bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of Mycobacterium avium complex isolates giving discordant results in AccuProbe tests by PCR-restriction enzyme analysis, 16S rRNA gene sequencing, and DT1-DT6 PCR
Authors: Devallois A , Picardeau M , Paramasivan C N , Vincent V , Rastogi N
Published: January 2020

Based on cultural and biochemical tests, a total of 84 strains (72 clinical and 12 environmental isolates from the Caribbean Isles, Europe, and the Indian subcontinent) were identified as members of the Mycobacterium avium complex (MAC). They were further characterized with MAC, M. avium, and M. intracellulare probes of the AccuProbe system, and this was followed by selective amplification of DT6 and DT1 sequences. Seventy isolates gave concordant results; 63 were identified as M. avium, 5 were identified as M. intracellulare, and 24 remained untypeable by both methods. Fourteen isolates gave discrepant results, as they were DT1 positive but gave negative results by the M. intracellulare AccuProbe test. Consequently, a detailed molecular analysis of all DT1-positive isolates (14 discrepant strains plus 5 M. intracellulare strains) was performed by PCR-restriction analysis (PRA) of the hsp65 gene and 16S rRNA gene sequencing. The results confirmed the reported heterogeneity of M. intracellulare, as only 6 of 19 isolates (32%) gave PRA results compatible with published M. intracellulare profiles while the rest of the isolates were grouped in four previously unpublished profiles. 16S rRNA gene sequencing showed that only 8 of 19 isolates (42%) were related to M. intracellulare IWGMT 90247 (EMBL accession no. X88917), the rest being related to MCRO19 (EMBL accession no. X93030) and MIWGTMR10 (EMBL accession no. X88915). In conclusion, we have characterized a significant number of MAC isolates which were not identified by the AccuProbe test, PRA, or 16S rRNA sequencing. However, all of them were identifiable by DT1-DT6 PCR (they were DT6 negative and DT1 positive) and could be tentatively identified as M. intracellulare based on previously published observations. It is noteworthy that the majority of such isolates (14 of 19) were from the Indian subcontinent, with 12 of 14 being environmental isolates. Our study confirms the marked heterogeneity of M. intracellulare isolates and shows the utility of in-house DT1 PCR to detect this group of isolates, which would otherwise have been missed by the AccuProbe system in a routine clinical microbiology laboratory.
Molecular epidemiology of varicella-zoster virus in East London, England, between 1971 and 1995
Authors: Hawrami K , Hart I J , Pereira F , Argent S , Bannister B , Bovill B , Carrington D , Ogilvie M , Rawstorne S , Tryhorn Y , Breuer J
Published: January 2020

The molecular epidemiology of varicella-zoster virus in London, England, between 1971 and 1995 was examined by using two informative polymorphic markers, variable repeat region R5 and a BglI restriction site in gene 54. Viruses from 105 cases of chickenpox and 144 of zoster were typed. Two alleles of R5, A and B, were found at prevalences of 89 and 6%, respectively. No difference in allele frequency between the zoster and chickenpox cases was found, and no change in the frequencies of these alleles was observed to occur over time. By contrast, a BglI restriction site (BglI+) was found with increasing frequency over time among cases of varicella (P < 0.005) and, to a lesser extent, cases of zoster. The BglI+ polymorphism was strongly associated (P < 0.0005) with zoster in subjects who had immigrated to the United Kingdom from countries with low adult immunity to varicella (LAIV). Sixty-three percent of the subjects with zoster who had emigrated from countries with LAIV carried the BglI+ virus, in contrast to 10% of adults who had grown up in countries with high adult immunity to varicella. The significance of these data, in view of the changing epidemiology of chickenpox, is discussed.
Antigenic lipopolysaccharide components of Legionella pneumophila recognized by monoclonal antibodies: possibilities and limitations for division of the species into serogroups
Authors: Helbig J H , Kurtz J B , Pastoris M C , Pelaz C , Lück P C
Published: January 2020

Legionella pneumophila accounts for the majority of cases of Legionnaires' disease. By using rabbit antisera, the species has been divided into 14 numbered and 1 unnumbered serogroups. To recognize the antigenic diversity of the lipopolysaccharide (LPS) responsible for this classification, the Dresden Legionella LPS MAb panel, containing 98 monoclonal antibodies (MAbs), was created. Each serogroup reference strain possesses at least one specific epitope not found on any other reference strain and therefore designated the serogroup-specific epitope. When the appropriate MAbs were used for serotyping of 1,064 human and environmental isolates, 1,045 (98%) could be placed into the known serogroups. In most cases (97%), this was in agreement with the polyclonal typing. Of the 29 isolates that showed strong cross-reactivities with the rabbit antiserum panel, 11 could be typed easily by MAbs; for the remaining 18, however, only serogroup-cross-reactive epitopes could be determined. Below the serogroup level, monoclonal subtypes were found for 11 serogroups. Altogether, the Dresden Legionella LPS MAb panel was able to divide the 1,064 isolates tested into 64 phenons, indicating its usefulness for both serogrouping and subgrouping of L. pneumophila strains. In order to compare the identities of patient and environmental isolates, testing their reactivity with MAbs should be the first step, especially if large numbers of colonies are to be typed. Only in cases of identical patterns are the more time consuming and expensive genetic fingerprints necessary. Moreover, the MAbs can also be used for specific antigen detection in respiratory specimens on the serogroup or subgroup level.
Surveillance and detection of erythromycin resistance in Bordetella pertussis isolates recovered from a pediatric population in the Intermountain West region of the United States
Authors: Korgenski E K , Daly J A
Published: January 2020

Forty-seven Bordetella pertussis isolates recovered from January 1985 to June 1997 at Primary Children's Medical Center were tested for erythromycin resistance. Agar dilution MICs were determined on Regan-Lowe agar. Forty-six isolates were found to be erythromycin susceptible (all MICs were less than or equal to 0.12 microg/ml). One isolate was found to be erythromycin resistant (MIC, 32 microg/ml). In addition, we compared Etest MIC results and disk diffusion zone diameter measurements, performed on commercially prepared Regan-Lowe agar, to the agar dilution MIC result. Etest MIC and/or disk diffusion testing on commercial Regan-Lowe agar appears to be an adequate method for erythromycin resistance screening of B. pertussis isolates.
Comparison of charcoal- and starch-based media for testing susceptibilities of Legionella species to macrolides, azalides, and fluoroquinolones
Authors: Pendland S L , Martin S J , Chen C , Schreckenberger P C , Danziger L H
Published: January 2020

We compared growth characteristics of 46 Legionella strains grown on buffered charcoal yeast extract alpha (BCYE alpha) agar and buffered starch yeast extract (BSYE) agar and MICs of macrolides, azalides, and fluoroquinolones for these organisms. Growth was poor and not reproducible on BSYE agar. Growth was excellent on BCYE alpha, and MICs were easy to interpret. BCYE alpha is superior to BSYE for testing susceptibilities of Legionella species by agar dilution.
IS6110 fingerprinting of drug-resistant Mycobacterium tuberculosis strains isolated in Germany during 1995
Authors: Niemann S , Rüsch-Gerdes S , Richter E
Published: January 2020

The epidemiological relatedness of drug-resistant Mycobacterium tuberculosis strains isolated in Germany in 1995 was evaluated by the standardized IS6110 fingerprinting method. Altogether, 196 M. tuberculosis isolates from 167 patients were analyzed. A large degree of IS6110 polymorphism was found, ranging from 1 to 20 copies. Multiple isolates from one patient generally remained stable over a period of up to 1 year. However, one strain showed an additional fragment 7 months after the first isolate was obtained. Isolates from 55 patients (33%) showed identical fingerprint patterns or fingerprint patterns that differed only in one band, and thus they were clustered in 22 fingerprint groups. Specific transmission links could be established between members of four groups, e.g., transmission by family contacts. In one case, transmission of a multidrug-resistant strain to a patient initially infected with a drug-susceptible strain could be shown. Besides these fingerprint groups, 30 of the 167 isolates (approximately 18%) could be grouped in two fingerprint clusters with a similarity of at least 78%. Approximately 60% of the patients of these two clusters were known to be immigrants from the former Soviet Union, and one patient is still living in Belarus. In conclusion, our results indicate that (i) transmission of drug-resistant strains contributes substantially to the emergence of drug-resistant tuberculosis in Germany and (ii) drug-resistant M. tuberculosis strains were presumably carried over from the former Soviet Union to Germany by immigrants.
Identification of Legionella species by lipopolysaccharide antigen pattern
Authors: Jürgens D , Fehrenbach F J
Published: January 2020

Electrophoretic analysis of lipopolysaccharide (LPS) extracts from 430 previously serotyped Legionella isolates and 28 American Type Culture Collection (ATCC) non-Legionella pneumophila Legionella reference strains representing different Legionella species and serogroups has been performed. LPS was prepared from Legionella suspensions by sonication and proteinase K digestion. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LPS bands were either stained with silver nitrate or transferred onto a nitrocellulose membrane and detected with rabbit antibodies raised against L. pneumophila serogroup 5, which was known to cross-react with L. pneumophila serogroups 1 to 14. Silver staining revealed that each of the 28 ATCC non-L. pneumophila Legionella strains possessed an individual and characteristic LPS banding pattern. The LPS profile was defined by the molecular weight of the visualized bands and/or the individual ladder-like LPS pattern. It was demonstrated by immunoblotting that non-L. pneumophila Legionella strains did not react with the serogroup 5 antiserum, thus allowing for the differentiation between L. pneumophila and non-L. pneumophila species.
Multicenter evaluation of the updated and extended API (RAPID) Coryne database 2.0
Authors: Funke G , Renaud F N , Freney J , Riegel P
Published: January 2020

In a multicenter study, 407 strains of coryneform bacteria were tested with the updated and extended API (RAPID) Coryne system with database 2.0 (bioMérieux, La-Balme-les-Grottes, France) in order to evaluate the system's capability of identifying these bacteria. The design of the system was exactly the same as for the previous API (RAPID) Coryne strip with database 1.0, i.e., the 20 biochemical reactions covered were identical, but database 2.0 included both more taxa and additional differential tests. Three hundred ninety strains tested belonged to the 49 taxa covered by database 2.0, and 17 strains belonged to taxa not covered. Overall, the system correctly identified 90.5% of the strains belonging to taxa included, with additional tests needed for correct identification for 55.1% of all strains tested. Only 5.6% of all strains were not identified, and 3.8% were misidentified. Identification problems were observed in particular for Corynebacterium coyleae, Propionibacterium acnes, and Aureobacterium spp. The numerical profiles and corresponding identification results for the taxa not covered by the new database 2.0 were also given. In comparison to the results from published previous evaluations of the API (RAPID) Coryne database 1.0, more additional tests had to be performed with version 2.0 in order to completely identify the strains. This was the result of current changes in taxonomy and to provide for organisms described since the appearance of version 1.0. We conclude that the new API (RAPID) Coryne system 2.0 is a useful tool for identifying the diverse group of coryneform bacteria encountered in the routine clinical laboratory.
Confirmation of suspicious cases of meningococcal meningitis by PCR and enzyme-linked immunosorbent assay
Authors: Saunders N B , Shoemaker D R , Brandt B L , Zollinger W D
Published: January 2020

A significant problem in efficacy trials of meningococcal vaccines has been accurate identification of all cases of meningococcal disease that occur in study populations. The accuracy of case determination would be improved by utilizing methods which confirm or disprove suspicious cases of meningococcal disease that are culture negative. A collection of serum and cerebrospinal fluid (CSF) samples from a meningococcal vaccine field trial performed in Iquique, Chile, were utilized to assess the status of patients for whom cultures, Gram stains, and clinical evaluations for meningococcal disease were available. Nested PCRs (nPCRs) for amplification of Neisseria meningitidis DNA in CSF samples and enzyme-linked immunosorbent assays (ELISAs) for quantification of serum immunoglobulin G antibodies specific for N. meningitidis were used in combination to confirm or eliminate cases classified by physicians as suspicious for meningococcal disease. Samples from 12 of 79 patients suspected of having meningococcal meningitis tested positive by both methods; specimens from 61 of the 79 were negative by both methods; and samples from 6 patients yielded ambiguous results, and these cases remained unconfirmed. Direct sequence analysis of amplified DNA from patients suspected of having meningococcal disease confirmed that 2 of the 12 newly confirmed cases were not attributable to the typical epidemic strain (B:15:P1.[7],3) while the others were due to the epidemic strain. A combination of nPCR and ELISA reduced the number of suspicious cases in this study from 79 to 6, thereby improving the potential for assessment of vaccine efficacy. Molecular identification by nPCR in conjunction with immunological assessment of patient response could be considered diagnostic of disease in future testing of meningococcal vaccines to improve efficacy analyses.
Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica)
Authors: Sjölander K B , Elgh F , Kallio-Kokko H , Vapalahti O , Hägglund M , Palmcrantz V , Juto P , Vaheri A , Niklasson B , Lundkvist A
Published: January 2020

Nephropathia epidemica (NE), Puumala (PUU) virus infection, is a febrile disease which is commonly associated with acute renal impairment. To differentiate NE from other acute febrile illnesses, a rapid and reliable serological diagnosis is important, and a number of different protocols have recently been introduced. In the present report we describe a comparative evaluation of six PUU virus immunoglobulin M (IgM) and seven IgG enzyme-linked immunosorbent assay (ELISA) protocols based on native, Escherichia coli-expressed, or baculovirus-expressed nucleocapsid protein (N). Neutralization and immunofluorescence assays were included for comparison. Equally high sensitivities and specificities were obtained with three mu-capture-based IgM ELISAs using native, baculovirus-expressed, and E. coli-expressed N antigens, respectively, and by an ELISA based on purified E. coli-expressed full-length N adsorbed to solid phase. The assays based on truncated amino-terminal N proteins, including a commercially available PUU virus IgM ELISA, all showed lower sensitivities. For detection of PUU virus-specific IgG, ELISAs based on monoclonal antibody-captured native or baculovirus-expressed N antigens showed optimal sensitivities and specificities, while the assays based on E. coli-expressed N did not detect all PUU virus IgG-positive serum samples. A commercially available PUU virus IgG ELISA based on E. coli-expressed amino-terminal N showed a significantly lower sensitivity than those of all other IgG assays.
GEMHEP multicenter quality control study of PCR detection of GB virus C/hepatitis G virus RNA in serum
Authors: Bogard M , Buffet-Janvresse C , Cantaloube J F , Biagini P , Duverlie G , Castelain S , Izopet J , Dubois M , Defer C , Lepot I , Coste J , Marcellin P , Martinot-Peignoux M , Halfon P , Gerolami V , Frangeul L , Pawlotsky J M , Roudot-Thoraval F , Dussaix E , Loiseau P , Ravera N , Lewin P , Lamoril J , Lerable J , Lebon P
Published: January 2020

PCR is, to date, the only available tool for the detection of GB virus C (GBV-C) and hepatitis G virus (HGV) RNAs. Twenty-two French laboratories participated in a quality control study to assess the sensitivity and specificity of their procedures. The panel included 13 positive controls and 7 negative controls. The laboratories used either in-house PCR techniques adapted from the literature or partly standardized commercial tests. Three laboratories performed faultlessly with the entire panel. Most laboratories had excellent specificity (100% in 20 of 22 laboratories). Sensitivity was acceptable (85 to 100%) in 15 centers and insufficient (38 to 77%) in 7. As with nonstandardized in-house PCR, the commercial assays gave discrepant performances in different laboratories. These results suggest that laboratories willing to use PCR for detection of GBV-C/HGV RNA for research or diagnostic purposes should participate in multicenter quality control trials.
Reproducibility of AMPLICOR enterovirus PCR test results
Authors: Rotbart H A
Published: January 2020

The reproducibility of AMPLICOR enterovirus PCR test results was determined with clinical samples of cerebrospinal fluid, serum, urine, and throat and rectal swabs. Among 608 samples from which duplicate aliquots were run simultaneously, only seven pairs gave discordant results. Among 104 samples from which duplicate aliquots were run in separate assays, no discordance was seen. Overall, the reproducibility of test kit results was 99% (705 of 712).
Detection of immunoglobulin G (IgG) and IgM antibodies to Toxoplasma gondii: evaluation of four commercial immunoassay systems
Authors: Hofgärtner W T , Swanzy S R , Bacina R M , Condon J , Gupta M , Matlock P E , Bergeron D L , Plorde J J , Fritsche T R
Published: January 2020

A comparative evaluation of the following commercial immunoassays for the determination of antibodies to Toxoplasma gondii was performed: Behring Diagnostics OPUS Toxo G and Toxo M, Abbott Diagnostics IMX Toxo-IgG 2.0 and Toxo-IgM, Sanofi Diagnostics Pasteur Platelia Toxo IgG and Toxo IgM, and bioMérieux Vitek VIDAS Toxo IgG and IgM. Of 676 specimens that were tested for Toxoplasma-specific immunoglobulin G (IgG) antibodies, 26% were reactive by all methods while 8% displayed some discrepancy. Of 718 specimens that were tested for Toxoplasma-specific IgM antibodies, 3% were reactive by all methods while 10% displayed some discrepancy. Analysis of discrepant specimens revealed performance shortcomings with all IgM-specific assays. The impact of such shortcomings is magnified in a population with a low prevalence of toxoplasmosis.
Reliability of mycobacteria growth indicator tube for testing susceptibility of Mycobacterium tuberculosis to ethambutol and streptomycin
Authors: Bergmann J S , Woods G L
Published: January 2020

The reliability of mycobacterial growth indicator tubes (MGIT) for testing susceptibility of Mycobacterium tuberculosis to ethambutol and streptomycin was evaluated by comparing MGIT results to those obtained by the radiometric BACTEC TB system and the method of proportion. The method of proportion was considered the reference method. To resolve discrepancies, all three testing methods were repeated. For the 74 isolates evaluated, initial ethambutol results agreed by all three methods for 64 (86.5%) of them; 58 were susceptible and 6 were resistant. MGIT and method-of-proportion results agreed for 67 isolates, and BACTEC results agreed with method-of-proportion results for 71 (P = 0.096). Initial streptomycin results obtained by all three methods agreed for 69 (93.2%) isolates: 55 were susceptible and 14 were resistant. MGIT and method-of-proportion results were concordant for 69 isolates, and BACTEC and method-of-proportion results agreed for 73 (P = 0.049). The mean times to MGIT results were 5.58 +/- 0.10 days (range, 3 to 9 days) for ethambutol and 5.47 +/- 0.11 days (range, 3 to 9 days) for streptomycin, compared to a mean of 7.41 +/- 0.20 days (range, 4 to 12 days) for both drugs with the BACTEC system (P < 0.001).
Rapid discrimination of Mycobacterium tuberculosis complex strains by ligation-mediated PCR fingerprint analysis
Authors: Prod'hom G , Guilhot C , Gutierrez M C , Varnerot A , Gicquel B , Vincent V
Published: January 2020

A ligation-mediated PCR (LMPCR) method for the amplification of sequences flanking the IS6110 of the Mycobacterium tuberculosis complex has been developed. The method uses one primer specific for IS6110 and a second specific for a linker ligated to SalI-restricted genomic DNA. LMPCR is a rapid screening method, valuable for the fingerprinting of M. tuberculosis complex strains.
Isolation of Cryptococcus neoformans var. gattii from Eucalyptus camaldulensis in India
Authors: Chakrabarti A , Jatana M , Kumar P , Chatha L , Kaushal A , Padhye A A
Published: January 2020

Cryptococcus neoformans var. gattii has an ecological association with five Eucalyptus species: E. blakelyi, E. camaldulensis, E. gomphocephala, E. rudis, and E. tereticornis. After human infections due to C. neoformans var. gattii were diagnosed in the states of Punjab, Himachal Pradesh, and Karnataka, India, a study was undertaken to investigate the association of C. neoformans var. gattii with Indian eucalypts, especially in the state of Punjab. A total of 696 specimens collected from E. camaldulensis, E. citriodora and E. tereticornis (hybrid) trees were examined for the presence of C. neoformans var. gattii. Flowers from two trees of E. camaldulensis in the Chak Sarkar forest and one from the village of Periana near the Ferozepur area yielded five isolates of C. neoformans var. gattii. The origin of the trees could be traced to Australia, thus providing evidence that the distribution of E. camaldulensis correlated with the distribution of human cryptococcosis cases caused by C. neoformans var. gattii in northern India.
Antigen detection: the method of choice in comparison with virus isolation and serology for laboratory diagnosis of herpes zoster in human immunodeficiency virus-infected patients
Authors: Dahl H , Marcoccia J , Linde A
Published: January 2020

Ninety-two adult human immunodeficiency virus (HIV)-infected patients with suspected herpes zoster were included in a study. The clinical diagnosis of herpes zoster was verified by examination of blister cell and fluid material or serum samples. Antigen detection by a direct immunofluorescence assay with a fluorescein isothiocyanate-labelled monoclonal antibody, virus isolation, and serologic methods (in-house varicella-zoster virus [VZV] immunoglobulin G [IgG] and IgM enzyme-linked immunosorbent assays and the commercial Enzygnost assay) were compared. The direct immunofluorescence assay was found to be the most sensitive method, diagnosing 85 of 92 infections (92%), while the sensitivity of virus isolation was 65% (60 of 92 patients). Despite the use of two different serological methods, only 60 of 92 patients (65%) had significant VZV IgG titer rises, and only 26 of 92 patients (28%) had detectable VZV IgM. The lack of a VZV IgG antibody titer rise was found to correlate with low CD4 counts in peripheral blood and high VZV IgG titers in the acute-phase serum sample. The frequency of IgM-positive sera was lower than that expected from reports of studies with patients without AIDS. This may be related to early antiviral treatment or deficient antibody production due to the HIV-related immunosuppression. There was no significant difference in CD4 counts between VZV IgM-positive and -negative patients.
Molecular epidemiology of Mycobacterium tuberculosis strains isolated from patients in a human immunodeficiency virus cohort in Switzerland
Authors: Strässle A , Putnik J , Weber R , Fehr-Merhof A , Wüst J , Pfyffer G E
Published: January 2020

From 1989 to 1995, 46 patients infected with the human immunodeficiency virus were diagnosed with tuberculosis at the University Hospital in Zurich. Using the IS6110 insertion sequence as a genetic marker, restriction fragment length polymorphism analyses were done for 52 Mycobacterium tuberculosis isolates. We have found a large degree of IS6110 polymorphism, ranging from 1 to 16 copies. For isolates from patients from whom multiple isolates had been available, the IS6110 pattern remained virtually stable over a period of up to 4 years, as well as during emerging drug resistance. In none of the cases was a reinfection of a patient with another strain detected. For isolates from 10 patients we detected identical patterns which could be associated with four clusters. In one of these, the strains exhibited a low IS6110 copy number (four bands), and the strains were further analyzed by hybridizing with (i) the polymorphic GC-rich repetitive sequence (PGRS) and (ii) the 36-bp direct-repeat (DR) cluster sequence. One of these isolates had a different pattern with the PGRS as well as with the DR sequence and could therefore be safely excluded from that cluster. These findings point to the importance of applying more than one genetic criterion in the molecular biological study of strain relatedness.
Clonal dissemination and colony morphotype variation of vancomycin-resistant Enterococcus faecium isolates in metropolitan Detroit, Michigan
Authors: Dunne W M , Wang W
Published: January 2020

Thirty-two isolates of vancomycin-resistant Enterococcus faecium (VRE) recovered from 25 patients hospitalized at six hospitals in the metropolitan Detroit, Mich., area over a 32-month period were examined for relatedness by repetitive-sequence PCR (rep-PCR). All isolates were shown to carry the vanA gene by PCR. The rep-PCR patterns generated from each isolate showed that the first three VRE isolates obtained from hospital A between June 1992 and February 1994 were distinct strains. Thereafter, all VRE isolates originating from hospital A and those collected from five other area hospitals had identical rep-PCR patterns. On detailed examination, subcultures of 25 of the 32 VRE isolates produced two distinct colony types characterized phenotypically by a rough and a smooth appearance, respectively. Both colony types retained the vanA locus and the rep-PCR pattern of the primary isolate. These data suggest that a single strain of VRE with the capacity to produce two colonial variants has been disseminated to several Detroit-area hospitals. The clinical significance of the colonial morphotypes is unclear.
A Mycobacterium tuberculosis IS6110 preferential locus (ipl) for insertion into the genome
Authors: Fang Z , Forbes K J
Published: January 2020

A 267-nucleotide Mycobacterium tuberculosis genomic sequence (ipl, the IS6110 preferential locus) which can harbor the insertion sequence IS6110 at six alternative locations has been identified in some three-quarters of the isolates tested. Only one IS6110 copy was observed at this locus in the ipl::IS6110(+)-containing isolates tested, and all insertions had the same orientation. The implications of this finding for IS6110 fingerprint typing methods is discussed in this work.
Serologic markers in early stages of African horse sickness virus infection
Authors: Martínez-Torrecuadrada J L , Díaz-Laviada M , Roy P , Sánchez C , Vela C , Sánchez-Vizcaíno J M , Casal J I
Published: January 2020

Fifteen horses were experimentally infected with African horse sickness virus (AHSV) serotype 4. To learn more about the time course of production and specificity of AHSV-specific antibodies, sera were analyzed by immunoblot analysis. Only animals that survived for more than 9 days were able to develop a humoral immune response detectable by immunoblotting. The earliest serological markers corresponded mainly to VP5, VP6, and NS2 and to a lesser extent to VP3, NS1, and NS3. Neutralizing antibodies to VP2 were not detected by immunoblotting, suggesting that they are mostly conformation dependent. VP7-specific antibodies were detected later in infection. These results make NS2 and VP6 the most attractive candidates for the rapid diagnosis of the infection.
Evaluation of the AnaeroPack system for growth of clinically significant anaerobes
Authors: Delaney M L , Onderdonk A B
Published: January 2020

The AnaeroPack (Mitsubishi Gas Chemical America, Inc., New York, N.Y.) system was compared with the GasPak (Becton Dickinson Microbiology Systems, Cockeysville, Md.) system and a conventional anaerobe chamber to evaluate the ability of the AnaeroPack system to support the growth of clinically significant anaerobes. The AnaeroPack system requires no catalyst or water, produces no hydrogen, and is oxygen absorbing and carbon dioxide generating. It is simple to use and reduces preparation time to a minimum. One hundred forty clinical isolates obtained from various anatomic sites and 10 American Type Culture Collection type strains were evaluated. Isolates were plated on various media, and bacterial growth was examined after 24, 48, 72, and 168 h of incubation. Criteria for evaluation and comparison of systems included rate and quality of growth, colonial morphology, hemolytic reactions, and pigment production. Results indicate that the AnaeroPack system is highly effective in creating an anaerobic atmosphere. The AnaeroPack system never failed to reduce the methylene blue indicator, while the GasPak system failed 15% of the time. The rate or quality of growth achieved by the AnaeroPack system compared with that of established anaerobic culturing techniques was similar and significantly better for several genera including the Bacteroides fragilis group, Fusobacterium, Clostridium, and Peptostreptococcus. The AnaeroPack system appears to be an excellent alternative to established methods for generating an environment for anaerobic incubation.
Isolator component responsible for inhibition of Mycobacterium avium-M. intracellulare in BACTEC 12B medium
Authors: Wasilauskas B L , Morrell R M
Published: January 2020

We previously reported an inhibitory effect on Mycobacterium avium-M. intracellulare (MAI) when blood collected and processed with the Isolator system was placed in BACTEC 12B bottles for radiometric monitoring. We sought to identify the specific component(s) of the Isolator lysis-anticoagulant reagent (LAR) responsible for the inhibitory effect. We added the three components of the LAR, saponin, polyanetholesulfonate, and polypropylene glycol (PPG), to triplicate sets of BACTEC bottles separately and in various combinations. These bottles were then seeded with 10(2) CFU of MAI. The growth index (GI) was observed over a 42-day period and was compared with the GI for a growth control bottle set with no reagents and with the GI for a bottle set containing an equivalent volume of LAR. Growth in all growth control bottles and those bottles containing no PPG reached the maximum GI (GI = 999) within 8 to 10 days. Growth in bottles containing PPG never reached the maximum GI before day 14. In addition, the GIs of one bottle containing all three reagent components as well as all of the bottles containing actual Isolator LAR failed to reach the maximum within 42 days. Our data suggest that PPG is the main cause of the inhibitory effect, but that a secondary synergistic interaction between all three of the reagents may also be present.
Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains
Authors: Gannon V P , D'Souza S , Graham T , King R K , Rahn K , Read S
Published: January 2020

PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred with serotype O?:H7, O19:H7, O121:H7, O88:H7, and O156:H7 strains. Despite these differences, the nucleotide sequences of the E. coli E32511 (O157:NM) and U5-41 (O1:H7) fliC genes were 97% homologous. Two PCR primer pairs synthesized on the basis of the E32511 H7 fliC sequence amplified specific DNA fragments from all E. coli H7 strains, but did not amplify DNA fragments from the other bacterial strains. The H7-specific primers were used in combination with other primers which target the Verotoxin 1(VT1) and VT2 genes and the E. coli O157:H7 eaeA gene in multiplex PCR assays. In these assays, vt and eaeA PCR products were observed with DNAs from the majority of EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from E. coli O157:H7 or NM strains. Only eaeA PCR products were present with DNA from enteropathogenic E. coli, and only vt PCR products occurred with VT-producing E. coli which are not EHEC. The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains.
An enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D salmonellae: a rapid screening prototype
Authors: Luk J M , Kongmuang U , Tsang R S , Lindberg A A
Published: January 2020

We describe a digoxigenin-based enzyme-linked immunosorbent assay (DIG-ELISA) following a PCR to detect the amplified lipopolysaccharide rfbS gene as a means for rapid screening of serogroup D salmonellae in stool specimens. For pure bacterial cultures, the sensitivity of the PCR DIG-ELISA was approximately 10 bacteria. In the presence of stool materials, the salmonellae were first isolated by an immunomagnetic separation technique with an O9-specific monoclonal antibody. MATy-O9, followed by PCR and DIG-ELISA. The corresponding sensitivity was about 10 to 100 bacteria. To evaluate the assay performance clinically, 203 stool samples from patients with diarrhea were subjected to the routine culture techniques and the PCR ELISA method with overnight enrichment. The conventional culture method identified 145 salmonellae (31 serogroup B, 27 serogroup C, 83 serogroup D, and 5 serogroup E isolates) and 58 non-salmonella bacteria. The PCR ELISA method correctly identified all 82 serogroup D salmonellae (A405 by ELISA, 2.54 +/- 0.74) but was negative for the other Salmonella serogroups (A405, 0.26 +/- 0.08; n = 63) and non-Salmonella isolates (A405, 0.16 +/- 0.04; n = 58). In order to obtain a visible result, the assay takes approximately 6 h (PCR, 4 h; ELISA, 2 h), along with brief enrichment cultivation of the samples (from 4 to 16 h). Thus, the PCR DIG-ELISA offers a fast, accurate, semiquantitative means of detecting infectious agents such as salmonellae, and future robotic automation is possible.
Simple diagnosis of Encephalitozoon sp. microsporidial infections by using a panspecific antiexospore monoclonal antibody
Authors: Enriquez F J , Ditrich O , Palting J D , Smith K
Published: January 2020

Microsporidia (phylum Microsproa) have recently become recognized as common opportunistic protozoans in the United States and worldwide, particularly affecting immunodeficient patients. Microsporidian organisms within the genus Encephalitozoon are the cause of nephrologic, ophthalmic, pneumologic, gastroenteric, and systemic infections. However, diagnosis of the small spores by light microscopy is difficult, even with newly developed and improved staining techniques. We have developed an anti-Encephalitozoon species monoclonal antibody-based immunoassay for easy diagnosis. A hybridoma was produced and selected following one main criterion: recognition by immunofluorescence of all known Encephalitozoon spores affecting humans. The selected monoclonal antibody-secreting hybridomas were characterized by enzyme-linked immunosorbent assay, immunofluorescence, Western blot, and immunoelectron microscopy using Encephalitozoon species from fresh and fixed samples from patients and from in vitro cultures. In the immunofluorescence assay, one monoclonal antibody, termed 3B6, strongly recognized Encephalitozoon cuniculi, E. hellem, and E. intestinalis. Monoclonal antibody 3B6 bound to other microsporidia (Nosema and Vairimorpha spp.) without cross-reacting with any other parasite, including Enterocytozoon bieneusi, fungus, or bacterium tested. In immunoelectron microscopy assays, monoclonal antibody 3B6 bound to the exospore of Encephalitozoon species, while in Western blot assays, it recognized three to seven antigens with molecular masses ranging from 34 to 117 kDa. We have developed a sensitive and specific monoclonal antibody-based immunoassay to diagnose common microsporidian infections, particularly with Encephalitozoon species. This is a new tool for identifying spores in bodily fluids and biopsy samples and is an efficient diagnostic test. Additionally, monoclonal antibody 3B6 can serve to assess the prevalence of microsporidial infections in immunodeficient and immunocompetent patients.
Uniform staining of Cyclospora oocysts in fecal smears by a modified safranin technique with microwave heating
Authors: Visvesvara G S , Moura H , Kovacs-Nace E , Wallace S , Eberhard M L
Published: January 2020

Cyclospora, a coccidian protist, is increasingly being identified as an important, newly emerging parasite that causes diarrhea, flatulence, fatigue, and abdominal pain leading to weight loss in immunocompetent persons with or without a recent travel history as well as in patients with AIDS. Modified Kinyoun's acid-fast stain is the most commonly used stain to identify the oocyst of this parasite in fecal smears. Oocysts of Cyclospora stain variably by the modified acid-fast procedure, resulting in the possible misidentification of this parasite. We examined fecal smears stained by six different procedures that included Giemsa, trichrome, chromotrope, Gram-chromotrope, acid-fast, and safranin stains. We report on safranin-based stain that uniformly stains oocysts of Cyclospora a brilliant reddish orange, provided that the fecal smears are heated in a microwave oven prior to staining. This staining procedure, besides being superior to acid-fast staining, is fast, reliable, and easy to perform in most clinical laboratories.
Standardized peripheral blood mononuclear cell culture assay for zidovudine susceptibility testing of clinical human immunodeficiency virus type 1 isolates: effect of reducing the numbers of replicates and concentrations
Authors: Marschner I C , Mayers D L , Erice A , Smeaton L , Johnson V A , Richman D D , Reichelderfer P , Japour A J
Published: January 2020

Zidovudine susceptibility was assessed for 525 clinical human immunodeficiency virus type 1 isolates, before and after reducing the number of replicates and zidovudine concentrations in the standardized consensus peripheral blood mononuclear cell culture assay. We conclude that omitting the 0.001 microM concentration and using duplicate rather than triplicate wells are valid and cost-effective modifications of this expensive assay.
Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant
Authors: Gonzalez I , Grant K A , Richardson P T , Park S F , Collins M D
Published: January 2020

A PCR method for the rapid identification and discrimination of thermophilic Campylobacter jejuni and Campylobacter coli was developed by using a gene encoding a protein involved in siderophore transport (ceuE). A nucleotide sequence divergence of approximately 13% in the ceuE genes of C. jejuni and C. coli facilitated the design of two species-specific PCR primer sets. The specificity of the PCR amplification reactions was confirmed by using two nonradioactively labelled species-specific internal oligonucleotide hybridization probes for each of these species.
Dot blot assay for the serotyping of pneumococci
Authors: Fenoll A , Jado I , Vicioso D , Casal J
Published: January 2020

To simplify the serotyping of Streptococcus pneumoniae, a dot bot assay has been developed and compared with the standard quellung reaction in 1,082 isolates. The technique has been demonstrated to be sensitive, specific, easy to perform, and inexpensive. The dot blot assay could be useful when large numbers of pneumococci have to be studied.
Septicemia caused by Streptococcus canis in a human
Authors: Bert F , Lambert-Zechovsky N
Published: January 2020

We describe a case of septicemia due to Streptococcus canis in a 77-year-old man. The organism was presumably transmitted from a domestic animal. Ulcers of the lower limbs were the likely portals of entry. The differentiation between Streptococcus canis and Streptococcus dysgalactiae was based on biochemical properties and DNA macrorestriction analysis by pulsed-field gel electrophoresis.
Recurrent CDC group IVc-2 bacteremia in a human with AIDS
Authors: Anderson R R , Warnick P , Schreckenberger P C
Published: January 2020

A 30-year-old man with AIDS developed separate episodes of bacteremia with multiple organisms including CDC group IVc-2, related to an indwelling central venous catheter. Recovery at each interval followed antibiotic therapy and replacement of the central venous access. This is the first reported case of infection with this organism in a patient with AIDS. A review of the biochemical features of this bacterium as well as pertinent literature is presented.
Use of nucleic acid probes to identify mycobacteria directly from Difco ESP-Myco bottles
Authors: Labombardi V J , Carter L , Massarella S
Published: January 2020

Mycobacterial isolates were identified directly from positive ESP-Myco bottles by use of nucleic acid probes. Retrospective analysis of 360 cultures which grew either Mycobacterium tuberculosis, M. avium complex, or M. gordonae showed that 87% were identified by direct testing of an aliquot obtained at the time a positive culture was detected. Another 12% of these cultures gave results in the equivocal range, with only 1% of the isolates yielding negative results on initial testing.
Isolation of Lautropia mirabilis from sputa of a cystic fibrosis patient
Authors: Ben Dekhil S M , Peel M M , Lennox V A , Stackebrandt E , Sly L I
Published: January 2020

An aggregate-forming coccus, isolated twice as the predominant microorganism in sputa from a cystic fibrosis patient on consecutive days, was shown to belong to the species Lautropia mirabilis on the bases of similarities of 16S rRNA gene sequences and phenotype. These isolates of L. mirabilis appear to be the first reported from a patient with cystic fibrosis and outside of Denmark.
Effect of zinc concentration in Mueller-Hinton agar on susceptibility of Pseudomonas aeruginosa to imipenem
Authors: Daly J S , Dodge R A , Glew R H , Soja D T , DeLuca B A , Hebert S
Published: January 2020

The susceptibility of Pseudomonas aeruginosa to imipenem has been shown to vary according to zinc concentration in the media. MICs of imipenem for 68 unique clinical isolates of P. aeruginosa were determined in media supplemented with zinc at concentrations between 0.5 and 6.0 micrograms/ml. In agar containing up to 3 micrograms of zinc/ml, 75 to 82% of the strains were susceptible to imipenem at an MIC of < or = 4 micrograms/ml. In agar supplemented to contain 6 micrograms of zinc/ml, however, only 40% of the strains were susceptible to imipenem. Manufacturers should ensure that the concentration of zinc in commercial media is below 3 micrograms/ml to avoid false classification of isolates as resistant to imipenem.
Comparison of four different sampling methods for detecting pharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae in children
Authors: Rapola S , Salo E , Kiiski P , Leinonen M , Takala A K
Published: January 2020

Samples from 96 children with acute respiratory infection were obtained simultaneously with nasal, nasopharyngeal, and oropharyngeal swabs and by nasopharyngeal aspiration and were cultured on chocolate and blood agar plates. The rates of isolation of Streptococcus pneumoniae and Haemophilus influenzae detected by the four sampling methods were compared. Nasopharyngeal aspirates were optimal for the detection of both S. pneumoniae (isolation rate, 33%) and H. influenzae (isolation rate, 31%). When a nasopharyngeal aspirate is not available, such as for healthy children or children with no obtainable secretions, the nasopharyngeal swab seems optimal for the detection of both S. pneumoniae and H. influenzae among children younger than 13 months of age. Among older children, similarly, the nasopharyngeal swab seems optimal for the detection of S. pneumoniae; however, for H. influenzae, the oropharyngeal swab seems optimal.
Age-specific prevalence of Escherichia coli with localized and aggregative adherence in Venezuelan infants with acute diarrhea
Authors: González R , Díaz C , Mariño M , Cloralt R , Pequeneze M , Pérez-Schael I
Published: January 2020

To evaluate the epidemiological significance of HEp-2 cell-adherent Escherichia coli isolates in diarrheal disease, we performed a study with 513 Venezuelan infants with diarrhea and 241 age-matched controls to determine the prevalence of enteropathogenic E. coli (enteroadherent E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and enterohemorrhagic E. coli) and their correlation with O:H serotypes. E. coli isolates exhibiting localized and aggregative adherence in the HEp-2 cell assay were significantly more frequently isolated from the patients (8.5 and 26.9%, respectively) than from the controls (1.7 and 15%, respectively). This difference was significant for the group 0 to 2 months of age but for older infants. Regardless of age, E. coli isolates with diffuse adherence were found at similar frequencies in both the patients and the controls. A striking correlation between classic O serogroups and localized adherence was also observed. These findings confirm the pathogenic role of E. coli with localized and aggregative adherence in diarrheal disease, as well as the epidemiological importance of O:H serotyping for characterizing localized-adhering E. coli.