bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous Approach for Nonculture PCR-Based Identification and Serogroup Prediction of
Neisseria meningitidis
Authors: Taha Muhamed-Kheir
Published: December 2019

A nonculture PCR-based method to characterize
Neisseria meningitidis
was used to test 225 clinical specimens. PCR correctly identified and predicted the serogroups of
N. meningitidis
of culture-proven meningococcal diseases and confirmed this diagnosis in 35% of suspected samples. This approach could be useful when culture fails to isolate
N. meningitidis
.
Clinical Evaluation of the BDProbeTec ET System for Rapid Detection of
Mycobacterium tuberculosis
Authors: Bergmann John S. , Keating William E. , Woods Gail L.
Published: December 2019

The performance of the BDProbeTec ET system (BD Biosciences, Sparks, Md.) for direct detection of
Mycobacterium tuberculosis
complex (MTBC) in respiratory specimens was evaluated by comparing results to those of conventional mycobacterial culture performed with the BACTEC 460 TB system and Middlebrook 7H11 biplates. Patients known to have been on antituberculous therapy were excluded from the analysis. Of 600 evaluable specimens (4 specimens were excluded from the analysis due to failure of the internal amplification control [IAC]) from 332 patients, 57 grew mycobacteria; 16 were MTBC (from 12 patients), and 41 were nontuberculous mycobacteria. Of the 16 MTBC culture-positive specimens, 12 were smear positive and 4 were smear negative. BDProbeTec ET detected 14 of the 16 MTBC culture-positive specimens, resulting in initial overall sensitivity, specificity, and positive and negative predictive values of 87.5, 99.0, 70.0, and 99.7%, respectively. After resolution of discrepancies by review of medical records and retesting of samples yielding discordant MTBC culture and BDProbeTec ET results, the revised overall sensitivity, specificity, and positive and negative predictive values of the BDProbeTec ET were respectively 93.8, 99.8, 93.8, and 99.8% by specimen and 91.7, 99.7, 91.7, and 99.7% by patient. The BDProbeTec ET System offers the distinct advantage of including an IAC in the specimen well. These data suggest that the test performance is very good, especially for smear-positive samples. However, the number of patients with tuberculosis in our study, especially those with smear-negative disease, was small; therefore, additional studies are needed.
Survival of Aerobic and Anaerobic Bacteria in Purulent Clinical Specimens Maintained in the Copan Venturi Transystem and Becton Dickinson Port-a-Cul Transport Systems
Authors: Citron Diane M. , Warren Yumi A. , Hudspeth Marie K. , Goldstein Ellie J. C.
Published: December 2019

Recovery of aerobic and anaerobic bacteria from clinical specimens maintained in the Copan Venturi Transystem and the Becton Dickinson Port-a-Cul transport was assessed. Of 54 anaerobes, 53 were recovered after 4 h, and 52 were recovered after 24 h, from both systems. After 48 h, 45 and 50 were recovered from the two systems, respectively.
Epidemiology and Microbiology of Surgical Wound Infections
Authors: Giacometti A. , Cirioni O. , Schimizzi A. M. , Del Prete M. S. , Barchiesi F. , D'Errico M. M. , Petrelli E. , Scalise G.
Published: December 2019

This study included 676 surgery patients with signs and symptoms indicative of wound infections, who presented over the course of 6 years. Bacterial pathogens were isolated from 614 individuals. A single etiologic agent was identified in 271 patients, multiple agents were found in 343, and no agent was identified in 62. A high preponderance of aerobic bacteria was observed. Among the common pathogens were
Staphylococcus aureus
(191 patients, 28.2%),
Pseudomonas aeruginosa
(170 patients, 25.2%),
Escherichia coli
(53 patients, 7.8%),
Staphylococcus epidermidis
(48 patients, 7.1%), and
Enterococcus faecalis
(38 patients, 5.6%).
Identification of
Helicobacter pylori
and Other
Helicobacter
Species by PCR, Hybridization, and Partial DNA Sequencing in Human Liver Samples from Patients with Primary Sclerosing Cholangitis or Primary Biliary Cirrhosis
Authors: Nilsson Hans-Olof , Taneera Jalal , Castedal Maria , Glatz Elisabeth , Olsson Rolf , Wadström Torkel
Published: December 2019

Helicobacter pylori
was identified in human liver tissue by PCR, hybridization, and partial DNA sequencing. Liver biopsies were obtained from patients with primary sclerosing cholangitis (
n
= 12), primary biliary cirrhosis (
n
= 12), and noncholestatic liver cirrhosis (
n
= 13) and (as controls) normal livers (
n
= 10). PCR analyses were carried out using primers for the
Helicobacter
genus,
Helicobacter pylori
(the gene encoding a species-specific 26-kDa protein and the 16S rRNA),
Helicobacter bilis
,
Helicobacter pullorum
, and
Helicobacter hepaticus
. Samples from patients with primary biliary cirrhosis and primary sclerosing cholangitis (11 and 9 samples, respectively) were positive by PCR with
Helicobacter
genus-specific primers. Of these 20 samples, 8 were positive with the 16S rRNA primer and 9 were positive with the 26-kDa protein primer of
H. pylori
. These nine latter samples were also positive by Southern blot hybridization for the amplified 26-kDa fragment, and four of those were verified to be
H. pylori
by partial 16S rDNA sequencing. None of the samples reacted with primers for
H. bilis
,
H. pullorum
, or
H. hepaticus
. None of the normal livers had positive results in the
Helicobacter
genus PCR assay, and only one patient in the noncholestatic liver cirrhosis group, a young boy who at reexamination showed histological features suggesting primary sclerosing cholangitis, had a positive result in the same assay.
Helicobacter
positivity was thus significantly more common in patients with cholestatic diseases (20 of 24) than in patients with noncholestatic diseases and normal controls (1 of 23) (
P
= <0.00001). Patients positive for
Helicobacter
genus had significantly higher values of alkaline phosphatases and prothrombin complex than
Helicobacter
-negative patients (
P
= 0.0001 and
P
= 0.0003, respectively). Among primary sclerosing cholangitis patients,
Helicobacter
genus PCR positivity was weakly associated with ulcerative colitis (
P
= 0.05). Significant differences related to blood group or HLA status were not found.
Touchdown Enzyme Time Release-PCR for Detection and Identification of
Chlamydia trachomatis
,
C. pneumoniae
, and
C. psittaci
Using the 16S and 16S-23S Spacer rRNA Genes
Authors: Madico Guillermo , Quinn Thomas C. , Boman Jens , Gaydos Charlotte A.
Published: December 2019

Three touchdown enzyme time release (TETR)-PCR assays were used to amplify different DNA sequences in the variable regions of the 16S and 16S-23S spacer rRNA genes specific for
Chlamydia trachomatis
,
Chlamydia pneumoniae
, and
Chlamydia psittaci
as improved tests for sensitive diagnosis and rapid species differentiation. The TETR-PCR protocol used 60 cycles of amplification, which provided improved analytical sensitivity (0.004 to 0.063 inclusion-forming unit of
Chlamydia
species per PCR). The sensitivity of TETR-PCR with primer set CTR 70-CTR 71 was 96.7%, and the specificity was 99.6%, compared to those of the AMPLICOR PCR for the detection of
C. trachomatis
in vaginal swab samples. TETR-PCR for
C. pneumoniae
with primer set CPN 90-CPN 91 was 90% sensitive and 93.3% specific compared with a nested PCR with primer set CP1/2-CPC/D for clinical respiratory samples. TETR-PCR for
C. psittaci
with primer set CPS 100-CPS 101 showed substantial agreement with cell culturing (κ, 0.78) for animal tissue samples. Primer sets were then combined into a single multiplex TETR-PCR test. The respective 315-, 195-, and 111-bp DNA target products were precisely amplified when DNA from each of the respective
Chlamydia
species or combinations of them was used. Multiplex chlamydia TETR-PCR correctly identified one strain of each of the 15 serovars of
C. trachomatis
, 22 isolates of
C. pneumoniae
, and 20 isolates of
C. psittaci
. The primer sets were specific for each species. No target products were amplified when DNA from
C. pecorum
or a variety of other microorganisms was tested for specificity. TETR-PCR with primers selected for specific sequences in the 16S and 16S-23S spacer rRNA genes is a valuable test that could be used either with individual primers or in a multiplex assay for the identification and differentiation of
Chlamydia
species from culture isolates or for the detection of chlamydiae in clinical samples.
Single-Tube Balanced Heminested PCR for Detecting
Mycobacterium tuberculosis
in Smear-Negative Samples
Authors: García-Quintanilla Albert , Garcia Lourdes , Tudó Griselda , Navarro Maria , González Julià , Jiménez de Anta Maria T.
Published: December 2019

In order to achieve more sensitive and specific results for the rapid diagnosis of tuberculosis, we have developed a new method, named balanced heminested PCR, which avoids the inconvenience of asymmetric amplification and has the advantages of single-tube heminested PCR. This was achieved by replacing the outer primer that participates in both rounds of amplification in the standard heminested technique by another primer containing the sequence of the inner primer attached at its 5′ end. When both techniques were tested for the IS
6110
target of
Mycobacterium tuberculosis
complex in 80 smear-negative culture-positive sputum samples and 60 control samples, the results showed 100% specificity for both techniques and sensitivities of 60 and 75% for heminested PCR and balanced heminested PCR, respectively (
P
= 0.02). In conclusion, the balanced heminested technique shows a higher sensitivity than that of the standard heminested, and it could be applied to any PCR by attaching the inner primer at the 5′ end of the opposite outer primer. Thus, the balanced heminested technique provides a target for the inner primer in both strands, avoiding asymmetric amplification and thereby resulting in a more efficient amplification, and, in practice, a higher sensitivity without loss of specificity and with a minimum risk of cross-contamination.
Isolation and Molecular Characterization of
Clostridium difficile
Strains from Patients and the Hospital Environment in Belarus
Authors: Titov Leonid , Lebedkova Natalia , Shabanov Alexander , Tang Yajarayma J. , Cohen Stuart H. , Silva Joseph
Published: December 2019

Toxigenic
Clostridium difficile
is the most common etiologic agent of hospital-acquired diarrhea in developed countries. The role of this pathogen in nosocomial diarrhea in Eastern Europe has not been clearly established. The goal of this study was to determine the prevalence of
C. difficile
in patients and the hospital environment in Belarus and to characterize these isolates as to the presence of toxin genes and their molecular type.
C. difficile
was isolated from 9 of 509 (1.8%) patients analyzed and recovered from 28 of 1,300 (2.1%) environmental sites cultured. A multiplex PCR assay was used to analyze the pathogenicity locus (PaLoc) of all isolates, and strain identity was determined by an arbitrarily primed PCR (AP-PCR). The targeted sequences for all the genes in the PaLoc were amplified in all
C. difficile
strains examined. A predominantly homogenous group of strains was found among these isolates, with five major AP-PCR groups being identified. Eighty-three percent of environmental isolates were classified into two groups, while patient isolates grouped into three AP-PCR types, two of which were also found in the hospital environment. Although no data on the role of
C. difficile
infection or epidemiology of
C. difficile
-associated diarrhea (CDAD) in this country exist, the isolation of toxigenic
C. difficile
from the hospital environment suggests that this pathogen may be responsible for cases of diarrhea of undiagnosed origin and validates our effort to further investigate the significance of CDAD in Eastern Europe.
Diagnostic and Public Health Dilemma of Lactose-Fermenting
Salmonella enterica
Serotype Typhimurium in Cattle in the Northeastern United States
Authors: McDonough Patrick L. , Shin Sang J. , Lein Donald H.
Published: December 2019

The presence of lactose-fermenting
Salmonella
strains in clinical case materials presented to microbiology laboratories presents problems in detection and identification. Failure to detect these strains also presents a public health problem. The laboratory methods used in detecting lactose-fermenting
Salmonella enterica
serotype Typhimurium from six outbreaks of salmonellosis in veal calves are described. Each outbreak was caused by a multiply-resistant and lactose-fermenting strain of
S. enterica
serotype Typhimurium. The use of Levine eosin-methylene blue agar in combination with screening of suspect colonies for C8 esterase enzyme and inoculation of colonies into sulfide-indole-motility medium for hydrogen sulfide production was particularly effective for their detection. A hypothesis for the creation of lactose-fermenting salmonellae in the environment is presented. It is proposed that the environment and husbandry practices of veal-raising barns provide a unique niche in which lactose-fermenting salmonellae may arise.
Molecular Identification and Epidemiological Tracing of
Pasteurella multocida
Meningitis in a Baby
Authors: Boerlin Patrick , Siegrist Hans H. , Burnens André P. , Kuhnert Peter , Mendez Purita , Prétat Gérard , Lienhard Reto , Nicolet Jacques
Published: December 2019

We report a case of
Pasteurella multocida
meningitis in a 1-month-old baby exposed to close contact with two dogs and a cat but without any known history of injury by these animals. 16S rRNA gene sequencing of the isolate from the baby allowed identification at the subspecies level and pointed to the cat as a possible source of infection. Molecular typing of
Pasteurella
isolates from the animals, from the baby, and from unrelated animals clearly confirmed that the cat harbored the same
P. multocida
subsp.
septica
strain on its tonsils as the one isolated from the cerebrospinal fluid of the baby. This case stresses the necessity of informing susceptible hosts at risk of contracting zoonotic agents about some basic hygiene rules when keeping pets. In addition, this study illustrates the usefulness of molecular methods for identification and epidemiological tracing of
Pasteurella
isolates.
Simple Latex Agglutination Assay for Rapid Serodiagnosis of Human Leptospirosis
Authors: Smits Henk L. , van der Hoorn Menno A. W. G. , Goris Marga G. A. , Gussenhoven George C. , Yersin Claude , Sasaki David M. , Terpstra Wiepko J. , Hartskeerl Rudy A.
Published: December 2019

A newly developed latex agglutination assay for the detection of genus-specific
Leptospira
antibodies in human sera was evaluated. The assay is performed by mixing, on an agglutination card, serum with equal volumes of stabilized antigen-coated, dyed test and control latex beads and is read within 2 min. The latex agglutination test was evaluated with groups of serum samples from patients with leptospirosis and control patients from Hawaii, the Seychelles, Thailand, and The Netherlands. The mean overall sensitivity was 82.3%, and the mean overall specificity was 94.6%. The assay is easy to perform and does not require special skills or equipment. The reagents have a long shelf life, even at tropical temperatures. Together, these factors make the assay suitable for use even at the peripheral level of a health care system as a rapid screening test for leptospirosis.
Genetic and Antigenic Evidence Supports the Separation ofHepatozoon canisandHepatozoon americanumat the Species Level
Authors: Baneth Gad , Barta John R. , Shkap Varda , Martin Donald S. , Macintire Douglass K. , Vincent-Johnson Nancy
Published: December 2019

Recognition ofHepatozoon canisandHepatozoon americanumas distinct species was supported by the results of Western immunoblotting of canine anti-H. canisand anti-H. americanumsera againstH. canisgamonts. Sequence analysis of 368 bases near the 3′ end of the 18S rRNA gene from each species revealed a pairwise difference of 13.59%.
Identification by 16S rRNA Gene Analyses of a Potential Novel Mycobacterial Species as an Etiological Agent of Canine Leproid Granuloma Syndrome
Authors: Hughes M. S. , James G. , Ball N. , Scally M. , Malik R. , Wigney D. I. , Martin P. , Chen S. , Mitchell D. , Love D. N.
Published: December 2019

PCR amplifications of the 16S rRNA gene were performed on 46 specimens obtained from 43 dogs with canine leproid granuloma syndrome to help determine its etiology. Sequence capture PCR was applied to 37 paraffin-embedded specimens from 37 dogs, and nested PCR was attempted on DNA from 9 fresh tissue specimens derived from 3 of the 37 aforementioned dogs and from an additional 6 dogs. Molecular analyses of the paraffin-embedded tissues and fresh tissue specimen analyses were performed at separate institutions. PCR products with identical sequences over a 350-bp region encompassing variable regions 2 and 3 of the 16S rRNA gene were obtained from 4 of 37 paraffin-embedded specimens and from all 9 specimens of fresh tissue originating from 12 of the 43 dogs. Identical sequences were determined from amplicons obtained from paraffin-embedded and fresh specimens from one dog. The consensus DNA sequence, amplified from paraffin-embedded tissue and represented by GenBank accession no.
AF144747
, shared highest nucleotide identity (99.4% over 519 bp) with mycobacterial strain IWGMT 90413 but did not correspond exactly to any EMBL or GenBank database sequence. With a probe derived from the V2 region of the novel canine sequence, reverse cross blot hybridization identified an additional four paraffin-embedded specimens containing the same novel sequence. In total, molecular methodologies identified the proposed novel mycobacterial sequence in 16 of 43 dogs with canine leproid granuloma syndrome, indicating that the species represented by this sequence may be the principal etiological agent of canine leproid granuloma syndrome.
Comparison of Protein A Gene Sequencing with Pulsed-Field Gel Electrophoresis and Epidemiologic Data for Molecular Typing of Methicillin-Resistant
Staphylococcus aureus
Authors: Tang Yi-Wei , Waddington Michael G. , Smith Douglas H. , Manahan Janice M. , Kohner Peggy C. , Highsmith Leanne M. , Li Haijing , Cockerill Franklin R. , Thompson Rodney L. , Montgomery Stacy O. , Persing David H.
Published: December 2019

The epidemiologic relatedness of methicillin-resistant
Staphylococcus aureus
(MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the
S. aureus
protein A (
spa
) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the
spa
repeats. Molecular typing results obtained both by
spa
sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the
spa
typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power,
spa
sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information.
Molecular Evolution in a Multidrug-Resistant Lineage of
Streptococcus pneumoniae
: Emergence of Strains Belonging to the Serotype 6B Icelandic Clone That Lost Antibiotic Resistance Traits
Authors: Vilhelmsson Sigurdur E. , Tomasz Alexander , Kristinsson Karl G.
Published: December 2019

Since their first detection in 1988, penicillin-resistant
Streptococcus pneumoniae
isolates have rapidly spread in Iceland to account for close to 20% of all pneumococcal disease in that country by 1993. The major component (70%) of the resistant pneumococci identified from 1989 to 1992 was the progeny of a single multidrug-resistant clone (Icelandic clone) with a homogeneous chromosomal macrorestriction profile and identical multilocus enzyme type expressing serotype 6B and resistance to penicillin, tetracycline, chloramphenicol, erythromycin, and trimethoprim-sulfamethoxazole. The rest of the non-penicillin-susceptible isolates included bacteria with serotype 6A and serogroups 19 and 23. The unique geographic and epidemiological setting and the availability of a complete collection of all non-penicillin-susceptible isolates of
S. pneumoniae
in Iceland prompted us to carry out a molecular epidemiological study to monitor the fate of the Icelandic clone between 1989 and 1996; in addition, we wished to extend the characterization to representative groups of all non-penicillin-susceptible serotype 6B pneumococci which showed variations in antibiotype and which were recovered in Iceland between late 1989 and the end of 1996. Also included in the study were non-penicillin-susceptible isolates of serogroup 23. Pulsed-field gel electrophoresis of
Sma
I-restricted chromosomal DNA and Southern hybridization with the
lytA
DNA probe and probes specific for antibiotic resistance genes were used to characterize pneumococcal isolates. The results show that (i) the Icelandic clone remained the predominant type among penicillin-resistant
S. pneumoniae
through 1996; (ii) the emergence of variants of the Icelandic clone which had lost one or more of the antibiotic resistance phenotypes and/or resistant genes, singly or in combination, was documented during the surveillance period; and (iii) isolates belonging to the internationally spread multidrug-resistant serotype 23F clone were present in the Icelandic collection since late 1989 but did not increase in number during the subsequent years.
Quantitative Analysis of Human Herpesvirus 8 Viral Load Using a Real-Time PCR Assay
Authors: Lallemand Francis , Desire Nathalie , Rozenbaum Willy , Nicolas Jean-Claude , Marechal Vincent
Published: December 2019

We have developed a quantitative real-time PCR (TaqMan) assay aimed at measuring the cellular human herpesvirus 8 (HHV-8) DNA load in various clinical samples. Standard curves were obtained by serial dilutions of a control plasmid containing both HHV-8 (ORF73 gene) and the cellular target (human albumin gene). The assay appeared to be very sensitive (100% detection rate for at least 10 copies per well) and specific and was easily reproducible (less than 3% intra-assay variability, 5% interassay variability). This method allowed us to quantify precisely the average HHV-8 copy number per cell in various persistently HHV-8-infected cell lines (BBG-1 cells,
n
= 200; BC-1 cells,
n
= 59; BCBL-1 cells,
n
= 70). A retrospective study was also conducted to assess the HHV-8 DNA load in 12 human immunodeficiency virus-infected patients with either Kaposi's sarcoma (KS; seven patients monitored over a 3-month period) or multicentric Castleman's disease (MCD; five patients). The HHV-8 DNA load ranged from 0 to 9,171 copies/10
6
cells in low-risk KS patients (T0, I0, S0 according to the classification of the AIDS Clinical Trials group). We also measured the viral loads in MCD patients either during symptomatic periods or during remission. The results are in agreement with previously published data, with high viral loads correlating with clinical symptoms (1.3 × 10
6
copies/10
6
cells) and low viral loads correlating with asymptomatic periods (less than 5,000 copies/10
6
cells).
Characterization of Strains of
Mycoplasma mycoides
subsp.
mycoides
Small Colony Type Isolated from Recent Outbreaks of Contagious Bovine Pleuropneumonia in Botswana and Tanzania: Evidence for a New Biotype
Authors: March John B. , Clark Jason , Brodlie Malcolm
Published: December 2019

Four strains of
Mycoplasma mycoides
subsp.
mycoides
small colony type (MmmSC) isolated from recent outbreaks of contagious bovine pleuropneumonia (CBPP) in Africa have been investigated. One Botswanan strain, M375, displayed numerous and significant phenotypic differences from both contemporary field isolates and older field and vaccine strains (African, Australian, and European strains dating back to 1936). Differences include altered morphology, reduced capsular polysaccharide production, high sensitivity to MmmSC rabbit hyperimmune antisera in vitro, and unique polymorphisms following immunoblotting. While insertion sequence analysis using IS
1634
clearly indicates a close evolutionary relationship to west African strains, hybridization with IS
1296
shows the absence of a band present in all other strains of MmmSC examined. The data suggest that a deletion has occurred in strain M375, which may explain its altered phenotype, including poor growth in vitro and a relative inability to cause septicemia in mice. These characteristics are also exhibited by
Mycoplasma capricolum
subsp.
capripneumoniae
(causal agent of contagious caprine pleuropneumonia [CCPP]), against which M375 antiserum exhibited some activity in vitro (unique among the various MmmSC antisera tested). These findings may have evolutionary implications, since CCPP is believed to be lung specific and without a septicemic phase (unlike CBPP). Since M375 was isolated from a clinical case of CBPP, this novel biotype may be fairly widespread but not normally isolated due to difficulty of culture and/or a potentially altered disease syndrome. Bovine convalescent antisera (obtained from contemporary naturally infected cattle in Botswana) were active against strain M375 in an in vitro growth inhibition test but not against any other strains of MmmSC tested. There exists the possibility therefore, that strain M375 may possess a set of protective antigens different from those of other strains of MmmSC (including vaccine strains). These findings have implications for the control of the current CBPP epidemic in Africa.
Analysis of Plasmid and Chromosomal DNA of Multidrug-Resistant
Salmonella enterica
Serovar Typhi from Asia
Authors: Mirza S. , Kariuki S. , Mamun K. Z. , Beeching N. J. , Hart C. A.
Published: December 2019

Molecular analysis of chromosomal DNA from 193 multidrug-resistant (MDR)
Salmonella enterica
serovar Typhi isolates from 1990 to 1995 from Pakistan, Kuwait, Malaysia, Bangladesh, and India produced a total of five major different pulsed-field gel electrophoresis (PFGE) patterns. Even within a particular country MDR
S. enterica
serovar Typhi DNA was found to be in different PFGE groups. Similar self-transferable 98-MDa plasmids belonging to either incompatibility group incHI1 or incHI1/FIIA were implicated in the MDR phenotype in
S. enterica
serovar Typhi isolates from all the locations except Quetta, Pakistan, where the majority were of incFIA. A total of five different PFGE genotypes with six different plasmids, based on incompatibility and restriction endonuclease analysis groups, were found among these MDR
S. enterica
serovar Typhi isolates.
Comparison of a Baculovirus-Based VP2 Enzyme Immunoassay (EIA) to an
Escherichia coli
-Based VP1 EIA for Detection of Human Parvovirus B19 Immunoglobulin M and Immunoglobulin G in Sera of Pregnant Women
Authors: Jordan Jeanne A.
Published: December 2019

A split-sample study was conducted to evaluate the clinical performance of an enzyme immunoassay that detects the human parvovirus B19 virus (B19V) immunoglobulin M (IgM) or IgG in the sera of pregnant women. The initial study compared a baculovirus-expressed VP2 enzyme immunoassay (BVP2 EIA) (Biotrin International Inc., Dublin, Ireland) with the currently available and commonly used
Escherichia coli
-expressed VP1 enzyme immunoassay (EVP1 EIA) (MRL Diagnostics, Cypress, Calif.). There was a high degree of agreement between the two assays in the detection of IgM antibodies (283 of 307 [92.2%]) or IgG antibodies (279 of 311 [89.7%]), with the majority of discrepancies (IgM, 17 of 24 [71%]; IgG, 16 of 31 [50%]) being due to equivocal data obtained with the EVP1 EIA. Specimens with discordant BVP2 EIA and EVP1 EIA results (23 of 24 IgM and 32 of 32 IgG results) were analyzed further by baculovirus-based VP1 immunofluorescence assays (BVP1 IFAs) (Biotrin International). The BVP2 EIA and BVP1 IFA results for 20 of 23 and 28 of 32 specimens for IgM and IgG, respectively, were concordant. In contrast, the EVP1 EIA and BVP1 IFA data for only 3 of 23 and 4 of 32 specimens for IgM and IgG, respectively, were in agreement, despite the fact that the same capsid antigen was used. Both the BVP2 EIAs and BVP1 IFAs utilize a conformational viral capsid antigen, while the EVP1 EIA uses a denatured viral capsid antigen. In conclusion, the BVP2 EIAs produced far fewer equivocal results for IgM and IgG, correlating more closely to the confirmatory BVP IFAs, than did the EVP1 EIAs and proved to be more accurate for detecting B19V antibodies in the sera of pregnant women.
Development and Clinical Evaluation of a Recombinant-Antigen-Based Cytomegalovirus Immunoglobulin M Automated Immunoassay Using the Abbott AxSYM Analyzer
Authors: Maine G. T. , Stricker R. , Schuler M. , Spesard J. , Brojanac S. , Iriarte B. , Herwig K. , Gramins T. , Combs B. , Wise J. , Simmons H. , Gram T. , Lonze J. , Ruzicki D. , Byrne B. , Clifton J. D. , Chovan L. E. , Wachta D. , Holas C. , Wang D. , Wilson T. , Tomazic-Allen S. , Clements M. A. , Wright G. L. , Lazzarotto T. , Ripalti A. , Landini M. P.
Published: December 2019

A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1,608 specimens from random volunteer blood donors (
n
= 300), pregnant women (
n
= 1,118), transplant recipients (
n
= 6), and patients with various clinical conditions and disease states (
n
= 184) were tested during development and evaluation of this new assay. In a preliminary clinical evaluation we tested specimens collected prospectively from pregnant women (
n
= 799) and selected CMV IgM-positive archived specimens from pregnant women (
n
= 39). The results from the new CMV IgM immunoassay were compared to the results of a consensus interpretation of the results obtained with three commercial CMV IgM immunoassays. The results for specimens with discordant results were resolved by a CMV IgM immunoblot assay. The relative sensitivity, specificity, and agreement for the AxSYM CMV IgM assay were 94.29, 96.28, and 96.19%, respectively, and the resolved sensitivity, specificity, and agreement were 95.83, 97.47, and 97.37%, respectively. We also tested serial specimens from women who experienced seroconversion or a recent CMV infection during gestation (
n
= 17) and potentially cross-reactive specimens negative for CMV IgM antibody by the consensus tests (
n
= 184). The AxSYM CMV IgM assay was very sensitive for the detection of CMV IgM during primary CMV infection, as shown by the detection of CMV IgM at the same time as or just prior to the detection of CMV IgG. Specimens from individuals with lupus (
n
= 16) or parvovirus B19 infection (
n
= 6) or specimens containing hyper IgM (
n
= 9), hyper IgG (
n
= 8), or rheumatoid factor (
n
= 55) did not cross-react with the AxSYM assay. One specimen each from individuals infected with Epstein-Barr virus (
n
= 26), measles virus (
n
= 10), herpes simplex virus (
n
= 12), or varicella-zoster virus (
n
= 13) infection, one specimen from an influenza vaccinee (
n
= 14), and one specimen containing antinuclear antibody cross-reacted with the assay. The overall rate of cross-reactivity of the specimens with the assay was 3.3% (6 of 184). The AxSYM CMV IgM assay is a sensitive and specific assay for the detection of CMV-specific IgM.
Epidemiological Study of
pap
Genes among Diarrheagenic or Septicemic
Escherichia coli
Strains Producing CS31A and F17 Adhesins and Characterization of Pap
31A
Fimbriae
Authors: Bertin Yolande , Girardeau Jean-Pierre , Darfeuille-Michaud Arlette , Martin Christine
Published: December 2019

The association of the
pap
operon with the CS31A and F17 adhesins was studied with 255
Escherichia coli
strains isolated from calves, lambs, or humans with diarrhea. The three classes of PapG adhesin with different receptor binding preferences were also screened. The
pap
operon was associated with 50 and 36% of human strains that produced CS31A and ovine strains that produced F17, respectively. Among the bovine isolates, the
pap
operon was detected in 61% of the CS31A-positive isolates and 72% of the strains that produce both CS31A and F17. The class II adhesin gene was present in bovine (20%) and ovine (71%) isolates. Both class II and III adhesins were genetically associated with 36% of the human strains. The highest prevalence of the
pap
operon was observed among
E. coli
strains that produce additional adhesins involved in the binding of bacteria to intestinal cells. Among the bovine isolates, the reference strain for CS31A and F17c was found to be positive for the
pap
operon. Phenotypic and genotypic characterizations were undertaken. Pap
31A
appeared as fine and flexible fimbriae surrounding the bacteria but did not mediate adhesion to calf intestinal villi. Pap
31A
production was optimal with bacteria cultured on minimal growth media and repressed by addition of exogenous leucine. The deduced amino acid sequence of the PapA
31A
structural subunit showed 57 to 97% identity with the different P-related structural subunits produced by
E. coli
strains isolated from pigs with septicemia or humans with urinary tract infections. None of the three
papG
allelic variants was detected, but a homologous
papG
gene was present in the chromosome of strain 31A.
Emergence of Resistance of
Candida albicans
to Clotrimazole in Human Immunodeficiency Virus-Infected Children: In Vitro and Clinical Correlations
Authors: Pelletier René , Peter Joanne , Antin Cynthia , Gonzalez Corina , Wood Lauren , Walsh Thomas J.
Published: December 2019

Oropharyngeal candidiasis (OPC) is a common opportunistic infection in human immunodeficiency virus (HIV)-infected patients and other immunocompromised hosts. Clotrimazole troches are widely used in the treatment of mucosal candidiasis. However, little is known about the potential contribution of clotrimazole resistance to the development of refractory mucosal candidiasis. We therefore investigated the potential emergence of resistance to clotrimazole in a prospectively monitored HIV-infected pediatric population receiving this azole. Adapting the National Committee for Clinical Laboratory Standards M27-A reference method for broth antifungal susceptibility testing of yeasts to clotrimazole, we compared MICs in macrodilution and microdilution assays. We further analyzed the correlation between these in vitro findings and the clinical response to antifungal therapy. One isolate from each of 87 HIV-infected children was studied by the macrodilution and microdilution methods. Two inoculum sizes were tested by the macrodilution method (10
3
and 10
4
CFU/ml) in order to assess the effect of inoculum size on clotrimazole MICs. The same isolates also were tested using a noncolorimetric microdilution method. Clotrimazole concentrations ranged from 0.03 to 16 μg/ml. Readings were performed after incubation for 24 and 48 h at 35°C. For 62 (71.2%) of 87 clinical isolates, the MICs were low (≤0.06 μg/ml). The MIC for 90% of the strains tested was 0.5 μg/ml, and the highest MIC was 8 μg/ml. There was no significant difference between MICs at the two inoculum sizes. There was 89% agreement (±1 tube) between the microdilution method at 24 h and the macrodilution method at 48 h. If the MIC of clotrimazole for an isolate of
C. albicans
was ≥0.5 μg/ml, there was a significant risk (
P
< 0.001) of cross-resistance to other azoles: fluconazole, ≥64 μg/ml (relative risk [RR] = 8.9); itraconazole, ≥1 μg/ml (RR = 10). Resistance to clotrimazole was highly associated with clinically overt failure of antifungal azole therapy. Six (40%) of 15 patients for whom the clotrimazole MIC was ≥0.5 μg/ml required amphotericin B for refractory mucosal candidiasis versus 4 (5.5%) of 72 for whom the MIC was <0.5 μg/ml (
P
= 0.001; 95% confidence interval = 2.3 to 22; RR = 7.2). These findings suggest that an interpretive breakpoint of 0.5 μg/ml may be useful in defining clotrimazole resistance in
C. albicans
. The clinical laboratory's ability to determine MICs of clotrimazole may help to distinguish microbiologic resistance from the other causes of refractory OPC, possibly reducing the usage of systemic antifungal agents. We conclude that resistance to clotrimazole develops in isolates of
C. albicans
from HIV-infected children, that cross-resistance to other azoles may develop concomitantly, and that this resistance correlates with refractory mucosal candidiasis.
Evaluation of Phenotypic Markers for Selection and Identification of
Candida dubliniensis
Authors: Tintelnot Kathrin , Haase Gerhard , Seibold Michael , Bergmann Frank , Staemmler Maren , Franz Tatjana , Naumann Dieter
Published: December 2019

Candida dubliniensis
is often associated with
C. albicans
in cultures. Easy-to-perform selective isolation procedures for these closely related species do not exist. Therefore, we evaluated previously described discriminatory phenotypic markers for
C. dubliniensis
. A total of 150 oral rinses from human immunodeficiency virus (HIV)-infected patients were cultured on CHROMagar Candida. Dark green colonies described as being indicative of
C. dubliniensis
and other green colonies, 170 in total, were isolated. Chlamydospore formation, intracellular β-
d
-glucosidase activity, ability to grow at 42°C, carbohydrate assimilation pattern obtained by the API ID 32C, and Fourier transform infrared (FT-IR) spectroscopy were used for phenotypic characterization. Sequencing of the 5′ end of the nuclear large-subunit (26S) ribosomal DNA gene was used for definitive species identification for
C. dubliniensis. C. dubliniensis
was found in 34% of yeast-colonized HIV-infected patients. The color of the colonies on CHROMagar Candida proved to be insufficient for selecting
C. dubliniensis
, since only 30 of 53 proven
C. dubliniensis
isolates showed a dark green color in primary cultures. The described typical chlamydospore formation can give only some indication of
C. dubliniensis
. The assimilation pattern proved to be insufficient to discriminate
C. dubliniensis
from
C. albicans
. All
C. dubliniensis
strains showed no or highly restricted growth at 42°C and a lack of β-
d
-glucosidase activity. Unfortunately, atypical
C. albicans
strains can also exhibit these phenotypic traits. FT-IR spectroscopy combined with hierarchical clustering proved to be as reliable as genotyping for discriminating the two species.
Long-Term Persistence of a Discotheque-Associated Invasive
Neisseria meningitidis
Group C Strain as Proven by Pulsed-Field Gel Electrophoresis and
porA
Gene Sequencing
Authors: Riesbeck Kristian , Orvelid-Mölling Paula , Fredlund Hans , Olcén Per
Published: December 2019

A cluster of a
Neisseria meningitidis
serogroup C strain causing invasive disease was investigated. Five out of seven cases were associated with a particular discotheque. The strains were indistinguishable, as revealed by pulsed-field gel electrophoresis and sequencing of variable regions of the
porA
gene, but caused strikingly different clinical presentations during 5 months.
Molecular Typing and Exopolysaccharide Biosynthesis of
Burkholderia cepacia
Isolates from a Portuguese Cystic Fibrosis Center
Authors: Richau João A. , Leitão Jorge H. , Correia Manuela , Lito Luís , Salgado Maria José , Barreto Celeste , Cescutti Paola , Sá-Correia Isabel
Published: December 2019

This work describes the first epidemiological survey of
Burkholderia cepacia
involved in pulmonary infections among the Portuguese population with cystic fibrosis (CF) who attended the major CF treatment Center in Lisbon at Sta. Maria Hospital from 1995 to the end of 1997. The characterization of the genomic relatedness of the isolates was based on the analysis of their ribopatterns (with
Eco
RI) followed by construction of a ribotype-based phylogenetic tree. This study was complemented with macrorestriction fragment analysis by pulsed-field gel electrophoresis. After optimization of the solid growth medium, we found that exopolysaccharide (EPS) production by
B. cepacia
CF isolates is not as rare a phenomenon as was thought before; indeed, 70% of the isolates examined were EPS producers.
Detection of and Discrimination between Gram-Positive and Gram-Negative Bacteria in Intraocular Samples by Using Nested PCR
Authors: Carroll Nora M. , Jaeger Emma E. M. , Choudhury Sarah , Dunlop Anthony A. S. , Matheson Melville M. , Adamson Peter , Okhravi Narciss , Lightman Susan
Published: December 2019

A nested PCR protocol has been developed for the detection of and discrimination between 14 species of gram-positive and -negative bacteria in samples of ocular fluids. First-round PCR with pan-bacterial oligonucleotide primers, based on conserved sequences of the 16S ribosomal gene, was followed by a gram-negative-organism-specific PCR, which resulted in a single 985-bp amplification product, and a multiplex PCR which resulted in two PCR products: a 1,025 bp amplicon (all bacteria) and a 355 bp amplicon (gram-positive bacteria only). All products were detected by gel electrophoresis. The sensitivity of the assay was between 10 fg and 1 pg of bacterial DNA, depending on the species tested, equivalent to between 24 and 4 live bacteria spiked in water. The identification was complete in 3.5 h. The molecular techniques were subsequently applied to four samples of intraocular fluid, (three vitreous and one aqueous) from three patients with clinical signs of bacterial endophthalmitis (test samples) and two samples of vitreous from a patient with chronic intraocular inflammation (control samples). In all culture-positive samples (two of three vitreous and one of one aqueous), a complete concordance was observed between molecular methods and culture results. PCR correctly identified the gram stain classification of the organisms. The bacterial etiology was also identified in a culture-negative patient with clinical history and signs highly suggestive of bacterial endophthalmitis. Furthermore, control samples from a patient with chronic intraocular inflammation remained PCR negative. In summary, this protocol has demonstrated potential as a rapid diagnostic test in confirming the diagnosis of infection and also determining the Gram status of bacteria with high specificity and sensitivity.
Immunoblot Analysis of Humoral Immune Response to
Helicobacter pylori
in Children with and without Duodenal Ulcer
Authors: Rocha Gifone A. , Oliveira Andreia M. R. , Queiroz Dulciene M. M. , Carvalho Anfrisina S. T. , Nogueira Ana M. M. F.
Published: December 2019

Several studies have demonstrated that enzyme-linked immunosorbent assay is not a sensitive and specific method to diagnose
Helicobacter pylori
infection in children, especially in the younger ones. Since serum immune response can also be determined by immunoblotting and it permits the detection of antibodies to virulence factors such as CagA and VacA, we evaluated the accuracy of a commercial immunoblotting test to diagnose
H. pylori
infection and to assess the humoral immune response to different
H. pylori
antigens in 122 children who underwent upper gastrointestinal endoscopy. The presence of
H. pylori
was determined in antral biopsy specimens by culture, preformed urease test, and histological analysis.
H. pylori
was identified by microbiological and histopathological methods in 66 children (including all of the 21 who had duodenal ulcer). Antibodies to
H. pylori
were detected in 63 infected children and in 8 noninfected ones. The sensitivity, specificity, and positive and negative predictive values of the immunoblotting test were 95.5, 85.7, 88.7, and 94.1%, respectively. The number of immunoreactive bands increased with age (
P
= 0.003), and the bands of 35 kDa (
P
= 0.013); 89 kDa, the VacA antigen (
P
= 0.001); and 116 kDa, the CagA antigen (
P
= 0.00004) were more frequently observed in older children. The frequency of the bands of 89 kDa (
P
= 0.001) and 116 kDa (
P
= 0.03) was higher in children with duodenal ulcer than in
H. pylori
-positive children without the disease. In conclusion, the immunoblotting test appears to be useful for the diagnosis of
H. pylori
infection in children, even in the younger ones.
Sequence Diversity of the
Escherichia coli
H7
fliC
Genes: Implication for a DNA-Based Typing Scheme for
E. coli
O157:H7
Authors: Wang Lei , Rothemund Deborah , Curd Heather , Reeves Peter R.
Published: December 2019

Flagellar (H) antigens are mostly encoded by genes at the
fliC
locus in
E. coli
. We have sequenced 11 H7
fliC
genes from
Escherichia coli
strains that belong to seven O serotypes. These sequences, together with those of nine other H7
fliC
genes (from strains of three different O serotypes) sequenced recently (S. D. Reid, R. K. Selander, and T. S. Whittam, J. Bacteriol. 181:153–160, 1999), include 10 different sequences. The differences between these 10 sequences range from 0.06 to 3.12%. By comparison with other
E. coli
flagellin genes, we have identified primer length sequences specific for H7 genes in general and others specific for H7 genes of O157 and O55 strains: the specificity was confirmed by PCR testing the type strains for all 53
E. coli
H types. We have previously identified genes specific for the
E. coli
O157 antigen, and use of the combination of O157- and H7-specific primers allows the sensitive and rapid detection of O157:H7
E. coli
strains, which cause the majority of hemorrhagic colitis cases.
Development of a Fluorescence Polarization-Based Diagnostic Assay for Equine Infectious Anemia Virus
Authors: Tencza Sarah Burroughs , Islam Kazi R. , Kalia Vandana , Nasir Mohammad S. , Jolley Michael E. , Montelaro Ronald C.
Published: December 2019

The control of equine infectious anemia virus (EIAV) infections of horses has been over the past 20 years based primarily on the identification and elimination of seropositive horses, predominantly by a standardized agar gel immunodiffusion (AGID) assay in centralized reference laboratories. This screening for EIAV-seropositive horses has been to date hindered by the lack of a rapid diagnostic format that can be easily employed in the field. We describe here the development of a rapid solution-phase assay for the presence of serum antibodies to EIAV based on fluorescence polarization (FP) (patent pending). Peptides derived from antigenic regions of EIAV core and envelope proteins were initially screened for their utility as probes in an FP assay to select the best peptide antigen candidates. The FP assay was optimized to detect the presence of EIAV-specific antibodies by a change in the FP of a fluorescein-labeled immunoreactive peptide diagnostic antigen. The most sensitive and specific peptide probe was a peptide corresponding to the immunodominant region of the EIAV transmembrane protein, gp45. This probe was tested for its reactivity in the optimized FP assay with 151 AGID-positive horse sera and 106 AGID-negative serum samples. The results of these studies demonstrated that the FP assay reactivity correlated with reported AGID results in 106 of 106 negative serum samples (100% specificity) and in 135 of 151 positive serum samples (89.4% sensitivity). The FP assay was also found to have a very low background reactivity and to readily detect antibodies produced early in infection (≤3 weeks postinfection). The developed EIAV FP assay is rapid (5 to 20 min) and simple to perform and is equally suitable for use both in the field and in the diagnostic laboratory, thus providing the basis of an improved commercial diagnostic assay for EIAV infection of horses.