless, valid results were obtained for 98.6% of the specimens because 59.1% of the inhibitory specimens were not inhibitory when a second aliquot was tested. The COBAS AMPLICOR and AMPLICOR CT/NG tests for
C. trachomatis
exhibited equally high sensitivity and specificity with both urogenital swab and urine specimens and thus are well suited for screening for
C. trachomatis
infection.
Quantitative and Cost Comparison of Ultrasensitive Human Immunodeficiency Virus Type 1 RNA Viral Load Assays: Bayer bDNA Quantiplex Versions 3.0 and 2.0 and Roche PCR Amplicor Monitor Version 1.5
Authors: Elbeik Tarek , Charlebois Edwin , Nassos Patricia , Kahn James , Hecht Frederick M. , Yajko David , Ng Valerie , Hadley Keith
Published: December 2019

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA as a measure of viral load has greatly improved the monitoring of therapies for infected individuals. With the significant reductions in viral load now observed in individuals treated with highly active anti-retroviral therapy (HAART), viral load assays have been adapted to achieve greater sensitivity. Two commercially available ultrasensitive assays, the Bayer Quantiplex HIV-1 bDNA version 3.0 (bDNA 3.0) assay and the Roche Amplicor HIV-1 Monitor Ultrasensitive version 1.5 (Amplicor 1.5) assay, are now being used to monitor HIV-1-infected individuals. Both of these ultrasensitive assays have a reported lower limit of 50 HIV-1 RNA copies/ml and were developed from corresponding older generation assays with lower limits of 400 to 500 copies/ml. However, the comparability of viral load data generated by these ultrasensitive assays and the relative costs of labor, disposables, and biohazardous wastes were not determined in most cases. In this study, we used matched clinical plasma samples to compare the quantification of the newer bDNA 3.0 assay with that of the older bDNA 2.0 assay and to compare the quantification and costs of the bDNA 3.0 assay and the Amplicor 1.5 assay. We found that quantification by the bDNA 3.0 assay was approximately twofold higher than that by the bDNA 2.0 assay and was highly correlated to that by the Amplicor 1.5 assay. Moreover, cost analysis based on labor, disposables, and biohazardous wastes showed significant savings with the bDNA 3.0 assay as compared to the costs of the Amplicor 1.5 assay.
Species Identification and Subtyping of
Ureaplasma parvum
and
Ureaplasma urealyticum
Using PCR-Based Assays
Authors: Kong Fanrong , Ma Zhenfang , James Gregory , Gordon Susanna , Gilbert Gwendolyn L.
Published: December 2019

There is good evidence that the organism currently known as
Ureaplasma urealyticum
should be divided into two species—
U. parvum
(previously
U. urealyticum
biovar 1) and
U. urealyticum
(previously
U. urealyticum
biovar 2). In this study, we designed a series of primers, targeting the 16S rRNA gene and 16S rRNA-23S rRNA intergenic spacer regions, the urease gene subunits, and the 5′ ends of the multiple-banded antigen (MBA) genes, to identify and subtype these
Ureaplasma
species. All of the species-specific primer pairs could distinguish the two species, but only subtype-specific primer pairs targeting the MBA genes could distinguish subtypes within each species.
U. parvum
was separated into three subtypes, represented by serovars 1, 3/14, and 6.
U. urealyticum
was also separated into three subtypes by PCR and/or direct sequencing. Subtype 1 consisted of serovars 2, 5, 8, and 9; subtype 2 contained serovars 4, 10, 12, and 13; and subtype 3 contained serovars 7 and 11. A selection of primer pairs was used to identify and subtype 78 clinical ureaplasma isolates from vaginal swabs of pregnant women and to identify and subtype ureaplasmas directly in 185 vaginal swabs in which they had been previously detected.
U. parvum
was identified in 228 (87%) of 263 isolates or specimens, and
U. urealyticum
was identified in 50 (19%) (both were present in 6%). Serovars 3/14 (48%) and 1 (43%) were most common among
U. parvum
isolates, and subtypes 2 (62%) and 1 (34%) were most common among
U. urealyticum
isolates. This new PCR-based typing system will facilitate future studies of the relationship between individual
Ureaplasma
species or subtypes and human disease.
Detection and Identification of Human Parainfluenza Viruses 1, 2, 3, and 4 in Clinical Samples of Pediatric Patients by Multiplex Reverse Transcription-PCR
Authors: Aguilar Jose C. , Pérez-Breña María P. , García María L. , Cruz Nieves , Erdman Dean D. , Echevarría Juan Emilio
Published: December 2019

We describe a multiplex reverse transcription-PCR (m-RT-PCR) assay that is able to detect and differentiate all known human parainfluenza viruses (HPIVs). Serial dilution experiments with reference strains that compared cell culture isolation and m-RT-PCR showed sensitivities ranging from 0.0004 50% tissue culture infective dose (TCID
50
) for HPIV type 4B (HPIV-4B) to 32 TCID
50
s for HPIV-3. As few as 10 plasmids containing HPIV PCR products could be detected in all cases. When 201 nasopharyngeal aspirate specimens from pediatric patients hospitalized for lower respiratory illness were tested, m-RT-PCR assay detected 64 HPIVs (24 HPIV-3, 23 HPIV-1, 10 HPIV-4, and 7 HPIV-2), while only 42 of them (21 HPIV-1, 14 HPIV-3, 6 HPIV-2, and 1 HPIV-4 isolates) grew in cell culture. Our m-RT-PCR assay was more sensitive than either cell culture isolation or indirect immunofluorescence with monoclonal antibodies for the detection of HPIV infections. Also, HPIV-4 was more frequently detected than HPIV-2 in this study, suggesting that it may have been underestimated as a lower respiratory tract pathogen because of the insensitivity of cell culture.
Detection of a Previously Unamplified Spacer within the DR Locus of
Mycobacterium tuberculosis
: Epidemiological Implications
Authors: Filliol Ingrid , Sola Christophe , Rastogi Nalin
Published: December 2019

Spoligotyping, a method based on the variability of distribution of the 43 inter-direct repeat (DR) spacers of
Mycobacterium tuberculosis
and
Mycobacterium bovis
BCG, is useful to study the molecular epidemiology of bovine and human tuberculosis. Recently, a major family of
M. tuberculosis
clinical isolates named the Haarlem family, which did not contain spacers 31 and 33 to 36, was reported in a multicenter study. Independently, a data bank containing all the published spoligotypes showed that the two most prevalent spoligotypes in the world differed only by the presence or absence of spacer 31. A careful analysis of the DR locus sequence led us to hypothesize that spacer 31 may not have been amplified in some isolates with the primer sets DRa and DRb currently used for spoligotyping. Consequently, a modified spoligotyping method based on different combinations of the 36-bp DR and IS
6110
primers was devised that was able to discriminate between the left and the right parts of the DR locus and demonstrated the presence of the previously unamplified spacer 31 for some of the clinical isolates. By analogy, we suggest that a single-spacer difference in some epidemiologically linked cases of tuberculosis may simply arise due to the insertion of an extra copy of IS
6110
within the DR locus, leading to its asymmetrical disruption and subsequent lack of the DRa or DRb targets. The influence of the IS
6110
preferential insertion sites within the DR locus on spoligotyping results should be further investigated.
Multiple Clones within Multidrug-Resistant
Salmonella enterica
Serotype Typhimurium Phage Type DT104
Authors: Markogiannakis Antonis , Tassios Panayotis T. , Lambiri Maria , Ward Linda R. , Kourea-Kremastinou Jenny , Legakis Nicholas J. ,   The Greek Nontyphoidal Salmonella Study Group, Vatopoulos Alkiviadis C.
Published: December 2019

Six distinct clones were present among Greek multidrug-resistant
Salmonella enterica
serotype Typhimurium phage type DT104, since isolates belonging to resistance phenotypes including the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) core could be distinguished with respect to their pulsed-field gel electrophoresis patterns,
int
1 integron structures, and presence or absence of antibiotic resistance genes
ant
(3")-Ia,
pse
-1, and
tem
-1.
Genotyping of
Mycoplasma pneumoniae
Clinical Isolates Reveals Eight P1 Subtypes within Two Genomic Groups
Authors: Dorigo-Zetsma J. Wendelien , Dankert Jacob , Zaat Sebastian A. J.
Published: December 2019

Three methods for genotyping of
Mycoplasma pneumoniae
clinical isolates were applied to 2 reference strains and 21 clinical isolates. By a modified restriction fragment length polymorphism (RFLP) analysis of PCR products of the
M. pneumoniae
cytadhesin P1 gene, 5 subtypes were discriminated among 13 P1 type 1 strains and 3 subtypes were discriminated among 8 P1 type 2 strains. Sequence analysis of the 16S-23S rRNA gene spacer region and part of the 23S rRNA gene revealed one nucleotide difference in the intergenic spacer region in 3 of the 21 isolates. In the 23S rRNA gene sequence of the 8 P1 type 2 strains an extra adenosine was present, but it was absent from the 13 P1 type 1 strains. On the basis of
M. pneumoniae
genome sequence data, primers were designed to amplify large interrepeat fragments by long PCR, and these fragments were subsequently analyzed by RFLP analysis. Only two types, long PCR types 1 and 2, could be discriminated among the
M. pneumoniae
isolates. All P1 type 1 strains were assigned to long PCR type 1, and all P1 type 2 strains were assigned to long PCR type 2. These data obtained by three independent typing methods thus confirm the existence of two distinct
M. pneumoniae
genomic groups but expand the possibility of strain typing on the basis of variations within their P1 genes.
Identification of the Major Spanish Clones of Penicillin-Resistant Pneumococci via the Internet Using Multilocus Sequence Typing
Authors: Zhou Jiaji , Enright Mark C. , Spratt Brian G.
Published: December 2019

Multilocus sequence typing was used to characterize isolates of the major Spanish clones of penicillin-resistant and multiple-antibiotic-resistant
Streptococcus pneumoniae
. Isolates of the multidrug-resistant Spanish serotype 23F clone and serotype variants of this clone either had identical allelic profiles or their allelic profiles differed from this typical allelic profile at only one of the seven housekeeping loci. Similarly, isolates of the Spanish serotype 6B and 14 clones and the penicillin-resistant serotype 9V clone (and serotype variants of this clone) each had the same allelic profiles or profiles that differed at a single locus. Multilocus sequence typing therefore allows resistant pneumococci to be assigned to the Spanish clones if they have the typical allelic profile of the clone or if their profiles differ from that profile at a single locus. A few resistant isolates that had allelic profiles typical of that of a Spanish clone or whose profiles differed from that of the typical profile at only a single locus possessed penicillin-binding protein
pbp1a
,
pbp2b
, or
pbp2x
genes that differed from those that are characteristic of the clone. In most cases these isolates could be assigned as variant members of the clone. Since almost all serotype 9V isolates have very similar genotypes, independently emerging penicillin-resistant clones of this serotype will inevitably appear to be similar by molecular typing procedures. Analysis of the
pbp
genes, in addition to multilocus sequence typing (or any other molecular typing procedure), is therefore required to assign isolates unambiguously to the penicillin-resistant Spanish serotype 9V clone.
Early Aqueous Humor Analysis in Patients with Human Ocular Toxoplasmosis
Authors: Garweg Justus G. , Jacquier Patrick , Boehnke Matthias
Published: December 2019

To evaluate the diagnostic sensitivity of a panel of laboratory tests for ocular toxoplasmosis performed at the time of presentation, paired samples of aqueous humor and serum were collected from 49 consecutive episodes of ocular toxoplasmosis with a clinical course of less than 3 weeks. Total immunoglobulin G (IgG) andToxoplasma gondii-specific IgG, IgM, and IgA were quantified by enzyme-linked immunosorbent assay. The avidity ofT. gondii-specific IgG was determined, and DNA extracted from aqueous humor was amplified for detection of a glycoprotein B gene sequence ofT. gondii. The diagnosis was confirmed for 73% (36 of 49) of the patients; this rate rose to 79.5% if data from a later analysis of aqueous humor derived from five of the negative patients were included. The analysis of serum (detection ofT. gondii-specific IgM and analysis of consecutive serum samples) alone did not contribute to the diagnosis. Calculation of local antibody production lacked diagnostic sensitivity when it was determined less than 3 weeks after the manifestation of clinical symptoms (28 of 49 patients [57%]), but this rose to 70% after an analysis of a second aqueous humor sample. The antibody avidity index attained diagnostic significance in only 8 of 43 instances (19%), andT. gondiiDNA was amplified from no more than 6 of 39 (16%) aqueous humor samples. However,T. gondii-specific IgA was found within the aqueous humors of 11 of 43 patients (26%); measurement of theT. gondii-specific IgA level thus contributed substantially to the diagnostic sensitivity of the laboratory tests.
Mitochondrial Cytochrome
b
Gene Analysis of
Aspergillus fumigatus
and Related Species
Authors: Wang Li , Yokoyama Koji , Miyaji Makoto , Nishimura Kazuko
Published: December 2019

Nucleotide sequences of 426 bp from the mitochondrial (mt) cytochrome
b
genes of six anamorph species and two species of
Neosartorya
teleomophs of
Aspergillus
section
Fumigati
were determined. These sequences were used to build nucleotide- and amino acid-based trees for phylogenetic analysis. Thirteen strains of
A. fumigatus
including 10 clinical isolates of
A. fumigatus
, 1 type culture of
A. fumigatus
var.
fumigatus
, 1 type culture of
A. fumigatus
var.
ellipticus
, and 1 strain of
A. fumigatus
var.
albus
, had the same nucleotide sequences. One strain of
A. fumisynnematus
, two strains labeled
A. neoellipticus
, two strains of
A. viridinutans
, and one strain of
A. duricaulis
had distinct nucleotide and amino acid sequences. Two strains of
A. brevipes
were divided into two types. One produced a 1,500-bp fragment that included an intron. The nucleotide sequences of its two exons were similar to those of the
A. fumigatus
, and the derived amino acid sequence was the same as that for
A. fumigatus
. The other produced a 426-bp fragment and had the same nucleotide and amino acid sequences as
A. unilateralis. Neosartorya fischeri
var.
fischeri
and
N. stramenia
had nucleotide sequences that differed from that of
A. fumigatus
. These species possessed their own characteristic nucleotide sequences that differed from each other. In comparisons of homologous sequences from four other pathogenic species of
Aspergillus
, regions specific to section
Fumigati
were found. The mt cytochrome
b
gene analysis was valuable for the identification, classification, and phylogenetic analysis of isolates of section
Fumigati
.
Comparison of the Nucleotide Sequences of 16S rRNA, 444Ep-ank, andgroESLHeat Shock Operon Genes in Naturally OccurringEhrlichia equiand Human Granulocytic Ehrlichiosis Agent Isolates from Northern California
Authors: Chae Joon-seok , Foley Janet E. , Dumler J. Stephen , Madigan John E.
Published: December 2019

We examined 11 naturally occurring isolates ofEhrlichia equiin horses and two human granulocytic ehrlichiosis agent isolates in California for sequence diversity in three genes.Ehrlichia equiisolates were from Sierra (n= 6), Mendocino (n= 3), Sonoma (n= 1), and Marin (n= 1) counties, and human granulocytic ehrlichiosis (HGE) agent isolates were obtained from Humboldt county. PCR with specific primers for 16S rRNA, 444Ep-ankandgroESLheat shock operon genes successfully produced amplicons for all 13 clinical samples. The 444Ep-ankgene of the HGE agent andE. equiisolates from northern California is different from the eastern U.S. isolates BDS and USG3. The translated amino acid sequence of thegroESLheat shock operon gene fragment is identical amongE. equi, the HGE agent, andE. phagocytophila, with the exception of the northern Californian equine CASOLJ isolate. Microheterogeneity was observed in the 16S rRNA gene sequences of HGE agent andE. equiisolates from northern California. These results suggest thatE. equiand the HGE agent found in California are similar or identical but may differ from the isolates of equine and human origin found in the eastern United States.
Diagnosis and Differentiation ofMycoplasma hyopneumoniaeandMycoplasma hyorhinisInfections in Pigs by PCR Amplification of the p36 and p46 Genes
Authors: Caron J. , Ouardani M. , Dea S.
Published: December 2019

The genome ofMycoplasma hyopneumoniaeencodes several immunodominant proteins, including a cytosolic protein (p36), three membranous proteins (p46, p65, and p74), and an adhesin (p97). Cross-reactions withM. flocculareandM. hyorhinisreduce the specificity of conventional serological detection methods. However, certain antigenic determinants of the p36 and p46 proteins have been shown to be specific forM. hyopneumoniae. In the present study, pairs of oligonucleotide primers were designed to permit PCR amplification of entire p36 and p46 genes and of internal fragments of these genes. Specific amplicons could be obtained with as low as 0.5 to 50 pg of extracted chromosomal DNA. No amplification product was obtained when testing p36 and p46 primer pairs with genomic DNA or RNA from other mycoplasma species, bacteria, and viruses commonly associated with respiratory diseases in pigs. By using the single p36-PCR method, a positive reaction was demonstrated in 100% (30 of 30) of lungs from pigs that developed typical lesions associated with anM. hyopneumoniaeinfection, and no false-positive results were detected when 62 apparently normal lungs were tested. On the other hand, with the single p46-PCR method a sensitivity of 86.6% (26 of 30) and a specificity of 96.7% (60 of 62) were obtained in comparison with the necropsy findings. A mixed infection withM. hyorhiniswas diagnosed in 13.3% (4 of 30) of the cases by using species-specific primers for the heterologous p37 gene. The sensitivity of the single p36-PCR method for the detection ofM. hyopneumoniae, when tested on tracheobronchial swabs, was 100% (20 positive samples), with a specificity of 93.3% (14 of 15 negative samples), compared to the necropsy findings. Both expected amplicons were obtained with 86.6% (26 of 30) positive lungs when p36 and p46 primers were used simultaneously (multiplex PCR) to further increase the specificity of the PCR assay.
Development of a Highly Specific RecombinantToxocara canisSecond-Stage Larva Excretory-Secretory Antigen for Immunodiagnosis of Human Toxocariasis
Authors: Yamasaki Hiroshi , Araki Kunioki , Lim Patricia Kim Chooi , Zasmy Ngah , Mak Joon Wah , Taib Radzan , Aoki Takashi
Published: December 2019

The specificity of the recombinantToxocara canisantigen developed for the immunodiagnosis of human toxocariasis was compared with that of the excretory-secretory antigen fromT. canissecond-stage larvae (TES) by enzyme-linked immunosorbent assay. A total of 153 human serum samples from patients infected with 20 different helminths, including 11 cases of toxocariasis, were examined. No false-negative reactions were observed for the toxocariasis cases. When the TES was used at concentrations of 0.5 and 0.125 μg/ml, cross-reactions were observed in 79 (55.6%) and 61 (43.0%) of 142 cases, respectively. In contrast, when the recombinant antigen was tested at a concentration of 0.5 μg/ml, cross-reactions were observed in 19 (13.4%) of 142 cases. At a concentration of 0.125 μg/ml, however, the cross-reaction rate decreased sharply to only 2.1%, corresponding to 3 of 142 cases. The cross-reactions occurred with one case each of gnathostomiasis, paragonimiasis withParagonimus miyazakii, and spirometriasis, in which high antibody titers were detected. In addition, the recombinant antigen showed negative reactions with serum samples from patients infected withAscarisand hookworms, which are the most common parasites in the world. These findings are also supported by experiments with animals infected withAscarisand hookworm. From these results, the recombinant antigen is highly specific for toxocariasis and may provide more reliable diagnostic results than other methods.
Assessment of Morphology for Rapid Presumptive Identification of
Mycobacterium tuberculosis
and
Mycobacterium kansasii
Authors: Attorri Silvia , Dunbar Sherry , Clarridge Jill E.
Published: December 2019

Mycobacterium tuberculosis
often exhibits serpentine cording when grown in liquid medium, whereas
Mycobacterium kansasii
can be larger and cross-barred. We assessed the use of these morphologic characteristics as a cost-effective method for rapid presumptive identification of isolates from BACTEC bottles. Without specific training, using the Kinyoun acid-fast stain, definitive cording was found in 237 of 373 specimens positive for
M. tuberculosis
(64%) and cross-barring was recognized within 63 of 76 (83%) of the specimens positive for
M. kansasii
, giving sensitivities specificities, positive predictive values, and negative predictive values of 63.5, 96, 92, and 79%, respectively, for
M. tuberculosis
and 83, 95, 59, and 98%, respectively, for
M. kansasii
. With training and experience, these results improved to 74.5, 98, 96, and 84% and 93, 98, 79, and 98%, respectively. The major improvements were in distinguishing the pseudocording, or loose aggregation of
Mycobacterium avium
complex from
M. tuberculosis
and the long beaded forms of
Mycobacterium gordonae
from
M. kansasii. Mycobacterium asiaticum
and
Mycobacterium szulgai
, which rarely occur, are genetically related to
M. kansasii
and morphologically difficult to distinguish. In defined circumstances, serpentine cording and cross-barring can be used for rapid presumptive identification of
M. tuberculosis
and
M. kansasii
, respectively, and as guides for initial probe selection to reduce costs.
A Multisite Trial Comparing Two Cytomegalovirus (CMV) pp65 Antigenemia Test Kits, Biotest CMV Brite and Bartels/Argene CMV Antigenemia
Authors: St. George Kirsten , Boyd Molly J. , Lipson Steven M. , Ferguson David , Cartmell G. Frank , Falk Leon H. , Rinaldo Charles R. , Landry Marie L.
Published: December 2019

A total of 513 blood specimens, predominantly from organ transplant recipients, human immunodeficiency virus-positive patients, and bone marrow transplant recipients, were tested for cytomegalovirus (CMV) by culture and pp65 antigenemia across four test sites. Peripheral blood leukocytes were examined by using both the Biotest CMV Brite and the Bartels/Argene CMV Antigenemia kits. A total of 109 specimens were positive for CMV, 106 (97%) were positive by antigenemia, and 34 (31%) were positive by culture. According to the manufacturers' instructions, 150,000 cells were applied per slide for the Biotest kit and 200,000 cells per slide for the Bartels kit. A total of 93 specimens (88%) were positive by the Biotest kit, and 86 (81%) were positive by the Bartels kit. In specimens found to be positive by only one kit, the positive cell counts were low (median, 1; range, 1 to 7). When the data from all four sites were combined and analyzed, there was no statistical difference between the performance of the two kits; the Biotest and Bartels kits were found to be equivalent in sensitivity, specificity, and positive and negative predictive values for the detection of CMV pp65 antigenemia.
Quantitative Galactomannan Detection Is Superior to PCR in Diagnosing and Monitoring Invasive Pulmonary Aspergillosis in an Experimental Rat Model
Authors: Becker Martin J. , de Marie Siem , Willemse Diana , Verbrugh Henri A. , Bakker-Woudenberg Irma A. J. M.
Published: December 2019

Two diagnostic tests, an
Aspergillus
-specific PCR and an enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of galactomannan, were compared for diagnosing and monitoring invasive pulmonary aspergillosis. Persistently neutropenic rats with left-sided invasive pulmonary aspergillosis were sacrificed at regular intervals after inoculation. Blood samples and bronchoalveolar lavage (BAL) fluid were cultured and tested by PCR as well as by ELISA. Disseminated fungal infection in extrapulmonary organs was determined. The sensitivity of the ELISA was higher than that of the PCR on all days of measurements, in both blood and BAL fluid. Positive PCR or ELISA results in blood were not significantly associated with disseminated fungal infection. Serial testing in a separate group of rats showed consistently increasing concentrations of circulating galactomannan during the course of disease, while a positive PCR could be followed by negative results. The concentration of galactomannan was highly predictive for the time of survival (
P
< 0.0001). It was concluded that, in this model, quantitative galactomannan detection is superior to PCR in diagnosing and monitoring invasive pulmonary aspergillosis.
New Agar Medium for Testing Susceptibility of
Mycobacterium tuberculosis
to Pyrazinamide
Authors: Heifets Leonid , Sanchez Tracy
Published: December 2019

A new agar medium to perform pyrazinamide (PZA) susceptibility testing with
Mycobacterium tuberculosis
has been developed. This medium has an acidic pH of 6.0 instead of the usual for agar media, pH 6.8, to provide optimal conditions for PZA activity, and it also differs from conventional Middlebrook 7H10/7H11 agar in that animal serum (fetal or calf bovine or fetal equine serum) is used instead of oleic acid-albumin-dextrose-catalase to support good growth of
M. tuberculosis
at the low pH of 6.0. A critical concentration of 900 or 1,200 μg of PZA/ml in this medium made it possible to differentiate between PZA-susceptible and PZA-resistant clinical isolates. This agar medium has the following advantages compared to a liquid medium: it allows determination of the actual proportion of PZA-resistant bacteria in the isolate and it is simple and inexpensive. In addition, it has the potential of being used for a direct susceptibility test with PZA, but this approach will require further confirmation. Further studies to develop critical concentrations of other drugs for this low-pH medium, as well as to investigate the possibility of cultivation in regular (non-CO
2
) incubators, are in progress.
Development of Reverse Transcription-PCR Assays Specific for Detection of Equine Encephalitis Viruses
Authors: Linssen Bettina , Kinney Richard M. , Aguilar Patricia , Russell Kevin L. , Watts Douglas M. , Kaaden Oskar-Rüger , Pfeffer Martin
Published: December 2019

Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species. The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and IV), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes IAB, IC, ID, IE, and II. The RT-PCR–seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype IAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR. The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos. All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE-specific RT-PCR–seminested PCR.
Detection of the Agent of Heartwater,
Cowdria ruminantium
, in
Amblyomma
Ticks by PCR: Validation and Application of the Assay to Field Ticks
Authors: Peter Trevor F. , Barbet Anthony F. , Alleman Arthur R. , Simbi Bigboy H. , Burridge Michael J. , Mahan Suman M.
Published: December 2019

We have previously reported that the pCS20 PCR detection assay for
Cowdria ruminantium
, the causative agent of heartwater disease of ruminants, is more sensitive than xenodiagnosis and the pCS20 DNA probe for the detection of infection in the vector
Amblyomma
ticks. Here, we further assessed the reliability of the PCR assay and applied it to field ticks. The assay detected DNA of 37 isolates of
C. ruminantium
originating from sites throughout the distribution of heartwater and had a specificity of 98% when infected ticks were processed concurrently with uninfected ticks. The assay did not detect DNA of
Ehrlichia chaffeensis
, which is closely related to
C. ruminantium
. PCR sensitivity varied with tick infection intensity and was high (97 to 88%) with ticks bearing 10
7
to 10
4
organisms but dropped to 61 and 28%, respectively, with ticks bearing 10
3
and 10
2
organisms. The assay also detected
C. ruminantium
in collections of
Amblyomma hebraeum
and
Amblyomma variegatum
field ticks from 17 heartwater-endemic sites in four southern African countries. Attempts at tick transmission of infection to small ruminants failed with four of these collections. The pCS20 PCR assay is presently the most characterized and reliable test for
C. ruminantium
in ticks and thus is highly useful for field and laboratory epidemiological investigations of heartwater.