bluuebunny/crossref_metadata_2025 huggingface dataset:
Whole-cell protein electrophoresis for typing Mycobacterium tuberculosis
Authors: Millership S E , Want S V
Published: January 2020

A method of discriminating between strains of Mycobacterium tuberculosis by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins combined with a sensitive silver stain is described. Thirty-five isolates of M. tuberculosis and five isolates from other species of Mycobacterium were examined, including serial isolates from the same patients and isolates from a small cluster of hospital cases. Different species of Mycobacterium were clearly distinguished, and within the species M. tuberculosis, different fingerprints were obtained, allowing discrimination of some strains from different patients. The reproducibility and discrimination of the technique are discussed.
Use of a recombinant 170-kilodalton surface antigen of Entamoeba histolytica for serodiagnosis of amebiasis and identification of immunodominant domains of the native molecule
Authors: Zhang Y , Li E , Jackson T F , Zhang T , Gathiram V , Stanley S L
Published: January 2020

We expressed the gene that encodes one of the major surface antigens of Entamoeba histolytica, the 170-kDa protein (1,270 amino acids), as a glutathione S-transferase fusion protein containing amino acids 1 to 1202 (lacking the putative transmembrane and cytoplasmic regions) and as separate fusion proteins containing each of three major domains of the 170-kDa molecule. Lysates from bacteria induced to express one of these proteins were used as the target antigens in a Western blot (immunoblot) analysis to determine whether a recombinant 170-kDa antigen could serve as the basis for a serologic test used to detect invasive amebiasis and whether there are differences in humoral immunogenicity among the three major domains of the 170-kDa antigen. Among patients with invasive amebiasis from three major areas where the disease is endemic and two sites in the United States, 54 (90%) of 60 had antibodies to the recombinant 170-kDa protein. Among 37 patients from regions where the disease is endemic and 20 patients from the United States without amebic disease, 1 (2%) of 57 had antibodies to the recombinant 170-kDa protein. We found significant differences in seroreactivity to each of three major domains of the molecule among patients seropositive for the complete construct, ranging from 100% seroreactivity with the fusion protein containing the domain designated cysteine rich and 89% seropositivity with the fusion protein incorporating a portion of the region designated cysteine poor to only 9% seropositivity for the fusion protein containing the pseudorepeat domain. Our study indicates that a serologic test based on the recombinant 170-kDA antigen could serve as a highly sensitive and specific test for acute invasive amebiasis.
A monoclonal antibody for distinction of invasive and noninvasive clinical isolates of Entamoeba histolytica
Authors: Gonzalez-Ruiz A , Haque R , Rehman T , Aguirre A , Jaramillo C , Castañon G , Hall A , Guhl F , Ruiz-Palacios G , Warhurst D C
Published: January 2020

Approximately 10% of the world population is infected with Entamoeba histolytica, but only 10% of the carriers develop symptomatic amebiasis. This discrepancy could be explained by the genotypic differences between the morphologically indistinguishable invasive and noninvasive strains of E. histolytica currently identified by zymodeme analysis, a technique that is unsuitable for routine diagnostic laboratories. Here we report the production of a monoclonal antibody against E. histolytica and its use in an immunofluorescence assay to identify invasive isolates cultured from stool samples of infected patients in several regions where amebiasis is endemic: Bangladesh, Colombia, and Mexico. After testing a total of 88 E. histolytica isolates, the correlation between zymodeme characterization and the immunofluorescence assay with the invasive isolate-specific monoclonal antibody was 100%. The epitope detected by the invasive isolate-specific monoclonal antibody resides in a previously undescribed internal protein with molecular masses of 84 and 81 kDa in axenic and polyxenic E. histolytica strains, respectively.
Epidemiology of Tsutsugamushi disease in relation to the serotypes of Rickettsia tsutsugamushi isolated from patients, field mice, and unfed chiggers on the eastern slope of Mount Fuji, Shizuoka Prefecture, Japan
Authors: Kawamori F , Akiyama M , Sugieda M , Kanda T , Akahane S , Uchikawa K , Yamada Y , Kumada N , Furuya Y , Yoshida Y
Published: January 2020

A total of 59 strains of Rickettsia tsutsugamushi were isolated from patients (24 isolates), Apodemus speciosus mice (30 isolates), and unfed larvae of Leptotrombidium scutellare (2 isolates) and Leptotrombidium pallidum (3 isolates) in the Gotenba-Oyama District, Shizuoka Prefecture, Japan. All these isolates were classified into the three serotypes Karp, Kawasaki, and Kuroki based on reactivity with strain-specific monoclonal antibodies. Kawasaki- and Karp-type rickettsiae were isolated from L. scutellare and L. pallidum, respectively, and the geographic distribution of patients and rodents infected with these two types of rickettsiae coincided with the areas densely populated by the respective chiggers. From these results, we conclude that Kawasaki-type rickettsiae are transmitted by L. scutellare and Karp-type ones are transmitted by L. pallidum. Kawasaki-type rickettsial infections were prevalent in early autumn, and Karp-type infections showed a peak of occurrence in the late autumn, reflecting the seasonal fluctuations of L. scutellare and L. pallidum. Isolates of Kuroki-type rickettsiae were obtained only from four patients in October and November, and the relationship between this type of rickettsia and its vector species could not be fully defined.
Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction
Authors: Loeffelholz M J , Lewinski C A , Silver S R , Purohit A P , Herman S A , Buonagurio D A , Dragon E A
Published: January 2020

A rapid and sensitive polymerase chain reaction (PCR)-based assay for detection of Chlamydia trachomatis in cervical specimens is described. This assay consists of (i) sample preparation which avoids the use of heat, centrifugation, or organic extractions; (ii) rapid, two-temperature PCR amplification of C. trachomatis cryptic plasmid sequences; and (iii) capture and colorimetric detection of amplified DNA in microwell plates. PCR was compared with culture by using 503 cervical specimens. After resolution of discrepant specimens with a confirmatory PCR assay directed against the chlamydial major outer membrane protein gene, PCR had a sensitivity of 97% and a specificity of 99.7% while culture had a sensitivity of 85.7% and a specificity of 100%. In a separate study, PCR was compared with a direct specimen enzyme immunoassay (Chlamydiazyme; Abbott Diagnostics) by using 375 cervical specimens. After resolution of discrepant specimens, PCR had a sensitivity and specificity of 100%, while the enzyme immunoassay had a sensitivity of 58.8% and a specificity of 100%.
Rapid plasmid DNA isolation from mucoid gram-negative bacteria
Authors: Domenico P , Marx J L , Schoch P E , Cunha B A
Published: January 2020

Exopolysaccharides interfere with the isolation and characterization of plasmid DNA from gram-negative bacteria. To repress capsular polysaccharide production, bacteria were cultured in medium containing bismuth nitrate and sodium salicylate. Rapid removal of other contaminating bacterial surface components was achieved by mild acidic zwitterionic detergent extraction. After treatment, bacterial cells were more readily lysed in alkaline detergents. The resulting plasmid preparations contained virtually no capsular polysaccharide and relatively small quantities of lipopolysaccharide and protein, yet they produced yields of nucleic acids similar to those of conventional plasmid preparations. Conventional preparations from encapsulated organisms were largely insoluble and appeared as smears following agarose gel electrophoresis, with indefinite plasmid banding. Plasmids prepared by the new method were highly soluble in conventional buffers and exhibited high-resolution plasmid banding patterns in agarose gels. Plasmids as large as 180 kbp could be isolated and visualized, without apparent nicking, and were readily digested by restriction endonuclease enzymes. The method proved effective with encapsulated or mucoid strains of Klebsiella pneumoniae, Escherichia coli, Acinetobacter anitratus, Salmonella typhimurium, and Enterobacter species. The complete method for plasmid isolation was not suitable for Pseudomonas aeruginosa because of the inhibitory effects of bismuth. Thus, removal of contaminating bacterial surface structures enabled the rapid isolation and characterization of plasmids from mucoid clinical isolates, without the use of organic solvents, CsCl gradients, or expensive, disposable columns.
Characterization and comparison of Australian human spotted fever group rickettsiae
Authors: Baird R W , Lloyd M , Stenos J , Ross B C , Stewart R S , Dwyer B
Published: January 2020

The microbiological and molecular characteristics of the rickettsiae isolated from humans with Queensland tick typhus (QTT) caused by Rickettsia australis and the recently described Flinders Island spotted fever (FISF) were compared. Clinically and serologically, the diseases are similar. Cell culture reveals differences in the plaque-forming abilities of the isolates. Characterization of the gene encoding the genus-specific 17-kDa antigen of R. australis revealed a unique nucleotide sequence unlike those of the FISF isolate and Rickettsia rickettsii. Southern blot analysis of rickettsial DNA from the isolates with a 17-kDa-antigen gene probe revealed the presence of this gene in all isolates but no difference in banding patterns. When a probe for the rRNA genes was used, clear differences in banding patterns of isolates from patients with QTT and FISF were revealed. Thus, the rickettsiae isolated from patients with FISF differ from those from patients with QTT and may represent a new rickettsial species.
Evaluation of the autoSCAN-W/A rapid system for identification and susceptibility testing of gram-negative fermentative bacilli
Authors: York M K , Brooks G F , Fiss E H
Published: January 2020

The autoSCAN-Walk-Away (W/A) system for identification and susceptibility testing was evaluated for 400 gram-negative fermentative bacteria by using the API 20E (366 isolates) and/or tube biochemical tests as the reference identification system and a frozen microdilution MIC tray system for susceptibility testing. The W/A system performed well for identification of this group of organisms representing 14 genera and 30 species, showing a sensitivity of 96% and results available in 2 h. Of the 16 misidentifications, 6 were with Serratia liquefaciens. A total of 63 isolates (17%) required further tests to complete the identification, compared with 106 (29%) of the isolates which required additional tests for the API 20E identification. Approximately half (32) of the additional tests with the W/A system were required in order to separate Citrobacter diversus from C. amalonaticus. For susceptibility determinations, the W/A system demonstrated an overall agreement of 93% (4,102 determinations) with 40 major errors (0.98%). However, of the 906 resistant organism-drug combinations in the study, there were 115 very major errors, for a false-susceptibility rate of 12.7% of the resistance determinations. Among these very major errors, 80% occurred with piperacillin and the cephalosporins. The W/A system completed the MIC determinations in 7 h; however, the difficulty in detecting resistance with some antimicrobial agents limited the advantages of the rapid susceptibility testing.
Rapid determination of human cytomegalovirus susceptibility to ganciclovir directly from clinical specimen primocultures
Authors: Pepin J M , Simon F , Dussault A , Collin G , Dazza M C , Brun-Vezinet F
Published: January 2020

We were able to rapidly determine the susceptibility of human cytomegalovirus to ganciclovir using a late antigen synthesis reduction assay directly on primocultures of clinical specimens. This test was compared with a conventional susceptibility assay which was performed with cell-free virus obtained after cytomegalovirus isolation and in vitro passages. Both tests produced similar results. The rapid test, unlike conventional assays, is able to provide a result within 5 days after receipt of the specimen and could thus play a direct role in the therapeutic decision.
Analysis of relationships among isolates of Citrobacter diversus by using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reaction
Authors: Woods C R , Versalovic J , Koeuth T , Lupski J R
Published: January 2020

Oligonucleotide probes which match consensus sequences of the repetitive extragenic palindromic (REP) element hybridize to genomic DNA of diverse bacterial species. Primers based on the REP sequence generate complex band patterns with genomic DNA in the polymerase chain reaction (PCR), a technique named REP-PCR. We used REP-PCR with genomic DNA to fingerprint 47 isolates of Citrobacter diversus. Previously, 37 were assigned electrophoretic types (ETs) by multilocus enzyme electrophoresis and 35 were evaluated by using outer membrane protein profiles. Fingerprints were compared by visual inspection and by similarity coefficients (SimCs) based on the number of common bands versus total bands between two given isolates. DNA fingerprints were highly similar visually for patient pairs and outbreak-related sets. SimCs for these were > or = 0.952. Fingerprints of isolates with different ETs generally were distinctive. Among 21 unrelated isolates representing 15 ETs, only 6 of 210 comparisons had SimCs of > or = 0.952. REP-PCR rapidly generated DNA fingerprints which were highly similar for epidemiologically linked isolates of C. diversus and distinct for previously characterized strains within this species. The ability of this method to discriminate between C. diversus isolates with the same biotype was similar to that of multilocus enzyme electrophoresis and outer membrane protein profiles. REP-PCR may be useful in evaluation of apparent outbreaks of this or other bacterial species which possess these extragenic, repetitive elements.
Isolation of a fastidious Mycobacterium species from two AIDS patients
Authors: Jackson K , Sievers A , Ross B C , Dwyer B
Published: January 2020

Two strains of fastidious mycobacteria were isolated from two patients with AIDS and clinical disease suggestive of Mycobacterium avium complex infection. Acid-fast bacilli were isolated from blood and bone marrow of both patients in BACTEC 12B and/or 13A media. The acid-fast bacilli failed to grow on subculture to routine Löwenstein-Jensen medium containing pyruvate and egg yolk agar. After several attempts, the strain from one patient was finally cultured on Middlebrook 7H9 medium with agar, charcoal, and yeast extract 13 months after the initial specimens were received in the laboratory. The second patient's strain was cultured on the same medium 6 weeks postinoculation with fresh BACTEC fluid and 5 months after specimen collection. Routine biochemical and growth tests were performed on these isolates but failed to give definitive identifications. 16S rRNA gene sequencing suggested that the organisms share at least 98.9% homology with M. simiae. Even greater homology (99.86%) was found with the recently described species "M. genavense." Recognition of the fastidious nature of some mycobacteria that infect AIDS patients is important in the treatment of infections in these patients and in understanding the epidemiology of atypical mycobacterial infections. It is suggested that a liquid culture medium such as BACTEC be employed for primary isolation of mycobacteria from AIDS patients and that subculture to the charcoal medium described here be carried out for those organisms that fail to grow on subculture to routine media.
DNA probe for strain typing of Cryptococcus neoformans
Authors: Varma A , Kwon-Chung K J
Published: January 2020

A 7-kb linear plasmid, harbored by a URA5 transformant, hybridized to all the chromosomes of Cryptococcus neoformans separated by contour-clamped homogeneous electric field electrophoresis. Its linear maintenance was determined to have been facilitated by the presence of telomere-like sequences at its free ends. Hybridization of this plasmid to AccI-digested genomic DNAs of 26 C. neoformans strains generated 21 unique DNA fingerprints. The DNA fingerprints of isolates within the same serotype were more similar to one another than to those from different serotypes. An acapsular clinical isolate, strain 602, widely used in immunological studies and previously thought to be in serotype D, showed DNA fingerprints typical of serotype A isolates. Isogenic strains of C. neoformans exhibited DNA fingerprints that were identical to one another. The DNA fingerprints were stable and reproducible in spite of repeated transfers in the laboratory on either complex (1% yeast extract, 2% Bacto Peptone, 2% glucose) or minimal (yeast nitrogen base) medium. The DNA fingerprints of isolates recovered from primary blood and cerebrospinal fluid cultures of patients for whom AIDS had been diagnosed showed that the original infection in each of these patients contained a homogeneous population of C. neoformans. The DNA fingerprints of isolates recovered from different tissues of infected mice and from patients undergoing different drug therapy regimens were also found to be very stable.
Colonization of the female genital tract with Staphylococcus saprophyticus
Authors: Rupp M E , Soper D E , Archer G L
Published: January 2020

The prevalence of colonization by Staphylococcus saprophyticus of the urogenital tracts of 276 women from an outpatient gynecology practice was determined by using selective and enrichment culture techniques. Nineteen subjects (6.9%) were found to be colonized by S. saprophyticus. The rectum was the most frequent site of colonization and was responsible for 40% of the isolates; this was followed in decreasing order by the urethra, urine, and cervix. Women colonized by S. saprophyticus were more likely to have experienced a urinary tract infection in the previous 12 months (P = 0.058; odds ratio, 2.844; 95% confidence interval, 1.054 to 7.671). Patients colonized by S. saprophyticus tended to have had their menstrual periods more recently (P = 0.066), experienced sexual intercourse more recently (P = 0.168), and had a recent or concurrent diagnosis of vaginal candidiasis (P = 0.111; odds ratio, 2.393; 95% confidence interval, 0.877 to 6.528). A seasonal variation in colonization was observed, with colonization most likely occurring during the summer and fall. Follow-up for an average of 6.75 months failed to document any colonized woman progressing to symptomatic urinary tract infection. In addition, 21 women colonized by non-S. saprophyticus, novobiocin-resistant, coagulase-negative staphylococci were identified and characterized.
Immunoblot analysis of the humoral immune response to Pythium insidiosum in horses with pythiosis
Authors: Mendoza L , Nicholson V , Prescott J F
Published: January 2020

Reactions to Pythium insidiosum by sera from horses with active pythiosis were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Five strains of P. insidiosum were grown in nutrient broth and then sonicated. After centrifugation, supernatant antigens were separated by SDS-PAGE. An exoantigen of Conidiobolus coronatus was also tested. Bands with molecular weights between 97,000 and 14,000 were identified by Coomassie blue and silver staining. After being transferred to nitrocellulose, the antigens were reacted against sera from six horses with pythiosis, sera from four horses cured a year earlier by vaccination, and sera from five healthy horses. The sera from horses with pythiosis recognized at least 20 antigens in all strains. Three antigens with molecular weights of 32,000, 30,000, and 28,000 appeared to be immunodominant and specific. Sera from horses cured by immunotherapy showed only five very weak bands, three of them the 32,000-molecular-weight (32K), 30K, and 28K antigens. No bands were observed with sera from healthy horses or sera from horses with a variety of other infections. Sera from horses with pythiosis cross-reacted with the 44K antigen of C. coronatus. The immunodominant antigens described here may be useful for diagnostic purposes and in immunotherapy for this oomycotic infection in horses.
Differentiation of Shigella flexneri strains by rRNA gene restriction patterns
Authors: Faruque S M , Haider K , Rahman M M , Abdul Alim A R , Ahmad Q S , Albert M J , Sack R B
Published: January 2020

We studied the restriction endonuclease cleavage patterns of rRNA genes (ribotypes) of 72 clinical isolates of Shigella flexneri representing eight serotypes to determine whether ribotyping could be used to distinguish S. flexneri strains and to compare the discriminating ability of the method with that of serotyping. By using a cloned Escherichia coli rRNA operon as the probe, Southern blot hybridization of restriction endonuclease-digested total DNA was carried out. Ribotyping of the isolates with each of the five restriction endonucleases BamHI, EcoRI, HindIII, PstI, and SalI generated reproducible restriction patterns. However, HindIII produced the optimum digestion pattern of the rRNA genes and was more useful than the other enzymes used in differentiating strains. Analysis of the 72 isolates showed 11 different HindIII cleavage patterns of their rRNA genes. Four of these HindIII-generated ribotypes could be further differentiated into two to four subribotypes by using PstI. The results indicate that ribotyping has an application for differentiation of S. flexneri strains and can complement serotyping. Definition of strains in terms of both serotype and ribotype may be of greater use in epidemiological studies.
Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS patients by polymerase chain reaction
Authors: Parmley S F , Goebel F D , Remington J S
Published: January 2020

The polymerase chain reaction (PCR) was used to detect Toxoplasma gondii DNA in cerebrospinal fluid (CSF) from 14 patients with AIDS by amplification of the repetitive B1 gene. Positive PCRs were obtained in CSF from four of nine patients with toxoplasmic encephalitis. CSF samples from five control patients were negative for T. gondii DNA by PCR.
Evidence of serologic diversity within group C rotaviruses
Authors: Tsunemitsu H , Jiang B , Yamashita Y , Oseto M , Ushijima H , Saif L J
Published: January 2020

The Cowden strain of porcine group C rotavirus and the Shintoku strain of bovine group C rotavirus were classified as different serotypes by two-way cross-neutralization tests. Two neutralization patterns against the Cowden and Shintoku strains were observed when hyperimmune or convalescent-phase antisera to three noncultivatable porcine group C rotaviruses and a human group C rotavirus were used in one-way cross-neutralization tests. Antisera to two porcine group C rotaviruses and the human group C rotavirus neutralized the Cowden strain at high titers but did not neutralize the Shintoku strain, suggesting that these three strains are serotypically related to the Cowden strain. The remaining antisera to a porcine group C rotavirus (HF strain) reacted with the Cowden and Shintoku group C rotaviruses in cell culture immunofluorescence tests but did not neutralize either virus in one-way cross-neutralization, suggesting that the HF strain belongs to a third serotype. However, confirmation of these findings requires additional analysis by two-way cross-neutralization. Our findings support the existence of at least two distinct serotypes of group C rotaviruses, and possibly a third, among animals and humans. The serotypic similarity observed between the Cowden strain and a human group C rotavirus suggests that the cultivatable Cowden strain and antiserum to this virus may provide important reagents for the diagnosis of group C rotaviruses in humans.
Detection of diacetyl (caramel odor) in presumptive identification of the "Streptococcus milleri" group
Authors: Chew T A , Smith J M
Published: January 2020

The caramel odor associated with the "Streptococcus milleri" group was shown to be attributable to the formation of the metabolite diacetyl. Levels of diacetyl in the 22- to 200-mg/liter range were produced by 68 strains of the "S. milleri" group; apart from one strain of Streptococcus mutans, all 92 other strains of streptococci belonging to 12 species produced < 13 mg of diacetyl per liter. Quantitation of diacetyl levels from cultures of streptococci is suggested as a rapid presumptive test for the "S. milleri" group.
Salmonella serogroups C2 and C3 identified by agglutination using an immunoglobulin G3(kappa) monoclonal antibody (32-1-E3) reactive with a somatic factor 8-like polysaccharide antigen
Authors: Duffey P S , Kani J C , Lee J O , Hill W J , Kokka R
Published: January 2020

An immunoglobulin G3(kappa) monoclonal antibody (MAb), MAb 32-1-E3, which was prepared in BALB/c mice by using a heated, alcohol-acetone-extracted Salmonella newport CDC 50 antigen, reacted with protein-free lipopolysaccharides from Salmonella groups C2 (O:6,8) and C3 (O:-,8) but not with those from any other serogroup tested. Sodium periodate did not inhibit antigen reactivity, which was consistent with its identity as the abequose-containing disaccharide O:8 antigen. Reactivity was inhibited by competition with serogroup C2 (O:6,8) and C3 (O:-,8) antigens but not with non-O:8 antigens. Reactivity was also inhibited by preincubation of the antigen with polyclonal rabbit antiserogroup C2 or C3 antibodies but not with antisera to serogroup C1 or other Salmonella serogroups. The MAb agglutinated with all strains of Salmonella serogroups C2 and C3 tested but not with other bacteria. Agglutination was inhibited by preabsorbing the MAb with either of two serogroup C3 Salmonella strains, S. virginia CDC 189 or S. haardt MDL 83A4545, which contain only O:8, but not by preabsortion with O:8-negative S. cholerasuis MDL 81A7623 (group C1; O:6,7), S. paratyphi type B CDC 157 (group B; O:1,[4],5,12), or Escherichia coli (O:157) (which contains no Salmonella serogroup antigens). The MAb reacted strongly (4+ agglutination) with all 140 wild-type strains of group C2 and C3 Salmonella spp. tested and showed no reaction with any of 1,324 wild-type strains of non-C2 or non-C3 Salmonella spp. tested. The MAb is useful as a replacement for absorbed, polyclonal, single-factor O:8 antiserum to discriminate Salmonella serogroups C2 and C3 from serogroup C1.
Production of monoclonal antibody to a phenolic glycolipid of Mycobacterium tuberculosis and its use in detection of the antigen in clinical isolates
Authors: Cho S N , Shin J S , Daffe M , Chong Y , Kim S K , Kim J D
Published: January 2020

A monoclonal antibody (MAbIII604) specific to phenolic glycolipid Tb (PGL-Tb), a Mycobacterium tuberculosis-specific antigen, was produced and used in the detection of the antigen. MAbIII604 reacted with the PGL-Tb antigen but not with other phenolic glycolipids from Mycobacterium leprae, M. bovis, and M. kansasii, thus indicating the specificity of the monoclonal antibody to PGL-Tb. A dot enzyme-linked immunosorbent assay with MAbIII604 was employed to detect the PGL-Tb antigen in lipids purified from M. tuberculosis clinical isolates. Of 50 isolates, 32 (64.0%) showed clear evidence of the PGL-Tb antigen by the dot enzyme-linked immunosorbent assay, but there were marked variations in the intensities and sizes of spots. This suggests differences in PGL-Tb antigen production among M. tuberculosis strains even when they are grown in the same culture media and conditions. This was most evident from the fact that in only eight (16.0%) of the isolates examined was the PGL-Tb antigen detectable by thin-layer chromatography, which is much less sensitive for the detection of glycolipid antigens. This study shows that monoclonal antibodies specific to PGL-Tb are useful in detecting the antigen in lipid extracts and that there is a marked variation in the PGL-Tb production among M. tuberculosis clinical isolates.
Risk factors for sporadic Campylobacter infections: results of a case-control study in southeastern Norway
Authors: Kapperud G , Skjerve E , Bean N H , Ostroff S M , Lassen J
Published: January 2020

In 1989 and 1990, a case-control study designed to identify risk factors for sporadic infections with thermotolerant Campylobacter bacteria was conducted in three counties in southeastern Norway. The investigation was confined to infections which were acquired in Norway. A total of 52 bacteriologically confirmed cases and 103 controls matched by age, sex, and geographic region were interviewed. The following risk factors were found to be independently associated with illness in conditional logistic regression analysis: consumption of sausages at a barbecue (odds ratio [OR] = 7.64; P = 0.005), daily contact with a dog (OR = 4.26; P = 0.024), and eating of poultry which was brought into the house raw (frozen or refrigerated) (OR = 3.20; P = 0.024). The risk associated with consumption of sausages at a barbecue could not be attributed to cross-contamination from poultry products. By univariate analysis, consumption of poultry which was brought raw and frozen was associated with illness (OR = 2.42; P = 0.042), even though freezing substantially reduces the number of viable campylobacters. When poultry consumption was examined by country of origin, eating of poultry produced in Denmark or Sweden was strongly associated with illness (OR = 13.66; P = 0.014), whereas consumption of poultry produced in Norway was not (OR = 1.33; P = 0.41).
Antibody to a recombinant merozoite protein epitope identifies horses infected with Babesia equi
Authors: Knowles D P , Kappmeyer L S , Stiller D , Hennager S G , Perryman L E
Published: January 2020

Horses infected with Babesia equi were previously identified by the presence of antibodies reactive with a merozoite surface protein epitope (D. P. Knowles, Jr., L. E. Perryman, L. S. Kappmeyer, and S. G. Hennager. J. Clin. Microbiol. 29:2056-2058, 1991). The antibodies were detected in a competitive inhibition enzyme-linked immunosorbent assay (CI ELISA) by using monoclonal antibody 36/133.97, which defines a protein epitope on the merozoite surface. The gene encoding this B. equi merozoite epitope was cloned and expressed in Escherichia coli. The recombinant merozoite protein, designated equi merozoite antigen 1 (EMA-1), was evaluated in the CI ELISA. Recombinant EMA-1 bound antibody from the sera of B. equi-infected horses from 18 countries. The antibody response to EMA-1 was then measured in horses experimentally infected with B. equi via transmission by the tick vector Boophilus microplus or by intravenous inoculation. Anti-EMA-1 antibody was detected 7 weeks post-tick exposure and remained, without reexposure to B. equi, for the 33 weeks of the evaluation period. The data indicate that recombinant EMA-1 can be used in the CI ELISA to detect horses infected with B. equi.
Detection of serum Candida antigens by enzyme-linked immunosorbent assay and a latex agglutination test with anti-Candida albicans and anti-Candida krusei antibodies
Authors: Fujita S , Hashimoto T
Published: January 2020

Serum samples from 197 patients with and without candidiasis were assayed for Candida albicans mannan and Candida krusei mannan by an enzyme-linked immunosorbent assay (ELISA) and a latex agglutination test (LA) and for D-arabinitol by the enzymatic fluorometric method. Of the 43 patients positive for C. albicans mannan (> or = 0.2 ng/ml), 34 were infected with C. albicans and 9 were infected with Candida tropicalis. C. krusei mannan (> or = 0.3 ng/ml) was detected in 10 patients infected with Candida parapsilosis, 2 patients infected with Candida guilliermondii, and 1 patient infected with C. krusei. With both anti-C. albicans antibodies and anti-C. krusei antibodies, the sensitivities of ELISA and LA for detection of invasive candidiasis (58 patients) were 74 and 38%, respectively. No false-positive reactions were observed by the ELISA or the LA. The sensitivity and specificity of the D-arabinitol/creatinine ratio (> or = 1.5 mumol/mg) to invasive candidiasis were 50 and 91%, respectively. The ELISA with antibodies against both C. albicans and C. krusei may be useful in diagnosing invasive candidiasis caused by medically important Candida strains excluding Candida glabrata.
Collaborative comparison of broth macrodilution and microdilution antifungal susceptibility tests
Authors: Espinel-Ingroff A , Kish C W , Kerkering T M , Fromtling R A , Bartizal K , Galgiani J N , Villareal K , Pfaller M A , Gerarden T , Rinaldi M G
Published: January 2020

A collaborative comparison of macro- and microdilution antifungal susceptibility tests was performed in five laboratories. MICs of amphotericin B, fluconazole, flucytosine, and ketoconazole were determined in all five centers against 95 coded isolates of Candida spp., Cryptococcus neoformans, and Torulopsis glabrata. A standard protocol with the following National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Susceptibility Testing recommendations was used: an inoculum standardized by spectrophotometer, buffered (RPMI 1640) medium (pH 7.0), incubation at 35 degrees C, and an additive drug dilution procedure. Two inoculum sizes were tested (1 x 10(4) to 5 x 10(3) to 2.5 x 10(3) CFU/ml) and three scoring criteria were evaluated for MIC endpoint determinations, which were scored as 0 (optically clear), < or = 1 (slightly hazy turbidity), and < or = 2 (prominent decrease in turbidity compared with that of the growth control). Overall intra- and interlaboratory reproducibility was optimal with the low-density inoculum, the second-day readings, and MICs scored as either 1 or 2. The microdilution MICs demonstrated interlaboratory agreement with most of the four drugs higher than or similar to that of the macrodilution MICs. In general, there was good interlaboratory agreement with amphotericin B, fluconazole, and flucytosine; ketoconazole gave more variable results.
Comparison of nine antigen detection kits for diagnosis of urogenital infections due to Chlamydia psittaci in koalas
Authors: Wood M M , Timms P
Published: January 2020

Chlamydia psittaci is the major cause of infectious disease in the koala (Phascolarctos cinereus). It causes four disease syndromes in the koala, namely, conjunctivitis, rhinitis, cystitis, and infertility (females only). Diagnosis of chlamydial infections in koalas relies primarily on isolation of the organism in cell culture. Serology has generally not been useful, and little use has previously been made of the commercially available antigen detection kits. We examined the sensitivity, specificity, and usefulness of three direct fluorescent-antibody kits (Vet-IF [Cell Labs], IMAGEN [Celltech], Chlamydia-Direct IF [Bio Merieux]) and six antigen detection enzyme-linked immunosorbent assay (ELISA) kits (Clearview [Unipath], Surecell [Kodak], Pathfinder [Kallestad], Chlamydia-EIA [Pharmacia], Chlamydiazyme [Abbott], IDEIA [Celltech]) for the detection of urogenital infections in koalas. Laboratory studies showed that the direct fluorescent-antibody kits were the least sensitive in this case and did not detect fewer than 10(4) elementary bodies per ml, while most ELISA kits detected between 130 and 600 elementary bodies per ml. Field study results showed that the Clearview kit was the most sensitive (91%) compared with the IDEIA (88%) and the Surecell (73%) kits. All three kits were more sensitive than cell culture (36%), highlighting viability loss problems that occur during transport. This study showed that the Clearview kit is sensitive, specific, and easy to use for the detection of type II (urogenital) C. psittaci from koalas in the field and warrants further evaluation.
Value of extended agitation and subculture of BACTEC NR 660 aerobic resin blood culture bottles for clinical yeast isolates
Authors: Prevost-Smith E , Hutton N
Published: January 2020

From 10,351 blood cultures, we prospectively studied 1,000 BACTEC NR 660 aerobic resin blood culture bottles (26+ and Peds Plus) for patients suspected of having yeast septicemia to determine whether extended agitation and subculturing would increase the recovery of yeasts. Aerobic bottles were agitated continuously for 144 h. On day 7, 1,000 culture-negative aerobic bottles which had fungal blood culture requests were agitated for an additional 14 days. During this time they were subcultured twice and read twice by BACTEC NR 660. ON days 1 to 7, 81 bottles were cultured positive for yeasts from 36 patients, which included 44 isolates of Candida albicans, averaging 1.4 days to detection, and 12 isolates of Cryptococcus neoformans, averaging 3.8 days to detection. The average detection time for all yeasts was 2.2 days. On days 7 to 21, no yeasts were detected by BACTEC or recovered from the subcultures. We conclude that when continuously agitated for at least 5 full days (120 h), the BACTEC NR 660 aerobic resin bottles reliably isolate yeasts, and it is unnecessary to subculture or hold these bottles beyond 5 days. It also eliminates the need for an additional blood culture system for yeast detection, thus saving (i) confusion in the collection process, (ii) patients' blood and money, and (iii) laboratory technologists' time.
Genotypic identification and characterization of species and strains within the genus Candida by using random amplified polymorphic DNA
Authors: Lehmann P F , Lin D , Lasker B A
Published: January 2020

Random amplified polymorphic DNA (RAPD) was used to better characterize the genotypic relatedness among medically important Candida species. By using short oligomer primers (10-mers) with arbitrarily chosen sequences in the polymerase chain reaction, distinctive and reproducible sets of polymerase chain reaction products were observed for isolates of C. albicans, C. lusitaniae, C. tropicalis, and Torulopsis (Candida) glabrata. The RAPD analysis differentiated a physiologically homogeneous panel of C. parapsilosis into three distinct groups and showed genetic diversity within C. haemulonii. Intraspecies DNA-length polymorphisms were seen for RAPD profiles derived from different isolates of each species. Analysis of RAPDs from a panel of C. albicans, which included 16 laboratory derivatives of two reference strains, showed that the profiles of unrelated strains differed and that the derivatives of each reference strain were identifiable. Minor differences in the RAPD profiles, suggestive of mutations that had occurred during the long-term maintenance of the strains, were detected. Because of its ease and reliability, RAPD analysis should be useful in providing genotypic characters for taxonomic descriptions, for confirming the identities of stock isolates, for typing Candida species in epidemiologic investigations, and for use in the rapid identification of pathogenic fungi.
An enzyme-linked immunosorbent assay for enumeration of Pneumocystis carinii in vitro and in vivo
Authors: Durkin M M , Bartlett M S , Queener S F , Shaw M M , Lee C H , Smith J W
Published: January 2020

An enzyme-linked immunosorbent assay (ELISA) to quantitate Pneumocystis carinii organisms from culture supernatant and rat lung has been developed. A polyclonal antibody specific to P. carinii was produced in Sprague-Dawley rats by allowing P. carinii-infected animals to recover from infection. This antibody reacted strongly to P. carinii proteins of 50 to 55 kDa and weakly to those of 33 and 116 kDa. The ELISA used this convalescent-phase antibody to quantitate the number of P. carinii organisms in lung homogenates of infected rats and supernatants from infected tissue cultures which were used to screen drugs for P. carinii. The results of the ELISA were compared with those of direct microscopic counting of organisms, and the two methods were highly correlated (r > 0.9). Thus, the ELISA can be used as an alternative method for the quantitation of P. carinii organisms, and it is superior to the conventional microscopic method because it is easier to perform and less labor-intensive.
Purification of Pneumocystis carinii trophozoites and identification of their circulating antigens
Authors: Ishimaru T , Shimono N , Sawae Y , Niho Y
Published: January 2020

We have developed a new method to isolate Pneumocystis carinii trophozoites from experimental rat bronchoalveolar lavage specimens by using a Percoll discontinuous gradient and have identified the circulating antigens in experimental rat P. carinii pneumonia. The antigenic components of the trophozoites were compared with those of cysts by immunoblotting. A major immunoreactive band of 90,000 Da and some others of lower molecular mass were found in trophozoites. On the other hand, bands of 110,000, 50,000, and 45,000 Da were observed in cysts. The band of 50,000 Da was not identified when antitrophozoite rabbit serum preabsorbed with P. carinii-infected rat serum was used for immunoblotting. These results suggest that the molecule of 50,000 Da is the major circulating antigen in P. carinii-infected rats.
Novel coagglutination method for serotyping group B streptococci
Authors: Håkansson S , Burman L G , Henrichsen J , Holm S E
Published: January 2020

A group G streptococcal strain was coated with antibody against six different serotypes (Ia, Ib, II, III, IV, and V) of group B streptococci. The coagglutination patterns of 114 strains of group B streptococci were compared with the serotypes determined after immunoprecipitation. The specificity of the method was 100% and the sensitivity 97%. It was used for the typing of 89 invasive and 101 colonizing isolates. The new method is swift, specific, and highly sensitive. It consumes only minute amounts of antibody.
Evaluation of the Wellcolex Colour Salmonella Test for detection of Salmonella spp. in enrichment broths
Authors: Rohner P , Dharan S , Auckenthaler R
Published: January 2020

The Wellcolex Colour Salmonella Test was evaluated for detection of Salmonella spp. in enrichment broths of 1,010 stool samples. In 39 specimens, Salmonella spp. could be isolated from the selenite F broth (SF). Wellcolex agglutination indicative of the presence of Salmonella spp. was noted with the SF in 36 cases, 34 of which were in agreement with the subculture results. Therefore, relative to subculture, the sensitivity and specificity of the Wellcolex-selenite F procedure were 87 and 99%, respectively. Five false-negative results were noted. The gram-negative broth (GN) subculture revealed only 23 Salmonella spp. (59% sensitivity). The Wellcolex agglutination procedure applied to the GN indicated Salmonella spp. for 21 samples; its sensitivity was 70% and its specificity was 99% compared with GN subcultures. The Wellcolex agglutination procedure applied to the SF performed better than the agglutination of GN or direct plating procedures and detected 17 of the 39 Salmonella spp. at least 24 h earlier than did culture.
Detection of human immunodeficiency virus type 1 RNA in plasma samples from high-risk pediatric patients by using the self-sustained sequence replication reaction
Authors: Bush C E , Donovan R M , Peterson W R , Jennings M B , Bolton V , Sherman D G , Vanden Brink K M , Beninsig L A , Godsey J H
Published: January 2020

There is an urgent need for rapid and sensitive methods to assess human immunodeficiency virus (HIV) infection in infants and children. We evaluated an approach by using the self-sustained sequence replication reaction (3SR) to amplify HIV type 1 (HIV-1) RNA directly. The amplified RNA product was then detected by bead-based sandwich oligonucleotide capture hybridization and rare earth metal chelate time-resolved fluorescence. The sensitivity of this technology was determined to be less than 12 HIV-1 RNA copies with an amplification level of 10(10)-fold with purified HIV-1 RNA. Plasma samples from 19 high-risk pediatric patients younger than 5 years of age were examined, and results were compared with viral culture of patient plasma. Results from plasma culture and 3SR amplification agreed for 14 of these patients and disagreed for 5. Of the five samples which did not agree, four were positive by 3SR and negative by culture and one was positive by culture and negative by 3SR but became positive by 3SR at a subsequent testing. We conclude that 3SR amplification coupled with time-resolved fluorescence is a promising technology for investigating the relationship between the presence of HIV-1 RNA in plasma and progression of disease in HIV-infected pediatric patients. This technology should be important in the assessment of HIV-1 infection, in evaluating drug therapies, and in understanding the pathogenesis and transmission of the virus.
Linear antigenic regions of the structural proteins of human T-cell lymphotropic virus type I detected by enzyme-linked immunosorbent assays using synthetic peptides as antigens
Authors: Washitani Y , Kuroda N , Shiraki H , Itoyama Y , Sato H , Ohshima K , Kiyokawa H , Maeda Y
Published: January 2020

We synthesized 46 sequential peptides 21 to 39 amino acids long over the structural protein of human T-cell leukemia virus type I (HTLV-I; the p19 and p24 gag protein and the gp46 and p20E env proteins) and tested their reactivities against antibodies in sera from HTLV-I healthy carriers and patients diagnosed as having human T-cell leukemia-lymphoma (ATLL) and myelopathy (HAM) by using an enzyme-linked immunosorbent assay. Of the 46 synthetic peptides, 18 peptides (2 corresponding to the p19 gag protein, 2 corresponding to the p24 gag protein, 8 corresponding to the gp46 env protein, and 6 corresponding to the p20E env protein) reacted with antibodies in the sera from HTLV-I healthy carriers. In particular, the peptides comprising amino acids 100 to 119 and 119 to 130 of the gag and 175 to 199, 213 to 236, 253 to 282, and 288 to 317 of the env proteins reacted with antibodies in sera from more than 30% of HTLV-I healthy carriers. These peptides also showed high reactivities to the antibodies in the sera from patients with ATLL and HAM. The results indicate that the predominant antigenic regions of the structural protein of HTLV-I were located at the C-terminal end of the p19 gag protein and the C-terminal half of the gp46 env protein, and the corresponding peptides proved to be useful antigens in detecting antibodies in the sera from individuals infected with HTLV-I.