bluuebunny/crossref_metadata_2025 huggingface dataset:
Pretreatment with Restriction Enzyme or Bovine Serum Albumin for Effective PCR Amplification of Epstein-Barr Virus DNA in DNA Extracted from Paraffin-Embedded Gastric Carcinoma Tissue
Authors: Satoh Yukio , Takasaka Noriko , Hoshikawa Yoshiko , Osaki Mitsuhiko , Ohfuji Satoshi , Ito Hisao , Kaibara Nobuaki , Kurata Takeshi , Sairenji Takeshi
Published: December 2019

An association between Epstein-Barr virus (EBV) and gastric carcinoma has been studied through the EBV genome present in the carcinoma cells. Recently, we found that EBV DNA in paraffin-embedded gastric carcinoma tissue was detected effectively by PCR after pretreatment of the extracted DNA with a restriction enzyme,
Bam
HI or
Eco
RI. Here, we show that the PCR amplification was also enhanced by pretreatment of the DNA with other restriction enzymes or with bovine serum albumin and several other proteins. Treatment with these proteins may remove a PCR inhibitor(s) in the DNA samples extracted from the paraffin blocks.
Prevalence of Specific Antibodies to
Chlamydia pneumoniae
in Korea
Authors: Choi Tae Yeal , Kim Duck An , Kim Sin Kyung , Kang Jung Oak , Park Sung Soo , Jung Sung Ro
Published: December 2019

To clarify the endemic status of
Chlamydia pneumoniae
in Korea, the incidence of antibodies in 564 serum samples from healthy individuals, patients with respiratory infection, and cord blood specimens was evaluated. We conclude that
C. pneumoniae
infection is highly endemic in Korea and that this infection is associated with acute respiratory diseases.
Identification of Encephalomyocarditis Virus in Clinical Samples by Reverse Transcription-PCR Followed by Genetic Typing Using Sequence Analysis
Authors: Vanderhallen H. , Koenen F.
Published: December 2019

The objective of the present study was to gain a better understanding of the epidemiology of encephalomyocarditis virus (EMCV) infections in pigs by applying molecular techniques. The diagnostic potential of a reverse transcription-PCR (RT-PCR) targeting 286 nucleotides at the 3′ end of the gene which encodes the viral polymerase was assessed with experimental and field samples. In addition, the use of the amplified sequences for an epidemiological study was evaluated. The heart was clearly shown to be the most suitable organ. The detection limit was determined to be 1 viral particle in 100 mg of heart tissue. The sensitivity and specificity of the assay on the basis of the results obtained in this study were 94 and 100%, respectively. Phylogenetic analysis of the amplified sequences classified EMCVs in two distinct lineages. Group A consists of the reference strain ATCC 129B, all isolates collected between 1991 and 1994 in Belgium in association with reproductive failure, and all Greek isolates. All Belgian isolates collected since the first isolation of EMCV in relation to myocardial failure in fatteners in Belgium group together with the isolates from Cyprus (1996 and 1997), Italy (1986 to 1996), and France (1995) in group B irrespective of their pathogenicity. The analyzed part of the 3D gene differed by 13.0% between Groups A and B. In contrast to the sequence homogeneity of the Belgian isolates collected between 1991 and 1994, molecular diversity, which ranged between 0 and 2%, was observed among the Belgian isolates collected in 1995 and 1996. Among all Greek isolates the diversity ranged between 1 and 8%. However, this diversity does not seem to reflect geographical links between the outbreaks. A RT-PCR for the rapid and specific diagnosis of EMCV in a variety of clinical samples followed by nucleotide sequence analysis proved to be valuable for molecular epidemiological studies.
Acquisition and Transmission of the Agent of Human Granulocytic Ehrlichiosis by
Ixodes scapularis
Ticks
Authors: Hodzic Emir , Fish Durland , Maretzki Craig M. , De Silva Aravinda M. , Feng Sunlian , Barthold Stephen W.
Published: December 2019

The purpose of the present study was to investigate the transmission of a human isolate of the agent of human granulocytic ehrlichiosis (HGE agent) from infected mice to larval ticks and to examine the population kinetics of the HGE agent in different stages of the tick life cycle. The HGE agent was quantitated by competitive PCR with blood from infected mice and with
Ixodes scapularis
ticks. The median infectious dose for C3H mice was 10
4
to 10
5
organisms when blood from an infected severe combined immunodeficient mouse was used as an inoculum. Uninfected larval ticks began to acquire infection from infected mice within 24 h of attachment, and the number of HGE agent organisms increased in larval ticks during feeding and after detachment of replete ticks. Molted nymphal ticks, infected as larvae, transmitted infection to mice between 40 and 48 h of attachment. Onset of feeding stimulated replication of the HGE agent within nymphal ticks. These studies suggest that replication of the HGE agent during and after feeding in larvae and during feeding in nymphs is a means by which the HGE agent overcomes inefficiencies in acquisition of infection by ticks and in tick-borne transmission to mammalian hosts.
Rapid Diagnosis of Cholera Caused by
Vibrio cholerae
O139
Authors: Chaicumpa Wanpen , Srimanote Potjanee , Sakolvaree Yuwaporn , Kalampaheti Thareerat , Chongsa-Nguan Manas , Tapchaisri Pramuan , Eampokalap Boonchuay , Moolasart Pikul , Nair G. Balakrish , Echeverria Peter
Published: December 2019

Hybridomas secreting specific monoclonal antibodies (MAbs) to
Vibrio cholerae
serogroup O139 were produced. Six monoclones (hybridomas) secreting MAbs specific only to lipopolysaccharide of
V. cholerae
O139 strains and which did not cross-react to 137 strains of other enteric microorganisms were obtained. These clones were designated 12F5-G11, 12F5-G2, 15F5-H5, 5B9-F8, 14C9-D2, and 6D2-D8. The immunoglobulin (Ig) heavy chain isotypes secreted by these clones were IgG2b, IgG2b, IgG2b, IgM, IgG2b, and IgG3, respectively. Clone 12F5-G11 was selected for mass production of MAb, which was used as a detection reagent in the antigen detection assay for diagnosis of cholera caused by
V. cholerae
O139, and this assay was compared to the conventional bacterial isolation method. Five batches of rectal swab cultures in alkaline-peptone water were collected from 6,497 patients with watery diarrhea. These were 6,310 patients admitted to Bamrasnaradura Infectious Diseases Hospital, 16 patients from Krung Thon Hospital, 78 patients from Bangkok Children’s Hospital, 19 patients from Karen refugee camps, and 74 Indian patients from the National Institute of Cholera and Enteric Diseases, Calcutta, India. The
V. cholerae
O139 isolations from the rectal swab cultures and the antigen detection assays (i.e., the MAb-based dot-blot ELISA) were performed by different persons of different laboratories, and the results were revealed after all specimens had been tested. Of the 6,497 samples tested, the dot-blot ELISA correctly identified 42 of 42
V. cholerae
O139-positive samples and gave a result of positive for three samples which were culture negative for
V. cholerae
O139. The diagnostic sensitivity, specificity, and efficacy of the dot-blot ELISA were 100, 99.95, and 99.26%, respectively. The ELISA is easy to perform and relatively inexpensive. It can test multiple samples at a single time, does not require special equipment, and does not produce great quantities of contaminated waste. Most of all, it reduces the diagnostic time from at least 2 days for the bacterial isolation to less than 90 min. The assay is recommended as a rapid screening test of cholera cases caused by
V. cholerae
O139.
Evaluation of PCR for Detection of DNA Specific for
Aspergillus
Species in Sera of Patients with Various Forms of Pulmonary Aspergillosis
Authors: Yamakami Yuriko , Hashimoto Atsuro , Yamagata Eiji , Kamberi Perparim , Karashima Reiko , Nagai Hiroyuki , Nasu Masaru
Published: December 2019

Pulmonary aspergillosis is classified into invasive, saprophytic, and allergic forms. In this study, we evaluated the usefulness of PCR for differentiating between different forms of aspergillosis or in monitoring disease activity during treatment by detecting DNA specific for
Aspergillus
species in the serum. Nested PCR was used to detect
Aspergillus
DNA in the sera of 30 patients with various forms of pulmonary aspergillosis. The results were compared with those of latex agglutination tests for detecting galactomannan antigen. We also examined the serial changes in the results of nested PCR during and after treatment of a subgroup of patients with invasive pulmonary aspergillosis with amphotericin B. The highest proportion of positive nested PCR results were in patients with invasive aspergillosis (10 of 12; 83%), while patients with pulmonary aspergilloma had the lowest frequency of positive tests (1 of 9; 11%). These results suggested that the sensitivity of the nested PCR depends on the extent of invasion by
Aspergillus
species. Serial assays showed that the results of nested PCR became negative shortly after commencement of antifungal treatment and that such changes did not correlate with clinical responsiveness to treatment. Our results indicate the potential usefulness of nested PCR with serum samples for the diagnosis of invasive aspergillosis and the detection of a shift in the status of infection from a noninvasive type to invasive aspergillosis. However, the results of the nested PCR did not correlate with the response to antifungal treatment.
Critical Evaluation of 4-Week Incubation for Fungal Cultures: Is the Fourth Week Useful?
Authors: Labarca Jaime A. , Wagar Elizabeth A. , Grasmick Ann E. , Kokkinos Heidi M. , Bruckner David A.
Published: December 2019

To determine the benefit of a 4-week incubation for mycology cultures, we evaluated all positive cultures during the fourth week of incubation in a 1-year period. Of 3,855 positive mycology cultures (yeast, 82%; molds, 18%), 62 (1.6%) were positive during the fourth week (yeast, 42%; molds, 58%). Only 15 of the 62 cultures (24%) were considered clinically relevant (2 isolates from invasive fungal infection and 13 isolates from cutaneous mycosis). With the exception of those from skin samples, isolates recovered during the fourth week are rarely important for patient care.
Prevalence of Astrovirus Infection among Chilean Children with Acute Gastroenteritis
Authors: Gaggero Aldo , O’Ryan Miguel , Noel Jacqueline S. , Glass Roger I. , Monroe Stephan S. , Mamani Nora , Prado Valeria , Avendaño Luis F.
Published: December 2019

The frequency of astrovirus infection in 456 Chilean children with diarrhea was determined by enzyme-linked immunosorbent assay, reverse transcriptase PCR, and cell culture. Astrovirus was detected in 16.5% of rotavirus-negative and 7% of rotavirus-positive samples obtained from emergency rooms or hospitals and in 11% of samples from day care centers. HAst-1 was the predominant serotype identified.
Differentiation of
Corynebacterium amycolatum
,
C. minutissimum
, and
C. striatum
by Carbon Substrate Assimilation Tests
Authors: Renaud François N. R. , Dutaur Marianne , Daoud Salah , Aubel Dominique , Riegel Philippe , Monget Daniel , Freney Jean
Published: December 2019

We tested the carbon substrate assimilation patterns of 40
Corynebacterium amycolatum
strains, 19
C. minutissimum
strains, 50
C. striatum
strains, and 1
C. xerosis
strain with the Biotype 100 system (bioMérieux, Marcy-l’Étoile, France). Twelve carbon substrates of 99 allowed discrimination among the species tested. Additionally, assimilation of 3 of these 12 carbon substrates (maltose, N -acetyl-
d
-glucosamine, and phenylacetate) was tested with the API 20 NE identification system (bioMérieux). Since concordant results were observed with the two systems for these three carbon substrates, either identification system can be used as a supplementary tool to achieve phenotypic differential identification of
C. amycolatum
,
C. minutissimum
, and
C. striatum
in the clinical microbiology laboratory.
Toxoplasma gondii
-Associated Guillain-Barré Syndrome in an Immunocompetent Patient
Authors: Bossi Philippe , Caumes Eric , Paris Luc , Dardé Marie-Laure , Bricaire François
Published: December 2019

We report a case of Guillain-Barré syndrome associated with an acute, disseminated toxoplasmosis due to a new and unknown zymodeme (zymodeme 12) in an immunocompetent patient. The patient’s condition improved with pyrimethamine and sulfadiazine. Only the standard treatment for toxoplasmosis was effective, whereas intravenous immunoglobulins were ineffective.
Survival of Vancomycin-Resistant and Vancomycin-Susceptible Enterococci on Dry Surfaces
Authors: Wendt Constanze , Wiesenthal Bettina , Dietz Ekkehart , Rüden Henning
Published: December 2019

We compared the abilities of
Enterococcus faecium
strains (three vancomycin-resistant enterococci [VRE] and five vancomycin-susceptible enterococci [VSE]) and
Enterococcus faecalis
strains (one VRE and 10 VSE) to survive under dry conditions. Bacterial suspensions of the strains were inoculated onto polyvinyl chloride and stored under defined conditions for up to 16 weeks. All strains survived for at least 1 week, and two strains survived for 4 months. A statistical model was used to distribute the 19 resulting survival curves between two types of survival curves. The type of survival curve was not associated with the species (
E. faecalis
versus
E. faecium
), the source of isolation (patient versus environment), or the susceptibility to vancomycin (VRE versus VSE). Resistance to dry conditions may promote the transmissibility of a strain, but VRE have no advantages over VSE with respect to their ability to survive under dry conditions.
Serotyping Scheme for
Campylobacter jejuni
and
Campylobacter coli
Based on Direct Agglutination of Heat-Stable Antigens
Authors: Frost J. A. , Oza A. N. , Thwaites R. T. , Rowe B.
Published: December 2019

Campylobacter is now the most frequently reported cause of gastrointestinal disease in England and Wales, yet few isolates are characterized beyond the genus level. The majority of isolates are
Campylobacter jejuni
(90%), with most of the remainder being
Campylobacter coli
. We describe an adaptation of the Penner serotyping scheme in which passive hemagglutination has been replaced by detection of heat-stable antigens by direct bacterial agglutination; absorbed antisera are used where appropriate. This scheme has been used to type 2,407
C. jejuni
samples and 182
C. coli
samples isolated in Wales between April 1996 and March 1997. Forty-seven
C. jejuni
serotypes were identified, with the 10 most prevalent serotypes accounting for 53% of the isolates tested; 19% of the isolates were untypeable. Only fifteen
C. coli
serotypes were identified, with three serotypes accounting for 69% of the isolates. This scheme provides a baseline for epidemiological studies of
C. jejuni
and
C. coli.
Improved Diagnostic PCR Assay forActinobacillus pleuropneumoniaeBased on the Nucleotide Sequence of an Outer Membrane Lipoprotein
Authors: Gram Trine , Ahrens Peter
Published: December 2019

The gene (omlA) coding for an outer membrane protein ofActinobacillus pleuropneumoniaeserotypes 1 and 5 has been described earlier and has formed the basis for development of a specific PCR assay. The corresponding regions of all 12A. pleuropneumoniaereference strains of biovar 1 were sequenced. Alignment of the sequences revealed conserved terminal and variable middle regions, which divided the reference strains into four distinct groups. Primers were selected from the conserved 5′ and 3′ termini of the gene. A 950-bp amplicon was obtained from each of 102 tested field isolates ofA. pleuropneumoniaeobtained from lungs. Their identity was verified by sequencing approximately 500 bp of the amplification product from 50 of theA. pleuropneumoniaeisolates, which all showed the expected DNA sequence characteristic of the serotype. To test the specificity of the reaction, 23 other bacterial species related toA. pleuropneumoniaeor isolated from pigs were assayed. They were all found negative in the PCR, as were tonsil cultures from 50 pigs of anA. pleuropneumoniae-negative herd. The sensitivity assessed by agarose gel analysis of the PCR product was 102CFU/PCR test tube. The specificity and sensitivity of this PCR compared to those of culture suggest the use of this PCR for routine identification ofA. pleuropneumoniae.
Association of Deficiency in Antibody Response to Vaccine and Heterogeneity ofEhrlichia risticiiStrains with Potomac Horse Fever Vaccine Failure in Horses
Authors: Dutta Sukanta K. , Vemulapalli Ramesh , Biswas Biswajit
Published: December 2019

Ehrlichia risticiiis the causative agent of Potomac horse fever (PHF), which continues to be an important disease of horses. Commercial inactivated whole-cell vaccines are regularly used for immunization of horses against the disease. However, PHF is occurring in large numbers of horses in spite of vaccination. In a limited study, 43 confirmed cases of PHF occurred between the 1994 and 1996 seasons; of these, 38 (89%) were in horses that had been vaccinated for the respective season, thereby clearly indicating vaccine failure. A field study of horses vaccinated with two PHF vaccines indicated a poor antibody response, as determined by immunofluorescence assay (IFA) titers. In a majority of horses, the final antibody titer ranged between 40 and 1,280, in spite of repeated vaccinations. None of the vaccinated horses developed in vitro neutralizing antibody in their sera. Similarly, one horse experimentally vaccinated three times with one of the vaccines showed a poor antibody response, with final IFA titers between 80 and 160. The horse did not develop in vitro neutralizing antibody or antibody against the 50/85-kDa strain-specific antigen (SSA), which is the protective antigen of the original strain, 25-D, and the variant strain of our laboratory, strain 90-12. Upon challenge infection with the 90-12 strain, the horse showed clinical signs of the disease. The horse developed neutralizing antibody and antibody to the 50/85-kDa SSA following the infection. Studies of the newE. risticiiisolates from the field cases indicated that they were heterogeneous among themselves and showed differences from the 25-D and 90-12 strains as determined by IFA reactivity pattern, DNA amplification finger printing profile, and in vitro neutralization activity. Most importantly, the molecular sizes of the SSA of these isolates varied, ranging from 48 to 85 kDa. These studies suggest that the deficiency in the antibody response to the PHF vaccines and the heterogeneity ofE. risticiiisolates may be associated with the vaccine failure.
Antibody Tests for Identification of
Mycobacterium bovis
-Infected Bovine Herds
Authors: Amadori M. , Tameni S. , Scaccaglia P. , Cavirani S. , Archetti I. L. , Giandomenico R. Quondam
Published: December 2019

Antibody in sera from 560 cattle of tuberculosis (TB)-infected and TB-free herds was investigated by competition and indirect enzyme-linked immunosorbent assays using bovine purified protein derivative tuberculin as the antigen. Antibody was detected in sera from both types of herd, with a widely overlapping range of titers. However, a “tail” of high-titered sera was observed for the distribution of data for only those cattle from TB-infected herds.
DNA Fingerprinting of
Mycobacterium tuberculosis
Complex Culture Isolates Collected in Brazil and Spotted onto Filter Paper
Authors: Burger Marion , Raskin Salmo , Brockelt Sonia R. , Amthor Beate , Geiss Heinrich K. , Haas Walter H.
Published: December 2019

The usefulness of filter paper for preservation of bacterial cells was shown by mixed-linker DNA fingerprint analysis of
Mycobacterium tuberculosis
isolates from 77 Brazilian patients. DNA fingerprints of samples spotted onto filter paper and conventional culture material were identical. Thus, filter paper specimens analyzed by an amplification-based typing method provide a new resource for epidemiological studies of infectious diseases.
Comparison of Eight Methods To Detect Vancomycin Resistance in Enterococci
Authors: Endtz Hubert P. , Van Den Braak Nicole , Van Belkum Alex , Goessens Wil H. , Kreft Deborah , Stroebel A. Barnard , Verbrugh Henri A.
Published: December 2019

A collection of genetically unrelated vancomycin-resistant enterococci (VRE) including 50
vanA
, 15
vanB
, 50
vanC1
, and 30
vanC2
VRE were used to evaluate the accuracy of eight currently available susceptibility test methods (agar dilution, disk diffusion, E-test, agar screen plate, Vitek GPS-TA and GPS-101, and MicroScan overnight and rapid panels).
vanA
VRE were detected by all methods.
vanB
VRE were often not detected by Vitek GPS-TA and MicroScan rapid (sensitivities, 47 and 53%, respectively), though the new Vitek GPS-101 was found to be a significant improvement. E-test and the agar screen were the only two methods detecting all VRE, including the
vanC1/C2
VRE.
Genus Level Identification of Mycobacteria from Clinical Specimens by Using an Easy-To-Handle
Mycobacterium
-Specific PCR Assay
Authors: Stauffer Fritz , Haber Heinrich , Rieger Armin , Mutschlechner Robert , Hasenberger Petra , Tevere Vincent J. , Young Karen K. Y.
Published: December 2019

An easy-to-handle
Mycobacterium
-specific PCR assay for detection of the presence of a wide range of mycobacterial species in clinical samples was evaluated. The performance of the genus probe was compared with the performance of probes specific for
Mycobacterium tuberculosis
and
Mycobacterium avium
and with that of standard culture. In addition, the utility of an internal control in monitoring amplification inhibitors was studied. Of 545 respiratory and 325 nonrespiratory specimens (a total of 870 specimens), 58 (6.7%) showed the presence of amplification inhibitors, as determined by a negative result for the internal control. Of these 58 specimens, 31 (53%) were stool specimens; other material, even citrate blood after lysis of erythrocytes, did not pose a problem with regard to inhibition of PCR amplification. Eighty-one of the remaining 812 specimens had a positive
Mycobacterium
culture result. Of these culture-positive specimens, 58 (71.6%) showed a positive result with the
Mycobacterium
genus-specific probe. Seventy-two samples had a positive result with the
Mycobacterium
-specific probe but a negative culture result. Of these 72 samples, 26 samples were regarded as true positive, either because the
M. tuberculosis
- or
M. avium
-specific probe was also positive at the same time or because other specimens from the same patient taken at the same time were culture positive. The sensitivity of the
Mycobacterium
-specific probe was 78.5% and the specificity was 93.5%. This study showed that pretesting of clinical specimens for mycobacteria to the genus level with a
Mycobacterium
-specific probe offers the routine clinical laboratory the possibility of detecting tuberculous and nontuberculous mycobacteria with one test. Furthermore, specimens testing positive with the genus-specific probe can be immediately identified with species-specific probes.
Method for Reduction of Inhibition in a
Mycobacterium tuberculosis
-Specific Ligase Chain Reaction DNA Amplification Assay
Authors: Leckie Gregor W. , Erickson Dwight D. , He Qizhi , Facey Ingrid E. , Lin Bor-Chian , Cao Jianli , Halaka Folim G.
Published: December 2019

The present study describes the identification of inhibitors of a
Mycobacterium tuberculosis
-specific gap ligase chain reaction (LCR) DNA amplification assay as well as a method for their removal. A major contributor to inhibition was deduced to be a calcium phosphate precipitate, CaHPO
4
. The precipitate forms during
N
-acetyl-
l
-cysteine–sodium hydroxide (NALC-NaOH) decontamination, digestion, and concentration of respiratory specimens. The solubility product of CaHPO
4
precipitate at pH 7.8, the pH at which gap LCR is optimized, indicates that the precipitate releases an amount of phosphate ions sufficient to inhibit amplification. A method for removal of the precipitate was identified. The precipitate is dissociated by exposing it to a mildly acidic (pH 4.1) buffer during the first of two centrifugation steps; the inhibitory phosphate ions are removed by the centrifugation steps. When 100 NALC-NaOH respiratory sediments were tested by gap LCR, none of the sediments were inhibitory when the acidic buffer was used while 24 samples were inhibitory when TE buffer, pH 7.8, was used. In another study, when the acidic buffer wash was applied to 1,440 NALC-NaOH respiratory sediments, only 10 sediments were found to be inhibitory. None of the inhibited sediments were culture positive for
M. tuberculosis
. This work demonstrates that when inhibition mechanisms are identified, relatively simple protocols can be used to obtain low inhibition rates and to allow the use of larger volume equivalents in amplification reactions.
Diversity of DNA Fingerprints of
Mycobacterium tuberculosis
Isolates in the United States
Authors: Yang Zhenhua , Barnes Peter F. , Chaves Fernando , Eisenach Kathleen D. , Weis Stephen E. , Bates Joseph H. , Cave M. Donald
Published: December 2019

To investigate the diversity of IS
6110
fingerprints of
Mycobacterium tuberculosis
isolates in the United States and to determine if matching IS
6110
fingerprints represent recent interstate tuberculosis transmission, we performed restriction fragment length polymorphism analysis of
M. tuberculosis
isolates from 1,326 patients in three geographically separated states. Seven hundred ninety-five different IS
6110
fingerprint patterns were generated, and pattern diversity was similar in each state. Ninety-six percent of the fingerprint patterns were observed in only one state, demonstrating that most IS
6110
fingerprint patterns are confined to a single geographic location. Of the IS
6110
fingerprint patterns that were shared by isolates from more than one state, most isolates with 1 to 5 IS
6110
copies were separable by pTBN12 fingerprinting whereas those with >15 copies were not. One high-copy-number
M. tuberculosis
strain had identical IS
6110
and pTBN12 fingerprints and included 57 isolates from three states. Epidemiological data demonstrated significant recent transmission of tuberculosis within each city but not among the states. This suggests that identical fingerprints of isolates from geographically separate locations most likely reflect interstate tuberculosis transmission in the past, with subsequent intrastate spread of disease. Further evaluation of
M. tuberculosis
strains that cause outbreaks in different geographic locations will provide insight into the epidemiological and bacteriological factors that facilitate the spread of tuberculosis.
Evaluation of Four Methods for Rapid Identification of
Staphylococcus aureus
from Blood Cultures
Authors: Speers David J. , Olma Thomas R. , Gilbert Gwendolyn L.
Published: December 2019

The identification of
Staphylococcus aureus
directly from blood cultures is clinically relevant, but it requires a test that is both rapid and reliable. Previously, biochemical, immunological, tube coagulase, and thermostable-endonuclease methods have shown variable sensitivity and specificity. Testing directly from blood culture broth has not been described for the latex kit Staphaurex Plus (Murex Diagnostics Ltd.), and the modified conventional tests have not been used with the newer, continuously monitored blood culture systems. In addition, the commercial RAPIDEC staph kit (bioMerieux Vitek, Inc.) has been used to detect
S. aureus
directly from the Vital blood culture system (bioMerieux, Marcy l’Etoile, France), but its performance has not been evaluated with other continuously monitored systems. A total of 201 clinical blood cultures (BACTEC 9240 culture system; Johnston Laboratories, Inc.) in which a Gram stain showed gram-positive cocci resembling staphylococci were evaluated prospectively. The Staphaurex Plus kit, the tube coagulase test, the thermostable-endonuclease test, and the RAPIDEC staph kit were compared. The sensitivities were 23, 92, 85, and 98% and the specificities were 99, 100, 93, and 100%, respectively. The RAPIDEC staph kit was the most reliable test, with a diagnostic accuracy comparable to that of the best published results for any of the rapid tests. However, it was the most expensive of the tests and relatively labor-intensive. The tube coagulase test was also sensitive, the simplest to perform, and inexpensive.
Comparison of Culture- and Non-Culture-Based Methods for Quantification of Viral Load and Resistance to Antiretroviral Drugs in Patients Given Zidovudine Monotherapy
Authors: Tedder Richard S. , Kaye Steve , Loveday Clive , Weller Ian V. D. , Jeffries Don , Norman Jane , Weber Jonathan , Bourelly Michel , Foxall Russell , Babiker Abdel , Darbyshire Janet H.
Published: December 2019

Virological assays for human immunodeficiency virus type 1 load and drug resistance can broadly be divided into culture-based and molecular biology-based methods. Culture-based methods give a direct measure of infectious virus load and phenotypic drug resistance, whereas molecular biology-based methods are indirect, assaying nucleic acid levels to determine virus load and point mutations associated with drug resistance. We have compared culture-based and non-culture-based methods for patients enrolled in a placebo-controlled trial of zidovudine (the Concorde Trial). Virus loads were assayed by culture of peripheral blood mononuclear cells (PBMCs) or quantitative PCR, and drug resistance was assayed in culture or in a quantitative, PCR-based point mutation assay. The rates of detection of viremia and drug resistance were higher by PCR than by culture for this population of subjects. Comparison of the virus loads by the two measures showed a good correlation for virus loads in PBMCs but a poor correlation for virus loads in plasma. The latter result probably reflected the inaccuracies of culture in assaying plasma with the low infectious virus titers seen in the study population. The concordance of phenotypic and genotypic drug resistance methods was high, with all phenotypically resistant isolates having at least one resistance-associated mutation and with no mutations being found in a drug-sensitive isolate. Genomic resistance scores (weighted sums of levels of resistance mutations) showed good correlations with the levels of phenotypic resistance, and both resistance measures were observed to increase as the duration of exposure to drug increased. Overall, non-culture-based methods were shown to correlate well with culture-based methods and offer a low-cost, high-throughput alternative. However, culture-based methods remain the final arbiters of infectious virus load and phenotypic drug resistance and are unlikely to be superseded entirely.
Molecular Typing of
Staphylococcus aureus
Based on PCR Restriction Fragment Length Polymorphism and DNA Sequence Analysis of the Coagulase Gene
Authors: Hookey John V. , Richardson Judith F. , Cookson Barry D.
Published: December 2019

A typing procedure for
Staphylococcus aureus
was developed based on improved PCR amplification of the coagulase gene and restriction fragment length polymorphism (RFLP) analysis of the product. All coagulase-positive staphylococci produced a single PCR amplification product of either 875, 660, 603, or 547 bp. Those strains of epidemic methicillin-resistant
S. aureus
16 (EMRSA-16) studied all gave a product of 547 bp. PCR products were digested with
Alu
I and
Cfo
I, and the fragments were separated by gel electrophoresis. Ten distinct RFLP patterns were found among 85 isolates of methicillin-resistant
S. aureus
(MRSA) and 10 propagating strains (PS) of methicillin-sensitive
S. aureus
(MSSA) examined. RFLP patterns 1, 2, and 3 were specific to strains of EMRSA-3, -15, and -16, respectively. By contrast, RFLP patterns 4 and 5 were seen with a heterogeneous collection of strains, together with drug-resistant forms of
S. aureus
isolated in Europe and four propagating strains used for the international phage set. RFLP pattern 6 was given by the Airedale isolate and PS 95. RFLP pattern 7 encompassed EMRSA-2 (isolate 331), PS 94, and PS 96. An isolate from Germany gave RFLP pattern 8. Eight strains of MSSA gave patterns similar to those of methicillin-resistant strains (RFLP patterns 3, 4, 5, 6, and 7), but two, PS 42E and PS 71, gave unique RFLP patterns 9 and 10, respectively. The coagulase gene PCR products for 24 isolates of MRSA and two isolates of MSSA were sequenced for both strands. The sequences were aligned, and evolutionary lineages were inferred based on pairwise distances between isolates.
Development of PCR Assays for Species- and Type-Specific Identification of
Pasteurella multocida
Isolates
Authors: Townsend Kirsty M. , Frost Alan J. , Lee Chiang W. , Papadimitriou John M. , Dawkins Hugh J. S.
Published: December 2019

Genomic subtractive hybridization of closely related
Pasteurella multocida
isolates has generated clones useful in distinguishing hemorrhagic septicemia-causing type B strains from other
P. multocida
serotypes. Oligonucleotide primers designed during the sequencing of these clones have proved valuable in the development of PCR assays for rapid species- and type-specific detection of
P. multocida
and of type B:2 in particular. This study demonstrated that the primer pair designed from the sequence of the clone 6b (KTT72 and KTSP61) specifically amplified a DNA fragment from types B:2, B:5, and B:2,5
P. multocida
and that the primers KMT1T7 and KMT1SP6 produced an amplification product unique to all
P. multocida
isolates analyzed. It was also shown that PCR amplification performed directly on bacterial colonies or cultures represents an extremely rapid, sensitive method of
P. multocida
identification.
Rising Levels of Human Cytomegalovirus (HCMV) Antigenemia during Initial Antiviral Treatment of Solid-Organ Transplant Recipients with Primary HCMV Infection
Authors: Gerna Giuseppe , Zavattoni Maurizio , Percivalle Elena , Grossi Paolo , Torsellini Maria , Revello M. Grazia
Published: December 2019

In 7 of 18 solid-organ transplant recipients with primary human cytomegalovirus (HCMV) infection, HCMV antigenemia levels were unexpectedly found to rise significantly (
P
= 0.018) during a mean time of 7.3 ± 3.2 days after initiation of specific antiviral treatment, whereas corresponding levels of viremia dropped significantly (
P
= 0.043). Thus, shifting to an alternative antiviral drug based solely on increasing antigenemia levels is not justified in this group of patients.
Detection of JC Virus in Cerebrospinal Fluid (CSF) Samples from Patients with Progressive Multifocal Leukoencephalopathy but Not in CSF Samples from Patients with Herpes Simplex Encephalitis, Enteroviral Meningitis, or Multiple Sclerosis
Authors: Bogdanovic G. , Priftakis P. , Hammarin A.-L. , Söderström M. , Samuelson A. , Lewensohn-Fuchs I. , Dalianis T.
Published: December 2019

JC virus (JCV) DNA was detected in cerebrospinal fluid (CSF) samples from patients with progressive multifocal leukoencephalopathy (PML) but not in CSF samples from patients with herpes simplex encephalitis, enteroviral meningitis, or multiple sclerosis. This suggests that inflammatory processes in the brain do not necessarily reactivate JCV, which further supports the proposal that the presence of JCV DNA in the CSF is diagnostic for PML.
Different Hepatitis C Virus (HCV) RNA Load Profiles Following Seroconversion among Injecting Drug Users without Correlation with HCV Genotype and Serum Alanine Aminotransferase Levels
Authors: Beld Marcel , Penning Maarten , McMorrow Martin , Gorgels Jozef , van den Hoek Anneke , Goudsmit Jaap
Published: December 2019

Hepatitis C virus (HCV) infection often persists in association with chronic hepatitis. Different factors have been proposed to determine the clinical outcome of HCV infection. The aim of this study was to examine three different factors of HCV infection among injecting drug users. Nineteen untreated HCV seroconverters were tested longitudinally for the presence of HCV RNA by reverse transcriptase (RT) PCR, and results were quantified by the branched-DNA (bDNA) assay. HCV genotypes were determined with the first sample taken after HCV seroconversion. To assess the natural course of infection, serum alanine aminotransferase (ALT) levels were measured at three stages in every individual. The concordance between bDNA and RT-PCR was 98.9%. Three distinct patterns were found, according to the HCV RNA load after seroconversion during a mean follow-up period of 5 years (range, 1 to 8 years). HCV genotype 1a was predominant (52.6%). There was a significant increase in serum ALT levels (mean 55.5 U/liter) in the early phase of HCV infection, compared with basal serum ALT levels before HCV seroconversion and at the end of the follow-up period. Three distinct HCV RNA load profiles were found, without apparent relationship to genotype and serum ALT levels in the first 5 years of HCV infection.
Taxonomic Relationships among Spotted Fever Group Rickettsiae as Revealed by Antigenic Analysis with Monoclonal Antibodies
Authors: Xu Wenbin , Raoult Didier
Published: December 2019

The spotted fever group (SFG) is made up of more than 20 different rickettsial species and strains. Study of the taxonomic relationships among the group has been attempted by phenotypic, genotypic, and phylogenetic analyses. In this study, we determined taxonomic relationships among the SFG rickettsiae by comparative analysis of immunogenic epitopes reactive against a panel of monoclonal antibodies. A total of 98 monoclonal antibodies, which were directed against epitopes on the major immunodominant proteins or on the lipopolysaccharide-like antigens of strains ofRickettsia africae,Rickettsia conorii,Rickettsia massiliae,Rickettsia akari,Rickettsia sibirica, andRickettsia slovaca, were used in the study. The distribution and expression of the epitopes among 29 SFG rickettsiae andRickettsia belliiwere assessed by determination of reaction titers in a microimmunofluorescence assay. The results were scored as numerical taxonomic data, and cluster analysis was used to construct a dendrogram. The architecture of this dendrogram was consistent with previous taxonomic studies, and the implications of this and other findings are discussed.
Emergence of Multidrug Resistance in Ubiquitous and Dominant
Pseudomonas aeruginosa
Serogroup O:11
Authors: Tassios Panayotis T. , Gennimata Vassiliki , Maniatis Anthony N. , Fock Caroline , Legakis Nicholas J. , Group The Greek Pseudomonas aeruginosa Study
Published: December 2019

The serotypes of 88 nonreplicate nosocomial
Pseudomonas aeruginosa
isolates from 11 Greek hospitals were studied in relation to their antibiotic susceptibilities. Rates of resistance to β-lactams, aminoglycosides, and quinolones ranged from 31 to 65%, except for those to ceftazidime (15%) and imipenem (21%). Four serotypes were dominant: O:12 (25% of isolates), O:1 (17%), O:11 (16%), and O:6 (10%). Multidrug resistance rates in the major serogroups O:12 (91%) and O:11 (79%) were higher than those in serogroups O:1 (40%) and O:6 (43%). Further typing with respect to pulsed-field gel electrophoresis patterns following
Xba
I digestion of genomic DNA discriminated the isolates into 74 types. Pulsed-field gel electrophoresis revealed that the ubiquitous O:12 group was genetically homogeneous, since 95% of strains belonged to two clusters of genotypic similarity, while the O:11 strains, present in 8 of the 11 hospitals, were distributed among five such clusters. Therefore, apart from the already reported O:12 multidrug-resistant European clone, an O:11 population, characterized by a serotype known to be dominant in the environment and the hospital in several parts of the world, but previously not associated with multidrug resistance to antibiotics, has progressed to a multidrug-resistant state.
Diversity of
Helicobacter pylori vacA
and
cagA
Genes and Relationship to VacA and CagA Protein Expression, Cytotoxin Production, and Associated Diseases
Authors: Rudi Jochen , Kolb Christof , Maiwald Matthias , Kuck Dirk , Sieg Andreas , Galle Peter R. , Stremmel Wolfgang
Published: December 2019

The vacuolating cytotoxin and the cytotoxin-associated protein, encoded by
vacA
and
cagA
, respectively, are important virulence determinants of
Helicobacter pylori
. Sixty-five
H. pylori
strains were isolated from dyspeptic patients (19 with peptic ulcer disease, 43 with chronic gastritis, and 3 with gastric cancer) and studied for differences in the
vacA
and
cagA
genes and their relationship to VacA and CagA expression, cytotoxin activity, and the clinical outcome of infection. By PCR, fifty-four (83.1%) of 65 strains had the
vacA
signal sequence genotype s1 and only 10 (15.4%) had the type s2. After primer modification, the
vacA
middle-region types m1 and m2 were detected in 24 (36.9%) and 41 (63.1%) strains, respectively. The combinations s1-m2 (31 [47.7%]) and s1-m1 (23 [35.4%]) occurred more frequently than s2-m2 (10 [15.4%]) (
P
= 0.01). No strain with the combination s2-m1 was found. All 19 patients with peptic ulcers harbored type s1 strains, in contrast to 32 (74.4%) of 43 patients with gastritis (
P
= 0.02). The
vacA
genotype s1 was associated with the presence of
cagA
(
P
< 0.0001), VacA expression (
P
< 0.0001), and cytotoxin activity (
P
= 0.003). The
cagA
gene was detectable in 48 (73.8%) of 65 isolates and present in 16 (84.2%) of 19 ulcer patients and 29 (67.4%) of 43 patients with gastritis (
P
= 0.17). The
vacA
genotypes of German
H. pylori
isolates are identical to those previously reported.
H. pylori
strains of
vacA
type s1 are associated with the occurrence of peptic ulceration and the presence of
cagA
, cytotoxin activity, and VacA expression.
Flow Cytometric Determination of Ganciclovir Susceptibilities of Human Cytomegalovirus Clinical Isolates
Authors: McSharry James M. , Lurain Nell S. , Drusano George L. , Landay Alan , Manischewitz Jody , Nokta Mostafa , O’Gorman Maurice , Shapiro Howard M. , Weinberg Adriana , Reichelderfer Patricia , Crumpacker Clyde
Published: December 2019

A flow cytometric assay has been developed for the measurement of susceptibilities to ganciclovir of laboratory strains and clinical isolates of human cytomegalovirus (HCMV). The assay uses fluorochrome-labeled monoclonal antibodies to HCMV immediate-early and late antigens to identify HCMV-infected cells and flow cytometry to detect and quantitate the number of antigen-positive cells. By this assay, the 50 and 90% inhibitory concentrations (IC
50
and IC
90
, respectively) of ganciclovir for the AD169 strain of HCMV were 1.7 and 9.2 μM, respectively, and the IC
50
for the ganciclovir-resistant D6/3/1 derivative of the AD169 strain was greater than 12 μM. The ganciclovir susceptibilities of 17 HCMV clinical isolates were also determined by flow cytometric analysis of the effect of ganciclovir on late-antigen synthesis in HCMV-infected cells. The average IC
50
of ganciclovir for drug-sensitive HCMV clinical isolates was 3.79 μM (±2.60). The plaque-reduction assay for these clinical isolates yielded an average IC
50
of 2.80 μM (±1.46). Comparison of the results of the flow cytometry assays with those obtained from the plaque-reduction assays demonstrated acceptable bias and precision. Flow cytometric and plaque-reduction analysis of cells infected with ganciclovir-resistant clinical isolates failed to show a reduction in the percentage of late-antigen-positive cells or PFU, even at 96 μM ganciclovir. The flow cytometric assay for determining ganciclovir susceptibility of HCMV is quantitative, and objective, and potentially automatable, and its results are reproducible among laboratories.
Detection of
Mycobacterium tuberculosis
Complex in Sputum Specimens by the Automated Roche Cobas Amplicor Mycobacterium Tuberculosis Test
Authors: Rajalahti Iiris , Vuorinen Pauli , Nieminen Markku M. , Miettinen Ari
Published: December 2019

Three hundred twenty-four sputum specimens from 151 patients with suspected active pulmonary tuberculosis were tested for the presence of the
Mycobacterium tuberculosis
complex with auramine fluorochrome stain and automated PCR assay (Roche Cobas Amplicor Mycobacterium Tuberculosis Test [MTB]). The results were compared with those of the conventional Löwenstein-Jensen tube culture and the BACTEC radiometer liquid culture. A total of 76 specimens from 32 patients were culture positive for
M. tuberculosis
. In addition, 37 specimens from 15 patients were smear and culture positive for other
Mycobacterium
species but negative by the present nucleic acid amplification method and thus were not included in the comparison. Compared with culture, the sensitivities, specificities, and positive and negative predictive values for acid-fast smear were 67, 98, 93, and 91% and those for the Cobas Amplicor MTB were 83, 99, 97, and 95%, respectively. When three consecutive sputum specimens per patient could be obtained, the sensitivity of the Cobas Amplicor MTB improved to 91%, whereas the sensitivity of the acid-fast smear remained unchanged.
Bordetella parapertussis
Infection in Children: Epidemiology, Clinical Symptoms, and Molecular Characteristics of Isolates
Authors: Mastrantonio Paola , Stefanelli P. , Giuliano M. , Rojas Y. Herrera , Ciofi degli Atti M. , Anemona A. , Tozzi A. E.
Published: December 2019

The clinical trial conducted in Italy to evaluate the efficacy of acellular pertussis vaccines provided an opportunity to estimate the frequency of clinical infections with
Bordetella parapertussis
and to compare the clinical characteristics of children suffering from
Bordetella pertussis
illness with those of children with
B. parapertussis
illness. This study dealt with 76
B. parapertussis
infections diagnosed from a population of 15,601 children participating in the follow-up of suspected cases of pertussis. An overall incidence of 2.1 cases of laboratory-confirmed parapertussis per 1,000 person-years was observed. Children affected by
B. parapertussis
infections showed a less severe clinical picture both in the duration of symptoms and in the percentage of patients affected, even when compared with vaccinated children with pertussis. To characterize the isolated strains, we performed assays for susceptibility to erythromycin and sulfamethoxazole-trimethoprim, and we examined the genomic DNAs by pulsed-field gel electrophoresis. The results showed a high degree of genetic stability among
B. parapertussis
strains regardless of time of collection and geographical distribution.
Growth and Survival ofHelicobacter pyloriin Defined Medium and Susceptibility to Brij 78
Authors: Albertson N. , Wenngren I. , Sjöström J.-E.
Published: December 2019

The gastrointestinal pathogenHelicobacter pylorirequires supplementation with either fetal calf serum (FCS), bovine serum albumin (BSA), or (2,6-dimethyl)-β-cyclodextrin (CD) for growth in a complex or defined medium. Because the availability of medium in which all components were chemically defined would facilitate metabolic studies ofH. pylori, growth of the type strain, ATCC 43504, was compared in a defined medium with different growth additives. The dependency ofH. pylorigrowth on FCS or BSA in a defined medium could partially be replaced by dependency on CD and cholesterol when the last two components were both added to the defined medium. Growth and cell yield were not affected by the addition of glucose, but the culture viability (numbers of CFU per milliliter was extended. Because therapeutic antifoams are used to relieve gastrointestinal symptoms we studied whether the unique susceptibility ofH. pylorito the emulsifier polyoxyethylene-20-stearylether (Brij 78) was growth dependent or medium specific. The bactericidal activity exerted in buffer at pH 5 was independent of the preculture medium, and a 5-h exposure of the bacteria to 1.28 to 2.56 μg of Brij 78 per ml reduced the numbers of viable bacteria by >5 log10. The MICs (0.16 to 0.32 μg/ml) were lower than the corresponding minimal bactericidal concentrations in different growth media and were affected by FCS or BSA. In conclusion, CD plus cholesterol promotes the growth ofH. pyloriin a serum-free defined medium in which glucose enhances cell viability. The antibacterial activity exerted by Brij 78 is neither growth dependent nor medium specific.