bluuebunny/crossref_metadata_2025 huggingface dataset:
Mailed, Home-Obtained Urine Specimens: a Reliable Screening Approach for Detecting Asymptomatic
Chlamydia trachomatis
Infections
Authors: Morré Servaas A. , van Valkengoed Irene G. M. , de Jong Afke , Boeke A. Joan P. , van Eijk Jacques T. M. , Meijer Chris J. L. M. , van den Brule Adriaan J. C.
Published: December 2019

The use of mailed, home-obtained urine specimens could facilitate screening programs for the detection of asymptomatic
Chlamydia trachomatis
infections. Since transport time could have an adverse effect on the sensitivity of
C. trachomatis
detection by PCR, the influence of DNA degradation on amplification was monitored over the course of 1 week. Therefore, urine specimens were aliquoted on the day of collection or arrival. Two groups of urine specimens were investigated. Group I contains first-void
C. trachomatis
-positive and -negative urine samples. DNA degradation was monitored in group I samples for 7 days at room temperature (RT) and at 4°C by amplifying different lengths of the human β-globin gene and the
C. trachomatis
plasmid target. DNA degradation was observed only for the larger human β-globin fragments at days 5 to 7 at RT. In contrast, at 4°C all targets could be amplified. Urine specimens were also frozen and thawed before aliquoting to mimic freezing during transport. This resulted in a lower sensitivity for the detection of
C. trachomatis
after thawing and 3 to 4 days at RT. In addition, mailed, home-obtained
C. trachomatis
-positive urine specimens (group II) were analyzed for 7 days after arrival by two commercially available
C. trachomatis
detection systems (PCR and ligase chain reaction [LCR]). The
C. trachomatis
plasmid target in mailed, home-obtained urine specimens could be amplified by both PCR and LCR after 1 week of storage and/or transport at RT. In conclusion, our findings indicate that mailed, home-obtained urine specimens are suitable for the sensitive detection of asymptomatic
C. trachomatis
infections by amplification methods, even if the transport time is up to 1 week at RT. These findings support the feasibility and validity of screening programs based on mailed, home-obtained urine specimens. Larger studies should be initiated to confirm our results.
Rapid and Sensitive Quantification of
Borrelia burgdorferi
-Infected Mouse Tissues by Continuous Fluorescent Monitoring of PCR
Authors: Morrison Tom B. , Ma Ying , Weis John H. , Weis Janis J.
Published: December 2019

The quantity of
Borrelia burgdorferi
organisms in tissue samples is an important determinant for infection studies in the mouse model of Lyme disease. This report presents the development of a rapid and sensitive external-standard-based PCR assay for the absolute quantification of
B. burgdorferi
in mouse tissue samples. The assay uses a double-stranded DNA dye to continuously monitor product formation and in less than an hour was able to quantify samples ranging up to 6 log units in concentration. The PCR efficiencies of the sample and the standard were matched by using a standard composed of purified
B. burgdorferi
chromosome mixed with tissue-matched mouse genome lacking bacterial DNA. Normalization of
B. burgdorferi
quantities to the mouse
nidogen
gene allowed comparison of
B. burgdorferi
numbers in samples isolated from different tissues and strains. PCR analysis of the chromosomal gene
recA
in cultured
B. burgdorferi
was consistent with a single
recA
per bacterium. The parameters defined in this assay should be applicable to quantification of other organisms, even infectious agents for which no ready source of DNA standard is available. In summary, this report presents a rapid external-standard-based PCR method for the quantification of
B. burgdorferi
in mouse DNA samples.
Prevalence ofMycobacterium aviumin Slaughter Pigs in The Netherlands and Comparison of IS1245Restriction Fragment Length Polymorphism Patterns of Porcine and Human Isolates
Authors: Komijn Ruud E. , de Haas Petra E. W. , Schneider Margriet M. E. , Eger Tony , Nieuwenhuijs Jan H. M. , van den Hoek Remco J. , Bakker Douwe , van Zijderveld Fred G. , van Soolingen Dick
Published: December 2019

A significant increase in the incidence of caseous lesions in the lymph nodes of slaughter pigs prompted a large-scale investigation in five slaughterhouses in The Netherlands. In total, 158,763 pigs from 2,899 groups underwent gross examination. At least one pig with caseous lesions in the submaxillary and/or mesenteric lymph nodes was observed in each of 154 of the 2,899 groups examined (5%). In total, 856 pigs (0.5%) were affected. As many as five pigs in each of 141 of the 154 positive groups (91.5%) had lymph node lesions. Greater numbers of pigs with affected lymph nodes were found in 13 groups (8.5%). Four pigs had lesions in the kidneys, liver, or spleen. Acid-fast bacteria were detected by microscopic examination of 121 of 292 Ziehl-Neelsen-stained smears of caseous lesions (41%). In a follow-up study,Mycobacterium aviumcomplex (MAC) bacteria were isolated from 219 of 402 affected lymph nodes (54.2%). Ninety-one of the isolated strains were analyzed by restriction fragment length polymorphism (RFLP) typing with insertion sequence IS1245as a probe. All but 1 of these 91 strains contained IS1245DNA, indicating that pigs in The Netherlands carried almost exclusivelyM. aviumbacteria and no other bacteria of MAC. Only one pig isolate exhibited the bird-type RFLP pattern. MAC isolates from 191 human patients in The Netherlands in 1996 were also typed by RFLP analysis. Computer-assisted analysis showed that the RFLP patterns of 61% of the human isolates and 59% of the porcine isolates were at least 75% similar to the RFLP patterns of the other group of strains. This indicates that pigs may be an important vehicle forM. aviuminfections in humans or that pigs and humans share common sources of infection.
Detection and Species Determination of Malaria Parasites by PCR: Comparison with Microscopy and with ParaSight-F and ICT Malaria Pf Tests in a Clinical Environment
Authors: Tham Jill M. , Lee Szu Hee , Tan Theresa M. C. , Ting Robert C. Y. , Kara Ursula A. K.
Published: December 2019

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients.
Plasmodium
infections were identified with a genus-specific primer set, and species differentiation between
Plasmodium falciparum
and
Plasmodium vivax
was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed
P. falciparum-P. vivax
infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays for
P. falciparum
, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive for
P. vivax
at the time of discharge were later readmitted to the hospital with a recurrence of
P. vivax
infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria.
GB Virus C/Hepatitis G Virus Groups and Subgroups: Classification by a Restriction Fragment Length Polymorphism Method Based on Phylogenetic Analysis of the 5′ Untranslated Region
Authors: Quarleri J. F. , Mathet V. L. , Feld M. , Ferrario D. , della Latta M. P. , Verdun R. , Sánchez D. O. , Oubiña J. R.
Published: December 2019

A phylogenetic tree based on 150 5′ untranslated region sequences deposited in GenBank database allowed segregation of the sequences into three major groups, including two subgroups, i.e., 1, 2a, 2b, and 3, supported by bootstrap analysis. Restriction site analysis of these sequences predicted that
Hin
fI and either
Aat
II or
Aci
I could be used for genomic typing with 99.4% accuracy. cDNA sequencing and subsequent alignment of 21 Argentine GB virus C/hepatitis G virus strains confirmed restriction fragment length polymorphism patterns theoretically predicted. This method may be useful for a rapid screening of samples when either epidemiological or transmission studies of this agent are carried out.
Detection of Multidrug-Resistant
Salmonella enterica
Serotype
typhimurium
DT104 Based on a Gene Which Confers Cross-Resistance to Florfenicol and Chloramphenicol
Authors: Bolton Lance F. , Kelley Lynda C. , Lee Margie D. , Fedorka-Cray Paula J. , Maurer John J.
Published: December 2019

Salmonella enterica
serotype
typhimurium
(
S. typhimurium
) DT104 (DT104) first emerged as a major pathogen in Europe and is characterized by its pentadrug-resistant pattern. It has also been associated with outbreaks in the United States. The organism typically carries resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. The mechanism of chloramphenicol resistance in DT104 was determined by producing antibiotic-resistant
Escherichia coli
host strain clones from DT104 DNA. DNA from chloramphenicol-resistant clones was sequenced, and probes specific for the genes

flo
S. typhimurium

(

flo
St

),
int
,
invA
, and
spvC
were produced for colony blot hybridizations. One hundred nine
Salmonella
isolates, including 44 multidrug-resistant DT104 isolates, were tested to evaluate the specificities of the probes. The gene

flo
St

, reported in this study, confers chloramphenicol and florfenicol resistance on
S. typhimurium
DT104. Florfenicol resistance is unique to
S. typhimurium
DT104 and multidrug-resistant
S. typhimurium
isolates with the same drug resistance profile among all isolates evaluated. Of 44 DT104 isolates tested, 98% were detected based on phenotypic florfenicol resistance and 100% had the

flo
St

-positive genotype. Resistances to florfenicol and chloramphenicol are conferred by the gene

flo
St

, described in this paper. Presumptive identification of
S. typhimurium
DT104 can be made rapidly based on the presence of the

flo
St

gene or its resulting phenotype.
Comparison of Cytomegalovirus Loads in Plasma and Leukocytes of Patients with Cytomegalovirus Retinitis
Authors: Jabs Douglas A. , Forman Michael , Enger Cheryl , Jackson J. Brooks
Published: December 2019

Cytomegalovirus (CMV) DNA loads in paired leukocyte and plasma samples from 199 patient visits by 66 patients with CMV retinitis were determined. Leukocyte CMV load determinations had a greater range of values (mean, 24,587 copies/10
6
leukocytes; maximum, 539,000) than did plasma CMV load determinations (mean, 10,302 copies/ml; maximum, 386,000), and leukocyte viral loads were detectable in a greater proportion of patients at the time of diagnosis of CMV retinitis prior to initiation of anti-CMV therapy (82%) than were plasma viral loads (64%) (
P
= 0.0078). Agreement with CMV blood cultures was slightly better for plasma (κ = 0.68) than for leukocytes (κ = 0.53), due to a greater proportion of patients with detectable viral loads in leukocytes having negative blood cultures.
Methicillin-ResistantStaphylococcus aureusOutbreak in a Veterinary Teaching Hospital: Potential Human-to-Animal Transmission
Authors: Seguin Jennifer C. , Walker Robert D. , Caron John P. , Kloos Wesley E. , George Carol G. , Hollis Richard J. , Jones Ronald N. , Pfaller Michael A.
Published: December 2019

During a 13-month period, 11 equine patients visiting a veterinary teaching hospital for various diagnostic and surgical procedures developed postprocedural infections from which methicillin (oxacillin)-resistantStaphylococcus aureus(MRSA) strains were isolated. TheS. aureusisolates were identified by conventional methods that included Gram staining, tests for colonial morphology, tests for clumping factor, and tests for coagulase and urease activities and were also tested with the API STAPH IDENT system. Antimicrobial susceptibility tests were performed by the disk diffusion method. The biochemical profile and antibiogram of each isolate suggested that the isolates may have come from a common source. Because MRSA strains are very uncommon animal isolates but are rather common human isolates, a nasal swab specimen for culture was collected voluntarily from five persons associated with equine surgery and recovery in an attempt to identify a possible source of the organisms. MRSA strains were isolated from three of the five people, with one person found to be colonized with two biotypes of MRSA. The MRSA isolates from the people appeared to be identical to the isolates from horses. Further study of the isolates includedSmaI andEagI macrorestriction analysis by pulsed-field gel electrophoresis conducted in two different laboratories. The results indicated that both the equine and human isolates were members of a very closely related group which appear to have originated from a common source. On the basis of the pattern associated with the infection, it is speculated that the members of the Veterinary Teaching Hospital staff were the primary source of the infection, although the specific mode of transmission is unclear.
Mycoplasma penetrans
and Other Mycoplasmas in Urine of Human Immunodeficiency Virus-Positive Children
Authors: Hussain Althaf I. , Robson William Lane M. , Kelley Robin , Reid Tanya , Gangemi J. David
Published: December 2019

Urine samples from children with human immunodeficiency virus (HIV) infection and healthy controls were examined for mycoplasmas by culture. Standard biochemical assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and PCR (16S and 16S-23S spacer rRNA region) were used for identification of isolates. Mycoplasmas were identified from 13 (87%) of 15 HIV-positive patients and 3 (20%) of 15 HIV-negative control patients. The frequency and type of mycoplasma varied with the severity of HIV infection.
Mycoplasma penetrans
,
Mycoplasma pirum
,
Mycoplasma fermentans
, and
Mycoplasma genitalium
were isolated from patients with severe immunodeficiency.
Mycoplasma hominis
and
Ureaplasma urealyticum
were isolated more frequently from children in the early stages of HIV infection and from HIV-negative patients.
Mycoplasma penetrans
was isolated from one (50%) of two patients in Centers for Disease Control and Prevention (CDC) group B and from five (55.5%) of nine pediatric patients with AIDS (CDC group C). This is the first report that indicates that “AIDS-associated” mycoplasmas are more common in HIV-infected children than in HIV-negative controls.
Clinical and Pathologic Evaluation of ChronicBartonella henselaeorBartonella clarridgeiaeInfection in Cats
Authors: Kordick Dorsey L. , Brown Talmage T. , Shin KwangOk , Breitschwerdt Edward B.
Published: December 2019

HumanBartonellainfections result in diverse medical presentations, whereas many cats appear to tolerate chronic bacteremia without obvious clinical abnormalities. Eighteen specific-pathogen-free cats were inoculated withBartonella henselae- and/orBartonella clarridgeiae-infected cat blood and monitored for 454 days. Relapsing bacteremia did not correlate with changes in protein profiles or differences in antigenic protein recognition. Intradermal skin testing did not induce a delayed type hypersensitivity reaction to cat scratch disease skin test antigen. Thirteen cats were euthanatized at the end of the study. Despite persistent infection, clinical signs were minimal and gross necropsy results were unremarkable. Histopathology revealed peripheral lymph node hyperplasia (in all of the 13 cats), splenic follicular hyperplasia (in 9 cats), lymphocytic cholangitis/pericholangitis (in 9 cats), lymphocytic hepatitis (in 6 cats), lymphoplasmacytic myocarditis (in 8 cats), and interstitial lymphocytic nephritis (in 4 cats). Structures suggestive ofBartonellawere visualized in some Warthin-Starry stained sections, andBartonellaDNA was amplified from the lymph node (from 6 of the 13 cats), liver (from 11 cats) heart (from 8 cats), kidney (from 9 cats), lung (from 2 cats), and brain (from 9 cats). This study indicates thatB. henselaeorB. clarridgeiaecan induce chronic infection following blood transfusion in specific-pathogen-free cats and thatBartonellaDNA can be detected in blood, brain, lymph node, myocardium, liver, and kidney tissues of both blood culture-positive cats and blood culture-negative cats. Detection of histologic changes in these cats supports a potential etiologic role forBartonellaspecies in several idiopathic disease processes in cats.
Ability of a Rapid Serology Test To Detect Seroconversion to Herpes Simplex Virus Type 2 Glycoprotein G Soon after Infection
Authors: Ashley Rhoda L. , Eagleton Marie , Pfeiffer Natasha
Published: December 2019

Sera (
n
= 188) from 29 patients with first-episode genital herpes simplex virus type 2 (HSV-2) infections were tested by POCkit-HSV-2 and Western blot (WB) to determine the speed of seroconversion. The median time to detection of HSV-2 antibody was 13 days (range, 3 to 102 days) by the POCkit-HSV-2 test versus 13 days (range, 2 to 58 days) for WB.
Cytolethal Distending Toxin Genes in
Campylobacter jejuni
and
Campylobacter coli
Isolates: Detection and Analysis by PCR
Authors: Eyigor Aysegul , Dawson Karl A. , Langlois Bruce E. , Pickett Carol L.
Published: December 2019

Campylobacter jejuni
produces a toxin called cytolethal distending toxin (CDT). Knowledge of the prevalence and homogeneity of
Campylobacter
sp.
cdt
genes is incomplete. In this work, we identified four PCR primer pairs that collectively amplified
cdt
genes in all of the
C. jejuni
and
Campylobacter coli
strains tested. Restriction analyses of the
cdt
PCR products showed clear differences between the
cdt
genes of these two species, yet there were few heterogeneities noted between members of the same species. Consequently, it may be possible to speciate
C. jejuni
and
C. coli
isolates on the basis of restriction patterns within their
cdt
genes.
Assessment of Strain Relatedness among
Salmonella
Serotypes Salinatis, Duisburg, and Sandiego by Biotyping, Ribotyping, IS
200
Fingerprinting, and Pulsed-Field Gel Electrophoresis
Authors: Old David C. , Rankin Shelley C. , Crichton Pamela B.
Published: December 2019

Salinatis (antigenic formula, 4,12:d:eh:enz
15
) is a rare
Salmonella
serotype currently designated a triphasic variant of the diphasic serotype Duisburg (
1
,4,12,
27
:d:enz
15
) (underlining indicates that the O antigen is determined by phage lysogenization). Salinatis could also be related to serotype Sandiego (4,[5],12:eh:enz
15
), from which it might have been derived by loss of H-d flagellin genes. Nineteen
Salmonella
strains of serotypes Salinatis, Duisburg, and Sandiego were examined by biotyping,
Pvu
II and
Sma
I ribotyping, IS
200
fingerprinting, and pulsed-field gel electrophoretic profiling. Results from these methods, used alone or together, indicate that serotype Salinatis is more likely to be related to serotype Sandiego than to serotype Duisburg. For future lists of serotype names, it is recommended that Salinatis be considered a variant of Sandiego.
Use of a Murine O-Antigen-Specific Monoclonal Antibody To IdentifyAcinetobacterStrains of Unnamed Genomic Species 13 Sensu Tjernberg and Ursing
Authors: Pantophlet Ralph , Brade Lore , Brade Helmut
Published: December 2019

A monoclonal antibody against the O-antigenic polysaccharide chain of the lipopolysaccharide (LPS) ofAcinetobacterstrains belonging to the unnamed genomic species 13 Sensu Tjernberg and Ursing (13TU) was obtained after immunization of BALB/c mice with heat-killed bacteria and was characterized by enzyme immunoassay and Western blot analysis, by use of LPS and proteinase K-treated bacterial lysates, analyses in which the antibody was shown to be highly specific for the homologous antigen. In addition, when tested in dot and Western blots, reactivity was observed with 9 of 18Acinetobacterstrains of genomic species 13TU which had been isolated in Germany and Denmark; no reactivity was observed with strains of other genomic species, including the closely related genomic groups 1 (A. calcoaceticus), 2 (A. baumannii), and 3 (unnamed), or with other gram-negative bacteria. The antibody described here represents a convenient reagent for the simple, economical, and accurate differentiation of clinical isolates of genomic species 13TU from otherAcinetobacterstrains. Although the antibody does not identify all isolates of this genomic group, it is evident that it will be a useful reagent in the development of a serotyping scheme for clinical laboratories.
High Prevalence of TT Virus Infection in Healthy Children and Adults and in Patients with Liver Disease in Taiwan
Authors: Hsieh Sen-Yung , Wu Yin-Hua , Ho Yu-Pin , Tsao Kuo-Chien , Yeh Chau-Ting , Liaw Yun-Fan
Published: December 2019

A newly identified DNA virus, named TT virus (TTV), was found to be related to transfusion-associated hepatitis. We conducted the following experiments to evaluate its pathogenic role in liver disease and potential modes of transmission. We used PCR to detect TTV DNA in serum. The rates of TTV viremia in 13 patients with idiopathic acute hepatitis, 14 patients with idiopathic fulminant hepatitis, 22 patients with chronic hepatitis, and 19 patients with cirrhosis of the liver were 46, 64, 55, and 63%, respectively, and were not significantly different from those in 50 healthy control subjects (53%). PCR products derived from seven patients with liver disease and three healthy controls were cloned and then subjected to phylogenetic analyses, which failed to link a virulent strain of TTV to severe liver disease. TTV infection was further assessed in an additional 148 subjects with normal liver biochemical tests, including 30 newborns (sera collected from the umbilical cord), 23 infants, 16 preschool children, 21 individuals of an age prior to that of sexual experience (aged 6 to 15 years), 15 young adults (aged under 30 years), and 43 individuals older than 30 years. The rates of TTV viremia were 0, 17, 25, 33, 47, and 54%, respectively. These findings suggest that TTV is transmitted mainly via nonparenteral daily contact and frequently occurs very early in life and that TTV infection does not have a significant effect on liver disease.
Serotyping of Adenoviruses on Conjunctival Scrapings by PCR and Sequence Analysis
Authors: Takeuchi Satoshi , Itoh Norihiko , Uchio Eiichi , Aoki Koki , Ohno Shigeaki
Published: December 2019

To detect and identify adenovirus (Ad), we investigated hypervariable regions (HVRs) of Ad by using a combination of PCR and direct sequencing (PCR-sequence) method. Primers for nested PCR to amplify the conserved region in the hexon protein containing HVRs were designed based on hexon gene sequences derived from GenBank. These two primer sets amplified a DNA fragment of 7 HVRs from 16 prototypes of Ad, which were divided into five subgenera, including seven serotypes that are the predominant causative agents of acute conjunctivitis in Japan, and from 31 recent conjunctival scraping specimens from patients with adenoviral conjunctivitis. HVR DNA sequences were determined by means of universal sequence primers. Analysis of the predicted amino acid homology of HVRs among Ad prototypes suggested three regions, HVR4, -5, and -7, to be candidates for the neutralization epitopes. The clinical serotype of specimens was determined by the PCR-sequence method with reference to these three HVRs. The serotype determined according to this method was identical to that obtained by culture isolation and the neutralization test (NT) in all scraping samples, whereas the results of this method did not match PCR and restriction fragment length polymorphism (PCR-RFLP) analysis in five samples. It took only three days to detect Ad and to identify the serotype, in contrast to culture isolation-NT, which took at least 2 weeks. These findings indicate that our newly developed PCR-sequence method is applicable for the detection and serotyping of human Ads.
Evaluation of Three Nucleic Acid Amplification Methods for Direct Detection of
Mycobacterium tuberculosis
Complex in Respiratory Specimens
Authors: Wang S. X. , Tay L.
Published: December 2019

Two hundred thirty respiratory specimens from 230 patients were analyzed by using COBAS AMPLICOR PCR, Amplified
Mycobacterium tuberculosis
Direct Test, and ligase chain reaction methods. Results were compared with those of smear microscopy and radiometric culture (Bactec) methods. No significant differences were observed among the results of the three methods, which are acceptable for direct detection of
M. tuberculosis
complex in respiratory specimens.
Development of a High-Throughput Quantitative Assay for Detecting Herpes Simplex Virus DNA in Clinical Samples
Authors: Ryncarz Alexander J. , Goddard James , Wald Anna , Huang Meei-Li , Roizman Bernard , Corey Lawrence
Published: December 2019

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 10
8
copies of HSV DNA/20 μl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.
Characterization of
Bacteroides forsythus
Strains from Cat and Dog Bite Wounds in Humans and Comparison with Monkey and Human Oral Strains
Authors: Hudspeth M. K. , Gerardo S. Hunt , Maiden M. F. J. , Citron D. M. , Goldstein E. J. C.
Published: December 2019

Bacteroides forsythus
strains recovered from cat and dog bite wound infections in humans (
n
= 3), monkey oral strains (
n
= 3), and the human oral ATCC 43037 type strain were characterized by using phenotypic characteristics, enzymatic tests, whole cell fatty acid analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, PCR fingerprinting, and 16S rDNA (genes coding for rRNA) sequencing. All three bite wound isolates grew on brucella agar supplemented with 5% sheep blood, vitamin K
1
, and hemin. These strains, unlike the ATCC strain and previously described monkey oral and human clinical strains, did not require
N
-acetylmuramic acid supplementation for growth as pure cultures. However, their phenotypic characteristics, except for catalase production, were similar to those of previously identified strains. PCR fingerprinting analysis showed differences in band patterns from the ATCC strain. Also, SDS-PAGE and whole cell fatty acid analysis indicated that the dog and cat bite wound strains were similar but not identical to the human
B. forsythus
ATCC 43037 type strain and the monkey oral strains. The rDNA sequence analysis indicated that the three bite wound isolates had 99.93% homology with each other and 98.9 and 99.22% homology with the human ATCC 43037 and monkey oral strains, respectively. These results suggest that there are host-specific variations within each group.
Detection of Toscana Virus-Specific Immunoglobulins G and M by an Enzyme-Linked Immunosorbent Assay Based on Recombinant Viral Nucleoprotein
Authors: Ciufolini Maria Grazia , Fiorentini Cristiano , di Bonito Paola , Mochi Stefania , Giorgi Colomba
Published: December 2019

An enzyme-linked immunosorbent assay (ELISA) based on the recombinant Toscana virus nucleoprotein (rN) has been developed. Its sensitivity and specificity for the detection of virus-specific immunoglobulins G and M in human sera were similar to those of the ELISA that is based on an antigen extracted from infected mouse brain and that is routinely used for serodiagnosis.
Evaluation of the New MB Redox System for Detection of Growth of Mycobacteria
Authors: Cambau Emmanuelle , Wichlacz Claudine , Truffot-Pernot Chantal , Jarlier Vincent
Published: December 2019

We evaluated a new mycobacterial culture system, MB Redox, for recovery rate and time to detection of mycobacteria from 742 consecutive respiratory specimens and compared the results to those found with Löwenstein-Jensen (LJ) medium. Twenty specimens (2.7%) were positive for
M. tuberculosis
: 17 on LJ medium and 19 in MB Redox, with 16 specimens positive in both media. In addition, 24 specimens (3.2%) were positive for nontuberculous mycobacteria (NTM), 20 on LJ medium, 18 in MB Redox, and 14 in both media. For
M. tuberculosis
, the mean times to detection were 28.9 days on LJ medium and 23.6 days in MB Redox, and for NTM, the mean times to detection were 40.6 days on LJ medium and 32.3 days in MB Redox.
Times to Detection of Bacteria and Yeasts in BACTEC 9240 Blood Culture Bottles
Authors: Reisner Barbara S. , Woods Gail L.
Published: December 2019

A 7-day incubation protocol was instituted with the BACTEC 9240 system for a 1-year period to determine the times to detection of clinically relevant organisms. A total of 23,686 blood and 693 sterile body fluid cultures were received; some cultures were held longer by special request. Of 1,609 likely skin contaminants, 42 were recovered on day 5, 34 on day 6, 16 on day 7, and 5 on day 8. Of 2,803 usual pathogens, 34 were recovered on day 5, 24 on day 6, 15 on day 7 and 1 on day 8. Twenty-one of the latter organisms were considered significant laboratory isolates because they were the first isolates from the respective patients. Chart review showed that 10 of 21 were considered clinically significant, but only 3 (all yeasts) affected the treatment of the patient. Our data show that 4 days of incubation were sufficient to recover all clinically relevant bacteria and 6 days were required to recover all clinically relevant yeasts.
Southern Extension of the Range of Human Babesiosis in the Eastern United States
Authors: Eskow Eugene S. , Krause Peter J. , Spielman Andrew , Freeman Kathy , Aslanzadeh Jaber
Published: December 2019

We sought evidence of babesiosis in three residents of New Jersey who were suspected of local acquisition of
Babesia microti
infection. We tested serial blood samples from these residents for
B. microti
antibodies and amplifiable DNA by using immunofluorescent antibody and PCR techniques. All three residents experienced symptoms suggestive of acute babesiosis. The sera of each of the patients reacted against babesial antigen at a titer fourfold or higher in sequentially collected blood samples. PCR-amplifiable DNA, characteristic of
B. microti
, was detected in their blood. These data suggest that human
B. microti
infections were acquired recently in New Jersey, extending the range of this piroplasmosis in the northeastern United States.
Amplified-Fragment Length Polymorphism Analysis versus Macro-Restriction Fragment Analysis for Molecular Typing of
Streptococcus pneumoniae
Isolates
Authors: van Eldere Johan , Janssen Paul , Hoefnagels-Schuermans Annette , van Lierde Stefaan , Peetermans Willy E.
Published: December 2019

Forty-eight pneumococci were genotyped by on-line laser fluorescence amplified-fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE) analysis of chromosomal restriction fragments. Overall, the data generated by the two methods corresponded well. However, with AFLP, clusters were delineated at a higher similarity level, and isolate differentiation was more pronounced. AFLP and PFGE were equally efficient for assessing intraserotype diversity. We conclude that AFLP is a useful alternative to PFGE.
Typing of
Salmonella enterica
Serotype Paratyphi C Isolates from Various Countries by Plasmid Profiles and Pulsed-Field Gel Electrophoresis
Authors: Kariuki S. , Cheesbrough J. , Mavridis A. K. , Hart C. A.
Published: December 2019

Pulsed-field gel electrophoresis (PFGE) of 61
Salmonella enterica
serotype Paratyphi C isolates from six countries gave five distinct clusters. Twenty-four isolates from five countries were susceptible to 10 antimicrobials tested and gave similar restriction endonuclease digest patterns of the 38-MDa plasmid. In contrast, plasmid and PFGE profiles of 37 multidrug-resistant isolates from Zaire were different from those from other countries.
Detection of Antibodies to Hepatitis C Virus in Dried Blood Spot Samples from Mothers and Their Offspring in Lahore, Pakistan
Authors: Parker S. P. , Khan H. I. , Cubitt W. D.
Published: December 2019

Dried blood spot samples from mothers and their offspring attending the obstetric and pediatric departments of two hospitals in Lahore, Pakistan, were tested for antibodies to hepatitis C virus (HCV). The seroprevalence of HCV in the women was 6.7% (95% confidence interval [CI], 4.3 to 9.1), and that in the children was 1.3% (95% CI, 0.34 to 2.26). Four anti-HCV immunoglobulin G (IgG)-positive children had mothers that were anti-HCV IgG negative, which suggested that their infection was community acquired.
Relative Frequencies of G and P Types among Rotaviruses from Indian Diarrheic Cow and Buffalo Calves
Authors: Gulati Baldev R. , Nakagomi Osamu , Koshimura Yumi , Nakagomi Toyoko , Pandey Ramayan
Published: December 2019

While an increasing number of studies suggest that there is a high prevalence of rotaviruses with P8[11], a typical P type of bovine rotavirus (BRV), among human neonates or infants in India, no data are available on the distribution of G and P types of Indian BRVs. Thus, fecal specimens were collected from cow and buffalo calves under 1 month of age on organized dairy farms in India during the period between 1994 and 1997, and 36 rotavirus-positive specimens were used to determine the relative frequencies of the G and P types of Indian BRVs. As to the G type, G10 was predominant (83%), followed by G6 (6%). The majority (94%) of BRVs had P8[11], and only one isolate possessed P6[1]. The most common combination of G and P types was G10P8[11] (81%), followed by G6P6[1] (3%) and G6P8[11] (3%). The high prevalence of BRVs possessing P8[11] VP4s strongly supports the hypothesis that BRVs may cross the host species barrier and circulate among neonates in India.
PCR Amplification from Fixed Tissue Indicates Frequent Involvement of
Brachyspira aalborgi
in Human Intestinal Spirochetosis
Authors: Mikosza Andrew S. J. , La Tom , Brooke C. Josephine , Lindboe Christian F. , Ward Peter B. , Heine Ralf G. , Guccion John G. , de Boer W. Bastiaan , Hampson David J.
Published: December 2019

PCR procedures amplifying portions of the 16S rRNA and NADH oxidase genes of
Brachyspira aalborgi
and
Serpulina pilosicoli
were applied to DNA extracted from paraffin-embedded human colonic or rectal tissues from 30 Norwegian, Australian, and U.S. patients, 16 of whom had histologic evidence of intestinal spirochetosis (IS).
B. aalborgi
-specific sequences were identified by PCR in 10 of the IS patients (62.5%) but none of the others, while
S. pilosicoli
sequences were not detected in tissues from any patient. Direct sequencing of products from three of the positive samples provided further confirmation of the presence of
B. aalborgi. B. aalborgi
may be a more common cause of intestinal spirochetosis than has been previously thought.
Primary
Shewanella alga
Septicemia in a Patient on Hemodialysis
Authors: Iwata Morihiro , Tateda Kazuhiro , Matsumoto Tetsuya , Furuya Nobuhiko , Mizuiri Sonoo , Yamaguchi Keizo
Published: December 2019

We report the first Japanese case of primary septicemia with
Shewanella alga
and also describe the bacteriological characteristics of and results of antibiotic susceptibility tests of the isolate.
S. alga
was repeatedly isolated, at times simultaneously with
Escherichia coli
, from the blood of a 64-year-old female undergoing hemodialysis. The isolated organism was determined to be
S. alga
based on recently published identification criteria, such as hemolysis on sheep blood agar, no acid production from carbohydrates, and growth on agar containing 6.5% NaCl. Results of antibiotic susceptibility tests demonstrated that the isolate was sensitive to levofloxacin and cefpirome (MICs, ≤0.063) but resistant to cefazolin, ceftizoxime, and imipenem (MICs, >128, 64, and 8 μg/ml, respectively). Although the role of
S. alga
as a human pathogen has not been fully determined, accumulating data suggest that this organism may be a potential pathogen, especially in compromised hosts.
Mycobacterium bovis
BCG Causing Vertebral Osteomyelitis (Pott’s Disease) Following Intravesical BCG Therapy
Authors: Aljada Ibrahim S. , Crane John K. , Corriere Nancy , Wagle Datta G. , Amsterdam Daniel
Published: December 2019

We report a case of
Mycobacterium bovis
BCG vertebral osteomyelitis in a 79-year-old man 2.5 years after intravesical BCG therapy for bladder cancer. The recovered isolate resembled
M. tuberculosis
biochemically, but resistance to pyrazinamide (PZA) rendered that diagnosis suspect. High-pressure liquid chromatographic studies confirmed the diagnosis of
M. bovis
BCG infection. The patient was originally started on a four-drug antituberculous regimen of isoniazid, rifampin, ethambutol, and PZA. When susceptibility studies were reported, the regimen was changed to isoniazid and rifampin for 12 months. Subsequently, the patient was transferred to a skilled nursing facility for 3 months, where he underwent intensive physical therapy. Although extravesical adverse reactions are rare, clinicians and clinical microbiologists need to be aware of the possibility of disseminated infection by
M. bovis
BCG in the appropriate setting of clinical history, physical examination, and laboratory investigation.
Development of Amplified 16S Ribosomal DNA Restriction Analysis for Identification of
Actinomyces
Species and Comparison with Pyrolysis-Mass Spectrometry and Conventional Biochemical Tests
Authors: Hall Val , O’Neill G. L. , Magee J. T. , Duerden B. I.
Published: December 2019

Identification of
Actinomyces
spp. by conventional phenotypic methods is notoriously difficult and unreliable. Recently, the application of chemotaxonomic and molecular methods has clarified the taxonomy of the group and has led to the recognition of several new species. A practical and discriminatory identification method is now needed for routine identification of clinical isolates. Amplified 16S ribosomal DNA restriction analysis (ARDRA) was applied to reference strains (
n
= 27) and clinical isolates (
n
= 36) of
Actinomyces
spp. and other gram-positive rods. Clinical strains were identified initially to the species level by conventional biochemical tests. However, given the low degree of confidence in conventional methods, the findings obtained by ARDRA were also compared with those obtained by pyrolysis-mass spectrometry. The ARDRA profiles generated by the combination of
Hae
III and
Hpa
II endonuclease digestion differentiated all reference strains to the species or subspecies level. The profiles correlated well with the findings obtained by pyrolysis-mass spectrometry and by conventional tests and enabled the identification of 31 of 36 clinical isolates to the species level. ARDRA was shown to be a simple, rapid, cost-effective, and highly discriminatory method for routine identification of
Actinomyces
spp. of clinical origin.
Upper Respiratory Tract Disease in the Gopher Tortoise Is Caused byMycoplasma agassizii
Authors: Brown M. B. , McLaughlin G. S. , Klein P. A. , Crenshaw B. C. , Schumacher I. M. , Brown D. R. , Jacobson E. R.
Published: December 2019

Upper respiratory tract disease (URTD) has been observed in a number of tortoise species, including the desert tortoise (Gopherus agassizii) and the gopher tortoise (Gopherus polyphemus). Clinical signs of URTD in gopher tortoises are similar to those in desert tortoises and include serous, mucoid, or purulent discharge from the nares, excessive tearing to purulent ocular discharge, conjunctivitis, and edema of the eyelids and ocular glands. The objectives of the present study were to determine ifMycoplasma agassiziiwas an etiologic agent of URTD in the gopher tortoise and to determine the clinical course of the experimental infection in a dose-response infection study. Tortoises were inoculated intranasally with 0.5 ml (0.25 ml/nostril) of either sterile SP4 broth (control group;n= 10) or 108color-changing units (CCU) (total dose) ofM. agassizii723 (experimental infection group;n= 9).M. agassiziicaused clinical signs compatible with those observed in tortoises with natural infections. Clinical signs of URTD were evident in seven of nine experimentally infected tortoises by 4 weeks postinfection (p.i.) and in eight of nine experimentally infected tortoises by 8 weeks p.i. In the dose-response experiments, tortoises were inoculated intranasally with a low (101CCU;n= 6), medium (103CCU;n= 6), or high (105CCU;n= 5) dose ofM. agassizii723 or with sterile SP4 broth (n= 10). At all time points p.i. in both experiments,M. agassiziicould be isolated from the nares of at least 50% of the tortoises. All of the experimentally infected tortoises seroconverted, and levels of antibody were statistically higher in infected animals than in control animals for all time points of >4 weeks p.i. (P< 0.0001). Control tortoises in both experiments did not show clinical signs, did not seroconvert, and did not have detectableM. agassiziiby either culture or PCR at any point in the study. Histological lesions were compatible with those observed in tortoises with natural infections. The numbers ofM. agassizii723 did not influence the clinical expression of URTD or the antibody response, suggesting that the strain chosen for these studies was highly virulent. On the basis of the results of the transmission studies, we conclude thatM. agassiziiis an etiologic agent of URTD in the gopher tortoise.
Molecular Epidemiology of an Outbreak of Febrile Gastroenteritis Caused by
Listeria monocytogenes
in Cold-Smoked Rainbow Trout
Authors: Miettinen M. K. , Siitonen A. , Heiskanen P. , Haajanen H. , Björkroth K. J. , Korkeala H. J.
Published: December 2019

Febrile gastroenteritis in five healthy persons was associated with the consumption of vacuum-packed cold-smoked rainbow trout containing
Listeria monocytogenes. L. monocytogenes
isolates from the incriminated fish product lot and the stool samples were all of serotype 1/2a and were indistinguishable by pulsed-field gel electrophoresis employing
Asc
I and
Sma
I.
Chlamydia pneumoniae
in a Free-Ranging Giant Barred Frog (
Mixophyes iteratus
) from Australia
Authors: Berger Lee , Volp Kym , Mathews Sarah , Speare Rick , Timms Peter
Published: December 2019

The koala biovar of
Chlamydia pneumoniae
was identified in lung tissue from a sick, free-ranging giant barred frog (
Mixophyes iteratus
) by using electron microscopy,
C. pneumoniae
-specific fluorescent-antibody staining, cell culture, and sequencing of the
ompA
,
ompB
and 16S rRNA genes. This is the first report of a chlamydial strain infecting both a homeotherm and a poikilotherm and only the fourth host (in addition to humans, koalas, and horses) to be naturally infected with this species of
Chlamydia
. The frog had severe, chronic, mononuclear pneumonia and nonregenerative anemia and pancytopenia.
Typhoid Fever Due to
Salmonella
Kapemba Infection in an Otherwise Healthy Middle-Aged Man
Authors: Sarnighausen Hans-Eckart , Benz Claus , Eickenberg Martin , Bockemühl J. , Tschäpe H. , Riemann Jürgen F.
Published: December 2019

We report the case of a patient with a
Salmonella
Kapemba infection, who suffered, 3 weeks after a holiday in Israel, occurrences of high fever and lower back pain for 10 days and icterus for 2 days before admission. Laboratory findings revealed a slight cholestasis and elevation of acute phase protein levels. In the blood culture a
Salmonella
Kapemba-type organism was cultured. The patient was afebrile for 10 days after hospitalization and then suddenly developed a temperature of 40°C again. At the same time leukopenia, thrombocytopenia, and a rise of D-dimer levels were detected. The patient was admitted to the intensive care unit for a few days, because a disseminated intravascular coagulation was suspected. With magnetic resonance imaging and bone scintigraphy no osteomyelitis or abscess formation could be found. A transesophageal ultrasonography of the heart revealed no signs of endocarditis. In multiple stool cultures no salmonellas could be detected. After antibiotic treatment with ciprofloxacin the fever and lower back pain subsided, and the patient was discharged a fortnight later. This is the first reported case of typhoid fever due to the bacterium
Salmonella
Kapemba.
Genetic Basis for Immunological Aberrations in Poliovirus Sabin Serotype 3 Strains Imported in The Netherlands
Authors: Reimerink Johan H. J. , van der Avoort Harrie G. A. M. , van Loon Anton M. , Koopmans Marion P. G.
Published: December 2019

During the characterization of poliovirus type 3 strains imported in The Netherlands, Sabin serotype 3 strains that reacted with both specific antisera against Sabin-like (vaccine) and non-Sabin-like (wild-type) strains by the intratypic strain differentiation assay have been found. The present study was done to determine the pathogenic potential of these virus strains for humans. Characterization of these so-called double-reactive strains with neutralizing monoclonal antibodies (MAbs) against the major antigenic sites of serotype 3 Sabin virus led to the identification of two groups with different antigenic properties. Six of the seven strains were resistant to neutralization with MAbs against sites 2B and 3B and one strain was neutralized by all the MAbs in a manner similar to that for the Sabin serotype 3 virus. Partial sequencing of the coding regions confirmed the antigenic changes for all six antigenically distinct strains. By inoculation of these viruses into transgenic mice which express the human poliovirus receptor, one strain was identified as highly neurovirulent, three were identified as intermediate, and three were identified as attenuated. Sera from vaccinated persons efficiently neutralized the mutants. Our data suggest that some double-reactive strains are a potential risk to the unvaccinated community but not to the vaccinated population.