bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of amplified Q beta replicase and PCR assays for detection of Mycobacterium tuberculosis
Authors: An Q , Buxton D , Hendricks A , Robinson L , Shah J , Lu L , Vera-Garcia M , King W , Olive D M
Published: January 2020

Because of the long time required to isolate Mycobacterium tuberculosis in culture, there is an acute need for simple rapid methods for direct detection of M. tuberculosis from human sputum specimens. We have developed and characterized quantitative manual Q beta replicase and PCR assays for M. tuberculosis. The Q beta replicase assay was based on reversible target capture of M. tuberculosis 23S rRNA followed by amplification of a replicatable detector probe with Q beta replicase. For PCR assays, primers generating a 370-bp amplification product from the IS6110 insertion element were used in combination with a control plasmid containing an internal deletion in the IS6110 amplicon. Serial dilutions of M. tuberculosis were spiked into sputum and subjected to digestion and decontamination with N-acetyl-L-cysteine and NaOH. Assay conditions were optimized for hybridization and sample processing chemistries in order to maximize sample utilization. Following assay optimization, the sensitivities of the Q beta replicase and PCR assays of spiked sputum samples were 0.5 and 5.0 CFU per assay reaction, respectively. The effects of sputum matrix on each assay were examined by testing 20 patient sputum samples which had been cultured for M. tuberculosis. The culture-positive samples included smear-positive and smear-negative samples. The results of the Q beta replicase assay were not inhibited by sputum and were in 100% agreement with those of culture, including detection of 10 culture-positive specimens. However, using an internal control plasmid coamplified with each PCR as an indicator, we detected PCR inhibition in 9 of 20 samples tested. Decreasing the amount of sample assayed in the PCR 24-fold alleviated the inhibitory effects in all but two specimens, one of which was culture positive. The decreased sample utilization also resulted in a false-negative result with a third specimen which was culture positive for M. tuberculosis. Quantitative smear results and QB replicase assay estimates of the number of organisms present in these specimens were in close agreement. The QB replicase assay performed well in comparison with both culture and PCR and should offer a rapid means for detecting and controlling infection due to M. tuberculosis.
PCR typing of field isolates of Plasmodium falciparum
Authors: Contamin H , Fandeur T , Bonnefoy S , Skouri F , Ntoumi F , Mercereau-Puijalon O
Published: January 2020

We report on an analysis of the constraints of PCR typing of field Plasmodium falciparum isolates by using a few highly polymorphic markers, MSA-1, MSA-2, TRAP, and CS. We show that the reactions are specific for the P. falciparum species. The detection threshold (minimum number of parasites required to detect a visible band by ethidium bromide) differed from one marker to the other and, within one locus, from one primer combination to the other. Importantly, the various MSA-1 and MSA-2 reference alleles were amplified with the same efficiency. Amplification from reconstituted allele mixtures indicated that at certain allele ratios, the most abundant allele interfered with the amplification of the less abundant one. An analysis of nine isolates collected from patients with acute malaria in Dielmo, Senegal, during a transmission season when the inoculation rate was one infective bite every second night is presented and discussed. All samples contained more than one parasite type. A significant polymorphism was observed for the four markers. Novel TaqI restriction fragment length polymorphisms were found for the TRAP gene, and TRAP gene typing alone allowed a distinction between the various isolates. MSA-1 and MSA-2 gave multiple band patterns specific for each sample.
Oxygen as limiting nutrient for growth of Cryptococcus neoformans
Authors: Odds F C , De Backer T , Dams G , Vranckx L , Woestenborghs F
Published: January 2020

In microbroth cultures with RPMI 1640 medium, the growth yield of seven Cryptococcus neoformans isolates was unaffected by augmentation of the normal (0.2%) glucose concentration in the medium to 2%, and the addition of other potential carbon, nitrogen, and vitamin sources to the medium also failed to produce large changes in growth yield. However, macrobroth cultures of C. neoformans in RPMI 1640 that were agitated by rotation in air gave turbidities 6 to 37 times greater than those in identical cultures incubated statically, and similar levels of increase were seen whether the medium contained 0.2 or 2% glucose. Incubation of microplates under an oxygen atmosphere or with agitation by rotation led to an increase of up to twofold in growth turbidity of the yeast. The maximum increase was achieved by incubation with rotation and was dependent on the brand of microplate used. The findings implicate oxygen as a growth-limiting nutrient for C. neoformans. Incubation of microbroth cultures under conditions that enhance oxygen availability for antifungal susceptibility testing purposes may increase the speed of such tests and enhance the determination of MIC endpoints.
Genomic fingerprinting of Bartonella species by repetitive element PCR for distinguishing species and isolates
Authors: Rodriguez-Barradas M C , Hamill R J , Houston E D , Georghiou P R , Clarridge J E , Regnery R L , Koehler J E
Published: January 2020

Repetitive-element PCR (rep-PCR) with primers based on repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC) repeated DNA sequences was used for genomic finger-printing of Bartonella species. This technique was applied by using either extracted genomic DNA or preparations of whole bacterial cells directly. PCR fingerprints with either the REP-based primers (REP-PCR) or primers based on the ERIC repeat (ERIC-PCR) revealed species-specific band patterns for the various Bartonella isolates. DNA fingerprints obtained from rep-PCR of extracted genomic DNA or from preparations of whole cells yielded comparable patterns. ERIC-PCR banding patterns were less complex than those obtained by REP-PCR but allowed better discrimination between strains within species. By combining results of REP-PCR and ERIC-PCR, five different fingerprint profiles were identified among 17 isolates of Bartonella henselae, but only one profile was identified among the five isolates of Bartonella quintana. Other Bartonella species yielded distinct rep-PCR fingerprints. rep-PCR is a useful technique for identification of Bartonella organisms to the species level and offers the advantage of ease of performance, with only small quantities of cells needed for the whole-cell procedure. This technique also appears to be useful for subtyping B. henselae isolates.
Expression of human papillomavirus type 16 E6-E7 open reading frame varies quantitatively in biopsy tissue from different grades of cervical intraepithelial neoplasia
Authors: McNicol P , Guijon F , Wayne S , Hidajat R , Paraskevas M
Published: January 2020

The proteins encoded by the human papillomavirus type 16 E6-E7 open reading frame are essential for transformation of the host cell. Two mRNA species, E6*I and E6*II, generated by alternative splicing of a polycistronic pre-mRNA, encode truncated E6 proteins and the E7 protein. Our investigation assessed whether or not the level of expression of E6*I and E6*II varies quantitatively in relation to the grade of cervical intraepithelial neoplasia (CIN). We used a quantitative reverse transcription PCR assay to quantify these transcripts in concurrently collected biopsy tissue and exfoliated cervical cells from 22 women with a normal cervix or various grades of CIN. We evaluated transcription profiles in relation to CIN grade and specimen type. The expression levels of E6*I and E6*II in exfoliated cervical cells did not vary significantly in relation to the grade of CIN. However, expression of E6*II was significantly diminished or absent in biopsy tissue obtained from CIN grade II and III lesions (P = 0.014). Our findings suggest that quantification of E6*I and E6*II expression in biopsy tissue may be more clinically relevant than analysis of exfoliated cells. The identification of distinct patterns of expression in association with low- and high-grade CIN suggests that quantification of E6*I and E6*II expression in biopsy tissue may have prognostic value as an indicator of CIN progression.
Continuous quality improvement for introduction of automated blood culture instrument
Authors: Alfa M , Sanche S , Roman S , Fiola Y , Lenton P , Harding G
Published: January 2020

Despite the critical nature and high cost of blood cultures, hospitals rely on manufacturers' test site data. As a result, in-hospital testing and compliance evaluation of newly acquired instruments are seldom done. The goal of this study was to apply a continuous quality improvement approach and to develop assessment criteria for all stages from the purchase order, through the on-site instrument evaluation, to the compliance evaluation. Despite the introduction of an automated high-blood-volume instrument (BacT/Alert) in our hospital, 56% of adult patients had only one venipuncture and 89.5% had < or = 20 ml of total blood volume sampled. False positives were associated with overfilling of bottles. These problems occurred because the phlebotomists did not like to perform multiple venipunctures on ill patients; therefore, they were drawing 20 ml of blood from one venipuncture and splitting it between two bottles. Unknown to the staff, the vacuum in the bottles draws significantly more than 10 ml of blood; therefore, the first bottle in the set was frequently overfilled and the second bottle was frequently underfilled. A diagrammatic guideline for a new blood culture protocol based on two venipunctures, taken one immediately after the other, to inoculate three bottles was developed. Compliance evaluation demonstrated that within 1 month of starting the new protocol, 74% of patients had at least two or more venipunctures and 60% had > or = 30 ml of blood drawn per patient episode. This study demonstrates the need for continuous quality improvement, including compliance evaluation, to ensure that the potential benefits of newer blood culture technology are actually realized.
Isolation of sucrose late-fermenting and nonfermenting variants of Vibrio cholerae O139 Bengal: implications for diagnosis of cholera
Authors: Ansaruzzaman M , Rahman M , Kibriya A K , Bhuiyan N A , Islam M S , Albert M J
Published: January 2020

The sucrose-containing selective medium thiosulfate-citrate-bile salt-sucrose agar missed a sucrose nonfermenting and four sucrose late-fermenting variant strains of Vibrio cholerae O139 Bengal from diarrheal stools. These strains were, however, correctly identified as V. cholerae O139 on a sucrose-deficient selective medium, taurocholate-tellurite-gelatin agar.
Identification of Campylobacter fetus by PCR-DNA probe method
Authors: Blom K , Patton C M , Nicholson M A , Swaminathan B
Published: January 2020

A PCR method for rapid identification of Campylobacter fetus subsp. fetus was evaluated. A fragment of the gene coding for 16S rRNA was amplified from crude cell lysates of 18 C. fetus strains and 30 strains representing other Campylobacter species and subspecies. The amplicons were probed by dot blot hybridization with a digoxigenin-labeled C. fetus-specific oligonucleotide probe. The probe reacted only with C. fetus subsp. fetus and C. fetus subsp. venerealis and may be useful for rapid identification in clinical laboratories.
Seroepizootiology of Helicobacter pylori gastric infection in nonhuman primates housed in social environments
Authors: Dubois A , Fiala N , Weichbrod R H , Ward G S , Nix M , Mehlman P T , Taub D M , Perez-Perez G I , Blaser M J
Published: January 2020

We determined the seroepizootiology of Helicobacter pylori infection in rhesus monkeys. Plasma was obtained from 196 animals (age range, 1 to 22 years) that were housed in social environments, either in indoor gang cages, in outdoor corrals, or in free-ranging forested conditions. Plasma immunoglobulin G levels were determined with a specific enzyme-linked immunosorbent assay, and the cutoff immunoglobulin G value for H. pylori seropositivity was determined from a study of 25 monkeys whose infection status was assessed by light microscopy and culture. One-year-old animals of both genders in all housing conditions had the lowest rate of positivity (60% in monkeys 1 year old versus 81% in monkeys 2 to 10 years old, P = 0.026). In addition, females tended to have higher rates of positivity than males. Seroconversion during a 1-year observation period occurred in 7 (28%) of 25 seronegative animals. Seroreversion occurred in 3 (4%) of the 78 positive animals; all 3 of these animals had received antimicrobial agents during the year. These observations demonstrate that the epizootiology of H. pylori infection in rhesus monkeys may serve as a model for human infection.
Ability of commercial and reference antimicrobial susceptibility testing methods to detect vancomycin resistance in enterococci
Authors: Tenover F C , Swenson J M , O'Hara C M , Stocker S A
Published: January 2020

We evaluated the abilities of 10 commercially available antimicrobial susceptibility testing methods and four reference methods (agar dilution, broth microdilution, disk diffusion, and the agar screen plate) to classify enterococci correctly as vancomycin susceptible or resistant using 50 well-characterized strains of enterococci. There was a high level of agreement of category classification data obtained with broth-based systems (Sceptor, MicroMedia, Pasco, and Sensititre), agar dilution, and an antibiotic gradient method (E test) with data obtained by reference broth microdilution; no very major or major errors were seen, and minor errors were < or = 6%. Increased minor error rates were observed with disk diffusion (12%), Alamar (16%), Uniscept (16%), and conventional (overnight) MicroScan panels (16%). The errors were primarily with Enterococcus casseliflavus strains and organisms containing the vanB vancomycin resistance gene. Very major error rates of 10.3 and 20.7% were observed with Vitek and MicroScan Rapid (MS/Rapid) systems, respectively; however, only the MS/Rapid system produced major errors (13.3%). On repeat testing of discrepant isolates, the very major error rate with the Vitek system dropped to 3.4%, while the very major error rate with the MS/Rapid system increased to 27.6%; major errors with the MS/Rapid system were not resolved. Many of the commercial systems had only 4 dilutions of vancomycin, which resulted in up to 84% of values being off scale (e.g., Uniscept). Of the methods tested, most conventional broth- and agar-based methods proved to be highly accurate when incubation was done for a full 24 h, although several of the tests had high minor error rates. Automated systems continued to demonstrate problems in detecting low-level resistance.
Description of feline nonsuppurative meningoencephalomyelitis ("staggering disease") and studies of its etiology
Authors: Nowotny N , Weissenböck H
Published: January 2020

A spontaneous neurological disease in domestic cats is described. The clinical signs included staggering gait, hind limb ataxia, and paresis. Histologically, a nonsuppurative meningoencephalomyelitis with a characteristic distribution pattern was found, indicating a viral etiology. In serum samples from diseased cats, antibodies to Borna disease virus were demonstrated.
Antimicrobial activity of fusidic acid and disk diffusion susceptibility testing criteria for gram-positive cocci
Authors: Toma E , Barriault D
Published: January 2020

The in vitro activity of fusidic acid was assessed and was compared with those of cloxacillin, cefamandole, vancomycin, teicoplanin, ofloxacin, ciprofloxacin, pefloxacin, and fleroxacin against 500 gram-positive cocci: 151 Staphylococcus aureus, 197 coagulase-negative staphylococci, and 152 Enterococcus faecalis strains. All clinical isolates were concomitantly tested by disk diffusion and agar dilution procedures as outlined by the National Committee for Clinical Laboratory Standards. The results with fusidic acid were further analyzed by regression line and error rate-bounded methods. With control American Type Culture Collection organisms, the values were within the limits of the National Committee for Clinical Laboratory Standards or published limits. The incidence of resistance to fusidic acid was 0.7% for S. aureus, 2.5% for coagulase-negative staphylococci, and 99.3% for E. faecalis. The correlation coefficient between the results of disk diffusion and agar dilution tests with fusidic acid was 0.90. Current interpretive criteria for susceptibility to fusidic acid (i.e., MIC of < 2 micrograms/ml and inhibitory zone of 20 mm) gave 1% false susceptibility (all strains being E. faecalis). This error rate is practically eliminated if a zone diameter of 21 mm is considered the breakpoint for susceptibility.
Use of amplified fragment length polymorphism in molecular typing of Legionella pneumophila and application to epidemiological studies
Authors: Valsangiacomo C , Baggi F , Gaia V , Balmelli T , Peduzzi R , Piffaretti J C
Published: January 2020

A novel method for molecular typing of organisms, amplified fragment length polymorphism analysis, was tested for its suitability in epidemiological studies in medical microbiology. Amplified fragment length polymorphism analysis, originally developed for typing crop plants, consists of a simple restriction-ligation reaction and a subsequent PCR amplification. In a single-step reaction, the genomic DNA is digested and the restriction fragments are ligated to specially constructed adapters. PCR amplification of such tagged restriction fragments with primers complementary to the adapters allows the detection of restriction fragment length polymorphisms upon resolution on agarose gels. The method is fast, efficient, and reproducible for typing strains of Legionella pneumophila isolated from both humans and the environment. The accuracy of the method was tested by comparison with standard restriction fragment length polymorphism typing performed with both a ribosomal and a genomic probe.
Dynamic analysis of heterogeneous hepatitis C virus populations by direct solid-phase sequencing
Authors: Odeberg J , Yun Z , Sönnerborg A , Uhlén M , Lundeberg J
Published: January 2020

In the present study, we used a semiautomated solid-phase direct sequencing method to analyze sequence diversity and variation of the hypervariable E2/NS1 region in the hepatitis C virus (HCV) genome in isolates from patients seropositive for HCV. A total of 24 isolates of various origins were sequenced. Six of the patients, not subject to any antiviral therapy, were monitored longitudinally, and rapid sequence variations were observed over a period of 14 months. The nucleotide change rate was found to be 0.1 to 0.2 nucleotide substitution per genome site per year. Furthermore, isolates from five of the patients were used for a comparative study of the direct solid-phase sequencing approach versus the frequently used approach of sequencing individual reverse transcriptase PCR clones. The advantage of direct solid-phase sequencing for studying dynamic changes in heterogeneous populations of HCV is discussed.
Chromosomal DNA fingerprinting analysis using the insertion sequence IS6110 and the repetitive element DR as strain-specific markers for epidemiological study of tuberculosis in French Polynesia
Authors: Torrea G , Levee G , Grimont P , Martin C , Chanteau S , Gicquel B
Published: January 2020

The polymorphism of Mycobacterium tuberculosis strains was evaluated in French Polynesia, an area with a low incidence of tuberculosis and a population which has been geographically stable during recent decades. Nonrepetitive strains isolated from 64 patients during 1991 and 1992 were subjected to DNA restriction fragment length polymorphism (RFLP) analysis, using the insertion sequence IS6110 and the repetitive element DR as probes. Thirty-eight different IS6110 RFLP types were identified. They could be clustered in 11 groups. All the members of each group are identical or differ by one to three bands. All the other strains are gathered in the miscellaneous group. In some cases, transmission of strains with identical RFLP types between patients of the same family or between patients living in the same area was identified. Strains exhibiting similar IS6110 RFLP types also exhibited identical DR RFLP patterns, confirming that strains with similar types were genetically linked. Strains belonging to two different IS6110 clusters exhibited the same DR RFLP type. These data may also indicate a common origin for these strains and evolution to new IS6110 types. The results obtained in this study suggest that not only reactivation of latent tuberculous infections but also active transmissions are still occurring in French Polynesia.
Evaluation of DNA probes for specific detection of Vibrio cholerae O139 Bengal
Authors: Nair G B , Bag P K , Shimada T , Ramamurthy T , Takeda T , Yamamoto S , Kurazono H , Takeda Y
Published: January 2020

Two DNA probes, 2R1 and 2R3, prepared from a region in the chromosome specific for the lipopolysaccharide O side chains of Vibrio cholerae O139 (M.K. Waldor and J.J. Mekalanos, Lancet 343:1366, 1994) were examined for their specificity and sensitivity. Both probes did not hybridize with any strain of V. cholerae belonging to serogroups other than O139 and to any of the other species examined belonging to the family Vibrionaceae. Among the 126 strains of V. cholerae O139 examined, probe 2R1 hybridized with 125 strains while probe 2R3 hybridized with all 126 strains. Both probes were found to be highly specific and sensitive and can be used for the specific identification of V. cholerae O139.
Evaluation of a simplified HEp-2 cell adherence assay for Escherichia coli isolated from south Indian children with acute diarrhea and controls
Authors: Kang G , Mathan M M , Mathan V I
Published: January 2020

A simplified HEp-2 cell adhesion assay was performed with stored Escherichia coli isolates from 761 children with acute diarrhea and 531 matched controls, and the results were evaluated by means of fluorescent actin staining and hybridization with DNA probes. The prevalence rates of localized adherence and aggregative adherence were significantly higher for patients (9.9 and 7.6%, respectively) than for controls (3.7 and 3.9%, respectively).
Molecular epidemiology of an outbreak of meningococcal disease in a university community
Authors: Edmond M B , Hollis R J , Houston A K , Wenzel R P
Published: January 2020

Over a 2-month period, five cases of serogroup C meningococcal disease occurred in Iowa City, Iowa. Two patients were unacquainted university students who had independently visited another university with endemic meningococcal disease. Isolates from these patients had DNA fingerprints identical to those of the isolates responsible for infections on the other campus. Three cases for which the patients' isolates had a different DNA fingerprint were linked to visiting a local tavern. To disrupt the outbreak, the University of Iowa offered free meningococcal vaccine to all students. This report demonstrates that outbreaks of meningococcal disease may be due to more than one circulating strain and illustrates the utility of pulsed-field gel electrophoresis in defining the molecular epidemiology of meningococcal infections.
Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis
Authors: Yajko D M , Madej J J , Lancaster M V , Sanders C A , Cawthon V L , Gee B , Babst A , Hadley W K
Published: January 2020

A colorimetric method for quantitative measurement of the susceptibility of Mycobacterium tuberculosis to antimicrobial agents is described. The method utilizes an oxidation-reduction dye, Alamar blue, as an indicator of growth. By this method, MICs of isoniazid, rifampin, streptomycin, and ethambutol were determined for 50 strains of M. tuberculosis. Colorimetric MIC results were available on the 7th, 10th, or 14th day of incubation for 29 (58%), 14 (28%), and 7 (14%) of the 50 strains, respectively. When MIC susceptibility results were compared with results obtained by the agar proportion method, increased levels of resistance detected by agar proportion were associated with higher MICs obtained by the colorimetric method. Tentative interpretive criteria for colorimetric MIC results which showed good agreement with results obtained by the agar proportion method were established. Interpretive agreement between the two methods was 98% for isoniazid, rifampin, and ethambutol and 94% for streptomycin. Overall, there was agreement between the two methods for 194 of 200 test results (97%). The colorimetric method is a rapid, quantitative, nonradiometric method for determining the antimicrobial susceptibility of M. tuberculosis.
Analysis of strains of Burkholderia (Pseudomonas) cepacia isolated in a nosocomial outbreak by biochemical and genomic typing
Authors: Ouchi K , Abe M , Karita M , Oguri T , Igari J , Nakazawa T
Published: January 2020

We analyzed strains of Burkholderia (formerly Pseudomonas) cepacia isolated in a nosocomial outbreak by biochemical and genomic typing methods. One hundred isolates of B. cepacia were obtained from patients at several wards in a single hospital from March 1983 to February 1984. These isolates were classified into 12 groups by a new biochemical typing scheme on the basis of the production of six enzymes and the production of hemolytic substance and yellow pigment. Among them, 33 strains collected from the 12 groups were further characterized by DNA fingerprinting by pulse-field gel electrophoresis, ribotyping, and plasmid profile analysis. Forty-seven strains of B. cepacia of independent origins and 25 isolates from the same hospital obtained 10 years later for a follow-up study were also subjected to analysis. Both DNA fingerprinting and ribotyping clearly discriminated the isolates from different hospitals. Of interest, although the biochemical typing and plasmid profiles of the isolates obtained during 1983 to 1984 in a single hospital were variable, genomic typing identified the majority of the isolates (32 of 33 [97%]) as derivatives of a single strain. Furthermore, a follow-up study suggested the persistence of such derivatives among the isolates after a decade. These results clearly indicated that the outbreak of B. cepacia infection in the hospital was nosocomial in origin. Thus, the usefulness of genomic typing for epidemiological studies of B. cepacia infection was further demonstrated. The biochemical typing revealed the marked variability of phenotypes of B. cepacia.
Immunological detection of penicillin-binding protein 2' of methicillin-resistant staphylococci by using monoclonal antibodies prepared from synthetic peptides
Authors: Saito M , Sekiguchi K , Yajima R , Hina M , Doss R C , Kanno H
Published: January 2020

Two synthetic peptides 31 and 32 amino acids in length were prepared as deduced from a known amino acid sequence of penicillin-binding protein 2' (PBP2') of methicillin-resistant Staphylococcus aureus. Two monoclonal antibodies were generated from fused cells of myeloma cells and splenic cells of mice immunized with the synthetic peptides. Western blot (immunoblot) analysis demonstrated specific binding of the antibodies to PBP2' of a methicillin-resistant S. aureus strain. An immunoradiometric assay was developed by using these antibodies for simple detection of PBP2'.
Monoclonal antibodies for detection of Norwalk virus antigen in stools
Authors: Herrmann J E , Blacklow N R , Matsui S M , Lewis T L , Estes M K , Ball J M , Brinker J P
Published: January 2020

Monoclonal antibodies against the prototype 8FIIa strain of Norwalk virus were prepared and applied to an enzyme immunoassay (EIA) for detecting Norwalk virus in stool specimens. The monoclonal antibodies immunoprecipitated a 58-kDa protein which had been produced by in vitro transcription-translation of Norwalk virus cloned cDNA, and they reacted by EIA with recombinant Norwalk virus capsid protein at a sensitivity level of 1 ng/ml. The EIA detected virus in all tested samples from 15 different Norwalk virus-infected volunteers. No cross-reactions were seen in stools containing other caliciviruses or in stools containing rotaviruses, astroviruses, or enteric adenoviruses.
Biochemical investigations of biogroups and subspecies of Morganella morganii
Authors: Janda J M , Abbott S L , Khashe S , Robin T
Published: January 2020

We determined the subspecies and biogroup designations for 73 strains of Morganella morganii principally recovered from routine clinical specimens. On the basis of trehalose fermentation, 90% of all strains were identified as M. morganii subsp. morganii (trehalose negative), while the remaining 10% were designated M. morganii subsp. sibonii (trehalose positive). Using three tests (ornithine decarboxylase [ODC] and lysine decarboxylase [LDC] activities and susceptibility to tetracycline), we determined the biogroup designations for these 73 strains. Four of the seven recognized biogroups within the genus Morganella were found in the study, with biogroup A (ODC positive [ODC+], LDC negative [LDC-]) predominating (78%); all M. morganii subsp. sibonii strains were found to belong to biogroup G (ODC+, LDC-). Rapid glycerol fermentation (24 h) was linked to nonmotility and biogroup B strains (ODC+, LDC+). LDC activity but not tetracycline resistance appeared to be associated with the possession of a 40- to 45-MDa plasmid. The use of three commercial systems (API ZYM, API 50 CH, and Biolog GN) failed to detect any new biochemical tests useful for subspecies identification, with the possible exception of L-phenylalanine utilization as a sole carbon source in the Biolog GN system. No Morganella strain was found to invade either HEp-2 or Vero cell lines, but four of seven M. morganii subsp. morganii strains were cytotoxic on sheets of both cells. This cytotoxic activity appeared to correlate with the rapid expression of beta-hemolytic activity.
Variant colony surface antigenic phenotypes within mycoplasma strain populations: implications for species identification and strain standardization
Authors: Rosengarten R , Yogev D
Published: January 2020

Immunobinding assays with mycoplasma colonies on agar plates (immunofluorescence and immunoperoxidase techniques) or with imprints of colonies transferred to solid supports (colony immunoblotting) are widely used as standard diagnostic tests for serological species identification of mycoplasma isolates. However, in light of the high rate of variability of surface antigens in many mycoplasmas, diagnostic data obtained with these techniques require a more critical evaluation. In this report, we demonstrate with some examples that mycoplasma surface variability based on alterations in expression, in size, and in surface presentation of integral and peripheral membrane proteins may lead to misinterpretation of colony immunostaining reactions obtained by using specific monoclonal antibodies as well as conventional diagnostic hyperimmune sera. To more easily identify phenotypically mixed isolates or samples which contain more than one species, we have introduced some minor modifications of the colony immunoblot technique which provide sharp signals of positive as well as negative reactions and enable identification of cryptic epitopes. It is further demonstrated that because of the variability in colony surface antigenic phenotype, mycoplasma strains, including well-established reference and other prototype strains which are used under the same designation in many laboratories, can differ markedly in their antigen profiles and their potentially virulence-related surface properties, since they are usually purified by filter cloning and often propagated by subcultivation of randomly selected agar-grown subpopulations. We conclude from this study that because of this surface variability, the establishment of criteria for standardization of mycoplasma strains and diagnostic antisera is urgently required in order to obtain reproducible results in different laboratories.
Evaluation of new computer-enhanced identification program for microorganisms: adaptation of BioBASE for identification of members of the family Enterobacteriaceae
Authors: Miller J M , Alachi P
Published: January 2020

We report the use of BioBASE, a computer-enhanced numerical identification software package, as a valuable aid for the rapid identification of unknown enteric bacilli when using conventional biochemicals. We compared BioBASE identification results with those of the Centers for Disease Control and Prevention's mainframe computer to determine the former's accuracy in identifying both common and rare unknown isolates of the family Enterobacteriaceae by using the same compiled data matrix. Of 293 enteric strains tested by BioBASE, 278 (94.9%) were correctly identified to the species level; 13 (4.4%) were assigned unacceptable or low discrimination profiles, but 8 of these (2.7%) were listed as the first choice; and 2 (0.7%) were not identified correctly because of their highly unusual biochemical profiles. The software is user friendly, rapid, and accurate and would be of value to any laboratory that uses conventional biochemicals.
Demonstration of borna disease virus RNA in peripheral blood mononuclear cells derived from domestic cats in Japan
Authors: Nakamura Y , Asahi S , Nakaya T , Bahmani M K , Saitoh S , Yasui K , Mayama H , Hagiwara K , Ishihara C , Ikuta K
Published: January 2020

Borna disease virus (BDV) naturally infects horses, sheep, and several other species, including humans, and it is believed to be related to neurological disorders. BDV infection in domestic cats has also been demonstrated by serological assays. We demonstrated for the first time BDV RNA in peripheral blood mononuclear cells from 11 of 83 (13.3%) randomly selected domestic cats in Japan by nested reverse transcriptase-PCR. The BDVs from cats were similar to but slightly different from those from horses and humans, as shown by sequencing the reverse transcriptase-PCR products. None of the cats was positive for both BDV RNA and anti-BDV antibodies.
Rapid and sensitive detection of enteroviruses in specimens from patients with aseptic meningitis
Authors: Yerly S , Gervaix A , Simonet V , Caflisch M , Perrin L , Wunderli W
Published: January 2020

A 5-h PCR assay (Amplicor enterovirus test) was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid. Of the cerebrospinal fluid specimens collected during a summer outbreak of aseptic meningitis, 34% were positive by viral culture whereas 66% were positive by the Amplicor PCR, suggesting that this technique improves the diagnosis of enteroviral meningitis.
Detection and identification of mycobacteria in formalin-fixed, paraffin-embedded tissues by nested PCR and restriction enzyme analysis
Authors: Bascuñana C R , Belák K
Published: January 2020

A novel assay based on a nested PCR and restriction enzyme analysis of the PCR products was developed for the rapid detection and identification of Mycobacterium bovis and M. avium-M. intracellulare species in formalin-fixed, paraffin-embedded tissue (PET) specimens. On the basis of the nucleotide sequence data obtained in the present study, general nested primers were constructed to amplify a 424-bp segment of the gene encoding the 65-kDa surface antigen of mycobacteria. The nested PCR assay proved to be highly sensitive, since as little as 5 to 10 fg of extracted mycobacterial DNA was detected. The safety of the assay as a routine method for the diagnosis of M. bovis and M. avium-M. intracellulare in PET specimens was provided by taking various precautions. In order to prevent false positivity, specific tools and procedures were applied. To detect false-negative results and assess the efficiency of the PCR, an internal standard molecule of amplification was constructed. The digestion of the amplicons with the restriction endonuclease Sau96-I allowed the identification of M. bovis and M. avium-M. intracellulare in a large number of clinical specimens. The present results indicate that PCR combined with an internal control of amplification and restriction enzyme analysis of the amplicons provides a rapid, sensitive, and reliable method for routine diagnostic laboratories to detect and identify M. bovis and M. avium-M. intracellulare in PET specimens.
Hepatitis C virus detection by single-round PCR specific for the terminal 3' noncoding region
Authors: Umlauft F , Wong D T , Oefner P J , Underhill P A , Cheung R C , Wright T L , Kolykhalov A A , Gruenewald K , Greenberg H B
Published: January 2020

A single-round PCR method with primers specific for the 3' noncoding region (NCR) of hepatitis C virus (HCV) has been developed. Using a double RNAzol-B extraction, a high-temperature reverse-transcription step with SuperScript II reverse transcriptase, and a 40-cycle two-temperature PCR with a TaqStart antibody hot-start procedure, we were able to detect a 92-nucleotide fragment of the recently discovered 98-nucleotide highly conserved sequence at the 3' terminus of the HCV genome. Direct sequencing of the PCR products confirmed the specificity of the PCR and demonstrated conservation in this region. Only one nucleotide change in 14 specimens was found. End point dilution titration of sera with known viral RNA titers showed the sensitivity of the single-round 3' NCR PCR to be comparable to those of the established nested 5' NCR assays (fewer than 25 HCV genome equivalents). To evaluate specificity and sensitivity, a panel of 116 serum samples characterized by nested 5'-end PCR, genotyping, and quantitative assays was tested. A high degree of concordance (96%) between the 3' NCR and 5' NCR PCR results was found. The sequence conservation at the 3' end of the HCV genome among common genotypes and the savings in time, labor, and reagents from a single-round PCR make this assay a useful addition to the detection systems available to identify and monitor HCV infection.
Cokeromyces recurvatus isolated from pleural and peritoneal fluid: case report
Authors: Munipalli B , Rinaldi M G , Greenberg S B
Published: January 2020

The zygomycetous mold Cokeromyces recurvatus was isolated from the pleural and peritoneal fluids of a 64-year-old man with a history of peptic ulcer disease and alcohol abuse. The patient presented to the hospital with severe abdominal pain and a ruptured duodenal ulcer. This is one of the few times that C.recurvatus has been isolated from a human source, and the organism may have contributed to the death of this patient.
Diverse patterns of recognition of hepatitis C virus core and nonstructural antigens by antibodies present in Egyptian cancer patients and blood donors
Authors: Attia M A , Zekri A R , Goudsmit J , Boom R , Khaled H M , Mansour M T , de Wolf F , el-Din H M , Sol C J
Published: January 2020

Serum samples from 429 cancer patients, 82 unpaid blood donors, and 74 paid blood donors were tested for hepatitis C virus (HCV) markers in two commercially available enzyme immunoassays (EIAs). A total of 229 of 429 (53.4%) cancer patients were positive by the two EIAs. A total of 34 of 156 (21.8%) of the blood donors were positive by the EIAs, with a higher prevalence among paid blood donors (20/74; 27%) compared with that among the unpaid blood donors (14 of 82; 17%). EIA-positive sera were tested for confirmation of the results in an immunoblot assay (LiaTek) in which reactivities to four synthetic peptides representing the HCV core protein and two synthetic peptides representing nonstructural proteins 4 and 5 were measured. Of 243 first and/or second EIA-positive samples from cancer patients, 188 (77.2%) were confirmed to be positive in the synthetic peptide immunoblot. A total of 33 of 35 (94.3%) blood donor samples were confirmed to be positive. A great diversity in reactivity patterns was seen. However, all sera from the group of paid blood donors were exclusively reactive to core peptides 1 and 2. A subset of LiaTek assay-positive samples were tested by the four-antigen RIBA-2 assay. The sera from the paid blood donors were all nonreactive. A subset of the LiaTek-positive sera was analyzed for the presence of the HCV genome by reverse transcriptase-PCR. Eleven of the 20 serum samples with reactivity to LiaTek core peptides 1 and 2 only were HCV reverse transcriptase-PCR positive, as were the majority of the sera with other reactivity patterns by the LiaTek assay. The results confirm the very high prevalence of HCV infection in Egypt. Furthermore, the results indicate that there is circulating in Egypt, particularly in the group of blood donors paid for their donation, an HCV variant which elicits an immune response that is not detected by the RIBA-2 assay.
Multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis
Authors: Tran S D , Rudney J D
Published: January 2020

Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are strongly associated with periodontitis. However, little is known about their distribution in periodontally healthy individuals, because culturing techniques are not sufficiently sensitive. A modified multiplex PCR was developed to address that question. Our method uses two species-specific forward primers in combination with a single reverse primer. These primers target variable and conserved regions of the 16S rRNA gene. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans and P. gingivalis cells. Primer specificity was tested against (i) six A. actinomycetemcomitans strains and four P. gingivalis strains, (ii) seven different species of oral bacteria, and (iii) supra- and subgingival plaque from 20 subjects. The multiplex PCR had a lower limit of detection of 2 A. actinomycetemcomitans and 30 P. gingivalis cells. Species-specific amplicons were obtained for all A. actinomycetemcomitans and P. gingivalis strains tested and did not occur with seven other bacterial species unless A. actinomycetemcomitans and P. gingivalis were added. Neither target species was detected in supragingival plaque; A. actinomycetemcomitans was detected in one subgingival specimen, and P. gingivalis was detected in another. When plaque samples were spiked with 10 A. actinomycetemcomitans cells and 100 P. gingivalis cells, species-specific amplicons were detected. These findings show our multiplex PCR to be highly sensitive and specific while allowing simultaneous detection of A. actinomycetemcomitans and P. gingivalis. This assay has potential applications in epidemiological studies, diagnosis, treatment planning, and monitoring of periodontal pathogens.
Clonal distribution of penicillin-resistant Streptococcus pneumoniae 23F in France
Authors: Ferroni A , Nguyen L , Gehanno P , Boucot I , Berche P
Published: January 2020

We studied the clonality of clinical isolates of Streptococcus pneumoniae 23F, the serotype most often associated with penicillin resistance in France. Clinical isolates obtained between November 1992 and April 1993 from nasopharyngeal samples from children with acute otitis media from different regions of the country were analyzed. The genetic polymorphism of penicillin-susceptible and -resistant 23F isolates (MIC, 2 mg/liter) was studied by pulsed-field gel electrophoresis. The resistant isolates were closely related, whereas the susceptible isolates were genetically heterogeneous. PCR amplification and restriction of the genes encoding penicillin-binding proteins (PBPs) 1A, 2B, and 2X also showed that the 24 resistant isolates had similar patterns which were very different from those of the susceptible isolates. All resistant isolates gave the same PBP pattern, with low affinities of PBPs for penicillin. Our results indicate that, in contrast to penicillin-susceptible 23F isolates, the penicillin-resistant 23F isolates have a single clonal origin, suggesting the rapid clonal spread of a resistant epidemic strain throughout the country.
Genetic diversity of rRNA operons of unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid of neonates suffering from meningitis
Authors: Chatellier S , Huet H , Kenzi S , Rosenau A , Geslin P , Quentin R
Published: January 2020

The genetic diversity of a collection of 54 unrelated Streptococcus agalactiae strains isolated from the cerebrospinal fluid of neonates and of 60 unrelated carrier strains was evaluated by investigating the restriction fragment length polymorphism of the rRNA gene region. Three restriction enzymes were selected for use: PstI, HindIII, and CfoI. Clustering analysis revealed two phylogenetic groups of strains with 40% divergence. Group I contained two clusters, A and B, and group II contained three clusters, C, D, and E. Strains of serotype Ia were mostly distributed in cluster A, and strains of serotype Ib were mostly distributed in cluster E. Serotype III isolates did not cluster. Nevertheless, 37 of 39 isolates belonging to cluster B were serotype III. With HindIII, two rRNA gene banding patterns characterized 38 of the 39 strains of cluster B, which represents a high-virulence group. In addition, two rRNA gene banding patterns with each enzyme and/or a pair of CfoI fragments of 905 and 990 bp identified 81% of the invasive strains. On account of the genetic homogeneity of the cerebrospinal fluid strains, ribotyping is a powerful typing method for investigation of nosocomial or epidemic invasive infections only when all three enzymes are used or when PstI and HindIII or PstI and CfoI are combined with serotyping (index of discrimination, > 0.95).
Evaluation of automated COBAS AMPLICOR PCR system for detection of several infectious agents and its impact on laboratory management
Authors: Jungkind D , Direnzo S , Beavis K G , Silverman N S
Published: January 2020

We evaluated the COBAS AMPLICOR (CA) PCR system (Roche Diagnostic Systems) designed for automated PCR amplification and detection of nucleic acids from infectious agents in clinical samples. The Roche AMPLICOR microwell plate (MWP) PCR was the reference method. CA amplifies target nucleic acid, captures the biotinylated amplification products by using magnetic particles coated with specific oligonucleotide probes, and detects the bound products colorimetrically. For Mycobacterium tuberculosis, the correlation of the results of CA tests with those of MWP tests was 100% with 230 samples, including 20 culture-positive samples. For hepatitis C virus, the correlation was 100% with 214 samples, including 60 positive samples. MultiPlex CA analysis of 199 cervical specimens for Chlamydia trachomatis, Neisseria gonorrhoeae, and the internal control gave 100% concordance. These samples included 19 C. trachomatis and 3 N. gonorrhoeae culture-positive samples. Overall, the agreement between PCR methods for all 842 comparisons was 100%. Compared with culture, the sensitivities of the assays for C. trachomatis and M tuberculosis were > or = 95%. After spiking alternating amplification tubes in the CA system with 10(14) copies of the Chlamydia amplicon per ml, we were unable to demonstrate any carryover cross-contamination of negative samples. Using the criteria of the College of American Pathologists workload recording method, we found that the total hands-on time to produce CA PCR results was 4.4, 7.9, and 3.3 min for M. tuberculosis, hepatis C virus, and the MultiPlexed assay for chlamydia plus gonorrhea and an internal control, respectively. The CA system brings true PCR automation to laboratories. In addition to the accuracy of automated results, the CA system provides labor savings, provides containment of the amplification and detection components of PCR, and supports both MultiPlex amplification and sequential algorithm (ReFlex) detection of analytes.