bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes for Differentiation between Tuberculous and Nontuberculous Mycobacterium Species in Smears of Mycobacterium Cultures
Authors: Stender Henrik , Lund Kaare , Petersen Kenneth H. , Rasmussen Ole F. , Hongmanee Poonpilas , Miörner Håkan , Godtfredsen Sven E.
Published: December 2019

TB PNA FISH is a new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for differentiation between species of the
Mycobacterium tuberculosis
complex (MTC) and nontuberculous mycobacteria (NTM) in acid-fast bacillus-positive (AFB+) cultures is described. The test is based on fluorescein-labelled PNA probes that target the rRNA of MTC or NTM species applied to smears of AFB+ cultures for microscopic examination. Parallel testing with the two probes serves as an internal control for each sample such that a valid test result is based on one positive and one negative reaction. TB PNA FISH was evaluated with 30 AFB+ cultures from Denmark and 42 AFB+ cultures from Thailand. The MTC-specific PNA probe showed diagnostic sensitivities of 84 and 97%, respectively, and a diagnostic specificity of 100% in both studies, whereas the NTM-specific PNA probe showed diagnostic sensitivities of 91 and 64%, respectively, and a diagnostic specificity of 100% in both studies. The low sensitivity of the NTM-specific PNA probe in the Thai study was due to a relatively high prevalence of
Mycobacterium fortuitum
, which is not identified by the probe. In total, 63 (87%) of the cultures were correctly identified as MTC (
n
= 46) or NTM (
n
= 17), whereas the remaining 9 were negative with both probes and thus the results were inconclusive. None of the samples were incorrectly identified as MTC or NTM; thus, the predictive value of a valid test result obtained with TB PNA FISH was 100%.
Rapid Detection of Human Rhinoviruses in Nasopharyngeal Aspirates by a Microwell Reverse Transcription-PCR–Hybridization Assay
Authors: Blomqvist S. , Skyttä A. , Roivainen M. , Hovi T.
Published: December 2019

A rapid and sensitive microwell reverse transcription (RT)-PCR–hybridization assay was developed to detect human rhinoviruses in clinical specimens and cell culture suspensions. Two hundred three nasopharyngeal aspirates collected from children with symptoms of respiratory disease were analyzed by a classical rolling-tube cell culture method, microwell culture of HeLa Ohio cell monolayers, and RT-PCR with detection of the amplicons in a microwell hybridization assay. The RT-PCR was also done with harvests of the microwell cultures. RNA was extracted with a commercial kit, and the RT-PCR procedure was carried out with microtiter-format equipment. A confirmatory test that exploited a blocking oligonucleotide at the hybridization step was developed to reliably identify marginally positive specimens. Of the 203 nasopharyngeal aspirate specimens, rhinovirus or rhinoviral RNA was detected in 111 specimens (55%). Ninety-eight specimens (48%) were found to be positive by RT-PCR of the original nasopharyngeal aspirates, while the conventional rolling-tube cell culture method yielded 52 (26%) positive specimens. This RT-PCR method with solid-phase hybridization is easy to perform, sensitive, and specific and will be especially useful for analysis of large numbers of clinical specimens.
Elucidating the Origins of Nosocomial Infections with
Candida albicans
by DNA Fingerprinting with the Complex Probe Ca3
Authors: Marco F. , Lockhart S. R. , Pfaller M. A. , Pujol C. , Rangel-Frausto M. S. , Wiblin T. , Blumberg H. M. , Edwards J. E. , Jarvis W. , Saiman L. , Patterson J. E. , Rinaldi M. G. , Wenzel R. P. ,   The Nemis Study Group, Soll D. R.
Published: December 2019

Computer-assisted DNA fingerprinting with the complex probe Ca3 has been used to analyze the relatedness of isolates collected from individuals with nosocomial bloodstream infections (BSIs) and hospital care workers (HCWs) in the surgical and neonatal intensive care units (ICUs) of four hospitals. The results demonstrate that for the majority of patients (90%), isolates collected from commensal sites before and after collection of a BSI isolate were highly similar or identical to the BSI isolate. In addition, the average similarity coefficient for BSI isolates was similar to that for unrelated control isolates. However, the cluster characteristics of BSI isolates in dendrograms generated for each hospital compared to those of unrelated control isolates in a dendrogram demonstrated a higher degree of clustering of the former. In addition, a higher degree of clustering was observed in mixed dendrograms for HCV isolates and BSI isolates for each of the four test hospitals. In most cases, HCW isolates from an ICU were collected after the related BSI isolate, but in a few cases, the reverse was true. Although the results demonstrate that single, dominant endemic strains are not responsible for nosocomial BSIs in neonatal ICUs and surgical ICUs, they suggest that multiple endemic strains may be responsible for a significant number of cases. The results also suggest that cross-contamination occurs between patients and HCWs and between HCWs in the same ICU and in different ICUs. The temporal sequence of isolation also suggests that in the majority of cases HCWs are contaminated by isolates from colonized patients, but in a significant minority, the reverse is true. The results of this study provide the framework for a strategy for more definitive testing of the origins of
Candida albicans
strains responsible for nosocomial infections.
Epidemiology of Feline Foamy Virus and Feline Immunodeficiency Virus Infections in Domestic and Feral Cats: a Seroepidemiological Study
Authors: Winkler I. G. , Löchelt M. , Flower R. L. P.
Published: December 2019

Although foamy viruses (Spumaviruses) have repeatedly been isolated from both healthy and diseased cats, cattle, and primates, the primary mode of transmission of those common viruses remains undefined. A database of the feline foamy virus (FeFV) and feline immunodeficiency virus (FIV) antibody status, age, and sex of 389 domestic cats presented to veterinarians was assembled. A similar database for 66 feral (wild) cats was also assembled. That FeFV antibody status reflects infection was validated by PCR. Both FeFV and FIV infection rates were found to gradually increase with age, and over 70% of cats older than 9 years were seropositive for FeFV. In domestic cats, the prevalence of FeFV infection was similar in both sexes. In feral cats, FeFV infection was more prevalent in female cats than in male cats. Although both FeFV and FIV have been reported to be transmitted by biting, the patterns of infection observed are more consistent with an interpretation that transmission of these two retroviruses is not the same. The prevalence of FIV infection is highest in nondesexed male cats, the animals most likely to display aggressive behavior. The gradual increase in the proportion of FeFV-infected animals is consistent with transmission of foamy viruses by intimate social contact between animals and less commonly by aggressive behavior.
Monitoring of Epstein-Barr Virus DNA Load in Peripheral Blood by Quantitative Competitive PCR
Authors: Stevens Servi J. C. , Vervoort Marcel B. H. J. , van den Brule Adriaan J. C. , Meenhorst Pieter L. , Meijer Chris J. L. M. , Middeldorp Jaap M.
Published: December 2019

A competitive quantitative PCR (Q-PCR) assay combined with simple silica-based DNA extraction was developed for monitoring of Epstein-Barr virus (EBV) DNA load in unfractionated peripheral blood. The Q-PCR is based on competitive coamplification of a highly conserved 213-bp region of the EBNA-1 open reading frame with an internal standard (IS), added in a known concentration. The IS has the same amplicon length and base composition as the wild-type (WT) EBNA-1 amplicon but differs in 23 internally randomized bases. Competitive coamplification yields two PCR products that are quantified by enzyme immunoassay or by electrochemiluminescence detection, with probes specific for the 23 differing internal nucleotides. The Q-PCR has a sensitivity of 10 copies of either WT or IS plasmid DNA. The Q-PCR was validated by quantification of known amounts of plasmid containing the WT EBNA-1 target. Furthermore, we determined EBV genome copy numbers in different cell lines. For EBV quantification in clinical samples, DNA was isolated from lysed whole blood by silica-affinity purification. Forty-six percent of healthy donor peripheral blood samples were positive by Q-PCR. In most of these samples, viral load was less than 2,000 EBV copies/ml of blood. In peripheral blood samples from two AIDS-related non-Hodgkin’s lymphoma patients, elevated EBV loads (up to 120,000 copies/ml) were observed, which decreased upon therapy. In Burkitt’s lymphoma patients, up to 4,592,000 EBV genome copies/ml of blood were detected. In conclusion, the EBNA-1-based Q-PCR assay provides a reproducible, accurate, and easy method for studying the relationship between EBV load and clinical parameters.
Detection of
Leishmania infantum
in Dogs by PCR with Lymph Node Aspirates and Blood
Authors: Reale S. , Maxia L. , Vitale F. , Glorioso N. S. , Caracappa S. , Vesco G.
Published: December 2019

The PCR technique was applied to the diagnosis of leishmaniasis in dogs, both serologically negative and positive. DNA was taken from lymph node aspirates and blood. The primers 13a and 13b, derived from
Leishmania amazonies
and
Leishmania braziliensis
kinetoplast DNA (kDNA), also amplified
Leishmania infantum
IPT1 constant region of minicircle kDNA. The amplified fragment is 116 bp long. It was cloned and the sequence was determined. A 70-bp inner fragment was designed and used as a probe in dot blot hybridization. A group of 124 dogs was examined, 37 of which showed typical symptoms of disease. PCR was performed on 124 blood samples and 52 lymph node aspirates. Using microscopic examination as the “gold standard,” we calculated sensitivity, specificity, and positive and negative predictive values of 100% using lymph node aspirates and values of 85, 80, 95, and 57%, respectively, using blood samples. We found that 40% of the animals without lesions and 38% of the animals with clinical signs gave false-negative results by indirect immunofluorescence antibody testing. These animals could contribute to the spreading of infection among dogs, and represent a potential risk for human health as well.
Comparison of Susceptibility Testing Methods with
mecA
Gene Analysis for Determining Oxacillin (Methicillin) Resistance in Clinical Isolates of
Staphylococcus aureus
and Coagulase-Negative
Staphylococcus
spp
Authors: Kohner P. , Uhl J. , Kolbert C. , Persing D. , Cockerill F.
Published: December 2019

Ninety-nine clinical staphylococcal isolates (58 coagulase-negative
Staphylococcus
spp. [CoNS] and 41
Staphylococcus aureus
isolates) were evaluated for susceptibility to oxacillin. The following susceptibility testing methods, media, and incubation conditions were studied: agar dilution by using Mueller-Hinton (MH) medium (Difco) supplemented with either 0, 2, or 4% NaCl and incubation at 30 or 35°C in ambient air for 24 or 48 h; disk diffusion by using commercially prepared MH medium (Difco) and MH II agar (BBL) and incubation at 35°C in ambient air for 24 or 48 h; and agar screen (spot or swab inoculation) by using commercially prepared agar (Remel) or MH agar (Difco) prepared in-house, each containing 4% NaCl and 6 μg of oxacillin/ml (0.6-μg/ml oxacillin was also studied with MH agar prepared in-house for the agar swab method and CoNS isolates) and incubation at 35°C in ambient air for 24 or 48 h for swab inoculation and at 30 or 35°C in ambient air for 24 or 48 h for spot inoculation. The results for these methods were compared to the results for
mecA
gene detection by a PCR method. Given the ability to support growth and the results for susceptibility testing (the breakpoint for susceptible isolates was ≤2 μg/ml), the best methods for CoNS isolates were (i) agar dilution by using MH medium supplemented with 4% NaCl and incubation at 35°C for 48 h (no growth failures were noted, and sensitivity was 97.6%) and (ii) agar screen (swab inoculation) by using MH medium prepared in-house supplemented with 4% NaCl and containing 0.6 μg oxacillin/ml and incubation at 35°C for 48 h (one isolate that did not carry the
mecA
gene did not grow, and the sensitivity was 100%). All but one (agar dilution without added NaCl and incubation at 30°C for 48 h) of the methods tested revealed all oxacillin-resistant
S. aureus
isolates, and no growth failures occurred with any method. If the breakpoint for susceptibility was lowered to ≤1 μg/ml for agar dilution methods, more CoNS isolates with oxacillin resistance related to the
mecA
gene were detected when 0 or 2% NaCl agar supplementation was used. Only one CoNS isolate with
mecA
gene-associated resistance was not detected by using agar dilution and MH medium supplemented with 4% NaCl with incubation for 48 h. When the breakpoint for susceptibility was decreased 10-fold (from 6.0 to 0.6 μg of oxacillin per ml) for the agar swab screen method, fully 100% of the CoNS isolates that carried the
mecA
gene were identified.
Phenotypic Diversity of Enterotoxigenic
Escherichia coli
Strains from a Community-Based Study of Pediatric Diarrhea in Periurban Egypt
Authors: Peruski Leonard F. , Kay Bradford A. , El-Yazeed Remon Abu , El-Etr Sahar H. , Cravioto Alejandro , Wierzba Thomas F. , Rao Malla , El-Ghorab Nemat , Shaheen Hind , Khalil Sami B. , Kamal Karim , Wasfy Momtaz O. , Svennerholm Ann-Mari , Clemens John D. , Savarino Stephen J.
Published: December 2019

No past studies of diarrhea in children of the Middle East have examined in detail the phenotypes of enterotoxigenic
Escherichia coli
(ETEC) strains, which are important pathogens in this setting. During a prospective study conducted from November 1993 to September 1995 with 242 children under 3 years of age with diarrhea living near Alexandria, Egypt, 125 episodes of diarrhea were positive for ETEC. ETEC strains were available for 98 of these episodes, from which 100 ETEC strains were selected and characterized on the basis of enterotoxins, colonization factors (CFs), and O:H serotypes. Of these representative isolates, 57 produced heat-stable toxin (ST) only, 34 produced heat-labile toxin (LT) only, and 9 produced both LT and ST. Twenty-three ETEC strains expressed a CF, with the specific factors being CF antigen IV (CFA/IV; 10 of 23; 43%), CFA/II (5 of 23; 22%), CFA/I (3 of 23; 13%), PCFO166 (3 of 23; 13%), and CS7 (2 of 23; 9%). No ETEC strains appeared to express CFA/III, CS17, or PCFO159. Among the 100 ETEC strains, 47 O groups and 20 H groups were represented, with 59 O:H serotypes. The most common O serogroups were O159 (13 strains) and O43 (10 strains). O148 and O21 were each detected in five individual strains, O7 and O56 were each detected in four individual strains, O73, O20, O86, and O114 were each detected in three individual strains, and O23, O78, O91, O103, O128, and O132 were each detected in two individual strains. The most common H serogroups were H4 (16 strains), 12 of which were of serogroup O159; H2 (9 strains), all of which were O43; H18 (6 strains); H30 (6 strains); and H28 (5 strains); strains of the last three H serogroups were all O148. Cumulatively, our results suggest a high degree of clonal diversity of disease-associated ETEC strains in this region. As a low percentage of these strains expressed a CF, it remains possible that other adhesins for which we either did not assay or that are as yet undiscovered are prevalent in this region. Our findings point out some potential barriers to effective immunization against ETEC diarrhea in this population and emphasize the need to identify additional protective antigens commonly expressed by ETEC for inclusion in future vaccine candidates.
Simple and Accurate PCR-Based System for Typing Vacuolating Cytotoxin Alleles of
Helicobacter pylori
Authors: Atherton J. C. , Cover T. L. , Twells R. J. , Morales M. R. , Hawkey C. J. , Blaser M. J.
Published: December 2019

Alleles of the vacuolating cytotoxin gene (
vacA
) of
Helicobacter pylori
vary between strains, particularly in the region encoding the signal sequence (which may be type s1 or s2) and the midregion (which may be type m1 or m2). Using a PCR-based typing system developed in the United States, we showed that 36 strains from Asia and South America were all
vacA
signal sequence type s1; 3 were midregion type m1 and 11 were m2, but 22 could not be typed for the
vacA
midregion. All strains possessed
cagA
(cytotoxin-associated gene A), another virulence marker.
vacA
nucleotide sequence analysis showed that midregion typing failure was due to base substitutions at the primer annealing sites. Using the new sequence data, we developed two new PCR-based
vacA
midregion typing systems, both of which correctly typed 41 U.S. strains previously typed by the old system and successfully typed all 36 of the non-U.S. strains. All previously untypeable strains were
vacA
m1, other than one m1/m2 hybrid. In summary, we describe and validate a simple PCR-based system for typing vacuolating cytotoxin (
vacA
) alleles of
H. pylori
and show that this system correctly identifies the signal and midregion types of
vacA
in 77 strains from Asia and North and South America.
Colonization of Mexican Patients by Multiple
Helicobacter pylori
Strains with Different
vacA
and
cagA
Genotypes
Authors: Morales-Espinosa Rosario , Castillo-Rojas Gonzalo , Gonzalez-Valencia Gerardo , Ponce de León Sergio , Cravioto Alejandro , Atherton John C. , López-Vidal Yolanda
Published: December 2019

Helicobacter pylori
virulence determinants have not previously been studied in detail in Latin Americans with
H. pylori
infections. We characterized the
vacA
(vacuolating cytotoxin gene A) and
cagA
(cytotoxin-associated gene A) types of more than 400 single-colony isolates from 20 patients in Mexico City. For 17 patients
H. pylori
strains of two or more different
vacA
genotypes were isolated from gastric biopsy specimens, indicating infection with two or more strains of
H. pylori
. The most frequent
vacA
genotype was s1b/m1.
vacA
diversity was more marked than that described previously, in that isolates from seven patients had untypeable
vacA
midregions and isolates from nine patients had type s1 signal sequence coding regions which could not be further subtyped. Previously undescribed
vacA
type s2/m1 strains were found in five patients. All patients were infected with
cagA
-positive strains, but occasionally, these coexisted with small numbers of
cagA
-negative strains. In conclusion, coinfection with multiple
H. pylori
strains is common in Mexico, and
vacA
in these strains is genetically more diverse than has been described in other populations.
Evaluation of Two Rapid Assays for Detection of
Clostridium difficile
Toxin A in Stool Specimens
Authors: Fedorko Daniel P. , Engler Howard D. , O’Shaughnessy Elizabeth M. , Williams Esther C. , Reichelderfer Cynthia J. , Smith William I.
Published: December 2019

Rapid laboratory diagnosis of
Clostridium difficile
-associated diarrhea (CDAD) is highly desirable in the setting of hospital cost containment. We tested 654 stool specimens to compare the performance of two assays for rapid detection of toxin A, the Immunocard Toxin A test (Meridian Diagnostics, Inc.) and the Culturette Brand Toxin CD enzyme immunoassay (EIA) (Becton Dickinson Microbiology Systems), with a cytotoxin assay (Cytotoxi Test; Advanced Clinical Diagnostics) and culture on cycloserine-cefoxitin-fructose agar followed by determination of the production of toxins A and B. A chart review was performed for patients whose stool specimens provided positive results on one to three of the assays. With the “gold standard” of all four assays positive or chart review evidence of CDAD, 97 (14.8%) stool specimens were positive by one or more assays and 557 (85.2%) were negative by all methods. Total agreement for all assays was 90.5% (592 of 654). The sensitivity, specificity, positive predictive value, and negative predictive value for toxigenic culture were 94.7, 98.6, 87.1, and 99.5%, respectively, for toxigenic culture; 87.7, 98.6, 86.2, and 98.8%, respectively, for the cytotoxin assay; 71.9, 99.3, 91.1, and 97.3%, respectively, for the Immunocard; and 68.4, 99.1, 88.6, and 96.9%, respectively, for the Culturette EIA. While easy to perform and highly specific, these rapid assays do not appear to be sufficient for accurate diagnosis of CDAD.
Prevalence of Group A Rotavirus, Human Calicivirus, Astrovirus, and Adenovirus Type 40 and 41 Infections among Children with Acute Gastroenteritis in Dijon, France
Authors: Bon F. , Fascia P. , Dauvergne M. , Tenenbaum D. , Planson H. , Petion A. M. , Pothier P. , Kohli E.
Published: December 2019

Group A rotaviruses, human caliciviruses, astroviruses, and adenovirus types 40 and 41 were detected by enzyme immunoassay or reverse transcription-PCR in 61, 14, 6, and 3% of stool specimens from 414 children consulting for gastroenteritis between 1995 and 1998. These data highlight the importance of caliciviruses in infantile gastroenteritis. Among these, Norwalk-like viruses belonging to genogroup II were predominant.
Multicenter Evaluation of AMPLICOR and Automated COBAS AMPLICOR CT/NG Tests for
Neisseria gonorrhoeae
Authors: Martin David H. , Cammarata Cathy , Van Der Pol Barbara , Jones Robert B. , Quinn Thomas C. , Gaydos Charlotte A. , Crotchfelt Kimberly , Schachter Julius , Moncada Jeanne , Jungkind D. , Turner Buffy , Peyton Cynthia
Published: December 2019

The fully automated COBAS AMPLICOR CT/NG and semiautomated AMPLICOR CT/NG tests were evaluated in a multicenter trial for their ability to detect
Neisseria gonorrhoeae
infections. Test performance compared to that of culturing was evaluated for 2,192 matched endocervical swab and urine specimens obtained from women and for 1,981 matched urethral swab and urine specimens obtained from men. Culture-negative, PCR-positive specimens that tested positive in a confirmatory PCR test for an alternative target sequence within the
N. gonorrhoeae
16S rRNA gene were considered to be true positives. The overall prevalences of gonorrhea were 6.6% in women and 20.1% in men. The COBAS AMPLICOR and AMPLICOR formats yielded concordant results for 98.8% of the specimens and exhibited virtually identical sensitivities and specificities. The results that follow are for the COBAS AMPLICOR format. With the infected patient as the reference standard, the resolved sensitivities of PCR were 92.4% for endocervical swab specimens and 64.8% for female urine specimens. There were no significant differences in these rates between women with and without symptoms. Among symptomatic men, COBAS AMPLICOR sensitivities were 94.1% for urine and 98.1% for urethral swabs; for asymptomatic men, the results were 42.3 and 73.1%, respectively. In comparison, the sensitivities of culturing were 84.8% for endocervical specimens, 92.7% for symptomatic male urethral specimens, and only 46.2% for urethral specimens obtained from asymptomatic men. When PCR results were analyzed as if only a single test had been performed on a single specimen type, the resolved sensitivity was always higher. The resolved specificities of PCR were 99.5% for endocervical swab specimens, 99.8% for female urine specimens, 98.9% for male urethral swab specimens, and 99.9% for male urine specimens. The internal control revealed that 2.1% of specimens were inhibitory when initially tested. Nevertheless, valid results were obtained for 99.2% of specimens because 60.0% of the inhibitory specimens were not inhibitory when a second aliquot was tested. The COBAS AMPLICOR CT/NG test for
N. gonorrhoeae
exhibited high sensitivity and specificity with urethral swab and urine specimens from men and endocervical swab specimens from women and thus is well suited for diagnosing and screening for
N. gonorrhoeae
infection.
Serodiagnosis of Louse-Borne Relapsing Fever with Glycerophosphodiester Phosphodiesterase (GlpQ) from
Borrelia recurrentis
Authors: Porcella Stephen F. , Raffel Sandra J. , Schrumpf Merry E. , Schriefer Martin E. , Dennis David T. , Schwan Tom G.
Published: December 2019

Human louse-borne relapsing fever occurs in sporadic outbreaks in central and eastern Africa that are characterized by significant morbidity and mortality. Isolates of the causative agent,
Borrelia recurrentis
, were obtained from the blood of four patients during a recent epidemic of the disease in southern Sudan. The
glpQ
gene, encoding glycerophosphodiester phosphodiesterase, from these isolates was sequenced and compared with the
glpQ
sequences obtained from other relapsing-fever spirochetes. Previously we showed that GlpQ of
Borrelia hermsii
is an immunogenic protein with utility as a serological test antigen for discriminating tick-borne relapsing fever from Lyme disease. In the present work, we cloned and expressed the
glpQ
gene from
B. recurrentis
and used recombinant GlpQ in serological tests. Acute- and convalescent-phase serum samples obtained from 42 patients with louse-borne relapsing fever were tested with an indirect immunofluorescence assay (IFA) and an enzyme-linked immunosorbent assay (ELISA) that used whole cells of
B. recurrentis
and with immunoblotting to whole-cell lysates of the spirochete and
Escherichia coli
producing recombinant GlpQ. The geometric mean titers of the acute- and convalescent-phase serum samples measured by IFA were 1:83 and 1:575, respectively. The immunoblot analysis identified a high level of reactivity and seroconversion to GlpQ, and the assay was more sensitive than the whole-cell IFA and ELISA using purified, recombinant histidine-tagged GlpQ. Serum antibodies to GlpQ and other antigens persisted for 27 years in one patient. We conclude that assessment of anti-GlpQ antibodies will allow serological confirmation of louse-borne relapsing fever and determination of disease prevalence.
Spoligotyping and Polymorphic GC-Rich Repetitive Sequence Fingerprinting of
Mycobacterium tuberculosis
Strains Having Few Copies of IS
6110
Authors: Yang Z. H. , Ijaz K. , Bates J. H. , Eisenach K. D. , Cave M. D.
Published: December 2019

Several genetic loci have been utilized to genotype isolates of
Mycobacterium tuberculosis
. A shortcoming of the most commonly used method, IS
6110
fingerprinting, is that it does not adequately discriminate between isolates having few copies of IS
6110
. This study was undertaken to compare pTBN12 fingerprinting of polymorphic GC-rich repetitive sequence genes and spoligotyping of the direct repeat locus as secondary typing procedures for
M. tuberculosis
isolates having fewer than six copies of IS
6110
. A total of 88 isolates (100% of the isolates with fewer than six copies of IS
6110
isolated in Arkansas during 1996 and 1997) were included in this study. Among the 88 isolates, 34 different IS
6110
patterns were observed, 10 of which were shared by more than 1 isolate, involving a total of 64 isolates. The 64 isolates were subdivided into 13 clusters (containing 37 isolates) and 27 unique isolates based on a combination of IS
6110
and pTBN12 fingerprinting and into 11 clusters (containing 51 isolates) and 13 unique isolates based on a combination of IS
6110
fingerprinting and spoligotyping. Identical spoligotypes were found among isolates having different IS
6110
patterns, as well as among isolates showing different pTBN12 patterns. In contrast, all isolates that had different IS
6110
patterns were found to be unique by pTBN12 typing. The clustering rate was 73, 58, and 42%, respectively, for IS
6110
fingerprinting alone, IS
6110
fingerprinting and spoligotyping combined, and IS
6110
and pTBN12 combined fingerprinting. The data indicate that the pTBN12 method has greater discriminating power among low-copy-number isolates than does spoligotyping.
Use of a Repetitive DNA Probe To Type Clinical and Environmental Isolates of
Aspergillus flavus
from a Cluster of Cutaneous Infections in a Neonatal Intensive Care Unit
Authors: James Michael J. , Lasker Brent A. , McNeil Michael M. , Shelton Mark , Warnock David W. , Reiss Errol
Published: December 2019

Aspergillus flavus
is second to
A. fumigatus
as a cause of invasive aspergillosis, but no standard method exists for molecular typing of strains from human sources. A repetitive DNA sequence cloned from
A. flavus
and subcloned into a pUC19 vector, pAF28, was used to type 18 isolates from diverse clinical, environmental, and geographic sources. The restriction fragment length polymorphisms generated with
Eco
RI- or
Pst
I-digested genomic DNA and probed with digoxigenin-labeled pAF28 revealed complete concordance between patterns. Eighteen distinct fingerprints were observed. The probe was used to investigate two cases of cutaneous
A. flavus
infection in low-birth-weight infants in a neonatal intensive care unit (NICU). Both infants were transported by the same ambulance and crew to the NICU on the same day.
A. flavus
strains of the same genotype were isolated from both infants, from a roll of tape used to fasten their umbilical catheters, from a canvas bag used to store the tape in the ambulance, and from the tape tray in the ambulance isolette. These cases highlight the need to consider exposures in critically ill neonates that might occur during their transport to the NICU and for stringent infection control practices. The hybridization profiles of strains from a second cluster of invasive
A. flavus
infections in two pediatric hematology-oncology patients revealed a genotype common to strains from a definite case patient and a health care worker. A probable case patient was infected with a strain with a genotype different from that of the strain from the definite case patient but highly related to that of an environmental isolate. The high degree of discrimination and reproducibility obtained with the pAF28 probe underscores its utility for typing clinical and environmental isolates of
A. flavus
.
16S Ribosomal DNA Sequence Analysis of a Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates
Authors: Drancourt Michel , Bollet Claude , Carlioz Antoine , Martelin Rolland , Gayral Jean-Pierre , Raoult Didier
Published: December 2019

Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of ≥97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of ≥99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (
P
= 0.07). The 16S rDNA approach failed to identify
Enterobacter
and
Pantoea
isolates to the species level (
P
= 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria.
Molecular Surveillance of European Quinolone-Resistant Clinical Isolates ofPseudomonas aeruginosaandAcinetobacterspp. Using Automated Ribotyping
Authors: Brisse Sylvain , Milatovic Dana , Fluit A. C. , Kusters Karlijn , Toelstra Annet , Verhoef Jan , Schmitz Franz-Josef
Published: December 2019

Nosocomial isolates ofPseudomonas aeruginosaandAcinetobacterspp. exhibit high rates of resistance to antibiotics and are often multidrug resistant. In a previous study (D. Milatovic, A. Fluit, S. Brisse, J. Verhoef, and F. J. Schmitz, Antimicrob. Agents Chemother. 44:1102–1107, 2000), isolates of these species that were resistant to sitafloxacin, a new advanced-generation fluoroquinolone with a high potency and a broad spectrum of antimicrobial activity, were found in high proportion in 23 European hospitals. Here, we investigate the clonal diversity of the 155P. aeruginosaand 145Acinetobacterspp. sitafloxacin-resistant isolates from that study by automated ribotyping. Numerous ribogroups (sets of isolates with indistinguishable ribotypes) were found among isolates ofP. aeruginosa(n= 34) andAcinetobacterspp. (n= 16), but the majority of the isolates belonged to a limited number of major ribogroups. Sitafloxacin-resistant isolates (MICs > 2 mg/liter, used as a provisional breakpoint) showed increased concomitant resistance to piperacillin, piperacillin-tazobactam, ceftriaxone, ceftazidime, amikacin, gentamicin, and imipenem. The major ribogroups were repeatedly found in isolates from several European hospitals; these isolates showed higher levels of resistance to gentamicin and imipenem, and some of them appeared to correspond to previously described multidrug-resistant international clones ofP. aeruginosa(serotype O:12) andAcinetobacter baumannii(clones I and II). Automated ribotyping, when used in combination with more discriminatory typing methods, may be a convenient library typing system for monitoring future epidemiological dynamics of geographically widespread multidrug-resistant bacterial clones.
Rapid Quantification and Differentiation of Human Polyomavirus DNA in Undiluted Urine from Patients after Bone Marrow Transplantation
Authors: Biel Stefan S. , Held Thomas K. , Landt Olfert , Niedrig Matthias , Gelderblom Hans R. , Siegert Wolfgang , Nitsche Andreas
Published: December 2019

A combined PCR assay was developed for the detection and typing of human polyomavirus (huPoV) in clinical samples, consisting of (i) a qualitative seminested PCR assay (snPCR) to discriminate between huPoV BK and JC and (ii) a high-throughput, quantitative TaqMan PCR assay (TM-PCR) for the general detection of huPoV. The TM-PCR detects huPoV DNA in a linear range from 107to 101copies per assay. In reproducibility runs, the inter- and intra-assay variabilities were ≤60 and ≤50%, respectively. The snPCR assay uses a set of four primers for the same region of the BK and JC viral genomes. In the first round of amplification, two general primers were used; in the second round, one of these general primers and two additional, BK- or JC-specific primers were used simultaneously to produce amplicons of different sizes specific for BK virus (246 bp) and JC virus (199 bp), respectively. We tested different urine dilutions in order to determine the inhibitory effects of urine on PCR amplification. Furthermore, we compared the use of native urine with DNA purified by different preparation procedures. Our results show, that a 1:10 dilution of the urine led to complete reduction of the amplification inhibition found with 6% of undiluted urine samples. In a clinical study including 600 urine specimens, our assay turned out to be fast, cheap, and reliable in both qualitative and quantitative aspects.
Evaluation of the Etest Method for Determining Voriconazole Susceptibilities of 312 Clinical Isolates of
Candida
Species by Using Three Different Agar Media
Authors: Pfaller M. A. , Messer S. A. , Houston A. , Mills K. , Bolmstrom A. , Jones R. N.
Published: December 2019

Performance of the Etest for voriconazole susceptibility testing of 312 isolates of
Candida
spp. was assessed against that of the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose (RPG), Casitone agar (CAS), and antibiotic medium 3 (AM3) agar and were read after incubation for 48 h at 35°C. The
Candida
spp. isolates included
C. albicans
(
n
= 174),
C. glabrata
(
n
= 55),
C. tropicalis
(
n
= 31),
C. parapsilosis
(
n
= 39),
C. krusei
(
n
= 5),
C. lusitaniae
(
n
= 2), and
C. guilliermondii
(
n
= 6). The Etest results obtained using RPG correlated well with the reference MICs. Overall agreement ranged from 91% for
C. glabrata
to 100% for
C. tropicalis
,
C. parapsilosis
,
C. guilliermondii
,
C. krusei
, and
C. lusitaniae
. When CAS was used, agreement ranged from 80% for
C. krusei
to 100% for
C. parapsilosis
,
C. guilliermondii
, and
C. lusitaniae
. With AM3, agreement ranged from 58% for
C. glabrata
to 100% for
C. lusitaniae
and
C. guilliermondii
. The Etest method using RPG appears to be a useful method for determining voriconazole susceptibilities of
Candida
species.
Molecular and Antigenic Characterization of a Highly Evolved Derivative of the Type 2 Oral Poliovaccine Strain Isolated from Sewage in Israel
Authors: Shulman Lester M. , Manor Yosef , Handsher Rachel , Delpeyroux Francis , McDonough Michael J. , Halmut Tova , Silberstein Ilana , Alfandari Jacklyn , Quay Jacqueline , Fisher Tamar , Robinov Jana , Kew Olen M. , Crainic Radu , Mendelson Ella
Published: December 2019

An unusual, highly diverged derivative of the Sabin type 2 oral poliovaccine (OPV) strain was recovered from environmental samples during routine screening for wild polioviruses. Virus was cultivated in L20B cells and then passaged on BGM cells at 40°C (RCT [reproductive capacity at supraoptimal temperature]-positive marker) to select against most OPV strains. All but 1 of 25 RCT-positive OPV-derived environmental isolates were antigenically and genetically (>99.5% VP1 sequence match) similar to the respective Sabin strains. However, isolate PV2/4568-1/ISR98 (referred to below as 4568-1) escaped neutralization with Sabin 2-specific monoclonal antibodies and cross-adsorbed sera, and had multiple nucleotide substitutions (220 of 2,646; 8.3%) in the P1 capsid region. Fourteen of the 44 associated amino acid substitutions in the capsid mapped to neutralizing antigenic sites. Neutralizing titers in the sera of 50 Israeli children 15 years old were significantly lower to 4568-1 (geometric mean titer [GMT], 47) than to Sabin 2 (GMT, 162) or to the prototype wild strain, PV2/MEF-1/EGY42 (GMT, 108). Two key attenuating sites had also reverted in 4568-1 (A
481
to G in the 5′ untranslated region and the VP1 amino acid I
143
to T), and the isolate was highly neurovirulent for transgenic mice expressing the poliovirus receptor (PVR-Tg21 mice). The extensive genetic divergence of 4568-1 from the parental Sabin 2 strain suggested that the virus had replicated in one or more people for ∼6 years. The presence in the environment of a highly evolved, neurovirulent OPV-derived poliovirus in the absence of polio cases has important implications for strategies for the cessation of immunization with OPV following global polio eradication.
Phenotypic and Genetic Characterization of
Lactococcus garvieae
Isolated in Spain from Lactococcosis Outbreaks and Comparison with Isolates of Other Countries and Sources
Authors: Vela A. I. , Vázquez J. , Gibello A. , Blanco M. M. , Moreno M. A. , Liébana P. , Albendea C. , Alcalá B. , Mendez A. , Domínguez L. , Fernández-Garayzábal J. F.
Published: December 2019

The phenotypic and genetic analysis results for 84 isolates of
Lactococcus garvieae
(including 62 strains from trout with lactococcosis from four different countries, 7 strains from cows and water buffalos with subclinical mastitis, 3 from water, and 10 from human clinical samples) are presented. There was great phenotypic heterogeneity (13 different biotypes) based on the acidification of saccharose, tagatose, mannitol, and cyclodextrin and the presence of the enzymes pyroglutamic acid arylamidase and
N
-acetyl-β-glucosaminidase.
L. garvieae
also exhibited high genetic diversity by pulsed-field gel electrophoresis (PFGE), with 19 different pulsotypes among the isolates of
L. garvieae
studied. Only epidemiologically related strains, like the Spanish and Italian fish isolates and the cow and water buffalo isolates, displayed a close genetic relationship by PFGE, while the strains isolated from sporadic clinical cases, like the human isolates, were genetically unrelated. Overall, a general correlation between phenotypic and genetic data was observed. Epidemiological analysis of biotype and PFGE results indicated that the trout lactococcosis outbreaks in Spain and Portugal and those in France and Italy were produced by genetically unrelated clones. In Spain, two different clones were detected; the outbreaks diagnosed from 1995 onward were produced by a clone (biotype 2, pulsotype A1) which, although genetically related, was different from the one that was responsible for the outbreaks studied between 1991 and 1994 (biotype 1, pulsotype B). The Portuguese isolate had a biochemical profile identical to that of the Spanish strain isolated from 1995 onward and is also genetically closely related to this strain (pulsotype A2). There was a close relationship between the two pulsotypes (E and F) found in the Italian isolates. The French isolate (biotype 3, pulsotype D) was not genetically related to any other
L. garvieae
fish isolate. These results suggest the existence of diverse infection sources for the different lactococcosis outbreaks.
Utility of Tissue Culture for Detection of
Toxoplasma gondii
in Vitreous Humor of Patients Diagnosed with Toxoplasmic Retinochoroiditis
Authors: Miller Darlene , Davis Janet , Rosa Robert , Diaz Martha , Perez Edith
Published: December 2019

Laboratory recovery and confirmation of the etiologic agent in necrotizing retinochoroiditis are problematic. Tissue culture and intraocular antibody titers were compared as adjuncts to clinical diagnosis for toxoplasmic retinochoroiditis: the correlations were 91 and 67%, respectively. Isolation of
Toxoplasma gondii
may establish a definitive diagnosis in patients with toxoplasmic retinochoroiditis.
A Probable New
Helicobacter
Species Isolated from a Patient with Bacteremia
Authors: Tee Wee , Hinds Suzanne , Montgomery Janet , Dyall-Smith Michael L.
Published: December 2019

A probable new
Helicobacter
species was isolated from the blood of a 14-month-old aboriginal child who presented with vomiting, diarrhea, fever, and dry cough. The most similar 16S rRNA gene sequence was that of
Helicobacter fennelliae
CCUG 18820
T
but the new sequence differed from it by at least 32 base substitutions and by the presence of a large (353-nucleotide) intervening sequence.
Evaluation of the Alexon-Trend ProSpecT
Campylobacter
Microplate Assay
Authors: Tolcin Rita , LaSalvia Margaret M. , Kirkley Barbara A. , Vetter Emily A. , Cockerill Franklin R. , Procop Gary W.
Published: December 2019

We evaluated stool specimens known to contain or be free of
Campylobacter
by traditional culture, using the ProSpecT
Campylobacter
microplate assay (Alexon-Trend, Ramsey, Minn.). This rapid enzyme immunoassay for the detection of
Campylobacter
-specific antigens demonstrated 96% sensitivity and 99% specificity and is an acceptable alternative method of
Campylobacter
detection.
Community Acquisition of Gentamicin-Sensitive Methicillin-Resistant
Staphylococcus aureus
in Southeast Queensland, Australia
Authors: Nimmo Graeme R. , Schooneveldt Jacqueline , O'Kane Gabrielle , McCall Brad , Vickery Alison
Published: December 2019

Community-acquired methicillin-resistant
Staphylococcus aureus
(MRSA) susceptible to gentamicin has been reported in a number of countries in the 1990s. To study the acquisition of gentamicin-sensitive MRSA (GS-MRSA) in southeast Queensland and the relatedness of GS-MRSA to other strains of MRSA, 35 cases of infection due to GS-MRSA from October 1997 through September 1998 were examined retrospectively to determine the mode of acquisition and risk factors for MRSA acquisition. Thirty-one isolates from the cases were examined using a variety of methods (antibiotyping, phage typing, pulsed-field gel electrophoresis [PFGE] fingerprinting, and coagulase typing by restriction analysis of PCR products) and were compared with strains of local hospital-acquired gentamicin-resistant MRSA (GR-MRSA) and of Western Australian MRSA (WA-MRSA). Only 6 of 23 cases of community-acquired GS-MRSA had risk factors for MRSA acquisition. Twenty of 21 isolates from cases of community-acquired infection were found to be related by PFGE and coagulase typing and had similar phage typing patterns. Hospital- and nursing home-acquired GS-MRSA strains were genetically and phenotypically diverse. Community-acquired GS-MRSA strains were not related to nosocomial GR-MRSA or WA-MRSA, but phage typing results suggest that they are related to GS-MRSA previously reported in New Zealand.
Outbreak of Nosocomial Infections Due to Extended-Spectrum β-Lactamase-Producing Strains of Enteric Group 137, a New Member of the Family
Enterobacteriaceae
Closely Related to
Citrobacter farmeri
and
Citrobacter amalonaticus
Authors: Warren John R. , Farmer J. J. , Dewhirst Floyd E. , Birkhead Karen , Zembower Teresa , Peterson Lance R. , Sims Lola , Bhattacharya Mondira
Published: December 2019

A member of the
Enterobacteriaceae
initially identified as
Kluyvera cryocrescens
by the MicroScan Gram-Negative Combo 13 panel caused an outbreak of nosocomial infections in four patients (pneumonia,
n
= 2; urinary tract infection,
n
= 1; wound infection,
n
= 1) and urinary tract colonization in one patient. When the strains were tested by the Enteric Reference Laboratory of the Centers for Disease Control and Prevention, biochemical results were most compatible with
Yersinia intermedia
,
Kluyvera cryocrescens
, and
Citrobacter farmeri
but identification scores were low and test results were discrepant. However, when the biochemical test profile was placed in the computer database as a new organism, all strains were identified as the organism with high identification scores (0.999968 to 0.999997) and no discrepant test results. By 16S rRNA sequence analysis the organism clustered most closely with, but was distinct from,
Citrobacter farmeri
and
Citrobacter amalonaticus
. Based on its unique biochemical profile and rRNA sequence, this organism is designated Enteric Group 137. Restriction endonuclease analysis and taxonomic antibiograms of strains causing the outbreak demonstrated a single clone of Enteric Group 137, and antibiotic susceptibility testing revealed the presence of extended-spectrum β-lactamase (ESBL) resistance. Enteric Group 137 appears to be a new opportunistic pathogen that can serve as a source of ESBL resistance in the hospital.