bluuebunny/crossref_metadata_2025 huggingface dataset:
Microaerophilic Conditions Promote Growth of
Mycobacterium genavense
Authors: Realini L. , De Ridder K. , Palomino J.-C. , Hirschel B. , Portaels F.
Published: December 2019

Our studies show that microaerophilic conditions promote the growth of
Mycobacterium genavense
in semisolid medium. The growth of
M. genavense
at 2.5 or 5% oxygen was superior to that obtained at 21% oxygen in BACTEC primary cultures (Middlebrook 7H12, pH 6.0, without additives). By using nondecontaminated specimens, it was possible to detect growth with very small inocula (25 bacilli/ml) of 12 different
M. genavense
strains (from nude mice) within 6 weeks of incubation under low oxygen tension; conversely, with 21% oxygen, no growth of 8 of 12 (66.7%)
M. genavense
strains was detected (growth index, <10). The same beneficial effect of 2.5 or 5% oxygen was observed in primary cultures of a decontaminated clinical specimen. Low oxygen tension (2.5 or 5%) is recommended for the primary isolation of
M. genavense
. Microaerophilic cultivation of other atypical mycobacteria, especially slow-growing (e.g.,
Mycobacterium avium
) and difficult-to-grow (e.g.,
Mycobacterium ulcerans
) species, is discussed.
Performance of Transcription-Mediated Amplification and Ligase Chain Reaction Assays for Detection of Chlamydial Infection in Urogenital Samples Obtained by Invasive and Noninvasive Methods
Authors: Stary Angelika , Schuh Elisabeth , Kerschbaumer Martina , Götz Birgit , Lee Helen
Published: December 2019

Based on the amplification of chlamydia-specific rRNA sequences and the ligase chain reaction (LCR), the performance characteristics of the Gen-Probe
Chlamydia trachomatis
transcription-mediated amplification (TMA) assay were evaluated with endocervical, urine, and vulval specimens from women and urethral and urine specimens from men and were compared with those for cultures on endocervical, vulval, and urethral swabs. Of the 308 women and 240 men tested, 25 (8.1%) and 44 (18.3%), respectively, were shown to be infected. By using the infected individual as the expanded “gold standard” for calculations, the TMA assay and LCR gave similar performances for the sensitivity of male urethral (93.2%) and urine (88.6 and 86.4%) samples, while culture detected only half of the 44 infected men. In women, the sensitivities of the TMA assay for endocervical and vulval samples were 88 and 92%, respectively, compared to values of 92% for the LCR on both sample types and of 52 and 8%, respectively, for culture. By using first-void urine for chlamydial diagnosis in women, LCR detected 24 (96%) and TMA assay detected 19 (76%) infected individuals, showing a significantly lower sensitivity for urine in women (
P
= 0.0253). The results indicate a high overall agreement for both amplifying techniques for all examined specimen types, except for female urine. Furthermore, they confirm the previous observation that vulval swabs are an effective alternative noninvasive sample type for the detection of
C. trachomatis
infection in women by nucleic acid-based amplification technologies.
Clonal Expansion of
Staphylococcus epidermidis
Strains Causing Hickman Catheter-Related Infections in a Hemato-Oncologic Department
Authors: Nouwen Jan L. , van Belkum Alex , de Marie Siem , Sluijs Jacqueline , Wielenga Jenne J. , Kluytmans Jan A. J. W. , Verbrugh Henri A.
Published: December 2019

The detailed analysis of 411 strains of coagulase-negative staphylococci (CoNS) obtained from 40 neutropenic hemato-oncologic patients (61 Hickman catheter episodes) on intensive chemotherapy is described. By random amplification of polymorphic DNA (RAPD) analysis, a total of 88 different genotypes were detected: 51 in air samples and 30 in skin cultures prior to insertion, 12 in blood cultures after insertion, and only 5 involved in catheter-related infections (CRI). Two RAPD genotypes of
Staphylococcus epidermidis
predominated, and their prevalence increased during patient hospitalization. At insertion, these clones constituted 11 of 86 (13%) CoNS isolated from air samples and 33 of 75 (44%) CoNS isolated from skin cultures. After insertion, their combined prevalence increased to 33 of 62 (53%) in catheters not associated with CRI and 139 of 188 (74%) in catheters associated with CRI (
P
= 0.0041). These two predominant
S. epidermidis
clones gave rise to a very high incidence of CRI (6.0 per 1,000 catheter days) and a very high catheter removal rate for CRI, 70%, despite prompt treatment with vancomycin. A likely source of
S. epidermidis
strains involved in CRI appeared to be the skin flora in 75% of cases. The validity of these observations was confirmed by pulsed-field gel electrophoresis (PFGE) of
Sma
I DNA macrorestriction fragments of blood culture CoNS isolates. Again, two predominant CoNS genotypes were found (combined prevalence, 60%). RAPD and PFGE yielded concordant results in 75% of cases. Retrospectively, the same two predominant CoNS clones were also found among blood culture CoNS isolates from the same hematology department in the period 1991 to 1993 (combined prevalence, 42%) but not in the period 1978 to 1982. These observations underscore the pathogenic potential of clonal CoNS types that have successfully and persistently colonized patients in this hemato-oncology department.
Molecular Procedure for Rapid Detection of
Burkholderia mallei
and
Burkholderia pseudomallei
Authors: Bauernfeind Adolf , Roller Carsten , Meyer Detlef , Jungwirth Renate , Schneider Ines
Published: December 2019

A PCR procedure for the discrimination of
Burkholderia mallei
and
Burkholderia pseudomallei
was developed. It is based on the nucleotide difference T 2143 C (T versus C at position 2143) between
B. mallei
and
B. pseudomallei
detected in the 23S rDNA sequences. In comparison with conventional methods the procedure allows more rapid identification at reduced risk for infection of laboratory personnel.
Comparison of Vitek GNI and GNI+ Cards for Identification of Gram-Negative Bacteria
Authors: Bourbeau Paul P. , Heiter Barbara J.
Published: December 2019

The GNI+ card has been developed by bioMerieux Vitek as an improvement over the GNI card for the identification of certain species of aerobic and facultative anaerobic bacteria. In this study, we tested 304 organisms from 30 different species on both the GNI and GNI+ cards. The GNI card correctly identified 285 (93.8%) of the isolates tested, and the GNI+ card correctly identified 287 (94.4%) of the isolates tested. The average time to reporting was 4.1 h for the GNI+ card compared to 5.7 h for the GNI card (
P
< 0.001). Overall, the GNI and GNI+ cards were comparable in identifying the organisms in this study while the GNI+ card gave substantially faster final test results.
Detection of
Pneumocystis carinii
DNA in Blood Specimens from Human Immunodeficiency Virus-Infected Patients by Nested PCR
Authors: Rabodonirina Meja , Cotte Laurent , Boibieux André , Kaiser Karine , Mayençon Martine , Raffenot Didier , Trepo Christian , Peyramond Dominique , Picot Stéphane
Published: December 2019

The detection of
Pneumocystis carinii
DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of
P. carinii
DNA from dilutions of a
P. carinii
f. sp.
hominis
cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready
P. carinii
DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients,
P. carinii
DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven
P. carinii
pneumonia, 7 of 22 patients who were considered to be colonized with
P. carinii
, and 9 of 48 patients who were neither infected nor colonized with
P. carinii. P. carinii
DNA was not detected in blood specimens from the 35 immunocompetent patients.
P. carinii
DNA in blood might represent viable
P. carinii
organisms or DNA complexes released from pulmonary phagocytes. In conclusion,
P. carinii
DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of
P. carinii
remain to be established.
Influence of Incubation Time, Inoculum Size, and Glucose Concentrations on Spectrophotometric Endpoint Determinations for Amphotericin B, Fluconazole, and Itraconazole
Authors: Nguyen M. Hong , Yu Christine Y.
Published: December 2019

We addressed the influence of the incubation time (24 h versus 48 h), starting inoculum size (standard inoculum size, ∼10
3
CFU/ml, versus large inoculum size, ∼10
4
CFU/ml), and supplementation with 2% glucose of RPMI 1640 medium on the spectrophotometric determination of the MICs of amphotericin B, fluconazole, and itraconazole. We compared the MICs determined spectrophotometrically with those determined by the standard broth macrodilution method (National Committee for Clinical Laboratory Standards approved guideline M27-A). The agreement between the results of the spectrophotometric and standard methods for amphotericin B testing was 100%; this agreement was independent of the inoculum size and incubation time. On the other hand, the agreement for the results for fluconazole testing and itraconazole testing was dependent on the inoculum size and incubation time. With large inoculum size, excellent agreement can be achieved at 24 h. With standard inoculum size, acceptable agreement can be achieved only at 48 h. In contrast to previous observations, the addition of 2% glucose did not have any significant impact on the growth density at 24 h, nor did it improve the agreement with the standard method. Furthermore, supplemental glucose might falsely elevate the MIC at 48 h.
Nucleic Acid-Based Cross-Linking Assay for Detection and Quantification of Hepatitis B Virus DNA
Authors: Lai Vicky C. H. , Guan Richard , Wood Michael L. , Lo Su Kong , Yuen Man-Fung , Lai Ching-Lung
Published: December 2019

A nucleic acid photo-cross-linking technology was used to develop a direct assay for the quantification of hepatitis B virus (HBV) DNA levels in serum. Cross-linker-modified DNA probes complementary to the viral genomes of the major HBV subtypes were synthesized and used in an assay that could be completed in less than 6 h. The quantification range of the assay, as determined by testing serial dilutions of Eurohep HBV reference standards and cloned HBV DNA, was 5 × 10
5
to 3 × 10
9
molecules of HBV DNA/ml of serum. Within-run and between-run coefficients of variation (CVs) for the assay were 4.3 and 4.0%, respectively. The assay was used to determine HBV DNA levels in 302 serum samples, and the results were compared to those obtained after testing the same samples with the Chiron branched-DNA (bDNA) assay for HBV DNA. Of the samples tested, 218 were positive for HBV DNA by both assays and 72 gave results below the cutoff for both assays. Of the remaining 12 samples, 10 were positive for HBV DNA by the cross-linking assay only; the 2 other samples were positive by the bDNA assay only. Twenty-eight samples had to be retested by the bDNA assay (CV, >20% between the results obtained from the testing of each sample in duplicate), whereas only three samples required retesting by the cross-linking assay. The correlation between the HBV DNA levels, as measured by the two tests, was very high (
r
= 0.902;
P
= 0.01). We conclude that the cross-linking assay is a sensitive and reproducible method for the detection and quantification of HBV DNA levels in serum.
Usefulness of Leifson Staining Method in Diagnosis of
Helicobacter pylori
Infection
Authors: Piccolomini Raffaele , Di Bonaventura Giovanni , Neri Matteo , Di Girolamo Arturo , Catamo Giovanni , Pizzigallo Eligio
Published: December 2019

The Leifson staining method was used to diagnose
Helicobacter pylori
infection and was compared to histology, culture, and the rapid urease test (RUT). Histology gave the best sensitivity (98%), compared to Leifson staining (97%), culture (92%), and RUT (85%) (
P
< 0.005). Leifson staining is a sensitive, rapid, economical method for diagnosis of
H. pylori
infection in dyspeptic patients.
E Test versus Agar Dilution for Antimicrobial Susceptibility Testing of Viridans Group Streptococci
Authors: Rosser S. J. , Alfa M. J. , Hoban S. , Kennedy J. , Harding G. K.
Published: December 2019

Viridans group streptococci (VGS) are commonly isolated from the blood of hospitalized patients. The E test represents a convenient method for determining the MICs for VGS, but for this purpose it has not been well validated against reference methods. In this study, 180 unselected VGS isolates were identified to a species level, and the MICs of penicillin, cefuroxime, cefotaxime, and vancomycin were determined by both agar dilution and the E test. Available data regarding demographic and laboratory variables for each VGS bacteremic episode were collected, the significance of each VGS isolate was assessed, and the associations between and among laboratory and clinical variables were investigated. Among all VGS isolates, 68.3% (median of three runs) were found to be fully susceptible to penicillin by agar dilution. The E test and agar dilution showed average agreements (within ±1 dilution) of 92.2% for penicillin, 95.7% for cefuroxime 91.3% for cefotaxime, and 86.7% for vancomycin. Agreements over serial E tests and serial agar dilutions were excellent for β-lactam agents (intraclass correlation coefficients, >0.9) but less impressive for vancomycin. Very major error rates for the E test were ≤0.7%, and combined major and minor error rates were within acceptable limits for all antimicrobial agents tested. Lysis-centrifugation culture methods were more often associated with clinically insignificant VGS isolates; otherwise, no associations between clinical and laboratory variables were noted.
Molecular Epidemiology of Outbreaks of Gastroenteritis Associated with Small Round-Structured Viruses in East Anglia, United Kingdom, During the 1996–1997 Season
Authors: Maguire Alison J. , Green Jon , Brown David W. G. , Desselberger Ulrich , Gray James J.
Published: December 2019

During the winter season from November 1996 to May 1997, 550 fecal specimens were submitted from 94 outbreaks of gastroenteritis occurring in East Anglia, United Kingdom. These specimens were tested for the presence of small round-structured viruses (SRSVs) by electron microscopy, reverse transcriptase PCR, or both methods. SRSVs were shown to be associated with 64 of 94 (68%) of these outbreaks, of which 16 (25%) outbreaks occurred at a single location (Southend) within the region. Twenty-four specimens from 13 of the 16 SRSV-positive outbreaks occurring in Southend were available for genomic analysis, in which divergence within the RNA polymerase region of the SRSV genome was investigated. A further 27 specimens from 17 other SRSV-associated outbreaks, occurring at different locations within East Anglia but at the same time as those at Southend, were also studied. Fifty of the total of 51 (98%) specimens studied were shown to belong to genogroup II, and within this genogroup, 49 of 50 (98%) specimens were shown to be Grimsby-like viruses, with only one Mexico-like strain. Furthermore, phylogenetic analysis of the Grimsby-like viruses indicated clusterings according to the geographical location of the outbreak. One specimen contained a virus belonging to genogroup I, and this had the greatest sequence identity (83%) with Southampton virus.
Use of PCR in Detection of
Mycobacterium avium
Complex (MAC) Bacteremia: Sensitivity of the Assay and Effect of Treatment for MAC Infection on Concentrations of Human Immunodeficiency Virus in Plasma
Authors: MacGregor Rob Roy , Dreyer Kimberly , Herman Steve , Hocknell Peter K. , Nghiem Lan , Tevere Vincent J. , Williams Amy L.
Published: December 2019

We evaluated the sensitivity and specificity of a PCR-based qualitative test for the rapid diagnosis of
Mycobacterium avium-M. intracellulare
complex (MAC) bacteremia in patients with AIDS disease. Eleven subjects with newly culture-proven MAC bacteremia had the following tests performed at biweekly intervals during the first 8 weeks of therapy: blood culture,
Mycobacterium
-specific PCR, and quantitative human immunodeficiency virus (HIV) viral-load testing.
Mycobacterium
genus-specific biotinylated primers were used to amplify a sequence of approximately 582 nucleotides within the 16S rRNA genes of
M. avium
and
M. intracellulare
. Detection of the amplified product was performed with an oligonucleotide probe-coated microwell plate combined with an avidin-horseradish peroxidase–tetramethylbenzidine conjugate-substrate system. While not as sensitive as BACTEC culture, PCR detected 17 of 18 specimens which grew ≥40 organisms/ml (94.4% sensitivity) and 9 of 16 specimens which grew ≤40 organisms/ml (56.3% sensitivity). No clear change in HIV viremia occurred in response to successful treatment of patients’ MAC bacteremia. Use of the PCR test allowed detection of MAC bacteremia in 1 day, with a sensitivity similar to those of quantitative blood culture techniques, and it may prove useful for rapid screening of suspected cases. HIV viremia was unaffected by 8 weeks of MAC therapy.
Evaluation of New Quantitative Assays for Diagnosis and Monitoring of Cytomegalovirus Disease in Human Immunodeficiency Virus-Positive Patients
Authors: Pellegrin Isabelle , Garrigue Isabelle , Binquet Christine , Chene Genevieve , Neau Didier , Bonot Pascal , Bonnet Fabrice , Fleury Herve , Pellegrin Jean-Luc
Published: December 2019

Cobas Amplicor CMV Monitor (CMM) and Quantiplex CMV bDNA 2.0 (CMV bDNA 2.0), two new standardized and quantitative assays for the detection of cytomegalovirus (CMV) DNA in plasma and peripheral blood leukocytes (PBLs), respectively, were compared to the CMV viremia assay, pp65 antigenemia assay, and the Amplicor CMV test (P-AMP). The CMV loads were measured in 384 samples from 58 human immunodeficiency virus (HIV) type 1-infected, CMV-seropositive subjects, including 13 with symptomatic CMV disease. The assays were highly concordant (agreement, 0.88 to 0.97) except when the CMV load was low. Quantitative results for plasma and PBLs were significantly correlated (Spearman ρ = 0.92). For PBLs, positive results were obtained 125 days before symptomatic CMV disease by CMV bDNA 2.0 and 124 days by pp65 antigenemia assay, whereas they were obtained 46 days before symptomatic CMV disease by CMM and P-AMP. At the time of CMV disease diagnosis, the sensitivity, specificity, and positive and negative predictive values of CMV bDNA 2.0 were 92.3, 97.8, 92.3, and 97.8%, respectively, whereas they were 92.3, 93.3, 80, and 97.8%, respectively, for the pp65 antigenemia assay; 84.6, 100, 100, and 95.7%, respectively, for CMM; and 76.9, 100, 100, and 93.8%, respectively, for P-AMP. Considering the entire follow-up, the sensitivity, specificity, and positive and negative predictive values of CMV bDNA 2.0 were 92.3, 73.3, 52.1, and 97.1%, respectively, whereas they were 100, 55.5, 39.4, and 100%, respectively, for the pp65 antigenemia assay; 92.3, 86.7, 66.7, and 97.5%, respectively, for CMM; and 84.6, 91.1, 73.3, and 95.3%, respectively, for P-AMP. Detection of CMV in plasma is technically easy and, despite its later positivity (i.e., later than in PBLs), can provide enough information sufficiently early so that HIV-infected patients can be effectively treated. In addition, these standardized quantitative assays accurately monitor the efficacy of anti-CMV treatment.
Role of Genotypic Analysis of the Thymidine Kinase Gene of Herpes Simplex Virus for Determination of Neurovirulence and Resistance to Acyclovir
Authors: Lee N. Y. , Tang Y.-W. , Espy M. J. , Kolbert C. P. , Rys P. N. , Mitchell P. S. , Day S. P. , Henry S. L. , Persing D. H. , Smith T. F.
Published: December 2019

Mutations in the thymidine kinase (TK) gene of herpes simplex virus (HSV) have been associated with resistance to acyclovir (ACY) and possible recognition of neurotropic strains. We sequenced a 335-bp segment of the TK gene to determine the frequency of mutations in HSV strains recovered from dermal, genital, and cerebrospinal fluid (CSF) specimens (
n
= 200; 102 HSV type 1 [HSV-1] 98 HSV-2 strains). Four polymorphic sites were detected in HSV-1 strains; C513T, A528G, C575T, and C672T. Among the polymorphisms, only C575T resulted in a change of amino acid sequence (residue 192, Ala→Val). For HSV-2 strains, only one polymorphism (G420T) which resulted in an amino acid substitution (residue 139, Leu→Phe) was detected. Phenotypic determination of resistance to ACY by a plaque reduction assay of 48 HSV isolates was not correlated with the sequence results of 11 strains in that 7 of these with genotypic polymorphisms were susceptible to the drug in vitro. In addition, of 32 ACY-resistant HSV strains, 28 (87.5%) had no polymorphisms detected in the 335-bp amplicon of the TK gene. There was no statistical difference in the frequency of polymorphisms according to the source of the specimens. We conclude that the detection of nucleic acid polymorphisms in a previously implicated 335-bp segment of the TK gene cannot be interpreted as indicative of either ACY resistance or neurotropism of HSV strains from dermal, genital, and CSF sources.
Genotyping of Epidemic Methicillin-Resistant
Staphylococcus aureus
Phage Type 15 Isolates by Fluorescent Amplified-Fragment Length Polymorphism Analysis
Authors: Grady Ruth , Desai Meeta , O’Neill Gael , Cookson Barry , Stanley John
Published: December 2019

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was investigated for its ability to identify and subtype isolates of an epidemic methicillin-resistant phage type of
Staphylococcus aureus
, EMRSA-15. These isolates were also characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) of the coagulase gene and pulsed-field gel electrophoresis (PFGE). For FAFLP, DNA was double digested with restriction enzymes
Apa
I plus
Taq
I or
Eco
RI plus
Mse
I. Site-specific adaptors were ligated to one or the other set of restriction fragments, and PCR amplification was carried out with adaptor-specific primers. Amplified fragments separated on an ABI 377 automated sequencer and analyzed with Genescan version 2.1 software generated FAFLP profiles for all the isolates. The presence or absence of fragments was scored, similarity coefficients were calculated, and UPGMA (unweighted pair group method using arithmatic averages) cluster analysis was performed. Either enzyme-primer combination readily differentiated EMRSA-15 from other methicillin-resistant
S. aureus
(MRSA) isolates and also revealed heterogeneity within the phage type. The discriminatory power of FAFLP was high. By combining both enzyme-primer data sets, 24 isolates were divided into 11 profiles. PCR-RFLP did not discriminate among these EMRSA-15 isolates. PFGE could discriminate well between isolates but was not as reproducible as FAFLP. All
S. aureus
and MRSA isolates in this study were typeable by FAFLP, which was easy to perform, robust, and reproducible, with evident potential to subtype MRSA for purposes of hospital infection control.
Study of Restriction Fragment Length Polymorphism Analysis and Spoligotyping for Epidemiological Investigation of
Mycobacterium bovis
Infection
Authors: Costello Eamon , O’Grady Donnacha , Flynn Orla , O’Brien Rory , Rogers Mark , Quigley Frances , Egan John , Griffin John
Published: December 2019

Restriction fragment length polymorphism (RFLP) analysis with probes derived from the insertion element IS
6110
, the direct repeat sequence, and the polymorphic GC-rich sequence (PGRS) and a PCR-based typing method called spacer oligonucleotide typing (spoligotyping) were used to strain type
Mycobacterium bovis
isolates from the Republic of Ireland. Results were assessed for 452 isolates which were obtained from 233 cattle, 173 badgers, 33 deer, 7 pigs, 5 sheep, and 1 goat. Eighty-five strains were identified by RFLP analysis, and 20 strains were identified by spoligotyping. Twenty percent of the isolates were the most prevalent RFLP type, while 52% of the isolates were the most prevalent spoligotype. Both the prevalent RFLP type and the prevalent spoligotype were identified in isolates from all animal species tested and had a wide geographic distribution. Isolates of some RFLP types and some spoligotypes were clustered in regions consisting of groups of adjoining counties. The PGRS probe gave better differentiation of strains than the IS
6110
or DR probes. The majority of isolates from all species carried a single IS
6110
copy. In four RFLP types IS
6110
polymorphism was associated with deletion of fragments equivalent in size to one or two direct variable repeat sequences. The same range and geographic distribution of strains were found for the majority of isolates from cattle, badgers, and deer. This suggests that transmission of infection between these species is a factor in the epidemiology of
M. bovis
infection in Ireland.
Serodiagnosis of Epstein-Barr Virus Infection by Using Recombinant Viral Capsid Antigen Fragments and Autologous Gene Fusion
Authors: Hinderer Walter , Lang Dieter , Rothe Markus , Vornhagen Rolf , Sonneborn Hans H. , Wolf Hans
Published: December 2019

Using recombinant 15- to 30-kDa fragments and fusion with glutathione
S
-transferase (GST), we investigated the seroreactivity of three large structural proteins of Epstein-Barr virus (EBV), p150 (BcLF1, capsid), p143 (BNRF1, tegument), and gp125 (BALF4, membrane) in Western blots. None of 13 fragments tested, however, was qualified for diagnostic application. In contrast, the two small viral capsid antigens (VCA), p18 (BFRF3) and p23 (BLRF2), demonstrated sensitive (100%) EBV-specific immunoglobulin G (IgG) reactivities. While p18 additionally showed maximum sensitivity for IgM detection, the IgM sensitivity of p23 was restricted (44%). An autologous fusion protein, p23-p18, which consists N-terminally of full-length p23, followed by the carboxy half of p18, was constructed. This antigen was subjected to indirect VCA enzyme-linked immunosorbent assays (ELISAs), for IgG and IgM, as well as to a μ-capture (μc) IgM ELISA. All assays were found to be 100% specific when EBV-negative sera were tested. Using sera from previously infected individuals, the p23-p18 fusion revealed an improved IgG sensitivity of 99% compared to sensitivities of 97 and 93% for the single antigens p18 and p23, respectively. The sensitivity and specificity of the indirect IgM ELISA with samples of primary and past infections, respectively, were 100%. The μc principle for IgM overcame completely the interference by rheumatoid factors. Compared to the specificity of the indirect IgM version, the specificity with sera collected from rheumatoid arthritis patients increased from 48 to 100%. In summary, the p23-p18 IgG and μc IgM ELISAs showed excellent performances and are promising new diagnostic tests for the detection of EBV-specific antiviral capsid antibodies.
Case of
Staphylococcus schleiferi
Endocarditis and a Simple Scheme To Identify Clumping Factor-Positive Staphylococci
Authors: Leung Michael J. , Nuttall Nichalas , Mazur Margaret , Taddei Tania L. , McComish Michael , Pearman John W.
Published: December 2019

Staphylococcus schleiferi
is a coagulase-negative staphylococcus infrequently reported as a human pathogen. We report a case of prosthetic valve endocarditis attributed to this organism, contrast it to another
Staphylococcus
species that gives similar clumping factor results (
S. lugdunensis
), and propose a simple, effective identification scheme for identification of clumping factor-positive staphylococci.
Cost-Effective Methods for Isolation of
Salmonella enterica
in the Clinical Laboratory
Authors: Kelly Sinead , Cormican Martin , Parke Leanne , Corbett-Feeney Geraldine , Flynn John
Published: December 2019

Data from 8,717 fecal specimens indicate that primary inoculation of xylose lysine deoxycholate (XLD) agar may enhance the speed, but not the sensitivity, of isolation of
Salmonella enterica
over that achieved with Selenite enrichment only. Plating of Selenite broth onto both brilliant green and XLD agar offers no advantage over plating onto XLD alone.
Adenovirus Strains of Subgenus D Associated with Nosocomial Infection as New Etiological Agents of Epidemic Keratoconjunctivitis in Japan
Authors: Takeuchi Satoshi , Itoh Norihiko , Uchio Eiichi , Tanaka Keico , Kitamura Noriko , Kanai Hikaru , Isobe Kazumi , Aoki Koki , Ohno Shigeaki
Published: December 2019

Adenovirus strains of a new type were isolated from patients with epidemic keratoconjunctivitis. They were not completely neutralized by any antiserum against adenovirus prototypes. PCR followed by restriction endonuclease analysis demonstrated that they were type 8. PCR followed by sequencing revealed a high homology rate between them and type 9.
Analysis by Multiplex PCR of the Physical Status of Human Papillomavirus Type 16 DNA in Cervical Cancers
Authors: Yoshinouchi Mitsuo , Hongo Atsushi , Nakamura Keichiro , Kodama Junichi , Itoh Sachio , Sakai Hiroyuki , Kudo Takafumi
Published: December 2019

Integration of human papillomavirus (HPV) DNA occurs early in cancer development and is an important event in malignant transformation of cervical cancer. Integration of HPVs preferentially disrupts or deletes the E2 open reading frame, which results in the loss of its expression. The preferential disruption of the E2 gene causes the absence of the E2 gene sequences in the PCR product following integration. Twenty-two carcinomas positive for HPV type 16 (HPV-16) DNA were first tested for the disruption of the E2 gene by PCR. A specific fragment of the E2 gene was not amplified in 10 cases, suggesting integration of HPV DNA into the host genome. Next, multiplex PCR for the HPV E2 and E6 genes was carried out in the remaining 12 cases. Copy numbers of both genes should be equivalent in episomal forms, while the E2 gene copy number will be smaller than that for E6 following the preferential disruption of the E2 gene in concominant forms. Although relative ratios of HPV E2 to E6 PCR products (E2/E6 ratios) ranged from 1.40 to 2.34 in 10 of 12 cases, multiplex PCR products from 2 cases displayed extremely low ratios of 0.69 and 0.61. Southern blot hybridization with an HPV-16 probe revealed that only in these two cases was both episomal and integrated HPV DNA being carried simultaneously. Thus, multiplex PCR for the E2 and E6 genes of HPV-16 DNA following PCR for the E2 gene can distinguish the pure episomal form from a mixed form of episomal and integrated HPV DNA. Clinical application of this technique will help researchers to understand the implication of the integration of HPV DNA for cervical carcinogenesis and cervical cancer progression.
Evaluation of Protein A Gene Polymorphic Region DNA Sequencing for Typing ofStaphylococcus aureusStrains
Authors: Shopsin B. , Gomez M. , Montgomery S. O. , Smith D. H. , Waddington M. , Dodge D. E. , Bost D. A. , Riehman M. , Naidich S. , Kreiswirth B. N.
Published: December 2019

Three hundred and twenty isolates ofStaphylococcus aureuswere typed by DNA sequence analysis of the X region of the protein A gene (spa).spatyping was compared to both phenotypic and molecular techniques for the ability to differentiate and categorizeS. aureusstrains into groups that correlate with epidemiological information. Two previously characterized study populations were examined. A collection of 59 isolates (F. C. Tenover, R. Arbeit, G. Archer, J. Biddle, S. Byrne, R. Goering, G. Hancock, G. A. Hébert, B. Hill, R. Hollis, W. R. Jarvis, B. Kreiswirth, W. Eisner, J. Maslow, L. K. McDougal, J. M. Miller, M. Mulligan, and M. A. Pfaller, J. Clin. Microbiol. 32:407–415, 1994) from the Centers for Disease Control and Prevention (CDC) was used to test for the ability to discriminate outbreak from epidemiologically unrelated strains. A separate collection of 261 isolates form a multicenter study (R. B. Roberts, A. de Lencastre, W. Eisner, E. P. Severina, B. Shopsin, B. N. Kreiswirth, and A. Tomasz, J. Infect. Dis. 178:164–171, 1998) of methicillin-resistantS. aureusin New York City (NYC) was used to compare the ability ofspatyping to group strains along clonal lines to that of the combination of pulsed-field gel electrophoresis and Southern hybridization. In the 320 isolates studied,spatyping identified 24 distinct repeat types and 33 different strain types.spatyping distinguished 27 of 29 related strains and did not provide a unique fingerprint for 4 unrelated strains from the four outbreaks of the CDC collection. In the NYC collection,spatyping provided a clonal assignment for 185 of 195 strains within the five major groups previously described.spasequencing appears to be a highly effective rapid typing tool forS. aureusthat, despite some expense of specificity, has significant advantages in terms of speed, ease of use, ease of interpretation, and standardization among laboratories.
Use of BACTEC MGIT 960 for Recovery of Mycobacteria from Clinical Specimens: Multicenter Study
Authors: Tortoli Enrico , Cichero Paola , Piersimoni Claudio , Simonetti M. Tullia , Gesu Giampietro , Nista Domenico
Published: December 2019

The BACTEC MGIT 960 instrument is a fully automated system that exploits the fluorescence of an oxygen sensor to detect growth of mycobacteria in culture. Its performance was compared to those of the radiometric BACTEC 460 instrument and egg-based Lowenstein-Jensen medium. An identical volume of sample was inoculated in different media, and incubation was carried out for 6 weeks with the automatic systems and for 8 weeks on solid media. A total of 2,567 specimens obtained from 1,631 patients were cultured in parallel. Mycobacteria belonging to nine different taxa were isolated by at least one of the culture systems, with 75% of them being represented by
Mycobacterium tuberculosis
complex. The best yield was obtained with the BACTEC 460 system, with 201 isolates, in comparison with 190 isolates with the BACTEC MGIT 960 system and 168 isolates with Lowenstein-Jensen medium. A similar but not significant difference was obtained when the most-represented organisms, the
M. tuberculosis
complex,
Mycobacterium xenopi
, and the
Mycobacterium avium
complex, were analyzed separately and when combinations of a solid medium with the BACTEC MGIT 960 system and with the BACTEC 460 system were considered. The shortest times to detection were obtained with the BACTEC MGIT 960 system (13.3 days); 1.5 days earlier than that with the BACTEC 460 system (14.8 days) and 12 days earlier than that with Lowenstein-Jensen medium (25.6 days). The BACTEC MGIT 960 system had a contamination rate of 10.0%, intermediate between those of the radiometric system (3.7%) and the egg-based medium (17.0%). We conclude, therefore, that the BACTEC MGIT 960 system is a fully automated, nonradiometric instrument that is suitable for the detection of growth of tuberculous and other mycobacterial species and that is characterized by detection times that are even shorter than that of the “gold standard,” the BACTEC 460 system. The contamination rate was higher than that for the radiometric BACTEC 460 system and needs to be improved.
Identification of
Mycobacterium
Species by PCR-Restriction Fragment Length Polymorphism Analyses Using Fluorescence Capillary Electrophoresis
Authors: Hernandez S. Moises , Morlock Glenn P. , Butler W. Ray , Crawford Jack T. , Cooksey Robert C.
Published: December 2019

We developed a scheme for the rapid identification of
Mycobacterium
species based upon PCR amplification of polymorphic genetic regions with fluorescent primers followed by restriction and analysis by fluorescence capillary electrophoresis.
Mycobacterium
species were identified by restriction enzyme analysis of a 439-bp segment of the 65-kDa heat shock protein gene (labeled [both strands] at the 5′ end with 4,7,2′,7′-tetrachloro-6-carboxyfluorescein) using
Hae
III and
Bst
EII and of a 475-bp hypervariable region of the 16S rRNA gene (labeled [both strands] at the 5′ end with 6-carboxyfluorescein) using
Hae
III and
Cfo
I. Samples were analyzed on an automated fluorescence capillary electrophoresis instrument, and labeled fragments were sized by comparison with an internal standard. DNA templates were prepared with pure cultures of type strains. In all, we analyzed 180 strains, representing 22
Mycobacterium
species, and obtained distinctive restriction fragment length polymorphism (RFLP) patterns for 19 species. Three members of the
Mycobacterium tuberculosis
complex had a common RFLP pattern. A computerized algorithm which eliminates subjectivity from pattern interpretation and which is capable of identifying the species within a sample was developed. The convenience and short preparatory time of this assay make it comparable to conventional methodologies such as high-performance liquid chromatography and hybridization assays for identification of mycobacteria.
Simple and Rapid Identification of the
Mycobacterium tuberculosis
Complex by Immunochromatographic Assay Using Anti-MPB64 Monoclonal Antibodies
Authors: Abe Chiyoji , Hirano Kazue , Tomiyama Tetsuo
Published: December 2019

A newly developed immunochromatographic assay (MPB64-ICA) for identification of the
Mycobacterium tuberculosis
complex was evaluated with 20 reference strains of mycobacterial species and 111 clinical isolates. MPB64-ICA displayed a very strong reaction band with organisms belonging to the
M. tuberculosis
complex but not with mycobacteria other than
M. tuberculosis
(MOTT bacilli), except for one of four
M. marinum
strains tested and one
M. flavescens
strain, both of which gave very weak signals. The effectiveness of MPB64-ICA in combination with two liquid culture systems (MB-REDOX and MGIT) was tested. A total of 108 of 362 sputum specimens processed were positive for acid-fast bacilli. Samples taken from the cultures on the same days when either of the two culture systems became positive for mycobacteria were assayed with MPB64-ICA. Of 108 cultures with mycobacteria, 51 showed a positive signal with the test, in which the presence of the
M. tuberculosis
complex was demonstrated later by the Accuprobe for
M. tuberculosis
complex. In addition, MPB64-ICA could correctly detect the
M. tuberculosis
complex in mixed cultures of the
M. tuberculosis
complex and MOTT bacilli. These results indicate that MPB64-ICA can be easily used for rapid identification of the
M. tuberculosis
complex in combination with culture systems based on liquid media without any technical complexity in clinical laboratories.
Predicting Susceptibility of
Streptococcus pneumoniae
to Ceftriaxone and Cefotaxime by Cefuroxime and Ceftizoxime Disk Diffusion Testing
Authors: Williams-Bouyer Natalie , Hernandez Antonio , Reisner Barbara S.
Published: December 2019

In this study, disk diffusion testing with ceftizoxime and cefuroxime was evaluated for use in predicting the susceptibility of
Streptococcus pneumoniae
to ceftriaxone and cefotaxime. Of the 194 isolates included in this study, 138 were susceptible, 34 were intermediate, and 22 were resistant to cefotaxime by MIC testing; 138 isolates were susceptible, 35 were intermediate, and 21 were resistant to ceftriaxone by MIC testing. A zone of inhibition around the cefuroxime disk of ≥32 mm correctly categorized 101 of 138 isolates as susceptible to cefotaxime and ceftriaxone. A zone of inhibition around the ceftizoxime disk of ≥26 mm correctly categorized 111 of 138 isolates as susceptible to cefotaxime and 114 of 138 as susceptible to ceftriaxone. We conclude that disk diffusion can separate
S. pneumoniae
isolates susceptible to ceftriaxone and cefotaxime from those that are not susceptible. Isolates not falling into the susceptible category by disk diffusion require additional testing to determine the MIC.
A Serotype VIII Strain among Colonizing Group B Streptococcal Isolates in Boston, Massachusetts
Authors: Paoletti Leanne J. , Bradford Jessica , Paoletti Lawrence C.
Published: December 2019

Maternal colonization with group B
Streptococcus
(GBS) is a risk factor for neonatal GBS disease. Whereas serotypes Ia, Ib, II, III, and V are prevalent in the United States, types VI and VIII predominate in Japan. Recently, a serotype VIII strain was detected among 114 clinical GBS isolates from a Boston, Mass., hospital.
Diagnosis of Contagious Bovine Pleuropneumonia by PCR–Laser- Induced Fluorescence and PCR-Restriction Endonuclease Analysis Based on the 16S rRNA Genes of
Mycoplasma mycoides
subsp.
mycoides
SC
Authors: Persson Anja , Pettersson Bertil , Bölske Göran , Johansson Karl-Erik
Published: December 2019

As contagious bovine pleuropneumonia (CBPP) is spreading fast in many African countries, there is an increasing demand for rapid and sensitive diagnostic methods that can be used to confirm the initial diagnosis based on clinical symptoms or pathological findings. Two PCR-based diagnostic systems for identification of the infectious agent,
Mycoplasma mycoides
subsp.
mycoides
SC (
M. mycoides
SC), in various samples are presented. Both systems involve group-specific amplification of the two 16S rRNA genes from mycoplasmas of the
M. mycoides
cluster. The laser-induced fluorescence assay is based on a unique sequence length difference between the two 16S rRNA genes in
M. mycoides
SC. This region was amplified by PCR, and the products were separated by polyacrylamide gel electrophoresis in a DNA sequencer. The resulting electropherogram showed two peaks for strains of
M. mycoides
SC and one peak for all other members of the
M. mycoides
cluster. The second system was based on restriction endonuclease analysis and agarose gel electrophoresis. Restriction of amplicons from a region containing a polymorphism, which is found in
M. mycoides
SC only, resulted in an extra band on the agarose gel because an
Alu
I site is lacking in the
rrnA
operon. Specimens from cows with postmortem signs of CBPP were analyzed with the two PCR systems.
M. mycoides
SC was clearly identified in pleural fluid and lung tissue, and the methods were found to be robust and rapid. The results were in agreement with those obtained by conventional diagnostic techniques.
Multilocus Sequence Typing and Antigen Gene Sequencing in the Investigation of a Meningococcal Disease Outbreak
Authors: Feavers Ian M. , Gray Stephen J. , Urwin Rachel , Russell Joanne E. , Bygraves Jane A. , Kaczmarski Edward B. , Maiden Martin C. J.
Published: December 2019

Multilocus sequence typing and antigen gene sequencing were used to investigate an outbreak of meningococcal disease in a university in the United Kingdom. The data obtained showed that five distinct
Neisseria meningitidis
strains belonging to the ET-37 complex were present in the student population during the outbreak. Three of these strains were not associated with invasive disease, and two distinct strains caused invasive disease, including several fatalities. The initial case of the disease cluster was caused by a strain distinct from that responsible for at least two subsequent cases and two cases remote from the university, which were epidemiologically linked to the outbreak. These observations were consistent with pulsed-field gel electrophoresis data, but the sequence data alone were sufficient to resolve the strains involved in the disease cluster. Interpretation of the nucleotide sequence data was more straightforward than interpretation of the fingerprint patterns, and the sequence data provided information on the genetic differences among the isolates.
Genetic Analysis of
Borrelia garinii
OspA Serotype 4 Strains Associated with Neuroborreliosis: Evidence for Extensive Genetic Homogeneity
Authors: Marconi R. T. , Hohenberger S. , Jauris-Heipke S. , Schulte-Spechtel U. , LaVoie C. P. , Rößler D. , Wilske B.
Published: December 2019

Infection with
Borrelia garinii
outer surface protein (Osp) A serotype 4 strains has been correlated with the development of neuroborreliosis in Lyme borreliosis patients in Europe. OspA serotype 4 isolates have been recovered primarily from human cerebrospinal fluid, suggesting a tropism for this environment. Previous studies with monoclonal antibodies directed against OspA and OspC demonstrated that OspA serotype 4 strains are antigenically closely related. In view of the pronounced antigenic and genetic variability that has been noted in the Osps of other
Borrelia
isolates, we sought to determine if OspA serotype 4 strains represent a recently emerged clonal lineage of
B. garinii
. Toward this goal, a representative group of OspA serotype 4 strains was analyzed for traits that typically exhibit hypervariability among isolates that cause Lyme borreliosis. The following criteria were assessed: (i)
ospC
sequences, (ii) plasmid composition, (iii) genomic restriction fragment length polymorphism (RFLP) patterns, and (iv) the RFLP patterns of the upstream homology box (UHB) element which flanks members of the UHB gene family at their 5′ end. Collectively, these analyses demonstrate genetic homogeneity, suggesting that OspA serotype 4 strains are a recently emerged clonal lineage with an apparent tropism for the central nervous system.
Demonstration of
Bartonella grahamii
DNA in Ocular Fluids of a Patient with Neuroretinitis
Authors: Kerkhoff F. T. , Bergmans A. M. C. , van der Zee A. , Rothova A.
Published: December 2019

We describe the clinical and laboratory features of a 55-year-old human immunodeficiency virus-negative female patient who presented with bilateral intraocular inflammatory disease (neuroretinitis type) and behavioral changes caused by a
Bartonella grahamii
infection. Diagnosis was based on the PCR analysis of DNA extracted from the intraocular fluids. DNA analysis of the PCR product revealed a 100% identity with the 16S rRNA gene sequence of
B. grahamii
. The patient was successfully treated with doxycycline (200 mg/day) and rifampin (600 mg/day) for 4 weeks. This is the first report that demonstrates the presence of a
Bartonella
species in the intraocular fluids of a nonimmunocompromised patient and that indicates that
B. grahamii
is pathogenic for humans.
Quantitative Evaluation of Ehrlichial Burden in Horses after Experimental Transmission of Human Granulocytic Ehrlichia Agent by Intravenous Inoculation with Infected Leukocytes and by Infected Ticks
Authors: Pusterla Nicola , Leutenegger Christian M. , Chae Joon-Seok , Lutz Hans , Kimsey Robert B. , Dumler J. Stephen , Madigan John E.
Published: December 2019

This paper describes the kinetics of the human granulocytic ehrlichiosis agent in the blood of horses experimentally infected by intravenous inoculation with infected leukocytes and by infected ticks as evaluated by using a real-time quantitative PCR assay. The data obtained indicated differences in the period of incubation, duration of rickettsemia, and initial and maximal ehrlichial loads between the two routes of infection.