bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Mycobacterial Species by Comparative Sequence Analysis of the RNA Polymerase Gene (
rpoB
)
Authors: Kim Bum-Joon , Lee Seung-Hyun , Lyu Mi-Ae , Kim Seo-Jeong , Bai Gill-Han , Kim Sang-Jae , Chae Gue-Tae , Kim Eui-Chong , Cha Chang-Yong , Kook Yoon-Hoh
Published: December 2019

For the differentiation and identification of mycobacterial species, the
rpoB
gene, encoding the β subunit of RNA polymerase, was investigated.
rpoB
DNAs (342 bp) were amplified from 44 reference strains of mycobacteria and clinical isolates (107 strains) by PCR. The nucleotide sequences were directly determined (306 bp) and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Comparative sequence analysis of
rpoB
DNAs provided the basis for species differentiation within the genus
Mycobacterium
. Slowly and rapidly growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. Pathogenic
Mycobacterium kansasii
was easily differentiated from nonpathogenic
M. gastri
; this differentiation cannot be achieved by using 16S rRNA gene (rDNA) sequences. By being grouped into species-specific clusters with low-level sequence divergence among strains of the same species, all of the clinical isolates could be easily identified. These results suggest that comparative sequence analysis of amplified
rpoB
DNAs can be used efficiently to identify clinical isolates of mycobacteria in parallel with traditional culture methods and as a supplement to 16S rDNA gene analysis. Furthermore, in the case of
M. tuberculosis
, rifampin resistance can be simultaneously determined.
CDC Group IV c-2: a New
Ralstonia
Species Close to
Ralstonia eutropha
Authors: Moissenet Didier , Goujon Christophe P. , Garbarg-Chenon Antoine , Vu-Thien Hoang
Published: December 2019

CDC group IV c-2, an environmental gram-negative bacillus recently proposed for inclusion in the genus
Ralstonia
, has been isolated in several human infections. Biochemical characterization and 16S ribosomal DNA (rDNA) sequencing with phylogenetic analysis were used to characterize eight clinical isolates and four type strains. Other typing tools, such as pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis, were also used. PFGE typing of clinical isolates was unsuccessful because the DNA was degraded, and RAPD analysis was poorly discriminatory. In contrast, the type strains were clearly distinguished with both PFGE and RAPD analysis. All of the 16S rDNA sequences were identical. Comparison of the 16S rDNA sequences to the GenBank sequences showed that they were consistent with CDC group IV c-2 belonging to the genus
Ralstonia
. The closest matches were obtained with
Ralstonia eutropha
. However, four differences in 32 biochemical tests separated
R. eutropha
from CDC group IV c-2, which suggests that CDC group IV c-2 is a new species of the genus
Ralstonia
.
Rapid Identification of Thermotolerant
Campylobacter jejuni
,
Campylobacter coli
,
Campylobacter lari
, and
Campylobacter upsaliensis
from Various Geographic Locations by a GTPase-Based PCR-Reverse Hybridization Assay
Authors: van Doorn Leen-Jan , Verschuuren-van Haperen Anita , Burnens André , Huysmans Michael , Vandamme Peter , Giesendorf Belinda A. J. , Blaser Martin J. , Quint Wim G. V.
Published: December 2019

Recently, a gene from
Campylobacter jejuni
encoding a putative GTPase was identified. Based on two semiconserved GTP-binding sites encoded within this gene, PCR primers were selected that allow amplification of a 153-bp fragment from
C. jejuni
,
C. coli
,
C. lari
, and
C. upsaliensis
. Sequence analysis of these PCR products revealed consistent interspecies variation, which allowed the definition of species-specific probes for each of the four thermotolerant
Campylobacter
species. Multiple probes were used to develop a line probe assay (LiPA) that permits analysis of PCR products by a single reverse hybridization step. A total of 320 reference strains and clinical isolates from various geographic origins were tested by the GTP-based PCR-LiPA. The PCR-LiPA is highly specific in comparison with conventional identification methods, including biochemical and whole-cell protein analyses. In conclusion, a simple method has been developed for rapid and highly specific identification of thermotolerant
Campylobacter
species.
Ultrasound-Enhanced Latex Immunoagglutination and PCR as Complementary Methods for Non-Culture-Based Confirmation of Meningococcal Disease
Authors: Gray Stephen J. , Sobanski Michael A. , Kaczmarski Edward B. , Guiver Malcolm , Marsh W. John , Borrow Raymond , Barnes Rosemary A. , Coakley W. Terence
Published: December 2019

Preadmission administration of antibiotics to patients with suspected meningococcal infection has decreased the likelihood of obtaining an isolate and has stimulated development of rapid and reliable non-culture-based diagnostic methods. The sensitivity of the conventional test card latex agglutination test (TCLAT) for detection of capsular polysaccharide has been reported to be suboptimal. In the United Kingdom meningococcal DNA detection by PCR has become readily available and is now used as a first-line investigation. Recently, the performance of latex antigen detection has been markedly improved by ultrasound enhancement. Three tests for laboratory confirmation of meningococcal infection, (i) PCR assays, (ii) TCLAT, and (iii) ultrasound-enhanced latex agglutination test (USELAT), were compared in a retrospective study of 125 specimens (serum, plasma, and cerebrospinal fluid specimens) from 90 patients in whom meningococcal disease was suspected on clinical grounds. Samples were from patients with (i) culture-confirmed meningococcal disease, (ii) culture-negative but PCR-confirmed meningococcal disease, and (iii) clinically suspected but non-laboratory-confirmed meningococcal disease. USELAT was found to be nearly five times more sensitive than TCLAT. Serogroup characterization was obtained by both PCR and USELAT for 44 samples; all results were concordant and agreed with the serogroups determined for the isolates when the serogroups were available. For 12 samples negative by USELAT, the serogroup was determined by PCR; however, for 12 other specimens for which PCR had failed to indicate the serogroup, USELAT gave a result. USELAT is a rapid, low-cost method which can confirm a diagnosis, identify serogroups, and guide appropriate management of meningococcal disease contacts. A complementary non-culture-based confirmation strategy of USELAT for local use supported by a centralized PCR assay service for detection of meningococci would give the benefits of timely information and improved epidemiological data.
PCR-Restriction Enzyme Analysis for Detection of
Candida
DNA in Blood from Febrile Patients with Hematological Malignancies
Authors: Morace Giulia , Pagano Livio , Sanguinetti Maurizio , Posteraro Brunella , Mele Luca , Equitani Francesco , D’Amore Giuseppina , Leone Giuseppe , Fadda Giovanni
Published: December 2019

Blood samples were drawn daily from 72 patients who had hematological malignancies, neutropenia, and fever and who had failed to respond to broad-spectrum antibiotics. Each sample was used for conventional fungal blood cultures and for detection and identification of
Candida
DNA by a PCR method with subsequent restriction enzyme analysis (REA) recently developed in our laboratory. The PCR method was able to detect five CFU of
Candida
spp. per ml of blood, and subsequent REA of the amplicons allowed the identification of the
Candida
species most commonly implicated in cases of candidiasis. Thirty-one patients were PCR-REA positive, and four of these patients were also culture positive. The ultimate diagnosis for 13 of these patients and 1 patient who was PCR-REA negative was disseminated candidiasis (confirmed by clinical data, multiple cultures, histology, autopsy, and/or ultrasonographic evidence of hepatosplenic candidiasis). The molecular method is significantly more sensitive than conventional fungal blood cultures and has a high negative predictive value (97.5%) for the development of disseminated candidiasis in neutropenic patients.
Genetic Features of
Streptococcus agalactiae
Strains Causing Severe Neonatal Infections, as Revealed by Pulsed-Field Gel Electrophoresis and
hylB
Gene Analysis
Authors: Rolland Karine , Marois Corinne , Siquier Veronique , Cattier Blandine , Quentin Roland
Published: December 2019

A collection of 114 independent
Streptococcus agalactiae
strains, including 54 strains isolated from the cerebrospinal fluid (CSF) samples of neonates and 60 strains from asymptomatic patients, was characterized by pulsed-field gel electrophoresis (PFGE) of DNA restricted with
Sma
I and by PCR analysis of the
hylB
gene. All strains were previously studied by multilocus enzyme electrophoresis (MLEE) (R. Quentin, H. Huet, F.-S. Wang, P. Geslin, A. Goudeau, and R. K. Selander, J. Clin. Microbiol. 33:2576–2581, 1995). Among these 114 strains, there were 92 PFGE patterns. Eleven genetic groups (A to K) were identified with 38% divergence. A more homogeneous group (PFGE group A) was defined, consisting of 73% of the strains previously identified as belonging to a particular MLEE phylogenetic group. A 162-kb fragment was identified as a marker of strains that invaded the central nervous system of neonates. It was detected in 69% of the PFGE patterns obtained with CSF isolates and in only 1.8% of the PFGE patterns obtained with carrier strains. The
hylB
gene encoding hyaluronate lyase was amplified for all strains in our collection. Ten of 15 isolates belonging to an MLEE subgroup, previously described as being likely to cause invasive infection, had an insertion in the
hylB
gene (IS
1548
).
Phenotypic and Genotypic Characterization of
Mycobacterium africanum
Isolates from West Africa
Authors: Frothingham Richard , Strickland Percy L. , Bretzel Gisela , Ramaswamy Srinivas , Musser James M. , Williams Diana L.
Published: December 2019

The
Mycobacterium tuberculosis
complex includes
M. tuberculosis
,
M. bovis
,
M. africanum
, and
M. microti
. Most clinical isolates are
M. tuberculosis
or
M. bovis
. These species can be distinguished by phenotypes and genotypes. However, there is no simple definition of
M. africanum
, and some authors question the validity of this species. We analyzed 17 human isolates from Sierra Leone, identified as
M. africanum
by biochemical and growth characteristics. We sequenced polymorphic genes and intergenic regions. We amplified DNA from six loci with variable numbers of tandem repeats (VNTRs) and determined the exact number of repeats at each locus in each strain. All
M. africanum
isolates had the ancestral CTG Leu at
katG
codon 463. Drug-resistant
M. africanum
isolates had
katG
and
rpoB
mutations similar to those found in drug-resistant
M. bovis
and
M. tuberculosis
. Fourteen Sierra Leone
M. africanum
isolates (designated group A) had
katG
codon 203 ACC Thr, also found in
M. africanum
T
(the T indicates type strain) from Senegal. Group A isolates clustered with
M. africanum
T
by VNTR analysis. Three
M. africanum
isolates (group B) had
katG
codon 203 ACT Thr, found in
M. tuberculosis
T
, and clustered with
M. tuberculosis
T
by VNTR analysis. Phenotypic identification of
M. africanum
yielded a heterogeneous collection of strains. Genotypic analyses identified a cluster (
M. africanum
group A) which included
M. africanum
T
and was distinct from the rest of the
M. tuberculosis
complex. Future studies of
M. africanum
should include both phenotypic and genotypic analyses.
Use of Base Excision Sequence Scanning for Detection of Genetic Variations in St. Louis Encephalitis Virus Isolates
Authors: Charrel Rémi N. , Lévy Nicolas , Tesh Robert B. , Chandler Laura J.
Published: December 2019

Twenty-two isolates of St. Louis encephalitis (SLE) virus of various geographical origins (Brazil, Argentina, Panama, Texas, Missouri, Maryland, California, and Florida) were examined for genetic variation by the base excision sequence scanning (BESS T-scan) method. A fragment was amplified in the envelope gene with the forward primer labeled in the PCR. The BESS T-scan method determined different clusters according to the profiles generated for the isolates and successfully grouped the isolates according to their geographical origins. Two major clusters, the North American cluster (cluster A) and the South and Central American cluster (cluster B), were defined. Two subgroups, the Texas-California subgroup (subgroup A1) and the Missouri-Maryland-Florida subgroup (subgroup A2), were distinguished within group A. Similarly, group B strains were subclustered to a South American subgroup (subgroup B1) and a Central American subgroup (subgroup B2). These results were consistent with those obtained by DNA sequencing analysis. The ability of the BESS T-scan method to discriminate between strains that present with high degrees of nucleotide sequence similarity indicated that this method provides reliable results and multiple applications for other virus families. The method has proven to be suitable for phylogenetic comparison and molecular epidemiology studies and may be an alternative to DNA sequencing.
Species-Specific Identification of
Mycobacterium leprae
by PCR-Restriction Fragment Length Polymorphism Analysis of the
hsp65
Gene
Authors: Rastogi Nalin , Goh Khye Seng , Berchel Mylene
Published: December 2019

PCR-restriction fragment length polymorphism analysis (PRA) of the
hsp65
gene present in all mycobacteria was used in the present investigation to characterize
Mycobacterium leprae
. Bacilli were extracted and purified from different organs from experimentally infected armadillos and nude mice (Swiss mice of
nu/nu
origin). A total of 15 samples were assayed in duplicate, and the results were compared with those obtained for a total of 147 cultivable mycobacteria representing 34 species. Irrespective of its origin or viability,
M. leprae
strains from all the samples were uniformly characterized by two fragments of 315 and 135 bp upon
Bst
EII digestion and two fragments of 265 and 130 bp upon
Hae
III digestion. PRA is a relatively simple method and permits the conclusive identification of
M. leprae
to the species level.
Tick-Borne Relapsing Fever Imported from West Africa: Diagnosis by Quantitative Buffy Coat Analysis and In Vitro Culture of
Borrelia crocidurae
Authors: van Dam Alje P. , van Gool Tom , Wetsteyn José C. F. M. , Dankert Jacob
Published: December 2019

West African tick-borne relapsing fever (TBRF) is difficult to diagnose due to the low number of spirochetes in the bloodstream of patients. Previously, the causative microorganism,
Borrelia crocidurae
, had never been cultured in vitro. TBRF was rapidly diagnosed for two patients returning from western Africa with fever of unknown origin by quantitative buffy coat (QBC) analysis. Diagnosis was confirmed by intraperitoneal inoculation of blood specimens from patients into laboratory mice. In vitro experiments showed that QBC analysis may be as much as 100-fold more sensitive than thick smear. Spirochetes were also cultured from blood samples from both patients in modified Kelly’s medium and were identified as
B. crocidurae
by partial sequencing of the PCR-amplified
rrs
gene.
Use of Dorset Egg Medium for Maintenance and Transport of
Neisseria meningitidis
and
Haemophilus influenzae
Type b
Authors: Wasas Avril D. , Huebner Robin E. , Klugman Keith P.
Published: December 2019

Studies of bacterial meningitis are hampered by the inability to maintain the viability of etiological agents during transport to reference laboratories. The long-term survival rate of 20 isolates of
Neisseria meningitidis
and
Haemophilus influenzae
type b (Hib) on Dorset egg medium, supplemented Columbia agar base medium, chocolate agar, and Amies medium was compared with that on 70% GC agar (chocolate) transport medium.
N. meningitidis
isolates were also inoculated onto 5% horse blood agar, and Hib was inoculated onto
Haemophilus
test medium. All of the
N. meningitidis
isolates remained viable on Dorset egg medium for 21 days; viability on the other media was poor after only 7 days. Recovery rates of Hib isolates were similar on Dorset egg and
Haemophilus
test media (100% after 21 days) and significantly better than on the other media. Dorset egg medium is inexpensive and easy to make and may be invaluable for studies of bacterial meningitis in developing countries.
Disk with High Oxacillin Content Discriminates between Methicillin-Resistant and Borderline Methicillin-Susceptible
Staphylococcus aureus
Strains in Disk Diffusion Assays Using a Low Salt Concentration
Authors: Petersson Ann Cathrine , Kamme Carl , Miörner Håkan
Published: December 2019

A separation between
mecA
+
strains of
Staphylococcus aureus
and strains lacking
mecA
was achieved by the disk diffusion assay and the agar dilution method, utilizing disks containing 5 μg of oxacillin and inocula of approximately 5 × 10
5
CFU/spot, respectively, provided that agar with 0 to 0.5% NaCl and incubation at 30°C were employed. The 5-μg oxacillin disks clearly discriminated between borderline methicillin-susceptible and
mecA
+
strains. The oxacillin MICs were more affected by the inoculum density and salt concentration than were the methicillin MICs, and oxacillin MICs of 4 to 16 μg/ml were obtained for strains lacking
mecA
. Significantly higher levels of β-lactamase production and reduced oxacillin susceptibilities were recorded for strains lacking
mecA
, in particular strains of phage group V, when agar with ≥2% NaCl was used than when agar with 0 to 0.5% NaCl was employed. The results indicate that the borderline methicillin-susceptible phenotype is a salt-dependent in vitro phenomenon of questionable clinical relevance.
Infection of Laboratory Mice with the Human Granulocytic Ehrlichiosis Agent Does Not Induce Antibodies to Diagnostically Significant
Borrelia burgdorferi
Antigens
Authors: Bunnell Joseph E. , Magnarelli Louis A. , Dumler J. Stephen
Published: December 2019

Laboratory diagnosis of
Borrelia burgdorferi
is routinely made by an enzyme-linked immunosorbent assay, with positive results confirmed by Western blot analysis. Concern has been raised that false-positive diagnoses may be made on the basis of serologic cross-reactivity with antibodies directed against other bacterial pathogens, in particular the agent of human granulocytic ehrlichiosis (HGE). The present study made use of a mouse model to ascertain the validity of these concerns. Two different strains of mice were inoculated with the HGE agent and assayed for production of polyclonal and monoclonal antibodies to antigens of both of these bacteria. Infection of mice with the HGE agent does not induce diagnostically significant
B. burgdorferi
serologic cross-reactions.
Isolation of DNA after Extraction of RNA To Detect the Presence of
Borrelia burgdorferi
and Expression of Host Cellular Genes from the Same Tissue Sample
Authors: Amemiya Kei , Schaefer Henry , Pachner Andrew R.
Published: December 2019

We are investigating the neuropathogenesis of Lyme disease caused by
Borrelia burgdorferi
in a nonhuman primate model. In the past, two separate pieces of tissue had to be used when both analyzing for the presence of the spirochete and examining the host response to infection. We have modified a procedure to purify DNA from the same sample after the extraction of RNA. The remaining material containing the DNA was precipitated, and residual organic reagent was removed prior to deproteinization and extraction of the DNA. This procedure now allows us to both assay for the presence of the Lyme microorganism and analyze the host response in the same tissue preparation.
Detection of Clinically Relevant Genotypes of Vancomycin-Resistant Enterococci in Nosocomial Surveillance Specimens by PCR
Authors: Jayaratne P. , Rutherford C.
Published: December 2019

This study evaluated a PCR method for the rapid detection of clinically significant genotypes of vancomycin-resistant enterococci (VRE) in nosocomial surveillance specimens. Detection of the
vanA
and
vanB
genes by multiplex PCR using 657 specimens that showed presumptive growth of VRE on bile esculin azide agar containing 6 mg of vancomycin/liter was compared to the conventional method. The diagnostic values for the PCR compared to the phenotypic method were as follows: 99.8% specificity, 95.4% sensitivity, 98.8% positive predictive value, and 99.3% negative predictive value. The average cost per test for PCR is $8.26, compared to $9.45 for the phenotypic method. The average turnaround time for detecting a VRE is 48 h for PCR, compared to 96 h for the conventional method.
Subtyping of
Haemophilus influenzae
Strains by Pulsed-Field Gel Electrophoresis
Authors: Saito Mitsumasa , Umeda Akiko , Yoshida Shin-ichi
Published: December 2019

A total of 200 isolates of
Haemophilus influenzae
were analyzed by serotyping, biotyping, and pulsed-field gel electrophoresis (PFGE). A total of 178 epidemiologically unrelated strains of
H. influenzae
demonstrated a variety of genome patterns by PFGE, and 165 genotypes were thus obtained in this study. PFGE typing proved to have a much stronger discriminatory power than either serotyping or biotyping. Six serotype b strains were all classified into discrete genotypes. A PFGE analysis of 18 strains obtained from the nasopharynx, blood, and cerebrospinal fluid of patients with meningitis also supported the hypothesis that invasive
H. influenzae
disseminates from the nasopharynx to the bloodstream and then subsequently to other body sites. PFGE typing of 10 other strains isolated from household contacts of patients with
H. influenzae
infection revealed that the strain that caused the
H. influenzae
infection often colonized the nasopharynges of household contacts. Our findings suggest that PFGE analysis is useful for the epidemiological study of
H. influenzae
infection, even when the invasive disease is caused by serotype b strains.
Most
Enterobacter aerogenes
Strains in France Belong to a Prevalent Clone
Authors: Bosi Claude , Davin-Regli Anne , Bornet Charleric , Mallea Monique , Pages Jean-Marie , Bollet Claude
Published: December 2019

The aim of this study was to determine the distribution in France of the
Enterobacter aerogenes
prevalent clone isolated in the hospitals of the Marseille area (A. Davin-Regli, D. Monnet, P. Saux, C. Bosi, R. Charrel, A. Barthelemy, and C. Bollet, J. Clin. Microbiol. 34:1474–1480, 1996). A total of 123
E. aerogenes
isolates were collected from 23 hospital laboratories and analyzed by random amplification of polymorphic DNA and enterobacterial repetitive intergenic consensus-PCR to determine their epidemiological relatedness. Molecular typing revealed that 21 of the 23 laboratories had isolated this prevalent clone harboring the plasmid encoding for extended-spectrum β-lactamase of the TEM-24 type. Most isolates were susceptible only to imipenem and gentamicin. Their dissemination seems to be clonal and was probably the result of the general use of broad-spectrum cephalosporins and quinolones. Four isolates showed an alteration of their outer membrane proteins, causing decrease of susceptibility to third-generation cephalosporins and imipenem and leading to the critical situation of having no alternative therapeutic. The large dissemination of the
E. aerogenes
prevalent clone probably results from its good adaptation to the antibiotics administered in France and the hospital environment, particularly in intensive care units.
Evaluation of the Discriminatory Power of Typing Methods forNeisseria gonorrhoeae
Authors: Van Looveren M. , Ison C. A. , Ieven M. , Vandamme P. , Martin I. M. , Vermeulen K. , Renton A. , Goossens H.
Published: December 2019

A panel of 18 strains ofNeisseria gonorrhoeae, known to be temporally and geographically diverse, was used to evaluate a number of typing systems, including conventional auxotyping and serotyping and the molecular methods of arbitrarily primed PCR (AP-PCR), amplified ribosomal-DNA restriction analysis (ARDRA),opatyping, and pulsed-field gel electrophoresis (PFGE). The discriminatory power of the different typing methods were determined with a collection of 87 clinical isolates from commercial sex workers in Indonesia, and Simpson’s index of diversity was calculated. Of the two traditional techniques, auxotyping and serotyping, the latter gives the highest discriminatory index (DI) (DI, 0.846). The combination of auxotyping and serotyping yields a high DI (DI, 0.928). D11344- and D8635-primed PCR showed low DIs of 0.608 and 0.622, respectively, but a combination of the two primers had a DI of 0.849. The combination of serotyping with D11344-primed or D8635-primed PCR resulted in DIs of 0.936 and 0.937, respectively. ARDRA revealed a low DI of 0.743 alone but a DI of 0.955 in combination with serotyping. PFGE using the restriction enzymeBglII andopatyping produced the highest discrimination (DIs, 0.997 and 0.996, respectively) for isolates ofN. gonorrhoeae.
Multiple Types of
Legionella pneumophila
Serogroup 6 in a Hospital Heated-Water System Associated with Sporadic Infections
Authors: Visca Paolo , Goldoni Paola , Lück P. Christian , Helbig Jürgen H. , Cattani Lorena , Giltri Giuseppe , Bramati Simone , Pastoris Maddalena Castellani
Published: December 2019

Five sporadic cases of nosocomial Legionnaires’ disease were documented from 1989 to 1997 in a hospital in northern Italy. Two of them, which occurred in a 75-year-old man suffering from ischemic cardiopathy and in an 8-year-old girl suffering from acute leukemia, had fatal outcomes.
Legionella pneumophila
serogroup 6 was isolated from both patients and from hot-water samples taken at different sites in the hospital. These facts led us to consider the possibility that a single clone of
L. pneumophila
serogroup 6 had persisted in the hospital environment for 8 years and had caused sporadic infections. Comparison of clinical and environmental strains by monoclonal subtyping, macrorestriction analysis (MRA), and arbitrarily primed PCR (AP-PCR) showed that the strains were clustered into three different epidemiological types, of which only two types caused infection. An excellent correspondence between the MRA and AP-PCR results was observed, with both techniques having high discriminatory powers. However, it was not possible to differentiate the isolates by means of ribotyping and analysis of
rrn
operon polymorphism. Environmental strains that antigenically and chromosomally matched the infecting organism were present at the time of infection in hot-water samples taken from the ward where the patients had stayed. Interpretation of the temporal sequence of events on the basis of the typing results for clinical and environmental isolates enabled the identification of the ward where the patients became infected and the modes of transmission of
Legionella
infection. The long-term persistence in the hot-water system of different clones of
L. pneumophila
serogroup 6 indicates that repeated heat-based control measures were ineffective in eradicating the organism.
Fluconazole Susceptibilities of Bloodstream
Candida
sp. Isolates as Determined by National Committee for Clinical Laboratory Standards Method M27-A and Two Other Methods
Authors: Cantón Emilia , Pemán Javier , Carrillo-Muñoz Alfonso , Orero Ana , Ubeda Pedro , Viudes Angel , Gobernado Miguel
Published: December 2019

The in vitro activity of fluconazole against 143
Candida
spp. obtained from the bloodstreams of 143 hospitalized patients from 1995 to 1997 was studied. Susceptibility tests were carried out by two macrodilution methods, the M27-A and a modified M27-A method (0.165 M, pH 7/morpholinepropanesulfonic acid-buffered RPMI 1640 medium supplemented with 20 g of
d
-dextrose per liter), and by the agar diffusion method (with 15-μg fluconazole [Neo-Sensitab] tablets). With 2 μg of fluconazole per ml, 96.92% of 65
C. albicans
isolates, 86.2% of 58
C. parapsilosis
isolates 7 of 8
C. tropicalis
isolates, and 1 of 6
C. glabrata
isolates were inhibited. Only one strain of
C. albicans
and one strain of
C. tropicalis
were resistant. The agreement between the two macrodilution methods was greater than 90% within ±2 log
2
dilutions for all strains except
C. glabrata
(83.3%) and
C. tropicalis
(87.5%). Generally, MICs were 1 log
2
dilution lower in glucose-supplemented RPMI 1640 medium. No correlation between zone sizes and MICs was found. All strains susceptible by the diffusion test were susceptible by the dilution method, but the converse was not necessarily true. Interestingly, inhibition zones were smaller for
C. albicans
, for which the geometric mean MIC was 0.29 μg/ml and the mean inhibition zone diameter was 25.7 mm, while for
C. parapsilosis
the geometric mean MIC was 0.96 μg/ml and the mean inhibition zone diameter was 31.52 mm. In conclusion, the two macrodilution methods give similar results. The modified M27-A method with 2% dextrose has the advantage of shortening the incubation time and simplifying the endpoint determination.
Validity of Interpretation Criteria for Standardized Western Blots (Immunoblots) for Serodiagnosis of Lyme Borreliosis Based on Sera Collected throughout Europe
Authors: Hauser Ulrike , Lehnert Gisela , Wilske Bettina
Published: December 2019

Western blotting (WB; immunoblotting) is a widely used tool for the serodiagnosis of Lyme borreliosis (LB), but so far, no generally accepted criteria for performance and interpretation have been established in Europe. The current study was preceeded by a detailed analysis of WB with whole-cell lysates of three species ofBorrelia burgdorferisensu lato (U. Hauser, G. Lehnert, R. Lobentanzer, and B. Wilske, J. Clin. Microbiol. 35:1433–1444, 1997). In that study, interpretation criteria for a positive WB result were developed with the data for 330 serum samples (from patients with LB in different stages [n= 189] and from a control group [n= 141]) originating mostly from southern Germany. In the present work, the interpretation criteria for strains PKo (Borrelia afzelii) and PBi (Borrelia garinii) developed in the previous study were reevaluated with 224 serum samples (from patients with LB in different stages [n= 97] and from a control group [n= 127]) originating from throughout Europe that were provided by the European Union Concerted Action on Lyme Borreliosis (EUCALB). De novo criteria were developed on the basis of the reactivities of the EUCALB sera and were evaluated with the data for the samples from southern Germany. Comparison of all results led to the following recommendations: For WB for immunoglobulin G (IgG), at least two bands among p83/100, p58, p43, p39, p30, OspC, p21, p17, and p14 for PKo and at least one band among p83/100, p39, p30, OspC, p21, and p17b for PBi; for WB for IgM, at least one band among p39, OspC, and p17 or a strong p41 band for PKo and at least one band among p39 and OspC or a strong p41 band for PBi. WB with PKo was the most sensitive, and this strain is recommended for use in WB for the serodiagnosis of LB throughout Europe.
Delayed versus Immediate Bedside Inoculation of Culture Media for Diagnosis of Vaginal Trichomonosis
Authors: Schwebke Jane R. , Venglarik Moira F. , Morgan Shellie C.
Published: December 2019

A comparison of delayed versus immediate inoculation of culture medium for the diagnosis of trichomonosis was conducted. The sensitivities of the two methods were 100 and 97.4%, respectively. Delayed inoculation of culture medium for women without evidence of trichomonosis on direct microscopic examination is a valid diagnostic procedure.
Cleavase Fragment Length Polymorphism Analysis of
Neisseria meningitidis
Basic Metabolic Genes
Authors: Tondella Maria Lucia C. , Reeves Mike W. , Popovic Tanja , Rosenstein Nancy , Holloway Brian P. , Mayer Leonard W.
Published: December 2019

Cleavase fragment length polymorphism (CFLP) is a subtyping system based on the property of the enzyme cleavase to recognize junctions between single- and double-stranded regions of DNA formed after denaturation and cooling. To assess the capacity of CFLP for discriminating
Neisseria meningitidis
serogroup B strains belonging to the electrophoretic type (ET) 5 (ET-5) complex from other serogroup B strains, 30 serogroup B
N. meningitidis
isolates were subtyped by CFLP with internal fragments of five housekeeping genes,
adk
,
aspC
,
carA
,
dhp
, and
glnA
. Two genes (
glnA
and
carA
) which demonstrated a high degree of diversity for the serogroup B isolates were then used to further evaluate the suitability of CFLP for screening 50 serogroup C
N. meningitidis
outbreak-associated and sporadic-case isolates with a single metabolic gene. The results were compared to those from multilocus enzyme electrophoresis (MEE), the current standard subtyping method. CFLP was able to distinguish the ET-5 complex isolates from other serogroup B isolates as efficiently as MEE. Furthermore, CFLP analysis of a single gene was sufficient to identify and cluster the serogroup C isolates belonging to the ET-37 complex from other, unrelated serogroup C isolates but was not capable of differentiating between the isolates of the major individual ETs of this complex (ET-17 and ET-24) causing most serogroup C meningococcal disease outbreaks in the United States. CFLP based on a single gene with a high degree of diversity but not under selective pressure can be applied to the rapid screening of a large number of isolates related to the recognized epidemic complex ET-5 or ET-37. Additionally, CFLP can be used as an initial screening tool to survey the amount of diversity in genes that might be used to develop a DNA sequence-based subtyping system.
Utility of Multiple-Stool-Specimen Ova and Parasite Examinations in a High-Prevalence Setting
Authors: Cartwright Charles P.
Published: December 2019

A retrospective analysis of the results of 2,704 ova and parasite (O & P) examinations performed on stool specimens collected from 1,374 patients between October 1996 and September 1997 was performed to evaluate the utility of performing O & P examinations on multiple, independently collected stool specimens in a high-prevalence setting. A total of 995 specimens (36.8%) examined during the study contained parasites; 546 (20.2%) contained pathogenic organisms. The positivity rate (54.5%) for the patients from whom three specimens were examined was significantly higher than for the patients from whom either two specimens (33.3%) or a single specimen (19.8%) was submitted for examination. For the group of patients from whom at least 3 specimens were submitted for O & P examination, 373 independent opportunities for diagnosing infection with intestinal parasites could be analyzed. The first stool specimen collected proved to be adequate in only 75.9% (283 of 373) of evaluated cases; however, examination of two specimens increased the sensitivity of O & P detection to 92% (343 of 373). The third specimen collected provided additional information on only 30 of 373 occasions (8%). These data indicate that in populations with a high prevalence of intestinal parasitic infections, two independently collected stool specimens should be subjected to O & P examination to ensure adequate diagnostic sensitivity.
Molecular Typing of Multiple-Antibiotic-Resistant
Salmonella enterica
Serovar Typhi from Vietnam: Application to Acute and Relapse Cases of Typhoid Fever
Authors: Wain John , Hien Tran T. , Connerton Phillippa , Ali Tahir , M. Parry Christopher , Chinh Nguyen T. T. , Vinh Ha , Phuong Cao X. T. , Ho Vo A. , Diep To S. , Farrar Jeremy J. , White Nicholas J. , Dougan Gordon
Published: December 2019

The rate of multiple-antibiotic resistance is increasing among
Salmonella enterica
serovar Typhi strains in Southeast Asia. Pulsed-field gel electrophoresis (PFGE) and other typing methods were used to analyze drug-resistant and -susceptible organisms isolated from patients with typhoid fever in several districts in southern Vietnam. Multiple PFGE and phage typing patterns were detected, although individual patients were infected with strains of a single type. The PFGE patterns were stable when the
S. enterica
serovar Typhi strains were passaged many times in vitro on laboratory medium. Paired
S. enterica
serovar Typhi isolates recovered from the blood and bone marrow of individual patients exhibited similar PFGE patterns. Typing of
S. enterica
serovar Typhi isolates from patients with relapses of typhoid indicated that the majority of relapses were caused by the same
S. enterica
serovar Typhi strain that was isolated during the initial infection. However, some individuals were infected with distinct and presumably newly acquired
S. enterica
serovar Typhi isolates.
Detection of Bovine Herpesvirus Type 1 in Blood from Naturally Infected Cattle by Using a Sensitive PCR That Discriminates between Wild-Type Virus and Virus Lacking Glycoprotein E
Authors: Fuchs Monika , Hübert Peter , Detterer Jan , Rziha Hanns-Joachim
Published: December 2019

In the present study, we report for the first time on the detection of bovine herpesvirus type 1 (BHV-1) in whole-blood samples derived from naturally infected cattle. Sensitive PCR assays specific for glycoprotein B (gB), gC, and gE of BHV-1 allow the detection of one BHV-1 DNA copy in 105to 107peripheral blood leukocytes (PBLs). The incidence of BHV-1-positive PBLs in naturally infected cattle appears to be quite high (92.2% positive PBLs among all samples tested), although in most cases only between 10−5and 10−7positive leukocytes were present. The results demonstrate that the viral DNA is detectable not only in the peripheral blood of acutely infected animals but, more importantly, also in the peripheral blood of subclinically infected cattle. The gE-specific PCR described in the report allows discrimination between wild-type (WT) virus-infected and vaccinated animals, which is of importance for control programs that use the recently introduced vaccination strategy with a gE-negative virus. The results further show that doubtful serological results can be verified or falsified and that individual animals can be monitored for the presence or absence of WT BHV-1 or gE-negative virus in cattle herds. The PCR protocols allow the detection of BHV-1 prior to seroconversion or in BHV-1-seronegative cattle. Finally, the results indicate the simultaneous presence of WT and gE-negative vaccine virus in the PBLs of several cattle. Therefore, investigations of viremia in naturally and experimentally infected cattle and on the identification of infected cell types of bovine PBLs can be now performed.
Genotype Dependence of Hepatitis C Virus Load Measurement in Commercially Available Quantitative Assays
Authors: Mellor Janet , Hawkins Anna , Simmonds Peter
Published: December 2019

Standardization and genotype independence of methods used to quantify hepatitis C virus (HCV) RNA in clinical specimens are necessary for accurate assessment of the role of HCV quantitation as a prognostic marker for HCV infection and monitoring of the response to antiviral treatment. Commercially available methods used to measure HCV loads include PCR-based (Roche Monitor) and hybridization-based (Quantiplex bDNA-2) methods. Recently, a new version of the Roche Monitor assay (version 2.0) has become available; it has been modified to achieve more equal quantitation of different HCV genotypes. Consistent with previous reports, Roche Monitor version 1.0 substantially underestimated concentrations of RNA transcripts of types 2b, 3a, 4a, 5a, and 6a and virus loads in individuals infected with genotypes 2 to 6 relative to reference tests. However, version 2.0 achieved equivalent quantitation of each genotype over a narrow quantitative range (10
3
to 5 × 10
5
copies of RNA/ml) but significantly underestimated RNA concentrations above this range. The assay showed an equivalent inability to quantify high levels of HCV RNA in plasma samples, and this was responsible for the falsely narrow range of virus loads detected in HCV-infected individuals. In contrast, the Chiron bDNA-2 assay could only measure RNA concentrations in the upper quantitative range (2 × 10
5
to 5 × 10
7
copies of RNA/ml) but showed equivalent sensitivity for genotypes 1 to 5; however, concentrations of type 6a RNA transcripts and virus loads in clinical specimens from individuals infected with type 6a were underestimated by a factor of 2 to 4. Differences were observed between PCR- and hybridization-based assays in their relative quantitation of HCV RNA transcripts and HCV genomic RNA, which may cause problems with the use of transcripts for interassay calibration.
Inhibition Enzyme-Linked Immunosorbent Assay for Serotyping of Group B Streptococcal Isolates
Authors: Arakere Gayathri , Flores Aurea E. , Ferrieri Patricia , Frasch Carl E.
Published: December 2019

Group B Streptococcus (GBS) is one of the most common organisms causing neonatal sepsis as well as serious infections in adults. Serotyping the organism is important in studying the epidemiology of the disease as well as deciding a course of treatment. There are several methods available for serotyping. Most of them need high-titered sera and are not quantitative. We are reporting a new inhibition enzyme-linked immunosorbent assay (ELISA) for serotyping which is sensitive and specific compared to the conventional methods but does not need high-titered serotype-specific antisera, as the specificity is controlled by the polysaccharide coating on the ELISA plates. The method can also be quantitative, and we have measured polysaccharide elaborated by different serotype V strains. Thus, the inhibition ELISA method will be useful in serotyping for epidemiological studies, assessing virulence, and performing strain selection for vaccine production.
Fluorescent Oligonucleotide Probes for Clinical and Environmental Detection of
Acanthamoeba
and the T4 18S rRNA Gene Sequence Type
Authors: Stothard Diane R. , Hay John , Schroeder-Diedrich Jill M. , Seal David V. , Byers Thomas J.
Published: December 2019

The first genus- and subgenus-specific fluorescent oligonucleotide probes for in situ staining of
Acanthamoeba
are described. Sequences of these phylogeny-based probes complement the 18S rRNA and the gene encoding it (18S rDNA). The genus-specific probe (GSP) is a fluorescein-labeled 22-mer specific for
Acanthamoeba
as shown here by its hybridization to growing trophozoites of all 12 known
Acanthamoeba
18S rDNA sequence types and by its failure to hybridize with amoebae of two other genera (
Hartmannella vermiformis
and
Balamuthia mandrillaris
), two human cell lines, and two bacteria (
Pseudomonas aeruginosa
and
Escherichia coli
). The sequence type T4-specific probe (ST4P) is a rhodamine-labeled 30-mer specific for
Acanthamoeba
18S rDNA sequence type T4, as shown here in hybridization tests with trophozoites of all 12 sequence types. T4 is the subgenus group associated most closely with
Acanthamoeba
keratitis (AK). GSP also was tested with corneal scrapings from 17 patients with a high index of clinical suspicion of AK plus 5 patient controls. GSP stained both trophozoites and cysts, although nonspecific cyst wall autofluorescence also was observed. Results could be obtained with GSP in 1 to 2 days, and based on results from cell culture tests, the probe correctly detected the presence or absence of
Acanthamoeba
in 21 of 24 specimens from the 22 patients. The use of GSP with cultured trophozoites and cysts from corneal scrapings has illustrated the suitability of using fluorescent oligonucleotide probes for identification of the genus
Acanthamoeba
in both environmental and clinical samples. In addition, the use of ST4P with cultured amoebae has indicated the potential of oligonucleotide probes for use in subgenus classification.
Comparative Performance of the RapID Yeast Plus System and the API 20C AUX Clinical Yeast System
Authors: Smith Michael B. , Dunklee Daisy , Vu Hangna , Woods Gail L.
Published: December 2019

The performance of the RapID Yeast Plus System (Innovative Diagnostic Systems, Norcross, Ga.), a 4-h micropanel using single-substrate enzymatic test reactions, was compared with that of the API 20C AUX Clinical Yeast System (bioMerieux Vitek, Hazelwood, Mo.), a 48- to 72-h carbohydrate assimilation panel. Two hundred twenty-five yeasts, yeast-like fungi, and algae, comprising 28 species and including 30 isolates of
Cryptococcus neoformans
, an important pathogen not tested in appreciable numbers in other comparisons, were tested by both methods. On initial testing, 196 (87.1%) and 215 (95.6%) isolates were correctly identified by the RapID and API systems, respectively. Upon repeat testing, the number of correctly identified isolates increased to 220 (97.8%) for the RapID system and 223 (99.1%) for the API system. Reducing the turbidity of the test inoculum to that of a no. 3 McFarland turbidity standard, which is below that recommended by the manufacturer, resulted in the correct identification of most of the isolates initially misidentified by the RapID system, including 10 of 30
C. neoformans
isolates. Concordance between the RapID and API results after repeat testing was 97.3%.
Molecular Detection and Identification of Intimin Alleles in Pathogenic
Escherichia coli
by Multiplex PCR
Authors: Reid Sean D. , Betting David J. , Whittam Thomas S.
Published: December 2019

A multiplex PCR was designed to detect the
eae
gene and simultaneously identify specific alleles in pathogenic
Escherichia coli
. The method was tested on 87 strains representing the diarrheagenic
E. coli
clones. The results show that the PCR assay accurately detects
eae
and resolves alleles encoding the α, β, and γ intimin variants.
Optimizing Voriconazole Susceptibility Testing of
Candida
: Effects of Incubation Time, Endpoint Rule, Species of
Candida
, and Level of Fluconazole Susceptibility
Authors: Lozano-Chiu M. , Arikan S. , Paetznick V. L. , Anaissie E. J. , Rex J. H.
Published: December 2019

Voriconazole is a new triazole antifungal agent that has potent activity against many isolates of
Candida
, including
Candida krusei
and
Candida glabrata
. In this work, we studied the impact of glucose supplementation, incubation time, agitation of the plates prior to reading, endpoint determination rule, visual versus spectrophotometric reading,
Candida
species, and fluconazole MIC on the MIC of voriconazole for
Candida
isolates tested by using the microdilution format assay of the National Committee for Clinical Laboratory Standards (NCCLS) M27-A antifungal susceptibility testing methodology. For both voriconazole and fluconazole, a spectrophotometric endpoint of 50% reduction in turbidity relative to the growth control correlated most closely with the NCCLS-defined visual endpoint of “prominent decrease in turbidity.” Correlation was generally better after 24 h of incubation than after 48 h. Supplementation of the medium to contain 20 g of glucose/liter did not alter the MIC significantly but did enhance growth and simplify visual readings. All
Candida
species appeared potentially susceptible to voriconazole, including isolates of
C. krusei
. For some isolates for which fluconazole MICs were markedly elevated voriconazole MICs were also elevated, but the clinical significance of these observations remains to be determined.