bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of
Burkholderia
spp. in the Clinical Microbiology Laboratory: Comparison of Conventional and Molecular Methods
Authors: van Pelt Cindy , Verduin Cees M. , Goessens Wil H. F. , Vos Margreet C. , Tümmler Burkhard , Segonds Christine , Reubsaet Frans , Verbrugh Henri , van Belkum Alex
Published: December 2019

Cystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways. Several species belonging to the genus
Burkholderia
are potential CF-related pathogens, but microbiological identification may be complicated. This situation is not in the least due to the poorly defined taxonomic status of these bacteria, and further validation of the available diagnostic assays is required. A total of 114 geographically diverse bacterial isolates, previously identified in reference laboratories as
Burkholderia cepacia
(
n
= 51),
B. gladioli
(
n
= 14),
Ralstonia pickettii
(
n
= 6),
B. multivorans
(
n
= 2),
Stenotrophomonas maltophilia
(
n
= 3), and
Pseudomonas aeruginosa
(
n
= 11), were collected from environmental, clinical, and reference sources. In addition, 27 clinical isolates putatively identified as
Burkholderia
spp. were recovered from the sputum of Dutch CF patients. All isolates were used to evaluate the accuracy of two selective growth media, four systems for biochemical identification (API 20NE, Vitek GNI, Vitek NFC, and MicroScan), and three different PCR-based assays. The PCR assays amplify different parts of the ribosomal DNA operon, either alone or in combination with cleavage by various restriction enzymes (PCR-restriction fragment length polymorphism [RFLP] analysis). The best system for the biochemical identification of
B. cepacia
appeared to be the API 20NE test. None of the biochemical assays successfully grouped the
B. gladioli
strains. The PCR-RFLP method appeared to be the optimal method for accurate nucleic acid-mediated identification of the different
Burkholderia
spp. With this method,
B. gladioli
was also reliably classified in a separate group. For the laboratory diagnosis of
B. cepacia
, we recommend parallel cultures on blood agar medium and selective agar plates. Further identification of colonies with a
Burkholderia
phenotype should be performed with the API 20NE test. For final confirmation of species identities, PCR amplification of the small-subunit rRNA gene followed by RFLP analysis with various enzymes is recommended.
Salmonella
Enteritidis Phage Types 1 and 4: Pheno- and Genotypic Epidemiology of Recent Outbreaks in Finland
Authors: Lukinmaa Susanna , Schildt Raili , Rinttilä Teemu , Siitonen Anja
Published: December 2019

In the 1990s,
Salmonella enterica
subsp.
enterica
serovar Enteritidis has caused 15 outbreaks in Finland; 12 of them were caused by phage type 1 (PT1) and PT4. Thus far, there has been no clear evidence as to the source of these
Salmonella
Enteritidis PT1 and PT4 strains, so it was necessary to try to characterize them further.
Salmonella
Enteritidis PT1 (
n
= 57) and PT4 (
n
= 43) isolates from different sources were analyzed by genomic pulsed-field gel electrophoresis (PFGE), plasmid profiling, and antimicrobial resistance testing to investigate the distribution of their subtypes in Finland. It was also hoped that this investigation would help in identifying the sources of the infections, especially the sources of the outbreaks caused by PT1 and PT4 in the 1990s. The results showed that both PFGE and plasmid profiling, but not antimicrobial susceptibility testing, were capable of differentiating isolates of
Salmonella
Enteritidis PT1 and PT4. By genotypic methods, it was possible to divide both PT1 and PT4 isolates into 12 subtypes. It could also be shown that all PT1 outbreak isolates were identical and, at least with this collection of isolates, that the outbreaks did not originate from the Baltic countries or from Russia, where this phage type predominates. It was also established that the outbreaks caused by PT4 all had different origins. Valuable information for future investigations was gained on the distribution of molecular subtypes of strains that originated from the tourist resorts that are popular among Finns and of strains that were isolated from livestock.
Nocardia
Thyroiditis: Unusual Location of Infection
Authors: Carriere Christian , Marchandin Helene , Andrieu Jean Michel , Vandome Anne , Perez Colette
Published: December 2019

Nocardia asteroides
complex is an important opportunistic agent in immunocompromised hosts. Usually, primary pulmonary infection occurs and is followed by dissemination of the pathogen to the central nervous system and soft tissues. As described in the literature, almost every organ can be infected, but to our knowledge,
Nocardia
has been described as a pathogen responsible for thyroid abscess in only one report, which was published in 1993. The present report is the second case report of
Nocardia
thyroiditis. The patient was under immunosuppressor treatment following a combined liver-kidney transplant and presented with a preexisting nodular goiter which was probably a predisposing factor to the start and development of the thyroid infection.
Prenatal Diagnosis of Parvovirus B19-Induced Hydrops Fetalis by Chemiluminescence In Situ Hybridization
Authors: Musiani M. , Pasini P. , Zerbini M. , Gentilomi G. , Roda A. , Gallinella G. , Manaresi E. , Venturoli S.
Published: December 2019

Parvovirus B19 can be transmitted transplacentally from the infected mother to the fetus during pregnancy, and hydrops fetalis, abortion, or stillbirth can result. In our study we explored the use of chemiluminescence in situ hybridization to detect B19 DNA on cord blood cells, amniotic fluid cells, and pleuric fluid cells from several cases of hydrops fetalis. B19 DNA was detected by using digoxigenin-labeled probes immunoenzymatically visualized with the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal emitted from the hybridized probes was detected, analyzed, and measured with a high-performance, low-light-level imaging luminograph connected to an optical microscope and to a personal computer for the quantification and localization of the chemiluminescent emission inside individual cells.
Clonal Diversity among Recently Emerged Strains of
Vibrio parahaemolyticus
O3:K6 Associated With Pandemic Spread
Authors: Bag Prasanta K. , Nandi Suvobroto , Bhadra Rupak K. , Ramamurthy T. , Bhattacharya S. K. , Nishibuchi M. , Hamabata T. , Yamasaki Shinji , Takeda Yoshifumi , Nair G. Balakrish
Published: December 2019

The genomes of the O3:K6 strains of
Vibrio parahaemolyticus
which abruptly emerged in Calcutta, India, in February 1996 and which demonstrated an unusual potential to spread and an enhanced propensity to cause infections were examined by different molecular techniques to determine clonality. No restriction fragment length polymorphism (RFLP) in the gene encoding the thermostable direct hemolysin was observed among the O3:K6 isolates of
V. parahaemolyticus
. Clonal diversity among the O3:K6 strains became evident by examining the RFLPs of the
rrn
operons and by the use of pulsed-field gel electrophoresis. Five ribotypes were distinguished among the O3:K6 strains examined, with ribotype R4 constituting the major type. Strains of O3:K6 isolated between June and August 1996 showed different pulsotypes compared to the pulsotypes of strains isolated before and after this period, indicating genetic reassortment among these strains, but those isolated between August 1996 and March 1998 showed identical or nearly similar pulsotypes. It is clear that there is a certain degree of genomic reassortment among the O3:K6 clones but that these strains are predominantly one clone.
Level of Human Immunodeficiency Virus DNA in Peripheral Blood Mononuclear Cells Correlates with Efficacy of Antiretroviral Therapy
Authors: McDermott Jennifer L. , Giri Adriana A. , Martini Isabella , Bono Maria , Giacomini Mauro , Campelli Antonio , Tagliaferro Luigi , Cara Andrea , Varnier Oliviero E.
Published: December 2019

A novel colorimetric assay was developed and validated for accurate quantitation of human immunodeficiency virus (HIV) DNA in peripheral blood mononuclear cells (PBMCs). We tested 318 sequential samples from 56 subjects, 53 of whom were undergoing dual or triple therapy. Patients were considered responders when viremia levels were below 5,000 HIV RNA copies/ml. The mean DNA copy numbers for untreated and responder subjects were similar (72 and 75, respectively), while it was 4.54-fold higher for nonresponders (339). This report provides strong evidence that HIV DNA levels in PBMCs correlate with therapeutic efficacy and suggests that DNA quantitation is a useful tool to monitor the decay of the HIV reservoir toward disease remission, especially when viremia is undetectable.
Evaluation of an Epstein-Barr Virus (EBV) Immunoglobulin M Enzyme-Linked Immunosorbent Assay Using a Synthetic Convergent Peptide Library, or Mixotope, for Diagnosis of Primary EBV Infection
Authors: Tranchand-Bunel D. , Gras-Masse H. , Bourez B. , Dedecker L. , Auriault C.
Published: December 2019

An immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) was developed by using a 24-amino-acid peptide of the 18-kDa Epstein-Barr virus (EBV) viral capsid antigen (VCAp18). IgM detection was increased by 23% by using this antigen. Detection of IgM antibodies to the EBV proteins in the new ELISA was 100% specific and 95% sensitive.
Molecular Analysis of
Mycobacterium avium
Isolates by Using Pulsed-Field Gel Electrophoresis and PCR
Authors: Pestel-Caron Martine , Graff Gabriel , Berthelot Gilles , Pons Jean-Louis , Lemeland Jean-François
Published: December 2019

Genetic relationships among 46 isolates of
Mycobacterium avium
recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS
1245
and IS
1311
. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of
M. avium
strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of
M. avium
strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of
M. avium
isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships.
TT Virus Infection in French Hemodialysis Patients: Study of Prevalence and Risk Factors
Authors: Gallian Pierre , Berland Yvon , Olmer Michel , Raccah Denis , de Micco Philippe , Biagini Philippe , Simon Sophie , Bouchouareb Dammar , Mourey Catherine , Roubicek Christa , Touinssi M’hammed , Cantaloube Jean-François , Dussol Bertrand , de Lamballerie Xavier
Published: December 2019

The TT virus (TTV) is a recently discovered DNA virus which was first identified in patients with non-A to -G hepatitis following blood transfusion. In this study, we tested 150 attendees of two hemodialysis (HD) units of the public hospitals of Marseilles, France, for the presence of TTV genome by using a PCR-based methodology. The overall prevalence of TTV viremia was 28% (compared to 5.3% in blood donors from the same region). We demonstrated the existence of chronic infections and superinfections by strains belonging to different genotypes. The prevalence of infection was higher in patients originating from Africa, in patients with previous blood transfusion or organ transplantation, in patients with antibody to hepatitis B core antigen, and in those with diabetes mellitus. A high prevalence of TTV infection (50%) was also observed in a population of patients with diabetes mellitus but without renal disease. No significant relationship was found between TTV viremia and hepatitis C virus or GB virus C, transaminases, age, sex, and duration of HD treatment. The PCR amplification products (located in open reading frame 1 of the TTV genome) were sequenced. These genomic sequences were submitted to phylogenetic analysis by using the Jukes-Cantor algorithm for distance determination and the neighbor-joining method for tree building. In several instances, sequences from viruses isolated in a HD unit were grouped in the same phylogenetic cluster. These results together with the different distribution of cases in the two HD units suggest there is viral transmission within each.
Comparison of
Ehrlichia chaffeensis
Recombinant Proteins for Serologic Diagnosis of Human Monocytotropic Ehrlichiosis
Authors: Yu Xue-Jie , Crocquet-Valdes Patricia A. , Cullman Louis C. , Popov Vsevolod L. , Walker David H.
Published: December 2019

Diagnosis of human monocytotropic ehrlichiosis (HME) generally depends on serology that detects the antibody response to immunodominant proteins of
Ehrlichia chaffeensis
. Protein immunoblotting was used to evaluate the reaction of the antibodies in patients’ sera with the recombinant
E. chaffeensis
120- and 28-kDa proteins as well as the 106- and the 37-kDa proteins. The cloning of the genes encoding the latter two proteins is described in this report. Immunoelectron microscopy demonstrated that the 106-kDa protein is located at the surfaces of ehrlichiae and on the intramorular fibrillar structures associated with
E. chaffeensis
. The 37-kDa protein is homologous to the iron-binding protein of gram-negative bacteria. Forty-two serum samples from patients who were suspected to have HME were tested by immunofluorescence (IFA) using
E. chaffeensis
antigen and by protein immunoblotting using recombinant
E. chaffeensis
proteins expressed in
Escherichia coli
. Thirty-two serum samples contained IFA antibodies at a titer of 1:64 or greater. The correlation of IFA and recombinant protein immunoblotting was 100% for the 120-kDa protein, 41% for the 28-kDa protein, 9.4% for the 106-kDa protein, and 0% for the 37-kDa protein. None of the recombinant antigens yielded false-positive results. All the sera reactive with the recombinant 28- or the 106-kDa proteins also reacted with the recombinant 120-kDa protein.
Evaluation of the PrimeCapture CMV DNA Detection Plate System for Detection of Cytomegalovirus in Clinical Specimens
Authors: Davoli Eugene H. , Lipson Steven M. , Match Mark E. , Shepp David H. , Morin Joseph W. , Curley Dennis M.
Published: December 2019

With the availability of anticytomegalovirus (CMV) therapeutic agents, rapid detection of CMV is important in the care and management of the immunosuppressed patient. The PrimeCapture CMV DNA Detection Plate System (PC-PCR) was evaluated for the detection of CMV in blood and cerebrospinal fluid (CSF). The resolution of discordant results was performed by consensus testing utilizing a combination of conventional cell culture (TC-CPE), the CMV-antigenemia (CMV-Ag) assay, one or more in-house CMV nested PCR assays, and/or patient evaluation and follow-up. Of 51 blood specimens from 34 patients, 23 (45%) were identified as true positives. PC-PCR was significantly more sensitive than the CMV-Ag assay, TC-CPE, or a combination of both tests. The sensitivities, specificities, positive predictive values (PPV), and negative predictive values (NPV) for PC-PCR, the CMV-Ag assay, TC-CPE, and a combination of CMV-Ag and TC-CPE were 78, 75, 72, 81%; 46, 100, 100, 70%; 39, 100, 100, 67%; and 58, 100, 100, 73%, respectively. CMV was not detected or isolated in CSF, resulting in a combined PC-PCR sensitivity, specificity, PPV, and NPV of 77, 90, 68, and 93%, respectively. Among those laboratorians considering the incorporation of molecular CMV diagnostics into their clinical microbiology or virology laboratories, the CMV PC-PCR offers a relatively simple-to-perform and sensitive assay system.
Comparison of Methods Based on Different Molecular Epidemiological Markers for Typing of
Mycobacterium tuberculosis
Complex Strains: Interlaboratory Study of Discriminatory Power and Reproducibility
Authors: Kremer K. , van Soolingen D. , Frothingham R. , Haas W. H. , Hermans P. W. M. , Martín C. , Palittapongarnpim P. , Plikaytis B. B. , Riley L. W. , Yakrus M. A. , Musser J. M. , van Embden J. D. A.
Published: December 2019

In this study, the currently known typing methods for
Mycobacterium tuberculosis
isolates were evaluated with regard to reproducibility, discrimination, and specificity. Therefore, 90
M. tuberculosis
complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays. In all methods, one or more repetitive DNA elements were targeted. The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method. To examine intralaboratory reproducibility, blinded duplicate samples were included. The specificities of the various methods were tested by inclusion of 10 non-
M. tuberculosis
complex strains. All five RFLP typing methods were highly reproducible. The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping. In contrast, the double repetitive element PCR (DRE-PCR), IS
6110
inverse PCR, IS
6110
ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible. The 90 strains were best discriminated by IS
6110
RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns). We conclude that for epidemiological investigations, strain differentiation by IS
6110
RFLP or mixed-linker PCR are the methods of choice. A strong association was found between the results of different genetic markers, indicating a clonal population structure of
M. tuberculosis
strains. Several separate genotype families within the
M. tuberculosis
complex could be recognized on the basis of the genetic markers used.
Determination of Hepatitis C Virus Genotype by Direct Sequence Analysis of Products Generated with the Amplicor HCV Test
Authors: Germer Jeff J. , Rys Paul N. , Thorvilson Jill N. , Persing David H.
Published: December 2019

Consistent with other members of the family
Flaviviridae
, hepatitis C virus (HCV) demonstrates a high degree of sequence variation throughout the coding regions of its genome. However, there is a high degree of sequence conservation found within the 5′ untranslated region (UTR) of the genome, making this region a target of choice for most nucleic acid amplification-based detection assays. In this study, the Amplicor HCV test, a commercially available assay which detects the 5′UTR, was used for the detection of HCV RNA in 669 serum samples obtained from a cohort of liver transplantation patients. Amplification products obtained from the HCV-positive cases were subjected to direct sequencing and genotyping based upon seven phylogenetically informative regions within the 5′UTR. Of the 669 specimens, 416 (62.2%) tested positive for the presence of HCV RNA. Of these, 372 (89.4%) specimens were successfully classified into 11 HCV genotypes and subtypes after computer-assisted analysis of the sequence data. Forty-four (10.6%) of the HCV RNA-positive specimens were not classifiable, the majority corresponding to low-titer specimens as determined by the Chiron Quantiplex HCV RNA 2.0 assay. Additional comparative studies targeting the NS-5 region of the viral genome generally confirmed the accuracy and sensitivity of the 5′UTR-based classifications, with the exception of the misclassification of a small number of type 1a cases as type 1b. We conclude that although the high sequence conservation within the 5′UTR results in the misclassification of a small number of HCV subtypes, the overall gains of efficiency, the shorter turnaround time, the inclusion of contamination control measures, and the low rate of test failure compared to that of methods based on the NS-5 gene together constitute significant advantages over other techniques.
Destructive Knee Joint Infection Caused by
Peptostreptococcus micros
: Importance of Early Microbiological Diagnosis
Authors: Riesbeck Kristian , Sanzén Lennart
Published: December 2019

Peptostreptococcus micros
is a commensal of the oral cavity and the genitourinary tract that rarely causes serious infections. A case of a destructive knee joint infection with rapid progress caused by
P. micros
is presented. The significance of the microbiological findings was initially not acknowledged, which contributed to a nonsuccessful clinical outcome.
PCR-Based Methods for Genotyping Viridans Group Streptococci
Authors: Alam Sharmin , Brailsford Susan R. , Whiley Robert A. , Beighton David
Published: December 2019

Standard repetitive extragenic palindromic (REP)-PCR, enterobacterial repetitive intergenic consensus-PCR, and
Salmonella enteritidis
repetitive element-PCR methods for bacterial strain typing were performed with DNA extracted by boiling members of each of the currently recognized species of human viridans group streptococci. Each of the methods was reproducible. The unique isolates (
n
= 72) from 15 species of viridans group streptococci were readily distinguishable, with no two isolates showing greater than 90% per cent similarity. The majority of strains exhibited much less than 90% similarity. Isolates identical by REP-PCR were also identical by the other two methods. These PCR-based typing methods, although they do not permit determination of the species of the isolates, are simple to perform and are suitable for clinical and ecological investigations of viridans group streptococci.
Impact of Reference Standard Sensitivity on Accuracy of Rapid Antigen Detection Assays and a Leukocyte Esterase Dipstick for Diagnosis of
Chlamydia trachomatis
Infection in First-Void Urine Specimens from Men
Authors: Chernesky M. , Jang D. , Krepel J. , Sellors J. , Mahony J.
Published: December 2019

A total of 128 previously frozen first-void urine (FVU) specimens from selected asymptomatic men were centrifuged and tested by three
Chlamydia trachomatis
rapid antigen detection tests and with a leukocyte esterase (LE) dipstick. When the results were compared to those of a reference standard of positivity determined by the Chlamydiazyme enzyme immunoassay as confirmed by a blocking assay, the sensitivities of the Testpack Chlamydia (Abbott), Clearview Chlamydia (Unipath), and Surecell Chlamydia (Kodak) tests and the LE dipstick test were 76.4, 76.4, 67.3, and 88.6%, respectively. Use of the ligase chain reaction (LCR), whose results were confirmed by direct fluorescent-antibody staining of elementary bodies, as the reference standard reduced the sensitivities to 70.9, 67.7, 62.9, and 87.5%, respectively. The specificities by use of LCR as the reference standard were 95.5, 95.5, 100, and 92.4%, respectively. These rapid chlamydial antigen tests performed reasonably well with FVU specimens, but the simple LE dipstick test, which had the highest sensitivity, would have enabled treatment of the greatest number of infected male patients.
Methicillin-Resistant
Staphylococcus aureus
Clonal Types in the Czech Republic
Authors: Melter O. , Santos Sanches I. , Schindler J. , Aires de Sousa M. , Mato R. , Kovárova V. , Zemlicková H. , de Lencastre H.
Published: December 2019

Molecular surveillance studies have documented the extensive spread of methicillin-resistant
Staphylococcus aureus
(MRSA) clones. Studies carried out by Centro de Epidemiologia Molecular-Network for Tracking Gram-Positive Pathogenic Bacteria (CEM/NET) led to the identification of two international multidrug-resistant strains, which were designated as the Iberian and Brazilian MRSA clones and which were defined by multiple genomic typing methods; these included
Cla
I restriction digests hybridized with
mecA
- and Tn
554
-specific DNA probes and pulsed-field gel electrophoresis (PFGE). The genotypic characteristics of these clones are distinct: the Iberian clone is defined as
mecA
type I, Tn
554
type E (or its variants), and PFGE pattern A (I:E:A), whereas the Brazilian clone is defined as
mecA
type XI (or its variants), Tn
554
type B, and PFGE pattern B (XI:B:B). In this study, we characterized 59 single-patient isolates of MRSA collected during 1996 and 1997 at seven hospitals located in Prague and five other cities in the Czech Republic by using the methodologies mentioned above and by using ribotyping of
Eco
RI and
Hin
dIII digests hybridized with a 16S-23S DNA probe. The Brazilian MRSA clone (XI:B:B) was the major clone (80%) spread in two hospitals located in Prague and one located in Brno; the Iberian MRSA clone (I:E:A or its variant I:DD:A), although less representative (12%), was detected in two hospitals, one in Prague and the other in Plzen. Almost all the strains belonging to clone XI:B:B (45 of 47) corresponded to a unique ribotype, E1H1, whereas most strains of the I:E:A and I:DD:A clonal types (6 of 7) corresponded to ribotype E2H2.
Somatic Serogroups, Capsular Types, and Species of Fecal
Klebsiella
in Patients with Ankylosing Spondylitis
Authors: Toivanen Paavo , Hansen Dennis S. , Mestre Francisca , Lehtonen Leena , Vaahtovuo Jussi , Vehma Mari , Möttönen Timo , Saario Riitta , Luukkainen Reijo , Nissilä Martti
Published: December 2019

The purpose of the present study was to find out whether patients with ankylosing spondylitis (AS) carry fecal
Klebsiella
strains that belong to serotypes or species specific for AS. Somatic serotypes (O groups), capsular (K) serotypes, and biochemically identified species were determined for fecal klebsiellae isolated from 187 AS patients and 195 control patients. The controls were patients with fibromyalgia or rheumatoid arthritis. The 638 isolates of
Klebsiella
that were obtained represented 161 strains; 81 from AS patients and 80 from the controls. The average number of
Klebsiella
strains per patient was 1.7 for the AS group and 1.5 for the control group. The most common O group was O1, which was observed for isolates from 23 of 187 AS patients and 24 of 195 control patients. Next in frequency was group O2, which was observed for isolates from 17 AS patients and 15 control patients. Regarding the K serotypes, 59 different types were identified, revealing a heterogeneous representation of
Klebsiella
strains, without a predominance of any serotype. By biochemical identification,
Klebsiella pneumoniae
was the most frequently occurring species, being found in 45 AS patients and 45 control patients. Next in the frequency was
K. oxytoca
, which was observed in 26 AS patients and in 29 control patients.
K. planticola
and
K. terrigena
occurred in only a minority of patients. Altogether, when analyzed either separately or simultaneously according to O groups, K serotypes, and biochemically identified species, no evidence of the existence of AS-specific
Klebsiella
strains was obtained. These findings do not indicate participation of
Klebsiella
in the etiopathogenesis of AS.
Isolation of Rifampin-Resistant Mutants of
Listeria monocytogenes
and Their Characterization by
rpoB
Gene Sequencing, Temperature Sensitivity for Growth, and Interaction with an Epithelial Cell Line
Authors: Morse Robert , O’Hanlon Karen , Virji Mumtaz , Collins Matthew D.
Published: December 2019

The sequence of the
rpoB
gene from
Listeria monocytogenes
was determined. Rifampin-resistant (Rif
r
) mutants arising from
L. monocytogenes
cultures exposed to rifampin were isolated, and by partial sequencing of their
rpoB
genes, seven different point mutations affecting five different amino acids (473Asp→Asn or Gly, 479Gly→Asp, 483His→Tyr or Leu, 528Ile→Phe, and 530Ser→Tyr), which led to MICs of 0.5 to 100 μg/ml for the organisms, were determined. These mutants showed various deficiencies for growth at 42°C, with only one being comparable to the wild-type strain. The interaction of these Rif
r
mutants with human Caco-2 cells was examined by using an immunofluorescence technique. Three mutants failed to interact, while three showed a reduced interaction compared to that of the wild type. It is believed that these pleiotropic phenotypes have arisen as a result of mutations within the DNA-dependent RNA polymerase holoenzyme.
Human Immunodeficiency Virus Type 1 Cloning Vectors for Antiretroviral Resistance Testing
Authors: Martinez-Picado Javier , Sutton Lorraine , De Pasquale Maria Pia , Savara Anu V. , D’Aquila Richard T.
Published: December 2019

Better detection of minority human immunodeficiency virus type 1 (HIV-1) populations containing gene mutations may improve the usefulness of antiretroviral resistance testing for clinical management. Molecular cloning of HIV-1 PCR products which might improve minority detection can be slow and difficult, and commercially available recombinant virus assays test drug susceptibility of virus pools. We describe novel plasmids and simple methods for rapid cloning of HIV-1 PCR products from patient specimens and their application to generate infectious recombinant virus clones for virus phenotyping and genotyping. Eight plasmids with differing deletions of sequences encoding HIV-1 protease, reverse transcriptase, or Gag p7/p1 and Gag p1/p6 cleavage sites were constructed for cloning HIV-1 PCR products. A simple HIV-1 sequence-specific uracil deglycosylase-mediated cloning method with the vectors and primers designed here was more rapid than standard ligase-mediated cloning. Pooled and molecularly cloned infectious recombinant viruses were generated with these vectors. Replicative viral fitness and drug susceptibility phenotypes of cloned infectious viruses containing patient specimen-derived sequences were measured. Clonal resistance genotyping analyses were also performed from virus isolates, plasma HIV-1 RNA, and infected cell DNA. Sequencing of a limited number of molecular clones detected minorities of resistant virus not identified in the pooled population PCR product sequence and linkage of minority mutations.
Outcome of Diagnostic Tests Using Samples from Patients with Culture-Proven Human Monocytic Ehrlichiosis: Implications for Surveillance
Authors: Childs James E. , Sumner John W. , Nicholson William L. , Massung Robert F. , Standaert Steven M. , Paddock Christopher D.
Published: December 2019

We describe the concordance among results from various laboratory tests using samples derived from nine culture-proven cases of human monocytic ehrlichiosis (HME) caused by
Ehrlichia chaffeensis
. A class-specific indirect immunofluorescence assay for immunoglobulin M (IgM) and IgG, using
E. chaffeensis
antigen, identified 44 and 33% of the isolation-confirmed HME patients on the basis of samples obtained at initial clinical presentation, respectively; detection of morulae in blood smears was similarly insensitive (22% positive). PCR amplifications of ehrlichial DNA targeting the 16S rRNA gene, the variable-length PCR target gene, and the
groESL
operon were positive for whole blood specimens obtained from all patients at initial presentation. As most case definitions of HME require a serologic response with compatible illness for a categorization of even probable disease, PCR would have been required to confirm the diagnosis of HME in all nine of these patients without the submission of a convalescent-phase serum sample. These data suggest that many, if not most, cases of HME in patients who present early in the course of the disease may be missed and underscore the limitations of serologically based surveillance systems.
Common Ancestry of
Borrelia burgdorferi
Sensu Lato Strains from North America and Europe
Authors: Postic D. , Ras N. Marti , Lane R. S. , Humair P.-F. , Wittenbrink M. M. , Baranton G.
Published: December 2019

Ten atypical European
Borrelia burgdorferi
sensu lato (
Borrelia
spp.) strains were genetically characterized, and the diversity was compared to that encountered among related
Borrelia
spp. from North America. Phylogenetic analyses of a limited region of the genome and of the whole genome extend existing knowledge about borrelial diversity reported earlier in Europe and the United States. Our results accord with the evidence that North American and European strains may have a common ancestry.
Discrimination of Multidrug-Resistant
Mycobacterium tuberculosis
IS
6110
Fingerprint Subclusters by
rpoB
Gene Mutation Analysis
Authors: Portugal Isabel , Maia Sara , Moniz-Pereira José
Published: December 2019

The
rpoB
gene mutations in a 69-bp region of the gene, resulting in resistance to rifampin, were used to discriminate between
Mycobacterium tuberculosis
IS
6110
fingerprint subclusters. These subclusters exhibited identical IS
6110
fragments or had one or two additional fragments. In the two major subclusters all the analyzed strains have the same variant
rpoB
allele but are different from each other, suggesting the occurrence of independent outbreaks.
Interactions between Methicillin and Vancomycin in Methicillin-Resistant
Staphylococcus aureus
Strains Displaying Different Phenotypes of Vancomycin Susceptibility
Authors: Howe Robin A. , Wootton Mandy , Bennett Peter M. , MacGowan Alasdair P. , Walsh Timothy R.
Published: December 2019

Vancomycin-sensitive, -intermediate, and -heterointermediate methicillin-resistant
Staphylococcus aureus
isolates were tested by using E-tests to explore the interaction of methicillin and vancomycin. For the vancomycin-intermediate and -heterointermediate strains both drugs showed antagonism at concentrations below their MICs but synergy at methicillin concentrations near the MIC. This property could be used to screen for heterointermediate
S. aureus
strains.
Peritonitis Caused by
Haemophilus parainfluenzae
in a Patient Undergoing Continuous Ambulatory Peritoneal Dialysis
Authors: Betriu Carmen , Coronel Francisco , Martin Pilar , Picazo Juan J.
Published: December 2019

We report a case of peritonitis in a patient undergoing continuous ambulatory peritoneal dialysis.
Haemophilus parainfluenzae
biotype III was recovered in pure culture from dialysis fluid.
Multicenter Evaluation of Epidemiological Typing of Methicillin-Resistant
Staphylococcus aureus
Strains by Repetitive-Element PCR Analysis
Authors: Deplano Ariane , Schuermans Annette , Van Eldere Johan , Witte Wolfgang , Meugnier Hèléne , Etienne Jerome , Grundmann Hajo , Jonas Daniel , Noordhoek Gerda T. , Dijkstra Jolanda , van Belkum Alex , van Leeuwen Willem , Tassios Panayotis T. , Legakis Nicholas J. , van der Zee Anneke , Bergmans Anneke , Blanc Dominique S. , Tenover Fred C. , Cookson Barry C. , O'Neil Gael , Struelens Marc J. ,   The European Study Group on Epidemiological Markers of the ESCMID
Published: December 2019

Rapid and efficient epidemiologic typing systems would be useful to monitor transmission of methicillin-resistant
Staphylococcus aureus
(MRSA) at both local and interregional levels. To evaluate the intralaboratory performance and interlaboratory reproducibility of three recently developed repeat-element PCR (rep-PCR) methods for the typing of MRSA, 50 MRSA strains characterized by pulsed-field gel electrophoresis (PFGE) (
Sma
I) analysis and epidemiological data were blindly typed by inter-IS
256
, 16S-23S ribosomal DNA (rDNA), and MP3 PCR in 12 laboratories in eight countries using standard reagents and protocols. Performance of typing was defined by reproducibility (
R
), discriminatory power (
D
), and agreement with PFGE analysis. Interlaboratory reproducibility of pattern and type classification was assessed visually and using gel analysis software. Each typing method showed a different performance level in each center. In the center performing best with each method, inter-IS
256
PCR typing achieved
R
= 100% and
D
= 100%; 16S-23S rDNA PCR,
R
= 100% and
D
= 82%; and MP3 PCR,
R
= 80% and
D
= 83%. Concordance between rep-PCR type and PFGE type ranged by center: 70 to 90% for inter-IS
256
PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS
256
PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility. None of these assays, however, was sufficiently reproducible for interlaboratory exchange of data.
Evaluation of the MicroScan Rapid Neg ID3 Panel for Identification of
Enterobacteriaceae
and Some Common Gram-Negative Nonfermenters
Authors: O'Hara Caroline Mohr , Miller J. Michael
Published: December 2019

The MicroScan Rapid Neg ID3 panel (Dade Behring, Inc., West Sacramento, Calif.) is designed for the identification of gram-negative bacilli. We evaluated its ability to accurately identify
Enterobacteriaceae
that are routinely encountered in a clinical laboratory and glucose nonfermenting gram-negative bacilli. Using 511 stock cultures that were maintained at −70°C and passaged three times before use, we inoculated panels according to the manufacturer's instructions and processed them in a Walk/Away instrument using version 22.01 software. The time to identification was 2 h and 30 min. All panel identifications were compared to reference identifications previously determined by conventional tube biochemicals. At the end of the initial 2.5-h incubation period, 405 (79.3%) identifications were correct. An additional 49 (9.6%) isolates were correctly identified after required additional off-line biochemical tests were performed. Thus, at 24 h, 88.8% of the 511 strains tested were correctly identified. Twenty-two (4.3%) were identified to the genus level only. Twenty-six (5.1%) strains were misidentified. Because the system is based on fluorogenics, there are no conventional tests readily available with which to compare possibly incorrect reactions. Of the 28
Salmonella
strains that were tested, 5 were incorrectly reported. The 21 remaining errors were scattered among the genera tested. Testing on nine strains gave a result of “no identification” (very rare biotype). The Rapid Neg ID3 panel in this study approached 89% accuracy for the identification of gram-negative organisms encountered in the hospital laboratory.
Detection of Borreliacidal Antibodies in Dogs after Challenge with
Borrelia burgdorferi
-Infected
Ixodes scapularis
Ticks
Authors: Callister Steven M. , Jobe Dean A. , Schell Ronald F. , Lovrich Steven D. , Onheiber Keysha L. , Korshus Jon B.
Published: December 2019

Detection of borreliacidal antibodies is an accurate serodiagnostic test for confirmation of Lyme disease in humans. In this study, 13 pathogen-free beagles, 12 to 26 weeks old, were infected with
Borrelia burgdorferi
by tick challenge. Dogs were monitored for clinical signs and symptoms of Lyme disease along with borreliacidal antibody production against
B. burgdorferi
sensu stricto isolates 297 and 50772. Ten (77%) dogs developed lameness in one or more legs within 210 days after attachment of
Ixodes scapularis
ticks. Eight (80%) of the lame animals had concurrent fever of ≥38°C. Spirochetes were also recovered from the skin and joints of 12 (92%) dogs, but rarely from other organs. Borreliacidal antibodies against
B. burgdorferi
isolate 297 were detected in only four (31%) dogs, and the levels of killing antibodies remained low for the duration of the infection. In contrast, borreliacidal antibodies against
B. burgdorferi
isolate 50772 were detected in 13 (100%) dogs within 21 days of infection. Furthermore, the borreliacidal antibody levels correlated with the severity of
B. burgdorferi
infection. Detection of borreliacidal antibodies, especially against
B. burgdorferi
isolate 50772, is also a reliable serodiagnostic test for detection of Lyme disease in dogs.
Comparison of Phenotypic and Genotypic Methods for Pyrazinamide Susceptibility Testing with
Mycobacterium tuberculosis
Authors: Davies A. P. , Billington O. J. , McHugh T. D. , Mitchison D. A. , Gillespie S. H.
Published: December 2019

Mycobacterium tuberculosis
converts pyrazinamide to its active form by using the enzyme pyrazinamidase. This enzyme is coded for on the
pncA
gene, and mutations in the
pncA
gene result in absence of active enzyme, conferring resistance to the drug pyrazinamide. We investigated 27 strains of
Mycobacterium tuberculosis
suspected of being multidrug resistant. Each isolate was tested for susceptibility to pyrazinamide by the BACTEC 460TB method, and 19 were pyrazinamide resistant. The presence of active pyrazinamidase enzyme was sought by using the Wayne assay, which was positive in all of the sensitive isolates and four of the resistant isolates. The
pncA
gene was amplified by PCR, and mutations were sought by single-strand conformation polymorphism (SSCP) analysis. We identified four isolates which were phenotypically resistant to pyrazinamide, but which had active pyrazinamide enzyme on the Wayne assay and normal
pncA
gene SSCP. MICs measured by BACTEC 460TB and susceptibility testing at a lower pH of 5.5 confirmed genuine resistance. The
pncA
gene was sequenced in these four isolates and found not to have any mutations. This implies that an alternative mechanism of resistance exists in these strains. We conclude that genotypic assessment of pyrazinamide resistance is unreliable, because it depends on the identification of a single resistance mechanism. Phenotypic methods such as the BACTEC 460TB technique remain the best methods for pyrazinamide susceptibility testing.
PCR Assessment of
Chlamydia trachomatis
Infection of Semen Specimens Processed for Artificial Insemination
Authors: Pannekoek Yvonne , Westenberg Steven M. , de Vries Jan , Repping Sjoerd , Spanjaard Lodewijk , Eijk Paul P. , van der Ende Arie , Dankert Jacob
Published: December 2019

In order to ascertain the microbiological quality of stored semen specimens processed for artificial insemination by a donor (AID), we developed a PCR assay targeting the chlamydial plasmid to detect
Chlamydia trachomatis
in semen. The lower limit of detection of this assay corresponded to 2.5 to 5 elementary bodies per μl of semen. A total of 669 cryopreserved ejaculates from 97 asymptomatic donors were tested for
C. trachomatis
infection. Twelve ejaculates, originating from four donors, were found to be positive, indicating a 4% prevalence of
C. trachomatis
infection among the donor population studied. Cross-contamination between the cryopreserved specimens in the storage container was studied by typing using sequence analysis of PCR-amplified
omp1
genes of the strains. Two donors were infected with serovar E, one was infected with serovar F, and one was infected with serovar K. For two donors, the duration of
C. trachomatis
positivity could be assessed. One donor donated
C. trachomatis
-positive semen for at least 4 successive months, and the other did so for at least 16 months. The occurrence of
C. trachomatis
infection in cryopreserved donor semen indicates that ejaculates from donors not tested for a
C. trachomatis
infection just prior to donation should be tested for infection by a direct test such as the PCR described here. Direct testing of semen specimens will detect not only donors with an active infection but also
C. trachomatis
-infected ejaculates already stored and will thus improve the microbiological quality of AID, since discrepancies in the presence of
C. trachomatis
in urine and semen specimens have been reported.
Rapid and Sensitive Plate Method for Detection of
Aspergillus fumigatus
Authors: Bauters T. G. M. , Nelis H. J.
Published: December 2019

The routine identification of
Aspergillus fumigatus
in clinical samples involves, apart from direct examination, the isolation of the organism on a plate followed by its microscopic characterization. This approach lacks sensitivity, specificity, and speed. A new procedure has been developed combining microcolony formation on a nylon membrane filter at 45°C with the detection of a specific 4-methylumbelliferyl-α-
l
-arabinopyranoside cleaving enzyme activity in digitonin permeabilized cells. The test takes approximately 14 h and has an efficiency of 98.2% and false-positive and -negative rates of 0 and 3.1%, respectively. When applied to 188 clinical samples taken from patients with proven or nonproven presence of
Aspergillus
species, a good agreement with the conventional plate-microscopy method was obtained.
Discrimination between Viable and DeadEncephalitozoon cuniculi(Microsporidian) Spores by Dual Staining with Sytox Green and Calcofluor White M2R
Authors: Green L. C. , LeBlanc P. J. , Didier E. S.
Published: December 2019

Microsporidia are obligate intracellular parasites, recognized as causing chronic diarrhea and systemic disease in AIDS patients, organ transplant recipients, travelers, and malnourished children. Species of microsporidia that infect humans have been detected in drinking-water sources, and methods are needed to ascertain if these microsporidia are viable and capable of causing infections. In this study, Calcofluor White M2R and Sytox Green stains were used in combination to differentiate between live (freshly harvested) and dead (boiled)Encephalitozoon cuniculispores. Calcofluor White M2R binds to chitin in the microsporidian spore wall. Dual-stained live spores appeared as turquoise-blue ovals, while dead spores appeared as white-yellow ovals at an excitation wavelength of 395 to 415 nm used for viewing the Calcofluor stain. Sytox Green, a nuclear stain, is excluded by live spores but penetrates compromised spore membranes. Dual-stained dead spores fluoresced bright yellow-green when viewed at an excitation wavelength of 470 to 490 nm, whereas live spores failed to stain with Sytox Green. After live and dead spores were mixed at various ratios, the number of viably stained spores detected in the dual-staining procedure correlated (P= 0.0025) with the expected numbers of viable spores. Spore mixtures were also assayed for infectivity in a focus-forming assay, and a correlation (P= 0.0002) was measured between the percentage of focus-forming microsporidia and the percentage of expected infectious spores in each mixture. By analysis of variance, no statistically significant differences were measured between the percentage of viably stained microsporidia and the percentage of infectious microsporidia (P= 0.964) in each mixture. These results suggest that Calcofluor White M2R and Sytox Green stains, when used together, may facilitate studies to identify viable microsporidia.
Use of Amplified Fragment Length Polymorphisms for Typing
Corynebacterium diphtheriae
Authors: De Zoysa Aruni , Efstratiou Androulla
Published: December 2019

Amplified fragment length polymorphism (AFLP) was investigated for the differentiation of
Corynebacterium diphtheriae
isolates. Analysis using Taxotron revealed 10 distinct AFLP profiles among 57 isolates. Strains with ribotype patterns D1, D4, and D12 could not be distinguished; however, the technique discriminated isolates of ribotype patterns D3, D6, and D7 further. AFLP was rapid, fairly inexpensive, and reproducible and could be used as an alternative to ribotyping.
Comparison of Six Commercial DNA Extraction Kits for Recovery of Cytomegalovirus DNA from Spiked Human Specimens
Authors: Fahle Gary A. , Fischer Steven H.
Published: December 2019

We evaluated six commercially available DNA extraction kits for their ability to recover DNA from various dilutions of cytomegalovirus (CMV) added to four different specimens: bronchoalveolar lavage, cerebral spinal fluid, plasma, and whole blood. The kits evaluated included the Puregene DNA isolation kit (PG), Generation Capture Column kit, MasterPure DNA purification kit, IsoQuick nucleic acid extraction kit, QIAamp blood kit, and NucliSens isolation kit (NS). All six kits evaluated effectively removed PCR inhibitors from each of the four specimen types and produced consistently positive results down to a spiked concentration of 200 PFU of whole CMV per ml. However, the NS and PG resulted in the most consistently positive results at the lowest concentrations of spiked CMV (4 and 0.4 PFU/ml) and, in this evaluation, offered the most sensitive methods for extracting CMV DNA from the four different spiked specimens. Processing time and cost were also evaluated.