bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of a bivalent (CVD 103-HgR/CVD 111) live oral cholera vaccine in adult volunteers from the United States and Peru
Authors: Taylor D N , Tacket C O , Losonsky G , Castro O , Gutierrez J , Meza R , Nataro J P , Kaper J B , Wasserman S S , Edelman R , Levine M M , Cryz S J
Published: January 2020

To provide optimum protection against classical and El Tor biotypes of Vibrio cholerae O1, a single-dose, oral cholera vaccine was developed by combining two live, attenuated vaccine strains, CVD 103-HgR (classical, Inaba) and CVD 111 (El Tor, Ogawa). The vaccines were formulated in a double-chamber sachet; one chamber contained lyophilized bacteria, and the other contained buffer. In the first study, 23 U.S. adult volunteers received CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(8), 10(7), or 10(6) CFU, CVD 111 alone at 10(7) CFU, or placebo. In the second study, 275 Peruvian adults were randomized to receive CVD 103-HgR at 10(9) CFU plus CVD 111 at 10(9) or 10(8) CFU, CVD 111 alone at 10(9) CFU, CVD 103-HgR alone at 10(9) CFU, or placebo. Three of 15 U.S. volunteers who received CVD 111 at 10(7) or 10(8) CFU developed mild diarrhea, compared to none of 4 who received CVD 111 at 10(6) CFU and 1 of 4 who received placebo. Twelve (63%) of 19 vaccine recipients shed the El Tor vaccine strain. All but one volunteer developed significant Ogawa and Inaba vibriocidal antibody titers. Volunteers who received CVD 111 at 10(7) CFU had geometric mean Ogawa titers four to five times higher than those of volunteers who received the lower dose. In the second study, all dosage regimens were well tolerated in Peruvians. About 20% of volunteers who received CVD 111 at the high dose excreted the El Tor organism, compared to 7% in the low-dose group. CVD 111 was detected in the stools of two placebo recipients, neither of whom had symptoms or seroconverted. In all vaccine groups, 69 to 76% developed fourfold rises in Inaba vibriocidal antibodies. Among those who received the bivalent vaccine, 53 to 75% also developed significant rises in Ogawa vibriocidal antibodies. We conclude that it is feasible to produce a single-dose, oral bivalent vaccine that is safe and immunogenic against both biotypes (El Tor and classical) and both serotypes (Inaba and Ogawa) of cholera for populations in both developed and developing parts of the world.
Utility of a respiratory virus panel containing a monoclonal antibody pool for screening of respiratory specimens in nonpeak respiratory syncytial virus season
Authors: McDonald J C , Quennec P
Published: January 2020

An indirect immunofluorescence respiratory virus panel containing monoclonal antibodies directed against respiratory syncytial virus, parainfluenza virus types 1, 2, and 3, adenovirus, and influenza viruses A and B was used to screen specimens in the nonpeak respiratory syncytial virus seasons in 1989 and 1990. The results indicate that the respiratory virus panel is fairly sensitive (79%) and very specific (99%) for the detection of respiratory viruses directly in clinical specimens during these time periods.
Prevalence of newly isolated, cytopathic small round virus (Aichi strain) in Japan
Authors: Yamashita T , Sakae K , Ishihara Y , Isomura S , Utagawa E
Published: January 2020

Cytopathic small round virus (Aichi strain), isolated from a patient with oyster-associated gastroenteritis, showed no reaction in the polymerase chain reaction method for enteroviruses or in the enzyme-linked immunosorbent assay (ELISA) for the five serotypes of astroviruses. Our ELISA was sensitive in detecting the Aichi strain antigen in stool samples, but there was no reaction in this ELISA with any non-Aichi strains of enteric viruses, with such origins as enterovirus, rotavirus, Norwalk virus, calicivirus, or astrovirus. In the ELISA, 13 of 47 stool samples from adult patients in five of nine oyster-associated gastroenteritis outbreaks were positive, but only 1 of 397 pediatric stool samples in Aichi Prefecture was positive. The prevalence rate for Aichi strain antibody was found to be 7.2% for persons aged 7 months to 4 years. The prevalence rate for antibody to Aichi strain increased with age, to about 80% in persons 35 years old. On the basis of the results of the present study, it was hypothesized that Aichi strain could be a new type of small round virus that mainly produces diarrhea in patients in the 15- to 34-year-old age group, 50 to 76% of whom possess neutralizing antibody.
Use of a monoclonal antibody in a dot immunobinding assay for detection of a circulating mannoprotein of Candida spp. in neutropenic patients with invasive candidiasis
Authors: De Bernardis F , Girmenia C , Boccanera M , Adriani D , Martino P , Cassone A
Published: January 2020

A dot immunobinding assay for the detection of a circulating mannoprotein (MP) antigen of Candida species in the sera of neutropenic patients in a hematological setting is described. The technique is based on the use of a monoclonal antibody which recognizes an oligomannoside epitope shared by distinct MP of pathogenic Candida species. The sensitivity of the assay for antigen detection in serum was between 2 and 5 ng/ml, and MPs from Candida albicans, Candida tropicalis, Torulopsis glabrata, and Candida parapsilosis, but not Candida krusei, could be detected. A retrospective analysis of sera from patients with proven invasive candidiasis versus sera from controls (Candida-colonized and noncolonized subjects) revealed that the novel assay has sufficient sensitivity, specificity, and predictive values to be of potential diagnostic significance.
Evaluation of Biolog for identification of members of the family Micrococcaceae
Authors: Miller J M , Biddle J W , Quenzer V K , McLaughlin J C
Published: January 2020

The Biolog Identification System (Biolog, Inc., Hayward, Calif.) was challenged at two separate laboratories with 113 coded isolates, including 33 type strains of staphylococci, 5 strains of Micrococcus spp., and 1 strain of Stomatococcus mucilaginosus. Test parameters between the sites were controlled as much as possible. Discrepancies were arbitrated by using conventional biochemicals. Overall accuracies (correct to the species level) upon initial testing were 47.7 and 59.3%, respectively, at the two laboratories. After repeat testing of isolates generating "no identification" responses or errors, the overall accuracies increased to 69.0 and 74.3% at the two sites, respectively, revealing no significant difference in the final results at the two laboratories (78 of 113 versus 84 of 113; P > 0.05). Error rates were 7.1% at one site and 9.7% at the other. The Biolog is not yet accurate enough to serve as a primary method for identifying staphylococci.
Comparison of extracellular enzymes of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme
Authors: Amoako K K , Goto Y , Shinjo T
Published: January 2020

A total of 10 strains each of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were tested for the production of 13 extracellular enzymes. DNase, alkaline phosphatase, and lipase were predominantly associated with all the strains of F. necrophorum subsp. necrophorum, with DNase not detected in any of the strains of F. necrophorum subsp. funduliforme. In addition, the strains of F. necrophorum subsp. necrophorum were generally more hemolytic than those of F. necrophorum subsp. funduliforme. Lecithinase, beta-lactamase, elastase, hyaluronidase, chondroitin sulfatase, and coagulase were not detected in any of the strains. DNase may be used to differentiate between the two subspecies.
Rapid detection and identification of Mycobacterium avium by amplification of 16S rRNA sequences
Authors: van der Giessen J W , Eger A , Haagsma J , van der Zeijst B A
Published: January 2020

An assay that is based on the amplification of 16S rRNA sequences and that was initially developed to detect Mycobacterium paratuberculosis in cattle was used to test 20 serotypes of the Mycobacterium avium complex (MAC) and atypical mycobacterial species not belonging to MAC. Only serotypes 1 to 6 and 8 to 11, designated M. avium, were detected by the assay, indicating that it can be used for the rapid detection and identification of M. avium. The results of the assay for clinical samples from animals suspected of having mycobacterial infections indicated that it can also be used directly on clinical samples.
Development of a recombinant enzyme-linked immunosorbent assay for detection of antibodies against Epstein-Barr virus nuclear antigens 2A and 2B
Authors: Geertsen R , Espander-Jansson A , Dobec M , Price P , Wunderli W , Rymo L
Published: January 2020

The baculovirus expression system was used to produce full-length Epstein-Barr virus nuclear antigens (EBNAs) 2A and 2B. Recombinant baculoviruses that contained the EBNA-2A- and EBNA-2B-encoding sequences were constructed. The proteins were expressed in Spodoptera frugiperda SF-9 cells infected with the recombinant viruses and were characterized by using monoclonal and human polyclonal antibodies by immunoblotting and immunofluorescence techniques. Partially purified extracts of the EBNA-2A- and EBNA-2B-infected insect cells were used to establish a new enzyme-linked immunosorbent assay for the detection of antibodies against EBNA-2A and EBNA-2B. Preferential reactivity toward the type A or type B EBNA-2 protein was observed in 36% of serum specimens from Swiss patients with acute infectious mononucleosis and in 81% of Swiss patients with latent Epstein-Barr virus infection. Of the patients in the latter group, sera from 76% reacted preferentially with EBNA-2A, sera from 5% reacted preferentially with EBNA-2B, sera from 12% showed similar reactivities against both antigens, and sera from 7% were nonreactive.
Survey of rotavirus G and P types associated with human gastroenteritis in São Paulo, Brazil, from 1986 to 1992
Authors: Timenetsky M do C , Santos N , Gouvea V
Published: January 2020

Rotavirus strains causing gastroenteritis in Brazilian children were characterized by PCR-based typing assays. In addition to strains bearing the major human G and P types, large numbers of strains bearing P3 (M37-like), P6 (HCR3-like), untypeable P and G types, and complex mixtures of P and G types not previously recognized were present in the community.
Epidemiologic investigation by macrorestriction analysis and by using monoclonal antibodies of nosocomial pneumonia caused by Legionella pneumophila serogroup 10
Authors: Lück P C , Helbig J H , Günter U , Assmann M , Blau R , Koch H , Klepp M
Published: January 2020

A 67-year-old woman was hospitalized with an acute pneumonia of the left lower lobe. Legionella pneumophila serogroup 10 was cultured from two sputum specimens taken on days 18 and 20 and was also detected by direct immunofluorescence assay by using a commercially available species-specific monoclonal antibody as well as serogroup 10-specific monoclonal antibodies. Antigenuria was detected in enzyme-linked immunosorbent assays by using serogroup 10-specific polyclonal and monoclonal antibodies. In the indirect immunofluorescence test rising antibody titers against serogroups 1, 4, 5, 8, 9, 10, 14, and 15 were found in serum, with the highest titers found against serogroups 8, 9, and 10. L. pneumophila serogroups 10 and 6 and a strain that reacted with serogroup 4 and 14 antisera were cultured from both central and peripheral hot water systems of the hospital. Macrorestriction analyses of the genomic DNAs by pulsed-field gel electrophoresis showed that the isolate from the patient was identical to the serogroup 10 strains from the hospital hot water system. In contrast, the genomic DNAs of 16 unrelated L. pneumophila serogroup 10 strains showed 12 different restriction patterns. Monoclonal antibody subtyping revealed only minor differences in L. pneumophila serogroup 10 strains isolated from different sources. In conclusion, macrorestriction analysis is a valuable tool for studying the molecular epidemiology of L. pneumophila serogroup 10.
Interpretive criteria for susceptibilities of Haemophilus influenzae to ampicillin, amoxicillin, and amoxicillin-clavulanic acid
Authors: Fuchs P C , Barry A L
Published: January 2020

One hundred fifty isolates of Haemophilus influenzae (including 30 beta-lactamase-positive strains and 23 beta-lactamase-negative, ampicillin-resistant strains) were tested for susceptibilities to ampicillin, amoxicillin, and amoxicillin-clavulanic acid (A/C) by the broth microdilution method in Haemophilus test medium (HTM) and in Mueller-Hinton medium with lysed horse blood and by the disk diffusion method on HTM agar. Our results support the use of HTM for susceptibility testing of H. influenzae but raise a number of questions regarding the interpretive criteria currently in use, particularly with respect to the fourfold difference in MIC susceptibility breakpoints for ampicillin and A/C and a resulting high proportion of A/C-susceptible beta-lactamase-negative, ampicillin-resistant strains.
Comparison of two panels of monoclonal antibodies for determination of Chlamydia trachomatis serovars
Authors: Ossewaarde J M , Rieffe M , de Vries A , Derksen-Nawrocki R P , Hooft H J , van Doornum G J , van Loon A M
Published: January 2020

A panel of monoclonal antibodies was developed for serovar typing of clinical isolates of Chlamydia trachomatis. The panel could distinguish all 15 established serovars from one another, although the hybridomas of the panel were developed by fusions of myeloma cells and spleen cells from mice immunized with antigen derived from the urogenital serovars D to L3. The typing assay was based on a dot enzyme immunoassay, and the monoclonal antibodies that were included in the panel reacted strongly in this assay. A collection of 289 clinical isolates from The Netherlands was typed. The observed serovar frequency distribution was 51 isolates of serovar D (17.6%), 103 isolates of serovar E (35.6%), 62 isolates of serovar F (21.5%), 28 isolates of serovar G (9.9%), 14 isolates of serovar H (4.8%), 2 isolates of serovar I' (0.7%), 20 isolates of serovar J (6.9%), and 9 isolates of serovar K (3.1%). These results were confirmed by typing these isolates with a panel of monoclonal antibodies purchased from the Washington Research Foundation, Seattle. No strain variation was observed within serovar D with both panels. However, restriction fragment length polymorphism analysis of the gene encoding the major outer membrane protein showed that 32 isolates were similar to the prototype D and 17 were similar to the variant D-. The two others showed a new restriction pattern. Our panel of monoclonal antibodies contained one monoclonal antibody that divided the serovar G isolates into two groups. This differentiation was confirmed by restriction fragment length polymorphism analysis, confining this difference to a known sequence variation in variable domain IV. These data support the subdivision of serovar G into serovars G (prototype strain UW-57) and Ga (prototype strain IOL-238).
Suppression of fungal growth exhibited by Pseudomonas aeruginosa
Authors: Kerr J R
Published: January 2020

Three surgery patients were monitored postoperatively, with particular reference to lung infection. In each case there was a clinical impression that Pseudomonas aeruginosa suppressed the growth of Candida albicans in patients with clinically significant lung infections from whom both of these organisms were isolated from serial sputum samples. Regrowth of C. albicans after P. aeruginosa eradication occurred in two patients, despite fluconazole therapy, to which both C. albicans isolates were susceptible. In all three patients, the strain of P. aeruginosa was found to inhibit the growth of the corresponding C. albicans strain in vitro. Further in vitro susceptibility studies revealed significant inhibition by 10 strains of P. aeruginosa of 11 strains of fungi known to infect humans; these were Candida krusei, Candida keyfr, Candida guillermondii, Candida tropicalis, Candida lusitaniae, Candida parapsilosis, Candida pseudotropicalis, Candida albicans, Torulopsis glabrata, Saccharomyces cerevisiae, and Aspergillus fumigatus.
Serodiagnosis of Chagas' disease by enzyme-linked immunosorbent assay using two synthetic peptides as antigens
Authors: Peralta J M , Teixeira M G , Shreffler W G , Pereira J B , Burns J M , Sleath P R , Reed S G
Published: January 2020

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against Trypanosoma cruzi. Two synthetic T. cruzi peptides, TcD and PEP2, were used. The specificity and sensitivity of the peptide ELISA were determined with 260 serum samples from individuals living in an area in which Chagas' disease is endemic. ELISAs were performed with the peptides singly or in combination. The evaluation of these tests showed that 168 (93.8%) of 179 serum samples from T. cruzi-infected patients were positive when TcD peptide was used as antigen; 164 (91.6%) samples were positive with PEP2, and 178 (99.4%) samples were positive when the two peptides were combined. Thus, the sensitivity of the ELISA using the two peptides exceeded 99%. The specificity was evaluated by using a panel of 118 serum samples that included samples from 81 individuals living in an area of endemicity with negative serology for Chagas' disease and from 37 patients from areas in which T. cruzi was not endemic but with other pathologies, such as leishmaniasis, tuberculosis, and leprosy. Only two false-positive serum samples were found in this group of individuals, giving a test specificity of more than 98%. Because these peptides can be synthesized and are very stable at room temperature, the use of such reagents can improve the standardization and reproducibility of ELISAs for the serodiagnosis of T. cruzi infection.
Restriction fragment length polymorphism analysis of Cryptococcus neoformans isolates from environmental (pigeon excreta) and clinical sources in New York City
Authors: Currie B P , Freundlich L F , Casadevall A
Published: January 2020

Restriction fragment length polymorphism analysis of environmental (pigeon excreta) and clinical Cryptococcus neoformans var. neoformans isolates in a limited geographic area distinguished 6 strains among 8 environmental isolates and 12 strains among 17 clinical isolates. Clusters of patients with three strains types accounted for 47% of clinical isolates. Despite this diversity, two strains were shared by environmental and clinical isolates.
Low-stringency PCR with diagnostically useful primers for identification of Leptospira serovars
Authors: de Caballero O L , Dias Neto E , Koury M C , Romanha A J , Simpson A J
Published: January 2020

Primers proposed for the diagnosis of the pathogenic spirochete Leptospira spp. (C. Gravekamp, H. V. D. Kemp, M. Franzen, D. Carrington, G.J. Schoone, G.J.J.M. Van Eys, C. O. R. Everard, R.A. Hartskeel, and W.J. Terpstra, J. Gen. Microbiol. 139:1691-1700, 1993) have been found to produce complex serovar-specific patterns under low-stringency PCR conditions. Such patterns obtained by low-stringency PCR, which maintain the specific band as an internal control, offer, an approach to the standardized identification of Leptospira serovars in clinical laboratories.
Performance of three commercially available monoclonal reagents for confirmation of Chlamydia pneumoniae in cell culture
Authors: Montalban G S , Roblin P M , Hammerschlag M R
Published: January 2020

We evaluated the performance of three commercially available monoclonal antibodies for confirmation of the presence of Chlamydia pneumoniae in cell culture by examining their abilities to stain inclusions of eight strains of C. pneumoniae. The antibodies tested were two unconjugated C. pneumoniae-specific monoclonal reagents and one conjugated genus-specific reagent. All three produced similar intensities of staining of C. pneumoniae, with some strain-to-strain variation. Methanol appeared to be a better choice of fixative than acetone, which greatly reduced the intensity of fluorescence with one of the species-specific antibodies.
Enzyme immunoassay with enhanced specificity for detection of antibodies to Chlamydia trachomatis
Authors: Ossewaarde J M , de Vries A , van den Hoek J A , van Loon A M
Published: January 2020

Two different methods for preventing the binding of cross-reacting antibodies to the genus-reactive chlamydial lipopolysaccharide (LPS) were used to improve the specificity of an enzyme immunoassay for the determination of antibodies to Chlamydia trachomatis. Coated elementary bodies were treated with either sodium periodate, to oxidize the antigenic sites of the LPS, or Triton X-100, to extract the LPS. By using these new enzyme immunoassays, the standard enzyme immunoassay, and the whole inclusion fluorescence (WIF) assay, antibodies to C. trachomatis were determined in sera from different groups of patients and controls. Paired serum samples from patients with culture-proven urogenital C. trachomatis infections showed similar responses in all three assays. Paired serum samples from patients with Chlamydia psittaci infections showed similar responses in the WIF assay and the standard enzyme immunoassay, whereas significantly reduced titers were obtained in the enzyme immunoassays with treated antigen, especially in the convalescent-phase serum samples. Serum samples from patients with symptoms suggestive of infection with C. trachomatis, pregnant women, and blood donors were evaluated by all three types of assays. Eighty percent of the significant reductions in immunoglobulin G (IgG), IgA, and IgM titers were observed in sera with WIF assay titers in the lower classes (IgG, 1: < or = 256; IgA, 1: < or = 32; IgM, 1: < or = 16). From these results we conclude that oxidation of the antigen by sodium periodate is a simple and effective method of producing an enzyme immunoassay with enhanced specificity that could be useful for diagnostic purposes and seroepidemiological studies.
General primer-mediated PCR for detection of Aspergillus species
Authors: Melchers W J , Verweij P E , van den Hurk P , van Belkum A , De Pauw B E , Hoogkamp-Korstanje J A , Meis J F
Published: January 2020

A PCR assay was developed for the diagnosis of invasive aspergillosis in immunocompromised patients. For this purpose, the complete nucleotide sequences of the genes encoding the 18S rRNA of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, and Aspergillus flavus were elucidated and aligned to the sequences of Aspergillus fumigatus and other clinically relevant prokaryotic and eukaryotic microorganisms. Genus-specific sequences could be identified in the V7 to V9 region of 18S rRNA. By using hot-start PCR, Southern blot hybridization, and restriction enzyme analysis, Aspergillus-specific and -sensitive determination was achieved. Five of six immunosuppressed mice experimentally infected with A. fumigatus developed infection, and rRNA could be detected in each case, even in livers with the absence of positive cultures. Aspergillus species were detected by PCR in four neutropenic patients with proven aspergillosis, although Aspergillus species had been isolated from only one bronchoalveolar lavage (BAL) fluid sample. Aspergillus species were detected by PCR in two more patients suspected of having infection. Positive PCR signals were obtained from the BAL samples of 3 of 8 neutropenic patients who had developed pulmonary infiltrates, but none were obtained from the samples of 14 nonimmunosuppressed patients. These results indicate the potential value of PCR to detect Aspergillus species in BAL samples and, therefore, to identify neutropenic patients at risk for invasive aspergillosis.
Rapid detection and identification of Candida albicans and Torulopsis (Candida) glabrata in clinical specimens by species-specific nested PCR amplification of a cytochrome P-450 lanosterol-alpha-demethylase (L1A1) gene fragment
Authors: Burgener-Kairuz P , Zuber J P , Jaunin P , Buchman T G , Bille J , Rossier M
Published: January 2020

PCR of a Candida albicans cytochrome P-450 lanosterol-alpha-demethylase (P450-L1A1) gene segment is a rapid and sensitive method of detection in clinical specimens. This enzyme is a target for azole antifungal action. In order to directly detect and identify the clinically most important species of Candida, we cloned and sequenced 1.3-kbp fragments of the cytochrome P450-L1A1 genes from Torulopsis (Candida) glabrata and from Candida krusei. These segments were compared with the published sequences from C. albicans and Candida tropicalis. Amplimers for gene sequences highly conserved throughout the fungal kingdom were first used; positive PCR results were obtained for C. albicans, T. glabrata, C. krusei, Candida parapsilosis, C. tropicalis, Cryptococcus neoformans, and Trichosporon beigelii DNA extracts. Primers were then selected for a highly variable region of the gene, allowing the species-specific detection from purified DNA of C. albicans, T. glabrata, C. krusei, and C. tropicalis. The assay sensitivity as tested for C. albicans in seeded clinical specimens such as blood, peritoneal fluid, or urine was 10 to 20 cells per 0.1 ml. Compared with results obtained by culture, the sensitivity, specificity, and efficiency of the species-specific nested PCR tested with 80 clinical specimens were 71, 95, and 83% for C. albicans and 100, 97, and 98% for T. glabrata, respectively.
Identification and elimination of DNA sequences in Taq DNA polymerase
Authors: Hughes M S , Beck L A , Skuce R A
Published: January 2020

This study confirms that different preparations of Taq DNA polymerase are contaminated with eubacterial DNA. The contaminants appeared to represent more than one strain or species but were not identified as Thermus aquaticus or Escherichia coli. Differences in microcentrifuge tube composition appeared to affect elimination of the contaminants.
Rapid detection of vancomycin-resistant enterococci
Authors: Edberg S C , Hardalo C J , Kontnick C , Campbell S
Published: January 2020

Campylobacter blood agar with clindamycin incubated in 6% CO2 served as a medium to both screen for vancomycin resistance and select for presumptive enterococci. Colonies that grew on the medium were specifically identified as enterococci within 30 min by the pyroglutamyl-beta-naphthylamide and rapid bile esculin tests. The combination of a selective medium plus rapid enzyme substrate tests offered an inexpensive means to enumerate vancomycin-resistant enterococci from specimens by using readily available reagents.
Comparison of PCR with culture for detection of Ureaplasma urealyticum in clinical samples from patients with urogenital infections
Authors: Teng K , Li M , Yu W , Li H , Shen D , Liu D
Published: January 2020

PCR was compared with culture for the detection of Ureaplasma urealyticum in 50 specimens, including sperm, urine, and prostate secretions, from hospital patients with urogenital infections. Five positive and a further four doubtful diagnoses were made by culture, whereas PCR detected U. urealyticum in 12 samples. PCR also was faster than culturing. The increased sensitivity and shorter time requirement of PCR support its further development for the diagnosis of U. urealyticum infection.
Use of UV ParaLens adapter for detection of acid-fast organisms
Authors: Patterson K V , McDonald C L , Miller B F , Chapin K C
Published: January 2020

Auramine-stained mycobacterial smears from 136 clinical specimens were interpreted by using the UV ParaLens adapter (Beckton Dickinson), and results were compared with smear interpretations using a traditional fluorescent microscope and culture. The sensitivity and specificity of the ParaLens were 84 and 93%, respectively. Smears yielding discrepant results were overstained by the Kinyoun method. Overall, the sensitivity of auramine-stained smears interpreted with the UV ParaLens was comparable to that of Kinyoun-stained smears.
Influence of volume of sample processed on detection of Chlamydia trachomatis in urogenital samples by PCR
Authors: Goessens W H , Kluytmans J A , den Toom N , van Rijsoort-Vos T H , Niesters B G , Stolz E , Verbrugh H A , Quint W G
Published: January 2020

In the present study, it was demonstrated that the sensitivity of the PCR for the detection of Chlamydia trachomatis is influenced by the volume of the clinical sample which is processed in the PCR. An adequate sensitivity for PCR was established by processing at least 4%, i.e., 80 microliters, of the clinical sample volume per PCR. By using this preparation procedure, 1,110 clinical samples were evaluated by PCR and by cell culture, and results were compared. After discordant analysis, cell culture resulted in a sensitivity of 79.1% and PCR resulted in a sensitivity of 92.7%. Furthermore, it was shown that treatment with antibiotics immediately resulted in negative cell culture results but that PCR could give positive results up to 2 weeks posttreatment.
Mycoleptodiscus indicus: a new etiologic agent of phaeohyphomycosis
Authors: Padhye A A , Davis M S , Reddick A , Bell M F , Gearhart E D , Von Moll L
Published: January 2020

Mycoleptodiscus indicus, a dematiaceous hyphomycete, was identified as the causal agent of subcutaneous infection in the knee of a 72-year-old male gardener residing in coastal South Carolina. The patient had Wegener's granulomatosis and immunodeficiency. Synovial fluid and biopsy tissue sections from the prepatellar bursa stained with hematoxylin and eosin, periodic acid-Schiff, and Gomori methenamine silver stains revealed branched, septate hyphae and many moniliform hyphal elements. When tissue sections were stained by the Fontana-Masson procedure, melanin pigment in the hyphal cell walls and at the septa was evident. A velvety, dematiaceous mold was isolated from both synovial fluid and the biopsy tissue. Sporulation was induced by exposure of slide cultures on potato dextrose agar to UV light for 12 h at 25 degrees C followed by incubation of the slide cultures at 25 degrees C in the dark for 4 weeks. Clypeate sporodochia consisting of ampulliform, compressed, phialidic conidiogenous cells produced curved, hyaline, one-celled conidia with setulae at one or both ends. Initial treatment with fluconazole for 7 days was not effective, and cultures were positive after treatment. Treatment with amphotericin B with concomitant irrigation and debridement of the affected area followed by treatment with itraconazole resulted in resolution of the infection.
Anti-purified protein derivative cell-enzyme-linked immunosorbent assay, a sensitive method for early diagnosis of tuberculous meningitis
Authors: Baig S M
Published: January 2020

A cell-enzyme-linked immunosorbent assay method was developed for the diagnosis of tuberculous meningitis by determining anti-purified protein derivative antibody production by cells derived from cerebrospinal fluid and peripheral blood. Of 20 patients with clinical histories and CSF findings suggestive of tuberculous meningitis, 18 showed high production of anti-purified protein derivative immunoglobulin antibody from CSF-derived lymphocytes, while none of the controls showed such response.
PCR and single-strand conformational polymorphism for recognition of medically important opportunistic fungi
Authors: Walsh T J , Francesconi A , Kasai M , Chanock S J
Published: January 2020

The application of PCR technology to molecular diagnostics holds great promise for the early identification of medically important pathogens. PCR has been shown to be useful for the detection of the presence of fungal DNA in both laboratory and clinical samples. Considerable interest has been focused on the utility of selecting universal primers, those that recognize constant regions among most, if not all, medically important fungi. Once an amplicon, or piece of amplified DNA determined by the unique pair of oligonucleotide primers, has been generated, several different methods may be used to distinguish between genera and between species. The two major approaches have utilized differences in restriction enzyme digestion patterns or hybridization with specific probe. We report the application of single-strand conformational polymorphism (SSCP) as a technique to delineate the differences between fungal species and/or genera. Minor sequence variations in small single-stranded DNA cause subtle changes in conformation, allowing these strands to be separated on polyacrylamide gels by SSCP. We used a 197-bp fragment amplified from the 18S rRNA gene, common to all medically important fungi. After amplification, the fragments were denatured and run on an acrylamide-glycerol gel at room temperature or 4 degrees C for 4.5 or 4 h, respectively. Under room temperature conditions, the SSCP patterns for Candida albicans, Candida tropicalis, and Candida parapsilosis were identical and all strains within each species demonstrated the same pattern. These patterns differed markedly from those of the genus Aspergillus. The SSCP patterns of major and minor bands at room temperature permitted distinction between strains of Aspergillus fumigatus and Aspergillus flavus. There also was consistency of the SSCP banding pattern among different strains of the same Aspergillus species. The SSCP patterns for other medically important opportunistic fungi, such as Cryptococcus neoformans, Pseudallescheria boydii, and Rhizopus arrhizus, were sufficiently unique to permit distinction from those of C. albicans and A. fumigatus. We conclude that the technique of PCR-SSCP provides a novel method by which to recognize and distinguish medically important opportunistic fungi and which has potential applications to molecular diagnosis, taxonomic classification, molecular epidemiology, and elucidation of mechanisms of antifungal drug resistance.
Molecular subtyping of human T-cell lymphotropic virus type 2 by single-strand conformation polymorphism analysis. Retrovirus Epidemiology Donor Study Group
Authors: Heneine W , Switzer W M , Busch M , Khabbaz R F , Kaplan J E
Published: January 2020

Molecular subtyping of human T-cell lymphotropic virus type 2 (HTLV-2) by the currently used method of restriction fragment length polymorphism analysis may not be sufficiently discriminatory for transmission studies because of the predominance of single restriction types in various HTLV-2-infected populations. The utility of single-strand conformations polymorphism (SSCP) analysis was evaluated as a tool to improve the sensitivity of the subtyping of HTLV-2. The assay was designed to target a highly variable region in the long terminal repeat and was shown to be able to detect single nucleotide changes in cloned HTLV-2 sequences. Analysis of 52 HTLV-2 samples, of which 32 were from 16 sex partner pairs (16 males, 16 females), showed nine different SSCP patterns. Identical SSCP results were obtained for each of the 16 couples, suggesting the presence of similar viral genotypes and, therefore, supporting the likelihood of sexual transmission of HTLV-2 in each of these couples. Furthermore, SSCP analysis of seven HTLV-2 samples of the same restriction type (b5) showed five different SSCP patterns. Nucleotide sequencing of two samples with distinct SSCP patterns confirmed the sequence differences. SSCP provides a facile and discriminatory tool for the differentiation of HTLV-2 strains, including those previously indistinguishable by restriction fragment length polymorphism.
Amplification of Mycobacterium tuberculosis from peripheral blood
Authors: Rolfs A , Beige J , Finckh U , Köhler B , Schaberg T , Lokies J , Lode H
Published: January 2020

To determine the value of the PCR assay of lymphomonocytic blood cells for the diagnosis of pulmonary tuberculosis, we compared, in a prospective study, PCR results with results of conventional diagnostic methods of Mycobacterium tuberculosis detection. Clinical investigators who were unaware of the PCR data classified 162 immunocompetent patients, who had been hospitalized because of suspected pulmonary tuberculosis, in accordance with the recommendations of the American Thoracic Society (ATS). By testing blood samples from these patients for mycobacterial DNA with three different PCR systems, we were able to demonstrate that nonquantitative PCR of peripheral blood leukocytes is of little value for the specific diagnosis of pulmonary tuberculosis. However, semiquantitative PCR assays might have some significance since we found an increasing level of mycobacterial DNA copies in blood from patients classified in ATS groups 2 to 5.
Recombinant antigen-based enzyme immunoassay for screening of Treponema pallidum antibodies in blood bank routine
Authors: Zrein M , Maure I , Boursier F , Soufflet L
Published: January 2020

This work reports a comparison of an enzyme immunoassay (EIA) using two major Treponema pallidum recombinant antigens with a T. pallidum hemagglutination (TPHA) assay and a nontreponemal Venereal Disease Reference Laboratory (VDRL) test. A total of 1,822 normal donor serum samples was tested for cardiolipin and T. pallidum antibodies, respectively, by the VDRL assay and EIA. Among these samples, 440 were further tested by TPHA technology. Four samples were found positive by EIA, while all were reported to be negative by both TPHA and VDRL routine assays. Subsequent testing of EIA-positive samples confirmed 100% (four of four samples) and 25% (one of four samples) positive results, respectively, by immunofluorescence assay and a Western blot (immunoblot) syphilis kit. The sensitivity of the recombinant EIA was estimated at virtually 100% with a reference panel of 50 syphilitic samples. According to this study, the newly developed EIA kit shows 100% sensitivity combined to a specificity greater than 99.8% for detecting treponemal immunoglobulin G antibodies in blood bank syphilis screening.
Molecular epidemiology of Candida isolates from AIDS patients showing different fluconazole resistance profiles
Authors: Lischewski A , Ruhnke M , Tennagen I , Schönian G , Morschhäuser J , Hacker J
Published: January 2020

Thirty Candida isolates obtained from the oropharynxes of three AIDS patients were genotypically characterized. In vitro fluconazole MIC determination revealed increasing fluconazole resistances during treatment, thereby confirming the in vivo situation. Pulsed-field gel electrophoresis karyotyping, randomly amplified polymorphic DNA analysis, and hybridizations with Candida albicans repetitive element 2 were used to determine possible genotypic changes. The isolates from two patients showed genetic homogeneity, suggesting the selection for resistant variants. One patient experienced a strain switch to Candida krusei. Horizontal spread of identical strains between the patients could be excluded. However, the molecular methods used might not be sufficient to detect the underlying genetic basis of resistance to fluconazole.
Identity of streptococcal blood isolates and oral isolates from two patients with infective endocarditis
Authors: Fiehn N E , Gutschik E , Larsen T , Bangsborg J M
Published: January 2020

The purpose of this study was to isolate streptococcal strains from the oral cavities of streptococcal endocarditis patients and compare these strains biochemically and genetically with the corresponding streptococcal blood isolates. Total identity was observed between the blood and oral cavity isolates from the two patients studied.
Long-term shedding and clonal turnover of enterohemorrhagic Escherichia coli O157 in diarrheal diseases
Authors: Karch H , Rüssmann H , Schmidt H , Schwarzkopf A , Heesemann J
Published: January 2020

To investigate the length of time that Shiga-like toxin-producing Escherichia coli O157 is excreted after the onset of diarrhea, 456 serial stool specimens were obtained from 53 children. E. coli O157 cells were identified by the use of DNA probes followed by agglutination with a specific antiserum. Specimens were collected until three consecutive stool samples (collected within 9 days) were negative for E. coli O157. The median durations of shedding were 13 days (range, 2 to 62 days) in patients with diarrhea or hemorrhagic colitis and 21 days (range, 5 to 124 days) in patients that developed hemolytic uremic syndrome. In 36 (68%) of the patients, only the first culture was O157 positive, and the three cultures that followed were negative. In 7 (13%) of the patients, E. coli O157 cells were shed for more than 32 days after the onset of diarrhea; these long-term shedders were clinically asymptomatic by the end of this period. In 12 patients, one or two serial O157-negative cultures, obtained up to 8 days after a positive culture, were followed by another positive culture. Comparison of the first and last E. coli O157 isolates by pulsed-field gel electrophoresis revealed that in three of the seven long-term shedders, pulsed-field gel electrophoresis types varied. In two cases, a Shiga-like toxin gene was apparently lost during infection. The observation of long-term shedding accompanied by genotypic turnover has epidemiological and diagnostic implications.
Immunoglobulin M capture immunoassay in investigation of coxsackievirus B5 and B6 outbreaks in South Australia
Authors: Goldwater P N
Published: January 2020

An immunoglobulin M (IgM) capture enzyme immunoassay was used to detect major overlapping outbreaks of disease in South Australia caused by coxsackieviruses B5 (CBV-5) and B6 (CBV-6). CBV-5-specific IgM was detected in patients presenting in spring 1992 with acute febrile illnesses, rash, severe acute respiratory disease, meningitis, myocarditis and/or pericarditis, while tests for other viruses were negative. CBV-5 was isolated from an early case. In December 1992 it was noted that CBV-6 had replaced CBV-5 as the major cause of disease. The CBV-6 epidemic continued until April 1993. Serum samples from 495 patients (276 inpatients) were submitted for testing. CBV-6 infection was associated with lower respiratory tract infection and persistent cough. This study demonstrated success of the IgM enzyme immunoassay and the need for diagnostic virology laboratories to look for CBV-6 infection in addition to the other five CBVs.
Comparison of multilocus enzyme electrophoresis and random amplified polymorphic DNA analysis for molecular subtyping of Cryptococcus neoformans. The Cryplococcal Disease Active Surveillance Group
Authors: Brandt M E , Hutwagner L C , Kuykendall R J , Pinner R W
Published: January 2020

We evaluated multilocus enzyme electrophoresis (MEE) and random amplified polymorphic DNA (RAPD) for their usefulness in subtyping 344 Cryptococcus neoformans clinical isolates obtained from four U.S. metropolitan areas in 1992 to 1994. MEE and RAPD with five primers both discriminated between the two varieties of C. neofromans. MEE divided C. neoformans var. neoformans isolates into 15 enzyme electrophoretic subtypes (ETs) arranged in three complexes. The predominant ET 1 complex contained 10 ETs, with isolates from 70% of patients in 1 ET. RAPD with five primers further sorted this predominant ET into 19 subtypes, with 60% of isolates sorting into three RAPD types. The ET 8 MEE complex, containing three ETs, could not be divided further by RAPD. The ET 7 complex (two ETs) included isolates from all serotype AD patients. Although both MEE and RAPD identified isolates of C. neoformans var. gattii, neither distinguished between serotypes B and C. These results showed that the two C. neoformans varieties could be identified by MEE or RAPD profile as well as by biochemical methods. RAPD improved the discriminatory power of MEE for isolates within the ET 1 complex but with other ETs offered little additional sensitivity over MEE and was less sensitive than MEE with isolates of C. neoformans var. gattii. This information will be useful in identifying particular environmental sources of disease-causing exposures, in seeking clusters of cases, and in determining whether an infecting strain changes over time.
Antigenic differences between a field isolate and vaccine strains of bovine viral diarrhea virus
Authors: Reddy J R , Xue W , Rivera S , Minocha H C
Published: January 2020

Antigenic diversity among the bovine viral diarrhea virus (BVDV) cytopathic strains 87-2552 (field isolate) and NADL and Singer (prototype strains) was demonstrated with monoclonal antibodies (MAbs) in enzyme-linked immunosorbent, immunofluorescence, virus neutralization, and immunoprecipitation assays. Two MAbs against BVDV strain 87-2552, designated D11 and B7, strongly neutralized this field strain and were specific for the 48-kDa glycoprotein of the virus. These two MAbs have different subisotypes, immunoglobulin G1 for D11 and immunoglobulin G3 for B7. MAbs against BVDV strains 87-2552 and NADL were specific for their respective strains in virus neutralization assays. The results indicated significant antigenic differences between BVDV strain 87-2552 and the NADL and Singer strains.
Evaluation of Alamar colorimetric broth microdilution susceptibility testing method for staphylococci and enterococci
Authors: Baker C N , Tenover F C
Published: January 2020

We compared the results of the Alamar broth microdilution susceptibility testing method with the results of the National Committee for Clinical Laboratory Standards reference broth microdilution method for 119 gram-positive organisms. The strains were tested for their susceptibilities to 20 antimicrobial agents. Only appropriate antimicrobial agents were evaluated for each species of bacteria. Absolute categorical agreement between the reference method and the test method was 91.5% for enterococci, 99.8% for oxacillin-susceptible staphylococci, and 97.4% for oxacillin-resistant staphylococci. Essential agreement (percent complete agreement plus percent minor errors) was > 99% for all organisms tested. The results for enterococci showed no very major errors, one major error with ofloxacin, and numerous minor errors with the quinolones. However, all except one of the minor errors were within +/- 1 log2 dilution of the reference result. For staphylococci, only 2 very major errors (one each with chloramphenicol and oxacillin), 1 major error (chloramphenicol), and 15 minor errors (multiple drugs) were observed. The Alamar colorimetric system was easy to use and the results were easy to read. It appears to be an acceptable method for antimicrobial susceptibility testing of staphylococci and enterococci.
Molecular epidemiology of Escherichia coli diarrhea in children in Hong Kong
Authors: Biswas R , Nelson E A , Lewindon P J , Lyon D J , Sullivan P B , Echeverria P
Published: January 2020

This pediatric hospital-based study of 388 diarrhea cases and 306 controls analyzed predominant E. coli colonies from primary culture (253 cases and 177 controls) with eight DNA probes for enteropathogenic, enterotoxigenic, enteroaggregative, and diffusely adherent E. coli. Only enteropathogenic E. coli adherence factor was identified significantly more frequently in cases (10) than in controls (0).