bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of PCR and pp65 antigenemia assay with quantitative shell vial culture for detection of cytomegalovirus in blood leukocytes from solid-organ transplant recipients
Authors: Storch G A , Buller R S , Bailey T C , Ettinger N A , Langlois T , Gaudreault-Keener M , Welby P L
Published: January 2020

This study compared PCR and an assay for cytomegalovirus (CMV) pp65 antigenemia (CMV-vue; INCSTAR Corp.) with a quantitative shell vial culture (QSVC) technique for the detection of CMV in serial blood specimens from 46 solid-organ transplant recipients. In a comparison based on 535 specimens tested by PCR and QSVC, CMV was detected by PCR in 41 and by QSVC in 37 of 43 recipients at risk of CMV infection. The mean number of days after transplantation of initial detection of CMV was 29.9 for PCR and 34.0 for QSVC (P = 0.01). The antigenemia assay was performed on 395 specimens, including 304 of those also tested by PCR. In these specimens, CMV was detected by the antigenemia assay, QSVC, and PCR in 30, 32, and 35 (respectively) of 38 patients at risk, with no statistically significant difference in the time to detection. Each of the assays detected CMV in similar proportions of patients with and without clinically significant CMV infection. PCR stayed positive longer after transplantation than the other assays but frequently returned to negative when more than 6 months had elapsed after transplantation. The antigenemia assay and PCR stayed positive longer after institution of antiviral therapy than QSVC. PCR can provide highly sensitive detection of CMV viremia, but a PCR assay for CMV is not yet available in kit form. The pp65 antigenemia assay and shell vial culture are quantifiable and comparable in sensitivity. Either is recommended for rapid detection of CMV in blood specimens from solid-organ transplant recipients.
Epidemiologic analysis of sporadic Salmonella typhi isolates and those from outbreaks by pulsed-field gel electrophoresis
Authors: Thong K L , Cheong Y M , Puthucheary S , Koh C L , Pang T
Published: January 2020

Pulsed-field gel electrophoresis (PFGE) was used to compare and analyze 158 isolates of Salmonella typhi from five well-defined outbreaks of typhoid fever in Malaysia and also isolates involved in sporadic cases of typhoid fever occurring during the same period. Digestion of chromosomal DNAs from these S. typhi isolates with the restriction endonucleases XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3') and then PFGE produced restriction endonuclease analysis (REA) patterns consisting of 11 to 24 DNA fragments ranging in size from 20 to 630 kbp. Analysis of the REA patterns generated by PFGE after digestion with XbaI and SpeI indicated that the S. typhi isolates obtained from sporadic cases of infection were much more heterogeneous (at least 13 different REA patterns were detected; Dice coefficient, between 0.73 and 1.0) than those obtained during outbreaks of typhoid fever. The clonal nature and the close genetic identities of isolates from outbreaks in Alor Setar, Penang, Kota Kinabalu, Johor Bahru, and Kota Bahru were suggested by the fact that only a limited number of REA patterns, which mostly differed by only a single band, were detected (one to four patterns; Dice coefficient, between 0.82 and 1.0), although a different pattern was associated with each of these outbreaks. Comparison of REA patterns with ribotyping for 18 S. typhi isolates involved in sporadic cases of infection showed a good correlation, in that 72% of the isolates were in the same group. There was no clear correlation of phage types with a specific REA pattern. We conclude that PFGE of s. typhi chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for comparing and differentiating S. typhi isolates for epidemiological purposes.
Humoral immune responses to Shiga-like toxins and Escherichia coli O157 lipopolysaccharide in hemolytic-uremic syndrome patients and healthy subjects
Authors: Greatorex J S , Thorne G M
Published: January 2020

Shiga-like-toxin (SLT)-producing Escherichia coli strains, especially serotype O157:H7, are important causes of bloody diarrhea and are associated with the development of hemolytic-uremic syndrome (HUS). Enzyme-linked immunosorbent assays (ELISAs) were developed for the serologic detection of immunoglobulin M (IgM), IgG, and IgA to Shiga toxin (ST) and SLT-I, IgG to SLT-II, and IgM and IgG reactive against E. coli O157 lipopolysaccharide (LPS). Serum samples were collected from 27 HUS patients (25 pediatric and 2 adult) and tested in the ELISAs. Of 27 patients, 10 (37%) were positive for at least one class of antibody to ST/SLT-I. None of the patients were positive for IgG antibody to SLT-II. Twenty-one of the 27 patients (78%) were positive for antibody to E. coli O157 LPS; 19 of 27 (70%) were positive for IgM, and 20 of 27 (74%) were positive for IgG. None of 48 control serum samples were positive in any of the toxin assays, and only 1 of 48 (2%) and 2 of 48 (4%) were positive for IgM and IgG, respectively, to E. coli O157 LPS. Twelve of the 24 patients (50%) from whom stool specimens were collected were positive by culture for E. coli O157. Overall, serology and culture produced confirmation of infection by SLT-producing organisms in 23 of 27 (85%) HUS patients. A combination of ELISA for antibodies to E. coli O157 LPS and culture provided evidence for 22 of 27 (82%) of these patients. The results indicate that while ELISAs for ST/SLT-I and SLT-II antibodies were of limited diagnostic value, the ELISAs for IgM and IgG to E. coli O157 LPS provided valuable and sensitive adjuncts to culture.
Detection and strain identification of Actinobacillus actinomycetemcomitans by nested PCR
Authors: Leys E J , Griffen A L , Strong S J , Fuerst P A
Published: January 2020

By using PCR, Actinobacillus actinomycetemcomitans strains were identified directly from plaque samples without the need to isolate or culture bacteria. DNA fragments were generated by a nested, two-step PCR amplification of the ribosomal spacer region between the 16S and 23S rRNA genes. For the first amplification, primers homologous to sequences common to all bacterial species were used. This was followed by a second amplification with primers specific to A. actinomycetemcomitans. The ribosomal DNA spacer region was amplified from as few as 10 bacterial cells within a total population of 10(8) cells (0.00001%), and cross-reactivity between species was not observed. DNA fragments specific for Porphyromonas gingivalis were generated from the same samples by using a P. gingivalis-specific primer, and equivalent sensitivity and specificity were observed. A. actinomycetemcomitans was detected in 60% and P. gingivalis was detected in 79% of 52 subjects tested. Sequence analysis of the spacer region DNA fragment for A. actinomycetemcomitans gave precise strain identification, producing unique sequences for seven reference strains and identification of nine plaque-derived isolates. A phylogenetic tree based on quantitative sequence relationships was constructed. Two-step PCR amplification directly from plaque samples combined with sequence analysis of the ribosomal DNA spacer region provides a sensitive assay for detection and strain identification of multiple species directly from a single plaque sample. This simplified approach provides a practical method for large-scale studies on the transmission and pathogenicity of periodontitis-associated bacteria.
Oligonucleotide (GTG)5 as a marker for Mycobacterium tuberculosis strain identification
Authors: Wiid I J , Werely C , Beyers N , Donald P , van Helden P D
Published: January 2020

Culture of Mycobacterium tuberculosis provides no information on the identity of a strain or the distribution of such a strain in the community. Strain identification of M. tuberculosis can help to address important epidemiological questions, e.g., the origin of an infection in a patient's household or community, whether reactivation of infection is endogenous or exogenous in origin, and the spread and early detection of organisms with acquired antibiotic resistance. To research this problem, strain identification must be reliable and accurate. Although genetic identification techniques already exist, it is valuable to have genetic identification techniques based on a number of genetic markers to improve the accurate identification of M. tuberculosis strains. We show that oligonucleotide (GTG)5 can be successfully applied to the identification of M. tuberculosis strains. This technique may be particularly useful in cases in which M. tuberculosis strains have few or no insertion elements (e.g., IS6110) or in identifying other strains of mycobacteria when informative probes are lacking.
Significance of highly positive c22-3 "indeterminate" second-generation hepatitis C virus (HCV) recombinant immunoblot assay (RIBA) and resolution by third-generation HCV RIBA
Authors: Pawlotsky J M , Fleury A , Choukroun V , Deforges L , Roudot-Thoraval F , Aumont P , Duval J , Dhumeaux D
Published: January 2020

Second-generation recombinant immunoblot assay (RIBA) is widely used for the validation of anti-hepatitis C virus (HCV) antibody detection. The aims of this work were (i) to determine, in terms of liver disease and HCV replication, the significance of a peculiar "indeterminate" second-generation RIBA pattern characterized by the presence of high titers of antibodies directed to c22-3, a protein bearing core epitopes and (ii) to determine whether a more advanced version of the same strip assay, namely a third-generation RIBA, may solve the problem of such indeterminate patterns. Sixty patients for which c22-3 indeterminate second-generation RIBAs were highly positive were studied. Forty-two of them (70%) were immunocompromised. Serum transaminases were increased in 46 cases (77%), and HCV RNA was detected by PCR in 50 cases (83%). Third-generation RIBA remained highly positive c22 indeterminate for 9 patients (15%) but was positive for 51 (85%), mostly because of increased sensitivity for the detection of both anti-c100 and anti-c33c antibodies. These results suggest that third-generation RIBA may achieve resolution of most of these cases but that highly positive c22 indeterminate third-generation RIBA may persist when used with some patients with very low titers of anti-HCV nonstructural protein antibodies.
Detection of cytomegalovirus DNA in sera of liver transplant recipients
Authors: Patel R , Smith T F , Espy M , Wiesner R H , Krom R A , Portela D , Paya C V
Published: January 2020

We prospectively studied the utility of the amplification of cytomegalovirus (CMV) DNA in the sera of liver transplant recipients in order to predict symptomatic CMV infection, thus enabling preemptive therapy with antiviral agents. Serum samples obtained at biweekly intervals from 20 sequential liver transplant recipients for at least 8 weeks following transplantation were tested by the PCR amplification procedure. Results were correlated with blood and urine cultures, histopathological findings from infected organs, and clinical manifestations. Six patients (30%) developed symptomatic CMV infection; in five (83%) of these patients, CMV DNA was detected prior to symptomatic CMV infection, and in one (17%) of these patients, CMV DNA was detected at the time of symptomatic CMV infection. CMV DNA was detected a mean of 13 days (range, 0 to 23 days) prior to the onset of symptomatic CMV infection. In addition, CMV DNA was detected in the sera of four of five patients with asymptomatic viremia and two patients with asymptomatic viruria. Lastly, the PCR was negative for sera from seven patients with no evidence of CMV infection. We found that PCR was able to detect the presence of CMV DNA in the sera of liver transplant recipients at a sensitivity of 92% and a specificity of 100% for CMV infection, while the sensitivity and specificity for symptomatic infection were 100 and 57%, respectively.
Comparison of Gen-Probe Group A streptococcus Direct Test with culture for diagnosing streptococcal pharyngitis
Authors: Pokorski S J , Vetter E A , Wollan P C , Cockerill F R
Published: January 2020

The Group A Streptococcus Direct Test (GP-ST test; Gen-Probe, Inc., San Diego, Calif.) was compared with culture for the detection of Streptococcus pyogenes from throat swabs of 767 patients with pharyngitis. Swabs were tested by the GP-ST test after inoculating a 5% sheep blood agar (SBA) plate. SBA plates were incubated at 35 degrees C in room air for 72 h. SBA plates with no evidence of beta-hemolytic colonies after 18 to 24 h of incubation were subcultured by taking a swipe across the primary inoculum from the SBA plate to an agar selective for Streptococcus spp. In a low-prevalence (11.9%) population and in comparison with the number of positive cultures detected by the 72-h single-culture method (SBA plate method), the GP-ST test had a sensitivity of 88.6%, a specificity of 97.8%, a positive predictive value of 83.9%, and a negative predictive value of 98.5%. In comparison with the growth of any colonies of S. pyogenes on the 72-h SBA plates plus a subculture onto selective blood agar, the sensitivities and specificities were as follows: 72-h SBA plate method, 96.7 and 100%, respectively; GP-ST test, 85.7 and 97.8%, respectively. The GP-ST test is an easy-to-perform, reliable test for batch screening of throat swabs for S. pyogenes.
Enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Escherichia coli Vero cytotoxin 1
Authors: Karmali M A , Petric M , Winkler M , Bielaszewska M , Brunton J , van de Kar N , Morooka T , Nair G B , Richardson S E , Arbus G S
Published: January 2020

The frequency of Vero cytotoxin 1 (VT1)-neutralizing antibody (NAb) in serum specimens from 790 age-stratified (0 to 70 years) control individuals from Toronto was 61 of 790 (7.7%), with a peak of 19% in the 20- to 30-year-old age group and a second peak of 16.7% in the 60- to 70-year-old age group. A total of 568 serum specimens, including 538 from the 790 Toronto control subjects, 21 from patients from three outbreaks of VT-producing Escherichia coli (VTEC) infection, and 9 known VT1-NAb-positive serum specimens from patients with hemolytic-uremic syndrome (HUS), were then tested for the presence of anti-VT1 immunoglobulin G (IgG) by an enzyme-linked immunosorbent assay (ELISA). The mean ELISA values of 522 VT1-NAb-negative serum specimens and 46 VT1-NAb-positive serum specimens were 0.09 +/- 0.06 (range, 0 to 0.56) and 0.78 +/- 0.66 (range, 0.16 to 2.91), respectively (P < 0.001; Student's t test). With a breakpoint of 0.21 (mean ELISA value of the VT1-NAb-negative sera + 2 standard deviations), the sensitivity, specificity, positive predictive value, and negative predictive value of the VT1 IgG ELISA compared with those of the VT1-NAb assay were, respectively, 95.7, 98.7, 86.3, and 99.6%. There were nine discrepant serum specimens, of which seven were anti-VT1 IgG positive and VT1-NAb negative and two were anti-VT1 IgG negative and VT1-NAb positive. The ELISA was also used for testing 238 control serum specimens from The Netherlands, Japan, and India and acute- and convalescent-phase serum specimens from 42 Toronto patients with HUS. The frequencies of anti-VT1 IgG (with VT1-NAb frequencies in parantheses) in control sera from the Netherlands, Japan, and India were 6% (3%), 1.1% (0%), and 12% (10%), respectively, with no age clustering. The frequencies of anti-VT1 IgG seropositivity in HUS patients were 5 of 14 (35.7%) in patients with unknown toxin exposure, 2 of 22 (9.1%) in individuals with known exposure to VT1 plus VT2 or VT1 alone, and 0 of 6 (0%) in patients exposed to only VT2. Development of serum anti-VT1 IgG response appears to be the exception rather than the rule in sporadic HUS patients infected with VTEC expressing VT1. However, in two family outbreaks associated with VTEC strains expressing VT1 alone and VT1 plus VT2, respectively, the presence of anti-VT1 IgG in virtually all exposed individuals who remained symptom free suggests that the presence of antibody was associated with protection.
Differential blocking of coagulation-activating pathways of Limulus amebocyte lysate
Authors: Zhang G H , Baek L , Buchardt O , Koch C
Published: January 2020

The coagulation of Limulus amebocyte lysate (LAL) can be activated through two pathways, one initiated by endotoxin and the other by beta-glucans. The two pathways join at the step of activation of the proclotting enzyme. We report here that the endotoxin-activated pathway can be differentially inhibited by two methods in a Limulus enzyme-linked immunosorbent assay (ELISA), either by the combined use of dimethyl sulfoxide and polymyxin B or by a monoclonal antibody against Limulus factor C. LAL reactivities to 10 different endotoxin preparations could be inhibited by the former method by a factor of 10(4) to 10(6) and could be blocked almost totally by the latter method, irrespective of the source of endotoxin. The sensitivity of the assay was approximately 50 pg/ml both for curdlan from Alcaligenes faecalis and for laminarin from Laminaria digitata. We also found that the beta-glucan-activated pathway could be totally blocked by laminarin (> 1 microgram/ml) without affecting the endotoxin-activated pathway, allowing endotoxin to be quantitated specifically by the Limulus ELISA with a detection limit of 0.005 endotoxin unit per ml. The use of uninhibited and differentially inhibited ELISAs demonstrated that different LAL preparations showed much greater variation in assaying beta-glucans than in assaying endotoxins. The LAL reactivity of normal human plasma was found to be due to the activation of the beta-glucan pathway, but not the endotoxin pathway, of LAL.
Multisite reproducibility of colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates
Authors: Pfaller M A , Vu Q , Lancaster M , Espinel-Ingroff A , Fothergill A , Grant C , McGinnis M R , Pasarell L , Rinaldi M G , Steele-Moore L
Published: January 2020

MICs of fluconazole and amphotericin B were determined independently for 100 coded yeast isolates by each of six laboratories to determine reproducibility of results by using a colorimetric oxidation-reduction-based broth microdilution test. In addition, each site tested five quality control isolates on at least four different occasions during the study. Results agreed within a three-dilution range (mode +/- 1 log2 dilution) for 96.2% of fluconazole tests and 92.7% of amphotericin B tests. Agreement among tests with the quality control isolates was 99.4% with fluconazole and 98.6% with amphotericin B. These results indicate that the colorimetric microdilution method is reproducible among laboratories.
Detection of Streptococcus pneumoniae DNA in blood cultures by PCR
Authors: Hassan-King M , Baldeh I , Secka O , Falade A , Greenwood B
Published: January 2020

We have developed a PCR assay, with primers derived from the autolysin (lyt) gene, for the detection of Streptococcus pneumoniae DNA in blood cultures. The predicted fragment of 247 bp was detected in all strains of pneumococci, embracing 12 different serotypes that were tested. Although DNA extracted from four viridans streptococci spp. Streptococcus oralis, Streptococcus mitis, Streptococcus sanguis, and Streptococcus parasanguis) gave amplification products, these were quite different from the predicted fragment for S. pneumoniae. Application of the assay for diagnosis of septicemia caused by S. pneumoniae showed concordance between PCR and culture results. However, on four occasions PCR was positive in supernatants from both paired culture bottles while pneumococci were cultured from only one. Performing PCR on negative cultures in controlled studies such as vaccine trials may provide a sensitive tool for increasing case detection.
Distinct genotypes of human and canine isolates of Campylobacter upsaliensis determined by 16S rRNA gene typing and plasmid profiling
Authors: Stanley J , Jones C , Burnens A , Owen R J
Published: January 2020

The utility of combined 16S rRNA (rrs) gene restriction fragment length polymorphism and plasmid profiles to differentiate between and within Campylobacter upsaliensis of human and canine origin was examined. Fourteen distinct rrs gene restriction fragment length polymorphs consisting of bands sized between 1.9 and 4.8 kb were observed. The copy number of the 16S rRNA gene was three in most strains of C. upsaliensis. Plasmids were found in almost 60% of the strains; ranging in size from 1.5 to 100 kb, they gave 15 distinct plasmid profiles. All isolates from humans contained one or more plasmids, as did strains isolated from dogs with sporadic diarrhea. The two commonest 16S ribotypes were divided into eight and nine subgroups by plasmid profiling. The genotyping of canine isolates from three veterinary surveys detected both multiple infections and reinfection of dogs. Except for one, each of the isolates from humans constituted a single and unique 16S ribotype, and these more frequently carried plasmids than did canine strains. Ribotypes of human strains were not found among canine isolates. These results suggest that host-specific genotypic differences may exist among strains of C. upsaliensis, for example, intraspecific clones or clone complexes pathogenic for humans.
Characterization and presumptive identification of Helicobacter pylori isolates from rhesus monkeys
Authors: Drazek E S , Dubois A , Holmes R K
Published: January 2020

We characterized 38 Helicobacter isolates, including 22 from gastric biopsy samples obtained from 14 rhesus monkeys and single isolates from 16 monkeys in a different colony. Biochemical profiles of these isolates were nearly identical to that of Helicobacter pylori ATCC 43504. Restriction fragment length polymorphism (RFLP) analysis indicated that each infected monkey harbored one to four strains. The 17 RFLP types found among these 22 isolates differed from all seven RFLPs found among the other 16 isolates. Thus, monkeys within a given colony are more likely to be infected by Helicobacter isolates with the same or a similar RFLP than are monkeys from different colonies. A 16S rRNA gene was amplified by PCR and cloned from the Helicobacter isolate from rhesus monkey 85D08. Ribotyping with this probe demonstrated less diversity among isolates from rhesus monkeys than was reported among isolates of H. pylori from humans, as did RFLP analysis of a PCR fragment of the ureA-ureB gene cluster. The DNA sequence of the cloned 16S rRNA gene was determined and compared with sequences reported for H. pylori and other Helicobacter species. Our analysis of 127 nucleotides (corresponding with residues 1240 to 1366 of the Escherichia coli 16S rRNA gene) indicated that the Helicobacter isolate from monkey 85D08 was 99.2 to 100% homologous to isolates of H. pylori from humans but only 83.5 to 96.9% homologous with other Helicobacter species in this region of the 16S rRNA gene. These data provide strong support for the presumptive identification of these isolates as H. pylori.
Use of PCR to study epidemiology of Serratia marcescens isolates in nosocomial infection
Authors: Liu P Y , Lau Y J , Hu B S , Shir J M , Cheung M H , Shi Z Y , Tsai W S
Published: January 2020

A method to characterize strains of Serratia marcescens based on the PCR amplification of enterobacterial repetitive intergenic consensus sequences has been developed. The PCR fingerprints were generated from boiled supernatants prepared directly from bacterial colonies without the need for DNA extraction. The technique was applied to isolates obtained during an outbreak of pneumonia from seven mechanically ventilated patients, and its result indicated that the outbreak was due to the spread of two epidemic strains. This technique was validated by comparison with rRNA gene restriction analysis. There was complete concordance between these two techniques in discriminating the outbreak-related strains from epidemiologically unrelated isolates. Typing with both biochemical profile and antibiogram profile, though simple, was found to be less reliable than genotyping. The results show that this enterobacterial repetitive intergenic consensus PCR provides a rapid and simple means of typing S. marcescens isolates for epidemiologic studies.
Enzymatic amplification of lactate dehydrogenase-elevating virus
Authors: van der Logt J T , Kissing J , Melchers W J
Published: January 2020

To improve the detection of lactate dehydrogenase-elevating virus (LDV), we developed a PCR assay. Primers were selected from ORF7, encoding nucleocapsid protein VP1. No specific amplification was observed with any other common murine virus or with RNAs from the closely related Lelystad virus and equine arteritis virus. In experimentally infected mice, LDV could be detected in plasma in both the acute and the persistent phases. LDV was also detected by the PCR in contaminated pools of Plasmodium berghei parasites which were maintained in mice, both by a direct analysis of the samples and by testing of plasma from mice inoculated with these pools. There was a complete agreement between the results of the PCR assay and the lactate dehydrogenase (LDH) enzyme assay of plasma from the inoculated mice. In contrast to the results of the LDH enzyme assay, no false-positive reactions were obtained in the PCR assay with negative control samples showing visible hemolysis. Storage of plasma samples at room temperature and at 4, -20, and -80 degrees C for up to 8 days did not influence the results of the PCR. These results show that the PCR is a valuable technique which may replace the LDH test as a diagnostic tool.
Biodegradable, effective substitute for xylene in the Ehrlich indole procedure
Authors: Miller J M , Miller M D , Driscoll P E , Miller P
Published: January 2020

Three extracting reagents were compared for effectiveness in the Ehrlich indole procedure: xylene (the recommended reagent), Hemo-De (a terpene-based product containing d-limonene), and Micro-Clear (an isoparaffinic hydrocarbon). Thirty-three strains representing 12 species of indole-positive aerobes or facultative anaerobes and 50 strains representing 11 species of indole-positive strict anaerobes were tested using the three reagents. Xylene extraction allowed indole detection in all of the isolates tested. Micro-Clear allowed detection in all of the aerobic isolates and in 49 of 50 anaerobes. Hemo-De allowed indole detection in 32 of 33 aerobes and in 49 of 50 anaerobes. There was no significant difference in the results among the reagents. Because Micro-Clear is biodegradable, nonflammable, noncarcinogenic, and odorless, we feel that this product should be considered a safe and effective substitute for xylene in the Ehrlich indole procedure.
Efficacy of microbial identification system for epidemiologic typing of coagulase-negative staphylococci
Authors: Birnbaum D , Herwaldt L , Low D E , Noble M , Pfaller M , Sherertz R , Chow A W
Published: January 2020

The lack of an adequate typing system hampers our understanding of the epidemiology of infections caused by coagulase-negative staphylococci (CoNS). CoNS have become recognized as important nosocomial pathogens and the principal cause of infections associated with invasive devices. Sensitive, specific, and convenient methods are needed to evaluate whether implementing infection control guidelines reduces the risk of nosocomial infections from CoNS and other pathogens. The Microbial Identification System (MIDI) (Microbial ID Inc., Newark, Del.), a semiautomated system for fatty acid methyl ester analysis, shows considerable promise for clinical and epidemiologic applications. Its predictive accuracy and reliability were tested by using epidemiologically related and replicated CoNS isolates as well as CoNS from epidemiologically unrelated clinical infections, which were obtained from five established hospital culture collections in diverse geographic locations. Two hundred isolates were fully characterized in 5 days by one person using MIDI, and the results were similar to those produced by more expensive and time-consuming conventional typing methods. MIDI appears to be a useful screening tool that could be used before more expensive and labor-intensive molecular methods. It offers important advantages to hospital epidemiologists and clinical microbiologists who must identify and type CoNS isolates.
Genital flora in pregnancy and its association with intrauterine growth retardation
Authors: Germain M , Krohn M A , Hillier S L , Eschenbach D A
Published: January 2020

A study of risk factors for intrauterine growth retardation (IUGR) was conducted among a cohort of 13,914 pregnant women enrolled in the multicenter Vaginal Infections and Prematurity Study. From 23 through 26 weeks of gestational age, cultures of specimens from the vagina and cervix were done for group B streptococci, Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Candida albicans, Gardnerella vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, and anaerobic gram-negative rods belonging to the genera Bacteroides, Porphyromonas, and Prevotella. Newborns who were small for their gestational age were delivered by 1,251 women, and infants of the appropriate weight for their gestational age were delivered by 10,332 women. When controlling for ethnicity and smoking and excluding women treated with antibiotics, the Mantel-Haenszel adjusted relative risk of IUGR was 1.16 for Bacteroides, Prevotella, and Porphyromonas spp. (95% confidence interval [95% CI], 1.01 to 1.33), 1.16 for M. hominis (95% CI, 1.04 to 1.29), 1.20 for U. urealyticum (95% CI, 1.05 to 1.38), and 1.22 for T. vaginalis (95% CI, 1.05 to 1.42). There was also a strong and significant trend for an increasing risk of IUGR with the number of these four microbes recovered. Among women colonized with all four isolates, the adjusted odds ratio of IUGR was 1.79 (95% CI, 1.27 to 2.52) in comparison with women not colonized with any of these microorganisms. Group B streptococci, N. gonorrhoeae, C. trachomatis, and C. albicans were not significantly associated with IUGR. These results suggest that infection is associated with some cases of IUGR and that specific microorganisms, alone or in combination, are involved. Since genital isolates are highly correlated with each other, the relative contribution of each microbe is difficult to determine.
Characterization of strains of Neisseria meningitidis recovered from complement-sufficient and complement-deficient patients in the Western Cape Province, South Africa
Authors: Orren A , Caugant D A , Fijen C A , Dankert J , van Schalkwyk E J , Poolman J T , Coetzee G J
Published: January 2020

Complement deficiency has been associated with increased susceptibility to meningococcal disease. In order to determine whether special meningococcal strains caused disease in complement-deficient (CD) patients, 17 Neisseria meningitidis strains recovered from patients in the Western Cape Province, South Africa, known to be CD were compared with 124 routine isolates obtained from patients living in the same area. Serogrouping of the strains from the CD subjects revealed that the common serogroups, particularly serogroup B, predominated. However, the prevalence of rare serogroups among isolates from CD subjects was significantly higher than that found among isolates from the control group. Sero- and subtyping of the class 1 and class 2 or 3 outer membrane proteins showed no significant difference between isolates from CD subjects and the routine clinical isolates. Multilocus enzyme electrophoresis of the 141 isolates revealed six clusters of electrophoretic types (ETs) and two unrelated ETs. The same degree of genetic diversity existed in ETs of isolates from CD subjects and the control group. However, the ET-5 complex, which is known to be associated with epidemic disease, was found in 22 (18%) of the routine clinical isolates but in none of the isolates from the CD subjects. This difference was marginally significant. What was highly significant was the finding that 8 of the 17 isolates from CD subjects were in one ET cluster, cluster F, which comprised a total of 20 isolates. Thus, our results show a difference in the clonal compositions of the strains that infect CD subjects in comparison with the clonal compositions of those that cause clinical infections in the population at large.
Detection of porcine reproductive and respiratory syndrome virus and efficient differentiation between Canadian and European strains by reverse transcription and PCR amplification
Authors: Mardassi H , Wilson L , Mounir S , Dea S
Published: January 2020

Two sets of oligonucleotide primers (1008PS-1009PR and 1010PLS-1011PLR) were designed according to the sequence of the nucleocapsid protein (N) gene of Quebec reference strain IAF-exp91 of porcine reproductive and respiratory syndrome virus (PRRSV). The primers were used in reverse transcription and PCR (RT-PCR) experiments for detection of viral genomic RNA either from infected porcine alveolar macrophages (PAM) or tissues from experimentally infected specific-pathogen-free pigs. Considering the high degree of variation detected between the nucleotide sequences of the N genes of IAF-exp91 and Lelystad virus (LV) strains of PRRSV, the primers 1008PS-1009PR were referred to as the specific primers, since they were chosen in such a manner that they could amplify only sequences from IAF-exp91 RNA and not from LV. On the other hand, the primer pair 1010PLS-1011PLR was common to both strains of PRRSV. When analyzed by agarose gel electrophoresis, the products of RT-PCR from each set of primers were resolved as single band of the predicted size, the specificity of amplified products being confirmed by Southern blotting with a specific IAF-exp91 N gene probe. No amplification was observed when RNA was extracted from uninfected PAM or from other porcine viruses. As expected, only the common primer pair was able to amplify RNA from the Quebec reference strain and two European strains (LV and Weybridge). The resulting bands displayed differences in electrophoretic mobilities due to the absence of 37 nucleotides in both European strains, thus allowing their differentiation from the IAF-exp91 strain. Most of the tissue culture-adapted Quebec isolates were detected with both primer pairs. The sensitivity of the enzymatic amplification method for detection of PRRSV from lung tissues was a 50% tissue culture infective dose of 5. RT-PCR was found to be more sensitive than indirect immunofluorescence assay for detection of PRRSV in tissues from experimentally infected pigs and as sensitive as virus isolation in PAM, especially when combined with Southern blotting with the digoxigenin-labeled N probe and chemiluminescence detection.
A 1-year evaluation of Syva MicroTrak Chlamydia enzyme immunoassay with selective confirmation by direct fluorescent-antibody assay in a high-volume laboratory
Authors: Chan E L , Brandt K , Horsman G B
Published: January 2020

TThe Syva MicroTrak Chlamydia enzyme immunoassay (EIA; Syva Company, San Jose, Calif.) with cytospin and direct fluorescent-antibody assay (DFA) confirmation was evaluated on 43,630 urogenital specimens over a 1-year period in the Provincial Laboratory in Regina, Saskatchewan, Canada. This was a two-phase study intended to define a testing algorithm for Chlamydia trachomatis that would be both highly accurate and cost-effective in our high-volume (> 3,000 tests per month) laboratory. The prevalence of C. trachomatis infection in our population is moderate (8 to 9%). In phase 1, we tested 6,022 male and female urogenital specimens by EIA. All specimens with optical densities above the cutoff value and those within 30% below the cutoff value were retested by DFA. This was 648 specimens (10.8% of the total). A total of 100% (211 of 211) of the specimens with optical densities equal to or greater than 1.00 absorbance unit (AU) above the cutoff value, 98.2% (175 of 178) of the specimens with optical densities of between 0.500 and 0.999 AU above the cutoff value, and 83% (167 of 201) of the specimens with optical densities within 0.499 AU above the cutoff value were confirmed to be positive. A total of 12% (7 of 58) of the specimens with optical densities within 30% below the cutoff value were positive by DFA. In phase 2, we tested 37,608 specimens (32,495 from females; 5,113 from males) by EIA. Only those specimens with optical densities of between 0.499 AU above and 30% below the cutoff value required confirmation on the basis of data from phase 1 of the study. This was 4.5% of all specimens tested. This decrease in the proportion of specimens requiring confirmation provides a significant cost savings to the laboratory. The testing algorithm gives us a 1-day turnaround time to the final confirmed test results. The MicroTrak EIA performed very well in both phases of the study, with a sensitivity, specificity, positive predictive value, and negative predictive value of 96.1, 99.1, 90.3, and 99.7%, respectively, in phase 2. We suggest that for laboratories that use EIA for Chlamydia testing, a study such as this one will identify an appropriate optical density range for confirmatory testing for samples from that particular population.
Comparison of plasma PCR and bronchoalveolar lavage fluid culture for detection of cytomegalovirus infection in adult bone marrow transplant recipients
Authors: Aspin M M , Gallez-Hawkins G M , Giugni T D , Tegtmeier B , Lang D J , Schmidt G M , Forman S J , Zaia J A
Published: January 2020

Plasma PCR for human cytomegalovirus (CMV) DNA was compared with bronchoalveolar lavage (BAL) fluid culture as an indicator for disseminated CMV infection. Thirteen (32.5%) of 40 consecutive bone marrow transplant (BMT) recipients were BAL fluid culture positive for CMV on day 35 post-BMT, and 9 (69%) of the 13 had positive plasma PCRs between days 28 and 49. Of the 27 with negative BAL fluid cultures, 2 (7%) had positive plasma PCRs (P < 0.001). Plasma CMV DNA in BMT recipients is a useful clinical marker for serious infection.
Opacification of Middlebrook agar as an aid in distinguishing Nocardia farcinica within the Nocardia asteroides complex
Authors: Carson M , Hellyar A
Published: January 2020

Among 58 aerobic actinomycetes isolated from different sources and geographical locations, none of 23 Nocardia asteroides isolates, at 18 N. farcinica isolates, 1 of 5 N. otitidiscaviarum isolates, and 1 of 4 Rhodococcus species isolates opacified Middlebrook 7H10 medium. Within the N. asteroides complex, this characteristic, together with growth at 45 degrees C and resistance to each of erythromycin, cefotaxime, and tobramycin, provides a simple means of distinguishing N. farcinica from N. asteroides.
Distribution of viral genotypes in Italy determined by hepatitis C virus typing by DNA immunoassay
Authors: Ravaggi A , Zonaro A , Marin M G , Puoti M , Albertini A , Cariani E
Published: January 2020

The distribution of hepatitis C virus (HCV) genotypes in Italy was investigated by PCR amplification of the E1 region and hybridization with type I- and type II-specific nonisotopic probes. Positive PCR results were obtained for 65 of 72 patients (90.3%). Type I was detected in 13 of 72 patients (18%), type II was detected in 39 patients (54.2%), and a mixed type I-type II infection was detected in 7 patients (9.7%). Six amplification products not classified by this method shared a low level of homology with HCV types I and II. HCV type I was significantly associated with human immunodeficiency virus, whereas HCV type II was detected in older subjects who were negative for human immunodeficiency virus markers. These results indicate different epidemiological distributions of HCV types I and II in Italy.
Quantitative colorimetric microneutralization assay for characterization of adenoviruses
Authors: Crawford-Miksza L K , Schnurr D P
Published: January 2020

A microneutralization assay that automates the reading and interpretation of viral infectivity and neutralization data was developed for the characterization of adenoviruses (AV). Virus, serum, and cells were added to 96-well plates, incubated for 7 days, and stained with vital stain. The A550 of solubilized dye was read in a plate reader interfaced to a personal computer which analyzed the results. Correlation of A550 values with visual observation of cytopathic effect was extremely good (r = 0.977625). Clinical isolates of 17 AV from 11 patients were characterized by colorimetric neutralization assay. Prototype AV titers tested were comparable to those determined by tube methods. Prototype homotypic antiserum titers were comparable to or greater than those determined by standard tube neutralization.
Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis
Authors: Vaneechoutte M , Dijkshoorn L , Tjernberg I , Elaichouni A , de Vos P , Claeys G , Verschraegen G
Published: January 2020

A total of 53 field and reference strains, including the type strains of the seven named species (nomenspecies) and belonging to the 18 described genomic species (DNA groups) of the genus Acinetobacter, were studied by amplified ribosomal DNA restriction analysis (ARDRA). Restriction analysis with the enzymes AluI, CfoI, MboI, RsaI, and MspI of the enzymatically amplified 16S rRNA genes allowed us to identify all species except the genomic species 4 (Acinetobacter haemolyticus) and 7 (A. johnsonii), 5 (A. junii) and 17, and 10 and 11, which clustered pairwise in three respective groups. Further analysis with the enzyme HaeIII, HinfI, NciI, ScrFI, or TaqI did not allow us to differentiate the species within these three clusters. However, use of a few additional simple phenotypic tests (hemolysis, growth at 37 degrees C, production of acid from glucose, and gelatin hydrolysis) can be used to differentiate between the species within these clusters. ARDRA proved to be a rapid and reliable method for the identification of most of the Acinetobacter genomic species, including the closely related DNA groups 1 (A. calcoaceticus), 2 (A. baumannii), 3, and 13. The results of this study suggest that ARDRA can be used for the identification of Acinetobacter species and as such may help to elucidate the ecology and clinical significance of the different species of this genus. Since ARDRA uses universal 16S rRNA gene primers, it is expected to be applicable to the identification of most bacterial species. Furthermore, ARDRA is less prone to contamination problems than PCR for detection, since the use of cultured organisms results in a large initial quantity of target DNA.
PCR amplification and restriction endonuclease analysis of a 65-kilodalton heat shock protein gene sequence for taxonomic separation of rapidly growing mycobacteria
Authors: Steingrube V A , Gibson J L , Brown B A , Zhang Y , Wilson R W , Rajagopalan M , Wallace R J
Published: January 2020

A total of 129 reference and clinical strains of rapidly growing mycobacteria (RGM) belonging to 10 taxonomic groups were studied for restriction fragment length polymorphism patterns from a PCR-amplified 439-bp segment of the 65-kDa heat shock protein (HSP) gene. Of 24 endonucleases evaluated, restriction fragment length polymorphism patterns produced by HaeIII and BstEII and then by AciI and CfoI gave the best separation. Sixty percent of all RGM taxa studied were differentiated by HaeIII digests alone. Single unique patterns were observed with HaeIII and/or BstEII for Mycobacterium fortuitum (100%), M. chelonae (94%), M. abscessus (96%), M. smegmatis (100%), M. mucogenicum (formerly the M. chelonae-like organism) (100%), and the sorbitol-negative third biovariant of M. fortuitum (100%). Evidence is presented in support of two subgroups within M. peregrinum, M. smegmatis, and the unnamed third biovariant of M. fortuitum (sorbitol positive and sorbitol negative). PCR-based technology provides a rapid, accurate system for the identification of clinically important species of RGM which should be particularly useful for reference laboratories.
Primary identification of Microbacterium spp. encountered in clinical specimens as CDC coryneform group A-4 and A-5 bacteria
Authors: Funke G , Falsen E , Barreau C
Published: January 2020

Over nearly two decades, 13 yellow- or orange-pigmented, fermentative gram-positive rods belonging to the genus Microbacterium were encountered in clinical specimens. All 13 strains, 10 of which came from blood cultures, were initially identified as CDC coryneform group A-4 and A-5 bacteria according to the scheme of Hollis and Weaver for the identification of gram-positive rods. The clinical isolates were compared with the type strains of the six species constituting the genus Microbacterium as well as with three Microbacterium strains isolated from hospital environments. By biochemical methods only 5 of 13 clinical isolates could be identified to species level. Peptidoglycan analysis proved to be a valuable tool for differentiation between Microbacterium spp. and related genera, whereas cellular fatty acid analysis did not allow species identification within the genus Microbacterium. The 22 Microbacterium strains studied were, in general, susceptible to antimicrobial agents used in the treatment of infections caused by gram-positive rods. This report is the first one concerning the isolation of Microbacterium strains from clinical specimens. The sources as well as the mode of transmission remain to be established.
Reliability of methods for hepatitis B virus DNA detection
Authors: Quint W G , Heijtink R A , Schirm J , Gerlich W H , Niesters H G
Published: January 2020

A quality assurance program has been established by the European Group for Rapid Viral Diagnosis and the European Expert Group on Viral Hepatitis for monitoring nucleic acid detection methods for hepatitis B virus (HBV) DNA in serum samples. Thirty-nine laboratories participated in this quality program and generated 43 data sets. Of the participating laboratories, all but one used the PCR technique to detect HBV DNA. A coded panel was tested that was composed of seven undiluted HBV DNA-positive serum samples and five HBV DNA-negative donor serum samples. Furthermore, two dilution series, one from a positive patient and one from a full-length recombinant DNA, were included. Twenty-six data sets (60.5%) had faultless results with both dilution series. Twelve data sets (27.9%) recognized the undiluted serum samples, and 19 data sets (44.2%) had false-negative and/or false-positive results. Ten data sets (23.3%) performed well with the entire panel of samples. From these results, it can be concluded that in a large group of laboratories HBV detection by PCR shows specificity and sensitivity problems; therefore, PCR test interpretation should be done with great care.
Torulopsis glabrata: azole susceptibilities by microdilution colorimetric and macrodilution broth assays
Authors: Tiballi R N , Zarins L T , He X , Kauffman C A
Published: January 2020

Fluconazole and itraconazole MICs were determined by both the standard macrodilution method of the National Committee for Clinical Laboratory Standards and a colorimetric broth microdilution method for 140 isolates of Torulopsis (Candida) glabrata obtained over a 15-year period. Using the method of the National Committee for Clinical Laboratory Standards the MICs at which 90% of isolates are inhibited (MIC50) for all isolates were 32 and 1.6 micrograms/ml for fluconazole and itraconazole, respectively. For fluconazole, the MIC90 rose from 16 to > 64 micrograms/ml when the MIC90s for isolates collected from July 1980 to June 1991 were compared with those for isolates collected from July 1991 to March 1995. For itraconazole, the MIC90s for isolates from the same time periods were 0.8 and 3.2 micrograms/ml, respectively. Although for isolates from some non-human immunodeficiency virus-infected patients the MICs rose, most of the high MICs were found for isolates from human immunodeficiency virus-infected patients who had been extensively treated with azole drugs for thrush. The colorimetric method yielded endpoints that were more definitive; concordances within 2 dilutions for the two methods were 87% for fluconazole and 86% for itraconazole.