bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of five commercial tests for detection of immunoglobulin M antibodies to human parvovirus B19
Authors: Bruu A L , Nordbø S A
Published: January 2020

The following commercial tests for detection of immunoglobulin M antibodies to human parvovirus B19 were evaluated: Ideia Parvovirus B19-IgM, MRL Diagnostics Human Parvovirus B19 IgM ELISA, Parvoscan-B19, and Biotrin Parvo B19 IgM EIA and IF. A total of 203 serum specimens from patients who probably have current B19 infections or have other viral infections and sera with rheumatoid factor were investigated. Between 75 and 79 of 102 serum samples from patients thought to have current B19 infections yielded positive results with the different tests. Ideia had the highest specificity (94.8%), while Parvoscan showed a specificity of only 70.1%. Our evaluation results show that Ideia, MRL, and Biotrin EIA and IF can be recommended for diagnostic purposes.
Identification of enteropathogenic Escherichia coli by PCR-based detection of the bundle-forming pilus gene
Authors: Gunzburg S T , Tornieporth N G , Riley L W
Published: January 2020

A rapid and simple method of detecting enteropathogenic Escherichia coli (EPEC) was developed. The procedure is based on amplifying by the PCR method a 326-bp region of the bundle-forming pilus gene of EPEC. The oligonucleotide DNA primers used in this procedure did not amplify DNA of any other bacterial enteropathogens tested. The procedure was 100% specific for EPEC strains that exhibit a characteristic pattern of attachment (localized adherence) to HeLa cells.
Comparison of ribotyping and pulsed-field gel electrophoresis for molecular typing of Acinetobacter isolates
Authors: Seifert H , Gerner-Smidt P
Published: January 2020

Seventy-three isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, including 26 isolates from 10 hospital outbreaks, were typed by ribotyping with EcoRI and ClaI and by pulsed-field gel electrophoresis (PFGE) of genomic DNA after digestion with ApaI. Ribotyping with EcoRI distinguished 31 ribopatterns. Digestion with ClaI generated another eight ribotypes. PFGE, in contrast, identified 49 distinct patterns with seven variants. Both methods detected all outbreak-related isolates. By ribotyping, nine epidemiologically unrelated strains could not be differentiated from outbreak strains, in contrast to only one isolate not identified by PFGE. Thus, PFGE was more discriminating than ribotyping. However, ribotyping is known to generate banding patterns specific to each DNA group in the A. calcoaceticus-A. baumannii complex that may be used for taxonomic identification of the strains. PFGE was shown to lack this property. Both methods are therefore useful for strain differentiation in epidemiological studies of Acinetobacter isolates.
Distribution of both rotavirus VP4 genotypes and VP7 serotypes among hospitalized and nonhospitalized Israeli children
Authors: Silberstein I , Shulman L M , Mendelson E , Shif I
Published: January 2020

Human rotaviruses belonging to genotype P9, of probable feline origin, which included both G3 and G1 serotypes, were detected in 3.8% of children shedding rotaviruses who attended sick fund clinics throughout Israel. None were detected in children admitted to hospitals because of severe diarrhea. In contrast, the relative prevalences of genotypes P8 and P4 were similar between the two groups.
Comparison of E test and agar dilution for antimicrobial susceptibility testing of Stenotrophomonas (Xanthomonas) maltophilia
Authors: Yao J D , Louie M , Louie L , Goodfellow J , Simor A E
Published: January 2020

Currently recommended dilution test methods for the determination of antimicrobial susceptibility of Stenotrophomonas (Xanthomonas) maltophilia are labor-intensive and often impractical in many clinical laboratories. We compared the E test with the agar dilution method for susceptibility testing of 176 clinical isolates of S. maltophilia against 16 antimicrobial agents. The MICs obtained by E test correlated well with those determined by the agar dilution method, with an overall agreement of 94%. Very major and major errors occurred infrequently (0.6 to 2.9%) when testing beta-lactam agents, tobramycin, trimethoprim-sulfamethoxazole, and fluoroquinolones. The E test was found to be accurate and easy to perform. For most antimicrobial agents tested against S. maltophilia, the E test is an acceptable alternative susceptibility test method.
Expression, self-assembly, and antigenicity of a snow mountain agent-like calicivirus capsid protein
Authors: Jiang X , Matson D O , Ruiz-Palacios G M , Hu J , Treanor J , Pickering L K
Published: January 2020

Virus-like particles were produced in insect cells infected with a recombinant baculovirus containing the capsid gene of MX virus, a Mexican strain of human calicivirus. These recombinant MX (rMX) particles were morphologically similar to recombinant Norwalk virus (rNV) particles as observed under an electron microscope and contained a single capsid protein with a molecular weight of 57,000, which was slightly smaller than that of rNV. This protein was immunoprecipitated by sera from volunteers infected with the Snow Mountain agent, but it reacted weakly with sera from volunteers infected with NV. This protein did not react with hyperimmune antisera from animals immunized with rNV in the rNV antigen enzyme immunoassay (EIA). Seroresponses were detected from volunteers infected with Snow Mountain agent and Hawaii agent when the rMX particles were used as antigen in an EIA. This EIA also detected an immune response in the sera of child from whom the MX virus was isolated, and a high prevalence of antibody to MX virus was found in the sera of a cohort of Mexican children.
Rapid bacterial antigen detection is not clinically useful
Authors: Perkins M D , Mirrett S , Reller L B
Published: January 2020

Latex agglutination (LA) of capsular polysaccharide bacterial antigen is a frequently performed laboratory procedure, but its use is controversial. To assess the clinical utility of this test, we reviewed all LA tests performed over a 10-month period at two sites, a major university-based referral center and a private specialty pediatric hospital. Samples were assayed either individually or as a panel for the group B streptococcus, Streptococcus pneumoniae, Haemophilus influenzae, and three sets of Neisseria meningitidis serogroups (A and Y, C and W135, and B and Escherichia coli K1). Of 5,169 assays performed on 1,268 clinical samples (786 urine and 478 cerebrospinal fluid, 3 pleural fluid, and 1 synovial fluid sample), 57 (1.1%) were positive, including 1.7% of urine and 0.3% of cerebrospinal fluid samples. All LA true-positive cerebrospinal fluid samples showed the causative microorganisms by Gram stain. Detailed chart review of these 57 positive samples showed that the LA result was false-positive in 31 (54%), true-positive in 22 (38%), and indeterminate in 4 (7%) samples. Therapy was not altered on the basis of any of the true-positive LA results. The 31 false-positive results led to additional cost, prolonged hospitalization, and some clinical complications. Total patient charges were $175,000 ($7,954 per true-positive), with no detectable clinical benefit. Our retrospective study does not support the current use of LA for rapid antigen detection. What, if any, specific indications exist for this test remain to be elucidated.
Arbitrarily primed PCR DNA fingerprinting of Escherichia coli O157:H7 strains by using templates from boiled cultures
Authors: Madico G , Akopyants N S , Berg D E
Published: January 2020

Arbitrarily primed PCR allows genetically different bacterial strains to be distinguished with great sensitivity and efficiency. We report that informative, reproducible arbitrarily primed PCR profiles can be obtained from Escherichia coli O157:H7 strains by using boiled stationary-phase cultures, without the need for time-consuming phenol extraction. This simple template preparation procedure should be especially useful in large epidemiologic studies when many strains must be typed.
Selective utilization of DNA probes for identification of Mycobacterium species on the basis of cord formation in primary BACTEC 12B cultures
Authors: Kaminski D A , Hardy D J
Published: January 2020

Primary BACTEC 12B cultures with serpentine cords observed in Kinyoun-stained smears were tested with a probe for Mycobacterium tuberculosis complex, while cultures without cords were tested with a probe for M. avium complex. The sensitivity, specificity, and positive and negative predictive values of cording for the presumptive identification of M. tuberculosis were 95, 95, 90, and 98%, respectively. With experience, the selection of a probe for testing of primary BACTEC 12B cultures on the basis of cord formation and history of tuberculosis can provide a rapid and reliable approach to the laboratory diagnosis of tuberculosis.
Identification of virulent Rhodococcus equi by amplification of gene coding for 15- to 17-kilodalton antigens
Authors: Takai S , Ikeda T , Sasaki Y , Watanabe Y , Ozawa T , Tsubaki S , Sekizaki T
Published: January 2020

During a survey of the prevalence of virulent Rhodococcus equi at horse-breeding farms by plasmid and protein profiles, cryptic plasmids of various sizes were found in 66 (3.8%) of 1,725 isolates from feces of horses and 129 (5.9%) of 2,200 isolates from soil. Twenty-two isolates, which contained cryptic plasmids of different sizes, were found by plasmid profiles, and their protein profiles and mouse pathogenicities were examined. Of the 22 isolates, 7 were virulent R. equi, contained both virulence and cryptic plasmids, and expressed 15- to 17-kDa antigens. The remaining 15 isolates were avirulent and did not express the antigens: 6 strains contained cryptic plasmids of two different sizes and 9 strains contained cryptic plasmids of various sizes. A PCR assay was developed for the rapid identification of virulence plasmids of R. equi. Oligonucleotide primers, derived from the sequence of a gene coding for the 15- to 17-kDa virulence-associated antigens of R. equi, amplified a 564-bp product from all the tested isolates harboring a virulence plasmid. This PCR product hybridized with virulence plasmid DNA in the Southern hybridization assay. Virulence plasmid-cured derivatives and all of the tested isolates harboring cryptic plasmids only were negative. The PCR is a rapid, sensitive, and specific test for the identification of virulent R. equi from environmental isolates compared with standard techniques, such as plasmid and protein profiles and the mouse pathogenicity test, and is considered to be a useful tool for epidemiological studies.
Prospective comparison of direct immunofluorescence and conventional staining methods for detection of Giardia and Cryptosporidium spp. in human fecal specimens
Authors: Alles A J , Waldron M A , Sierra L S , Mattia A R
Published: January 2020

In a prospective comparative study, 2,696 consecutive fresh stool specimens over the course of 1 year were examined for Giardia lamblia and Cryptosporidium parvum by using a direct immunofluorescent-monoclonal antibody stain (for unspun specimens) and conventional staining methods (chlorazol black E for Giardia cysts and modified Kinyoun acid-fast for Cryptosporidium oocysts). The direct immunofluorescent-monoclonal antibody method resulted in a significantly increased detection rate for both giardia (118 versus 79 specimens, 49.4%; P = 0.006) and cryptosporidia (39 versus 23 specimens, 69.6%; P = 0.055).
Intraerythrocytic presence of Bartonella henselae
Authors: Kordick D L , Breitschwerdt E B
Published: January 2020

Recent reports in the medical literature emphasize the risk of zoonotic disease and the high degree of prevalence of asymptomatic feline infection with Bartonella (Rochalimaea) henselae. While investigating Bartonella bacteremia in cats, we used transmission electron microscopy to demonstrate B. henselae in the erythrocytes of persistently bacteremic cats.
Arcobacter-specific and Arcobacter butzleri-specific 16S rRNA-based DNA probes
Authors: Wesley I V , Schroeder-Tucker L , Baetz A L , Dewhirst F E , Paster B J
Published: January 2020

The genus Arcobacter encompasses gram-negative, aerotolerant, spiral-shaped bacteria formerly designated Campylobacter cryaerophila. Two genus-specific 16S rRNA-based oligonucleotide DNA probes (23-mer and 27-mer) were developed. The probes hybridized with strains of Arcobacter butzleri (n = 58), Arcobacter cryaerophilus (n = 19), and Arcobacter skirrowii (n = 17). The probes did not cross-react with any of the reference strains of Campylobacter, Helicobacter, including "Flexispira rappini," or Wolinella. The 27-mer hybridized with 61 Arcobacter spp. field isolates originating from late-term aborted porcine (n = 54) and equine (n = 2) fetuses and humans with enteritis (n = 5). The species of Arcobacter isolates (n = 56) recovered from aborted livestock fetuses were determined by ribotyping and were as follows: A. cryaerophilus group 1A (11 of 56; 20%), A. cryaerophilus group 1B (37 of 56; 66%), A. butzleri (5 of 56; 9%), and unknown (3 of 56; 5%). The five human field strains were identified as A. butzleri. A species-specific DNA probe (24-mer) for A. butzleri was also developed since there is evidence that this organism may be a human pathogen. This probe hybridized with previously characterized strains of A. butzleri (n = 58), with 10 field strains identified as A. butzleri by ribotyping and with 2 strains having an indeterminate ribotype. The A. butzleri-specific probe did not cross-react with strains of A. skirrowii (n = 17) and A. cryaerophilus (n = 19).
Differentiation of human immunodeficiency virus type 1 (HIV-1) infections with HIV-2-cross-reacting antibody from mixed infections with HIV-1 and HIV-2 by serological absorption test
Authors: Imai M , Hayashi T , Kondo M , Saito T , Ito A , Hayami M , Nishioka K
Published: January 2020

The interpretation of dual seroreactivity with human immunodeficiency virus type 1 (HIV-1) and HIV-2 in blood samples is a serious problem facing AIDS researchers worldwide. Some samples of sera from HIV-1-infected patients showed a serological cross-reaction with HIV-2, causing confusion regarding the serodiagnosis. Therefore, we tried to differentiate these serum samples from those containing real mixed infections with both types of virus. Sera from patients with HIV-1 infections with HIV-2 cross-reacting antibody in Japan were distinguished from sera from patients with mixed infections with HIV-1 and HIV-2 in West Africa by our serological cross-absorption test, which proved to be highly specific and useful for serodiagnosis.
Preclinical evaluation of AMPLICOR hepatitis C virus test for detection of hepatitis C virus RNA
Authors: Nolte F S , Thurmond C , Fried M W
Published: January 2020

We compared a single-enzyme, combined reverse transcription-PCR (RT-PCR; AMPLICOR HCV Test; Roche Molecular Systems, Branchburg, N.J.) with an independent, two-enzyme, standard RT-PCR (SRT-PCR) assay for the detection of hepatitis C virus (HCV) RNA in serum and plasma. Test samples included a proficiency testing panel consisting of 10 undiluted plasma samples, three separate dilution series, and sera from 99 patients with chronic liver disease. The quantity of HCV RNA in each patient serum sample was determined by a branched DNA (bDNA) signal amplification assay (Quantiplex HCV-RNA assay; Chiron, Emeryville, Calif.). There was complete concordance between the results of the RT-PCR assays with the 10 undiluted plasma samples used for proficiency testing (3 positive and 7 negative samples). However, the analytical sensitivity of SRT-PCR was 4- to 10-fold greater than that of the AMPLICOR test in the dilution series. HCV RNA was detected in 44, 45, and 40 of the patient serum samples, by SRT-PCR, the AMPLICOR test, and the bDNA assay, respectively. There was 97% agreement between the results of the RT-PCR assays, with only three discrepancies. Review of the patients' medical records resolved all three discrepancies in favor of the AMPLICOR results (two false-negative SRT-PCR results and one false-positive SRT-PCR result). The quantity of HCV RNA in sera from five (11%) patients with viremia detected by AMPLICOR was below the bDNA assay cutoff (< 3.5 x 10(5) RNA equivalents per ml). AMPLICOR compared favorably with SRT-PCR, with key advantages of speed, ease of use, increased sample throughput, and protection against false-positive results because of amplicon carryover.
Differentiation of Bartonella-like isolates at the species level by PCR-restriction fragment length polymorphism in the citrate synthase gene
Authors: Norman A F , Regnery R , Jameson P , Greene C , Krause D C
Published: January 2020

The citrate synthase gene (gltA) of Bartonella henselae was cloned and sequenced to compare genetic divergence among alpha and gamma branches of the class Proteobacteria and to develop enhanced genotypic reagents for B. henselae identification. B. henselae gltA is 1,293 nucleotides in length and 63 to 66% homologous with corresponding gene sequences of Rickettsia prowazekii, Escherichia coli, and Coxiella burnetii. The observed genetic variability suggests that gltA sequences can provide a useful means for studying moderate divergence among related bacteria. Oligonucleotides specific for B. henselae gltA were evaluated for the ability to prime PCR amplification within the alpha and gamma branches of the proteobacteria. Under the conditions used, only B. henselae, Bartonella quintana, and R. prowazekii template DNAs yielded amplification products (approximately 380 bp). DNAs from 28 Bartonella-like isolates of feline origin were amplified by B. henselae primers and analyzed for restriction fragment length polymorphism. The resulting patterns for all 28 isolates were similar or identical to that of the recognized B. henselae strain. Current studies are aimed at optimization of PCR conditions for specificity and sensitivity of amplification of Bartonella sequences from clinical isolates.
Detection of penicillin-resistant Streptococcus pneumoniae with commercially available broth microdilution panels
Authors: Nolte F S , Metchock B , Williams T , Diem L , Bressler A , Tenover F C
Published: January 2020

We compared penicillin MICs obtained with three different commercially available broth microdilution panels (MicroScan, Sensititre, and Pasco) with MICs obtained with reference microdilution panels for 20 well-characterized pneumococci with decreased susceptibilities to penicillin (7 resistant and 13 intermediate). All panels were supplemented with 2 to 5% lysed horse blood (LHB) prepared in-house. Additional supplements included fastidious inoculum broth (FIB) for MicroScan panels and commercially prepared LHB (Difco) for Pasco panels. The percentages of penicillin-resistant strains (MIC 2 micrograms/ml) detected by the different methods follow: MicroScan-FIB, 0; MicroScan-LHB 0; Pasco in-house LHB, 71; and Sensititre-LHB, 100. The percentages of intermediate strains (MIC = 0.1 to 1.0 micrograms/ml) detected by the different methods follow: MicroScan-FIB, 31; MicroScan-LHB 23; Pasco in-house LHB, 46; and Sensititre-LHB, 85. Difco LHB supplement failed to support the growth of 86% of the strains in the Pasco panels. Of the commercially available panels evaluated, only Sensititre, supplemented with LHB prepared in-house could reliably detect penicillin-resistant pneumococci.
Evaluation of immunoglobulin G enzyme immunoassay for serodiagnosis of yaws
Authors: Backhouse J L , Hudson B J
Published: January 2020

A commercially available enzyme immunoassay (EIA), the Captia Syphilis-G immunoglobulin G (IgG) EIA, for the detection of IgG antibodies to Treponema pallidum was evaluated for use as a screening test for yaws (Treponema pallidum subsp. pertenue). The IgG EIA was compared with the fluorescent treponemal antibody absorption (FTA-ABS) test. All sera were also examined by the T. pallidum hemagglutination test and the Venereal Disease Research Laboratory test. Serum samples from 271 subjects (23 control serum samples from an area nonendemic for yaws, 58 control serum samples from an area endemic for yaws, and 190 serum samples from yaws patients and contacts) were investigated. The overall agreement between the IgG EIA and the FTA-ABS test was 90%, the sensitivity was 99%, and the specificity was 70.2%. The specificity fell as the endemicity of the disease increased: from 94.4% in the nonendemic area controls to 86.4% in the endemic area controls and to 52.3% in the yaws contacts. There was no difference in specificity between children and adults within each of the three groups. Fifteen children with clinical yaws were monitored for 9 months after treatment. The level of treponemal antibody fell consistently in 9 of the 15 children as measured by the antibody index (ratio of absorbance of the test serum to the mean absorbance of the low-titer-positive controls). Reinfection was seen in three children, with the antibody index rising with the Venereal Disease Research Laboratory test titer. The Captia Syphilis-G test is a sensitive assay for the detection of treponemal antibodies in yaws patients. However, the apparent low specificity of the test in the yaws endemic area limits its use as a screening test for yaws.
Initial use of a broth microdilution method suitable for in vitro testing of fungal isolates in a clinical microbiology laboratory
Authors: Hacek D M , Noskin G A , Trakas K , Peterson L R
Published: January 2020

Antifungal susceptibility testing methods currently lack a standardized procedure. Many factors, such as inoculum preparation, inoculum density, medium selection, pH, incubation time and temperature, and endpoint determination, affect results. We developed a workable procedure for fungal susceptibility testing, with a microtiter method based upon modifications of the proposed guidelines from the National Committee for Clinical Laboratory Standards, using two different growth media. For this procedure, the microtiter tray is prepared as a panel of 6 drugs (amphotericin B, flucytosine, fluconazole, ketoconazole, miconazole, and itraconazole) alone and in combination with amphotericin B. Eagle's minimal essential medium and RPMI 1640 are the two growth media. Two separate susceptibility trays are inoculated for each sensitivity test, with one tray incubated at 30 degrees C and the other incubated at 35 degrees C. After 48 h of growth, results for both temperatures and both media are recorded and interpreted. The four test environments (two media each at two temperatures) provided growth for 100 of the first 104 organisms that were submitted for testing. This approach provides a workable methodology for routine antifungal susceptibility testing in a clinical microbiology laboratory setting.
Quantitative culture of Mycobacterium tuberculosis from clinical sputum specimens and dilution endpoint of its detection by the Amplicor PCR assay
Authors: Yajko D M , Wagner C , Tevere V J , Kocagöz T , Hadley W K , Chambers H F
Published: January 2020

The minimum number of Mycobacterium tuberculosis CFU detectable in clinical sputum specimens by the Amplicor PCR test was estimated by performing the test on duplicate samples of quantitatively cultured serial dilutions of sputum. Positive PCR test results were obtained for all samples that contained 42 CFU of M. tuberculosis. The detection limits of the PCR assay for decontaminated (N-acetyl-L-cysteine [NALC]-NaOH) and nondecontaminated (NALC only) specimens were equivalent, even though the number of CFU cultured from decontaminated samples was only 11 to 20% of the number cultured from nondecontaminated samples. Thus, the 42 CFU that could be detected in nondecontaminated specimens by the Amplicor PCR test correspond to the approximately 8 CFU (0.20 x 42) that could be recovered in culture after decontamination with NALC-NaOH.
Human papillomavirus (HPV) type distribution and serological response to HPV type 6 virus-like particles in patients with genital warts
Authors: Greer C E , Wheeler C M , Ladner M B , Beutner K , Coyne M Y , Liang H , Langenberg A , Yen T S , Ralston R
Published: January 2020

Thirty-nine patients with condylomas (12 women and 27 men) attending a dermatology clinic were tested for genital human papillomavirus (HPV) DNA and for seroprevalence to HPV type 6 (HPV6) L1 virus-like particles. The L1 consensus PCR system (with primers MY09 and MY11) was used to determine the presence and types of HPV in sample specimens. All 37 (100%) patients with sufficient DNA specimens were positive for HPV DNA, and 35 (94%) had HPV6 DNA detected at the wart site. Three patients (8%) had HPV11 detected at the wart site, and one patient had both HPV6 and -11 detected at the wart site. Thirteen additional HPV types were detected among the patients; the most frequent were HPV54 (8%) and HPV58 (8%). Baculovirus-expressed HPV6 L1 virus-like particles were used in enzyme-linked immunosorbent assays to determine seroprevalence among the patients with warts. Seronegativity was defined by a control group of 21 women who were consistently PCR negative for HPV DNA. Seroprevalence was also determined for reference groups that included cytologically normal women who had detectable DNA from either HPV6 or HPV16 and women with HPV16-associated cervical intraepithelial neoplasia. Among the asymptomatic women with HPV6, only 2 of 9 (22%) were seropositive, compared with 12 of 12 (100%) female patients with warts. A similar trend in increased HPV6 seropositivity with increased grade of disease was found with the HPV16 DNA-positive women, whose seroprevalence increased from 1 in 11 (9%) in cytologically normal women to 6 in 15 (40%) among women with cervical intraepithelial neoplasia 1 or 3. However, only 4 of 25 (16%) male patients were seropositive. No factors examined, such as age, sexual behavior, or a history of warts, were found to definitively account for the gender difference in seroresponse.
Comparison of culture methods for monitoring Legionella species in hospital potable water systems and recommendations for standardization of such methods
Authors: Ta A C , Stout J E , Yu V L , Wagener M M
Published: January 2020

A lack of standardization of environmental monitoring techniques for Legionella spp. complicates the interpretation of results and comparisons of results from different institutions. A comparative assessment of techniques recommended by the Centers for Disease Control and Prevention, the Hygiene Institute (Graz, Austria), and our laboratory was performed. Variables investigated were sampling method (swabbing and collection of water samples [250 ml] before and after swabbing), method of concentration (none, filtration, and centrifugation), acid buffer treatment (no acid treatment, treatment for 3 min, and treatment for 15 min), and choice of medium (five formulations of buffered charcoal yeast extract agar with glycine, vancomycin, polymyxin B, anisomycin, or cycloheximide). Thirty-three sites in seven hospital buildings were studied. Recovery by swab correlated with recovery from water after swabbing (P < 0.05). However, the quantity of Legionella spp. recovered from swab specimens (mean, 3.0 x 10(4) CFU per swab) was greater than that recovered from water (mean, 4.7 x 10(3) CFU/250 ml). Filtration resulted in recovery rates (mean, 5.2 x 10(3) CFU/250 ml) higher than those by centrifugation (mean, 2.3 x 10(3) CFU/250 ml). Three minutes of acid buffer treatment to reduce overgrowth by commensal flora did not improve selectivity or sensitivity for Legionella spp. if glycine-containing selective media were used. Fifteen minutes of acid buffer treatment reduced recovery compared with that after a 3-min treatment. All glycine-containing media tested effectively inhibited background flora, but one selective medium containing dyes, glycine, vancomycin, and polymyxin B (DGVP) resulted in the greatest quantitative recovery of Legionella pneumophila. Use of buffered charcoal yeast extract agar and the acid buffer treatment gave the greatest recovery of non-pneumophila species. A standardized protocol with an emphasis on the culturing of swab samples is presented.
Immunomagnetic separation and PCR for detection of Helicobacter pylori in water and stool specimens
Authors: Enroth H , Engstrand L
Published: January 2020

The detection of Helicobacter pylori in clinical and environmental samples by PCR sometimes requires removal of polymerase inhibitors. We have used a magnetic immunoseparation technique as pre-PCR treatment to facilitate direct detection of H. pylori in stool and water specimens. Rabbit hyperimmune antiserum was produced and magnetic beads were coated with purified immunoglobulin G, which reacted with and bound to both coccoid and rod-shaped forms of H. pylori. When PCR was applied for the detection of H. pylori from cultured samples, the number of organisms that was required for positive scores varied significantly. For a 3-day culture of H. pylori, samples containing 10(2) bacteria per ml are needed for a positive score; for a 6-day culture, samples containing 10(4) bacteria per ml are needed; and for a 10-day culture, samples containing 10(6) bacteria per ml are needed. These results indicate that the coccoid forms of H. pylori may have a different antigenicity and DNA content and are therefore more difficult to detect by immunomagnetic separation and PCR than the rod-shaped forms. Spiked samples with the addition of feces, spiked water samples, and a patient stool specimen were all scored positive with this technique.
Comparison of preservation media for storage of stool samples
Authors: Wasfy M , Oyofo B , Elgindy A , Churilla A
Published: January 2020

Transportation of clinical samples and long-term recoverability of pathogens are critical to epidemiological studies, particularly when conditions do not permit immediate processing. This study confirms that Cary-Blair medium (CB) is suitable for the preservation of Salmonella and Shigella isolates for more than 2 weeks at 25, 4, or -70 degrees C. Campylobacter jejuni was not recovered after 2 days of storage in CB at 25 degrees C when an inoculum of 12 x 10(8) cells per ml was used. Lower temperatures supported the recovery of this organism for 6 days. When individual pathogens were preserved with stools in CB and incubated at 25, 4, or -70 degrees C, the Salmonella and Shigella concentrations dropped from 12 x 10(8) cells to 1 x 10(3) or 1 x 10(4) cells per ml within 2 days and then remained stable for the rest of the observation period (15 days). C. jejuni survived preservation with stools for 5 to 9 days. The addition of blood and glycerol to CB improved the recoverability of all enteropathogens, particularly C. jejuni, which was consistently detected for 7 to 9 days at the different preservation temperatures used. When trypticase soy broth-glycerol (freezing medium), with or without blood, was used, there was little or no decrease in the Salmonella and Shigella concentrations during 2 weeks of preservation with stools at -70 degrees C. C. jejuni demonstrated a relatively sustained high concentration in Trypticase soy broth-glycerol with 5% blood. The use of defibrinated, laked sheep blood as a long-term freezing medium supported the recovery of low concentrations of Salmonella and Shigella spp. (10(2) to 10(3) cells per ml) for more than 14 weeks. Recovery of C. jejuni was consistent for 7 weeks when an initial concentration of 10(6) cells per ml present in stools. Laked blood provided a simple, sterile, and inexpensive medium for the preservation of individual isolates and clinical samples.
Controlled evaluation of BacT/alert standard anaerobic and FAN anaerobic blood culture bottles for the detection of bacteremia and fungemia
Authors: Wilson M L , Weinstein M P , Mirrett S , Reimer L G , Feldman R J , Chuard C R , Reller L B
Published: January 2020

FAN medium was formulated to improve microbial recovery, particularly for fastidious microorganisms and for microorganisms causing sepsis in patients receiving antimicrobial therapy. In a controlled clinical evaluation performed at four university-affiliated hospitals, FAN anaerobic bottles were compared with standard anaerobic bottles for yield, speed of detection of microbial growth, and detection of septic episodes. A total of 10,431 blood culture sets were received; both anaerobic bottles of 7,694 blood culture sets were adequately filled with blood. Altogether, 925 isolates were recovered: 557 that were the cause of sepsis, 99 that were indeterminate as the cause of sepsis, and 269 contaminants. More Staphylococcus aureus (P < 0.001), coagulase-negative staphylococci (P < 0.001), Escherichia coli (P < 0.02), and all microorganisms combined (P < 0.005) were recovered from FAN bottles; more nonfermentative gram-negative bacilli (P < 0.05), Torulopsis glabrata (P < 0.001), and other yeasts (P < 0.01) were recovered from standard bottles. Growth of S. aureus (P < 0.001), coagulase-negative staphylococci (P < 0.001), Enterococcus faecalis (P < 0.025), streptococci other than Streptococcus pneumoniae (P < 0.01), and all microorganisms combined (P < 0.001) was detected earlier in standard bottles; growth of more isolates of E. coli (P < 0.05) and anaerobic bacteria (P < 0.01) was detected earlier in FAN bottles. The mean times to detection were 14.2 and 16.1 h for standard and FAN bottles, respectively. More septic episodes caused by S. aureus (P < 0.001), coagulase-negative staphylococci (P < 0.005), members of the family Enterobacteriaceae (P < 0.02), and all microorganisms combined (P < 0.02) were detected in FAN bottles; more septic episodes caused by nonfermentative gram-negative bacilli (P < 0.025) and yeasts (P < 0.005) were detected in standard bottles. In summary, more isolates (except for strict aerobes) were recovered from FAN bottles than from standard anaerobic bottles. Similarly, significant more septic episodes (except for those caused by strict aerobes) were detected with FAN bottles than with standard anaerobic bottles. With the exception of E. coli and anaerobic bacteria, growth of more isolates was detected earlier in standard anaerobic bottles.
Comparison of in vitro culture, immunohistochemical staining, and PCR for detection of Borrelia burgdorferi in tissue from experimentally infected animals
Authors: Lebech A M , Clemmensen O , Hansen K
Published: January 2020

An avidin-biotin-amplified immunophosphatase staining method with a purified polyclonal rabbit anti-Borrelia burgdorferi hyperimmune serum was developed for identification of B. burgdorferi in tissue specimens. The diagnostic efficacy was compared with those of in vitro culture and PCR with fresh and fixed, paraffin-embedded tissues. A nested PCR assay was developed for identification of a 276-bp fragment of the B. burgdorferi flagellin gene. The diagnostic sensitivities of the different techniques were evaluated with spleen, renal, and urinary bladder tissues from eight experimentally infected gerbils. A systemic infection was verified by positivity of 23 of 24 (96%) organ cultures. B. burgdorferi was visualized immunohistochemically in 9 of 23 (39%) of the specimens. Among these nine specimens, an average of 33% of the 15 sections examined were positive. The spirochetes accumulated in discrete clusters and were associated with focal lymphocytic infiltration. The diagnostic sensitivity obtained by PCR with fixed, paraffin-embedded tissue was 21%, considerably lower than that with fresh tissue (71%). Thus, the reliable demonstration of B. burgdorferi by immunohistochemical staining is possible but extremely laborious, and considering the fact that the density of B. burgdorferi in human tissue is even lower than that in experimentally infected animals, the method is not useful in a clinical setting. It may, however, still be valuable in pathogenetic research. Detection of B. burgdorferi DNA by PCR should be performed with fresh tissue specimens and not with fixed, paraffin-embedded specimens.
Evaluation of accuracy and reproducibility of E test for susceptibility testing of Streptococcus pneumoniae to penicillin, cefotaxime, and ceftriaxone
Authors: Skulnick M , Small G W , Lo P , Patel M P , Porter C R , Low D E , Matsumura S , Mazzulli T
Published: January 2020

We evaluated the reproducibility with which technologists perform and interpret the E test (AB Biodisk, North America, Inc., Piscataway, N.J.) for determining the susceptibility of Streptococcus pneumoniae to penicillin, cefotaxime, and ceftriaxone. Four technologists prepared E test assays to test 124 isolates of S. pneumoniae. Each technologist then interpreted the results of the E test blinded to the interpretation of the other technologists. In addition, E test results were compared with the reference method of broth microdilution. Intraobserver and interobserver agreement were assessed by use of the kappa statistic. Interpretation of the E test and broth microdilution results showed substantial to excellent agreement, with kappa values ranging from 0.878 to 0.987. Compared with broth microdilution, no very major errors and only four major errors were made with the E test. Most minor errors with penicillin and ceftriaxone occurred for isolates with intermediate or high-level resistance, whereas for cefotaxime the minor errors were more evenly distributed between susceptible and intermediate resistance and between intermediate and high-level resistance. These results indicate that there is good agreement between technologists for the interpretation of the E test when testing the susceptibility of S. pneumoniae to penicillin, cefotaxime, and ceftriaxone and that the results of the E test agree with those of broth microdilution.
Species-specific PCR detection of malaria parasites by microtiter plate hybridization: clinical study with malaria patients
Authors: Kimura M , Miyake H , Kim H S , Tanabe M , Arai M , Kawai S , Yamane A , Wataya Y
Published: January 2020

A simple and convenient PCR method that amplifies the 18S rRNA genes has been developed for the purpose of detecting and differentiating four species causing malaria in humans. The advantage of the assay is that the biotinylated PCR product is visualized following hybridization with specific probes which are immobilized on plate wells (microtiter plate hybridization). This method has been previously evaluated in a field study and was found to be sensitive and specific for the detection of Plasmodium falciparum and Plasmodium vivax. In the current study, the microtiter plate hybridization PCR method was evaluated by using blood specimens from malaria patients. All of 36 cases of falciparum malaria, 26 of 27 cases of vivax malaria, all of 11 cases of ovale malaria, and 2 cases of malariae malaria were diagnosed species specifically by the PCR method. There were four smear-negative, PCR-positive cases that seemed to correspond to the convalescent stage of malaria. In contrast, 30 cases for which the diagnosis of malaria has been excluded on the basis of microscopy and clinical courses showed negative PCR results. By comparing parasite densities and PCR results following antimalarial treatment of some patients, it was revealed that the PCR results largely paralleled the parasite densities and that PCR could detect as few as 10 parasites per microliter of blood. We conclude that this PCR method is highly sensitive and specific for the detection of all four parasite species and can serve as a useful supplement to microscopy for the clinical management of malaria.
Bartonella henselae prevalence in domestic cats in California: risk factors and association between bacteremia and antibody titers
Authors: Chomel B B , Abbott R C , Kasten R W , Floyd-Hawkins K A , Kass P H , Glaser C A , Pedersen N C , Koehler J E
Published: January 2020

The isolation of Bartonella henselae, the agent of cat scratch disease, from the blood of naturally infected domestic cats and the demonstration that cats remain bacteremic for several months suggest that cats play a major role as a reservoir for this bacterium. A convenience sample of 205 cats from northern California was selected between 1992 and 1994 to evaluate the B. henselae antibody and bacteremia prevalences and to determine the risk factors and associations between bacteremia and antibody titers. B. henselae was isolated from the blood of 81 cats (39.5%). Forty-two (52%) of these bacteremic cats were found to be infected with > or = 1,000 CFU/ml of blood. Impounded or former stray cats were 2.86 (95% confidence interval [CI] = 1.94, 4.22) times more likely to be bacteremic than the pet cats. Young cats ( < 1 year old) were more likely than adult cats to be bacteremic (relative risk = 1.64; (95% CI = 1.19, 2.28). Bacteremic cats were more likely than nonbacteremic cats to be infested with fleas (relative risk = 1.64; 95% CI = 1.38, 1.96). No association between B. henselae infection and feline immunodeficiency virus antibody prevalence was observed. Eighty-one percent of the cats (166 of 205) tested positive for B. henselae antibodies, and titers were higher in bacteremic than in nonbacteremic cats. Multiple logistic regression analysis indicated that younger age and seropositivity for B. henselae antibodies were associated with bacteremia. Serological screening for Bartonella antibodies may not be useful for the identification of bacteremic cats (positive predictive value = 46.4%), but the lack of antibodies to B. henselae was highly predictive of the absence of bacteremia (negative predictive value = 89.7%). Seronegative cats may be more appropriate pets for immunocompromised individuals who are at increased risk for developing severe B. henselae disease.
Antigenic properties and diagnostic potential of puumala virus nucleocapsid protein expressed in insect cells
Authors: Vapalahti O , Lundkvist A , Kallio-Kokko H , Paukku K , Julkunen I , Lankinen H , Vaheri A
Published: January 2020

Puumala virus (PUU) is a member of the genus Hantavirus in the family Bunyaviridae and the causative agent of nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome. Sera of nephropathia epidemica patients react specifically with PUU nucleocapsid (N) protein. In order to safely provide large quantities of antigen for diagnostic purposes, PUU Sotkamo strain N protein was expressed by using the baculovirus system in Sf9 insect cells to up to 30 to 50% of the total cellular protein. The recombinant N protein (bac-PUU-N) was solubilized with 6 M urea, dialyzed, and purified by anion-exchange liquid chromatography. In an immunoglobulin M mu-capture assay purified and unpurified bac-PUU-N antigen showed identical results compared with the results of a similar assay based on native PUU antigen grown in Vero E6 cells. An immunoglobulin G monoclonal antibody-capture assay based on unpurified bac-PUU-N also showed results identical to those of an assay with native PUU-N antigen. Moreover, a panel of monoclonal antibodies reactive with eight different epitopes showed identical reactivity patterns with both natural and bac-PUU-N antigen, while two epitopes in PUU-N expressed as a fusion protein in Escherichia coli were not recognized. Puumala hantavirus N protein expressed by the baculovirus system offers a safe and inexpensive source of specific antigen for large-scale diagnostic and seroepidemiological purposes.
Genotype profile of human polyomavirus JC excreted in urine of immunocompetent individuals
Authors: Agostini H T , Ryschkewitsch C F , Stoner G L
Published: January 2020

The human polyomavirus JC (JCV) causes the central demyelinating disease progressive multifocal leukoencephalopathy in about 5% of AIDS patients. To characterize the type profile of JCV in a control population in the United States, 54 females (10 to 79 years of age; average age, 43.4 years) and 51 males (18 to 94 years of age; average age, 47.9 years) were examined for the excretion of different genotypes of JCV in their urine by PCR followed by direct cycle sequencing. The group consisted of 89 patients of a general medical clinic in addition to 16 healthy volunteers. The overall incidence of JC viruria was 43 of 105 (40.9%) subjects, with a marked increase for those subjects above the age of 30 years. Two men were found to excrete two different types of JCV at the same time, indicating double infections. Of the three different genotypes of JCV identified to date, type 1 strains (European) were the most common in this cohort (64% of total strains) followed by type 2 (East Asian) (18%). No type 3 (East African) strains were detected. Indirect evidence for the existence of JCV type 3 was found in seven individuals (16%) in the form of a type 1/3 recombinant (also called type 4). In addition, a single example of JCV which differs from types 1, 2, and 3 and may represent a phylogenetically older type (type 5) was found in a 59-year-old African-American. Delineation of sequence variations between JCV types is essential for the design of primers for sensitive PCR with clinical samples.
Use of gen-probe probe competition assay as a supplement to probes for direct detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urogenital specimens
Authors: Woods G L , Garza D M
Published: January 2020

The potential for development of a cost-effective protocol for selective use of the Gen-Probe probe competition assay (PCA) in conjunction with PACE 2 for direct detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urogenital specimens that would not compromise patient care was investigated. To accomplish this, PCA data from testing performed over 12 months were retrospectively reviewed. Of 237 samples that were presumptively positive for C. trachomatis by initial probe assay and could be tested by PCA, positive PCA results were obtained for 100, 79, and 59%, respectively, of specimens that gave a signal of more than 1,500, 1,000 to 1,500, and less than 1,000 relative light units (RLU). For the 141 specimens that were presumptively positive for N. gonorrhoeae and could be tested by PCA, positive PCA results were obtained for 99, 80, and 42%, respectively, of samples with a signal of more than 1,500, 1,000 to 1,500, and less than 1,000 RLU. These data indicate that PCA should be a routine supplement to Gen-Probe PACE 2 for specimens with an initial signal by probe assay of less than 1,500 RLU and may not be necessary for samples yielding a signal of more than 1,500 RLU.
Genetic heterogeneity of M type 3 group A streptococci causing severe infections in Tayside, Scotland
Authors: Upton M , Carter P E , Orange G , Pennington T H
Published: January 2020

To explain the worldwide increase in the frequency of severe infections by group A streptococci, molecular techniques are increasingly being employed to evaluate the genetic relationships of strains. We used restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), the PCR, ribotyping, and DNA sequence analysis in a study of 13 group A streptococci isolated from a cluster of cases of serious infections over a 3-month period in Tayside, Scotland. Eight of the strains were M type 3; molecular characterization identified two subclones. The first, displaying PFGE profile 4, has been observed in Northern Scotland and has been circulating in New Zealand for over a decade. The second subclone has been documented only in the United Kingdom; it was first seen in 1993 in Scotland. Sequence analysis of emm-3 genes further differentiated the PFGE 4 subclone. DNA sequence analysis of virulence factors supports the suggestion that they may have recently been acquired by horizontal gene transfer.