bluuebunny/crossref_metadata_2025 huggingface dataset:
Infection with a plasmid-free variant Chlamydia related to Chlamydia trachomatis identified by using multiple assays for nucleic acid detection
Authors: An Q , Radcliffe G , Vassallo R , Buxton D , O'Brien W J , Pelletier D A , Weisburg W G , Klinger J D , Olive D M
Published: January 2020

Clinical samples in transport media from 40 patients exhibiting pathologies potentially caused by Chlamydia trachomatis infection were analyzed for chlamydial nucleic acid, and the results were compared with those of culture. Chlamydial culture was performed by a shell vial centrifugation method with HeLa 229 host cells. Polymerase chain reaction (PCR) assays were used to detect either regions on a 7.5-kb plasmid characteristic of C. trachomatis (plasmid-PCR) or a segment of the 16S rRNA genes (rRNA-PCR). All PCR results were confirmed by hybridization with probes for the specific amplified products in either a Southern or a dot blot format. An RNase protection (RNP) assay was used to detect genus-specific chlamydial 16S rRNA directly from the clinical samples. The PCR assays detected C. trachomatis but not other bacteria, including Chlamydia spp. C. trachomatis was isolated from six samples which were positive by the rDNA-PCR and plasmid-PCR assays. Five of the culture-positive specimens were positive by the RNP assay. Twenty-two samples were negative by all criteria. Surprisingly, nine samples were positive by rRNA-PCR and RNP assays only. Nucleic acid sequencing of the rRNA-PCR-amplified products indicated a close relationship between the variants and C. trachomatis. The data may indicate an unrecognized process in C. trachomatis infection or that these patients were infected by a variant strain of C. trachomatis which lacks the C. trachomatis-specific plasmid.
Detection and quantification of latently infected B lymphocytes in Epstein-Barr virus-seropositive, healthy individuals by polymerase chain reaction
Authors: Wagner H J , Bein G , Bitsch A , Kirchner H
Published: January 2020

We designed a highly sensitive and specific polymerase chain reaction assay for the detection of Epstein-Barr virus (EBV)-related sequences in B-cell DNA of EBV-seropositive healthy individuals. By using this assay, we were able to amplify at least 10 copies of a plasmid containing the BamHI-W region, which is repeated up to 11 times within the EBV genome, in the presence of 1 microgram of EBV-negative DNA, indicating that one virus genome was detectable from 150,000 cells. In 15 of 16 tested individuals, EBV-related sequences were found frequently when the DNA from 10(6) B lymphocytes was examined and 1 microgram of DNA was used in each polymerase chain reaction. When the results of amplifying the diluted plasmid were used as a semiquantitative standard, the number of EBV genomes detected could be estimated to range between 50 and less than 1 from 10(6) B lymphocytes. The results of our study will provide the basis for further investigations of the characteristics of the latent carrier state in healthy EBV-seropositive individuals, such as the determination of the number of virus copies per cell and expression of antigens.
Polymerase chain reaction-based detection of Trypanosoma cruzi DNA in serum
Authors: Russomando G , Figueredo A , Almirón M , Sakamoto M , Morita K
Published: January 2020

DNAs prepared from chagasic patients' sera were amplified by the polymerase chain reaction using oligonucleotide primers which anneal specifically to a highly repetitive sequence of Trypanosoma cruzi nuclear DNA. Samples from both acutely and chronically infected patients yielded positive results by this method. No significant difference was observed when either whole blood or serum samples of the patients were used. These results indicate that serum instead of whole-blood samples could be used for polymerase chain reaction-based detection of T. cruzi in field studies without the need of applying any special chemical treatment to the specimens. This would represent a considerable advantage due to the easier handling and transportation of serum as compared with whole-blood samples, especially in tropical climates.
A novel polymerase chain reaction method for detection of human immunodeficiency virus in dried blood spots on filter paper
Authors: Yourno J , Conroy J
Published: January 2020

A method for detection of proviral human immunodeficiency virus DNA in dried blood spots on filter paper by direct polymerase chain reaction (PCR) has been developed. To develop the method, a standard system was used which was prepared from cells each containing a single integrated provirus and titrated with normal donor blood. This rapid procedure provides virtually quantitative yields of nuclear DNA and exploits most of the standard methodology described for blood specimens. A nested PCR using SK38-SK39 gag as the internal primer pair was also designed; this PCR detected a single copy of provirus per filter at near theoretical frequency with SK19 probe. The utility of the procedure was demonstrated with clinical specimens. Blood spot filters from human immunodeficiency virus-infected and uninfected individuals were readily and unequivocally discriminated. The method is designed for ultimate use with large (1.5-ml) sample preparation tubes that are compatible as PCR tubes with thermal cyclers. This will permit convenient, direct single-tube PCR of dried blood specimens on filters. It should be adaptable to analysis of dried blood spots for a variety of infectious or genetic diseases.
Comparison of the Vitek Immunodiagnostic Assay System with three immunoassay systems for detection of cytomegalovirus-specific immunoglobulin G
Authors: Hopson D K , Niles A C , Murray P R
Published: January 2020

The Vitek Immunodiagnostic Assay System (VIDAS; bioMerieux Vitek Inc., Hazelwood, Mo.) was evaluated for its ability to detect anticytomegalovirus immunoglobulin G (IgG) and was compared with the following assay systems: Abbott IMx Cytomegalovirus IgG antibody assay (Abbott Laboratories, Abbott Park, Ill.), Whittaker Cytomegelisa II IgG assay (Whittaker Bioproducts, Walkersville, Md.), and Whittaker FIAX Cytomegalovirus IgG assay (Whittaker Bioproducts). Samples were considered positive if at least two of three (IMx, Cytomegelisa II, and FIAX) comparative assays gave positive results; a sample was considered negative if at least two of the three assays were negative. Of the 199 clinical serum samples tested, 194 gave concordant results among the three comparative assays (145 were positive and 49 were negative). The VIDAS results with two samples were persistently equivocal and were therefore excluded from the comparison study. The overall agreement between VIDAS and the established standard was 98.5%. The VIDAS assay yielded one false-positive and two false-negative results. The intra- and interassay precision studies indicate that the VIDAS assay is suitable for clinical use. The VIDAS system is rapid, self-contained, and fully automated and would be useful for screening of cytomegalovirus immune status.
Identification of a genetically distinct subspecies of Mycobacterium kansasii
Authors: Ross B C , Jackson K , Yang M , Sievers A , Dwyer B
Published: January 2020

To assess the usefulness of a specific DNA probe for Mycobacterium kansasii, 105 isolates from Australia, Belgium, Japan, South Africa, and Switzerland were collected and analyzed. Twenty of these isolates were probe negative, of which 18 were from Belgium and Switzerland. Analysis of all isolates by Southern blot hybridization indicated a lack of variability among probe-positive isolates, while probe-negative isolates were clearly distinct and showed greater diversity. Sequence analysis of the 250 nucleotides at the 5' end of the 16S rRNA gene revealed that 19 of the 20 probe-negative isolates had a sequence different from that of M. kansasii. A total of five nucleotide differences were present in a cluster consisting of two nucleotide deletions and three nucleotide substitutions. These results suggest the existence of a genetic subspecies of M. kansasii.
Comparison of two culture approaches, blind passage and dual observation, for detecting Chlamydia trachomatis in various prevalence populations
Authors: Zimmerman S J , Moses E , Sofat N , Bartholomew W R , Amsterdam D
Published: January 2020

Chlamydia trachomatis diagnosis in our laboratory consisted of dual inoculation of shell vials and detection of inclusions by using fluorescein-conjugated monoclonal antiserum; the second culture vial was conventionally used for blind passage when the first vial was negative. We compared the increase in positivity using blind passage with that of a strategy utilizing observation of two stained monolayers (dual observation) without blind passage, in an effort to reduce the reporting time and labor associated with the conventional approach. A total of 4,329 specimens were obtained from an obstetrics and gynecology (OB-GYN) clinic (2,563 specimens) and the sexually transmitted disease clinic (1,766 specimens). These specimens were used to compare the two strategies. Blind passage of 1,269 initially culture-negative specimens from the OB-GYN clinic resulted in an additional 6 positive chlamydial diagnoses. In comparison, a similar number of specimens (1,294) from the OB-GYN clinic collected subsequently to the first group were tested by dual observation. There were five additional positive findings. A similar evaluation of specimens from the sexually transmitted disease clinic was performed. Blind passage of 313 initially culture-negative specimens yielded 3 additional positive diagnoses, whereas dual observation of 1,435 similar specimens resulted in 9 positive diagnoses. On the basis of analysis of 4,332 specimens, sensitivity of dual observation is comparable to that of blind passage; labor, cost, and reporting time of dual observation are reduced in comparison to those of blind passage.
Characterization and distribution of Pasteurella species recovered from infected humans
Authors: Holst E , Rollof J , Larsson L , Nielsen J P
Published: January 2020

During a 3-year period, all Pasteurella strains recovered at the Clinical Microbiological Laboratory, Lund, Sweden, were studied biochemically with respect to their relationship to the recently described taxa of this genus. Of 159 strains recovered from 146 infected humans, 95 were identified as Pasteurella multocida subsp. multocida, 21 as Pasteurella multocida subsp. septica, 28 as Pasteurella canis, 10 as Pasteurella stomatis, and 5 as Pasteurella dagmatis. The homology within and between the Pasteurella species regarding cellular fatty acids and enzymatic activities was also studied. Strains of the different Pasteurella species were indistinguishable from each other regarding fatty acid composition; all strains contained major amounts of C14:0, C16:1, C16:0, and 3-OH-C14:0 acids and minor amounts of C18:2, C18:1, and C18:0 acids. Neither did the enzymatic activities distinguish between strains belonging to different species. In addition, of 56 strains examined, toxin production was demonstrated only in 1 strain each of P. multocida subsp. multocida and P. canis. Except for one severe case of necrotizing cellulitis involving P. dagmatis, P. multocida subsp. multocida or P. multocida subsp. septica was recovered in the more serious cases of infection. Except for P. canis, which in all cases was associated with dog bites, most Pasteurella strains were recovered in cases of infection associated with cat bites or scratches. Pasteurella strains occurred in four infected patients without evident connections with animals.
Experimental Borrelia burgdorferi infection of outbred mice
Authors: Masuzawa T , Beppu Y , Kawabata H , Yanagihara Y , Iwamoto Y , Shimizu T , Johnson R C
Published: January 2020

Infectivity of Borrelia burgdorferi strain 297 in normal outbred ddY mice was examined. Strain 297 was inoculated intraperitoneally in 3-week-old outbred ddY mice. B. burgdorferi was routinely cultured from the heart and urinary bladder 5 to 84 days postinoculation. The combined isolation rate from both heart and urinary bladder was significantly higher than the rate from spleen, kidney, liver, urine, and blood samples. Three- and 10-week-old mice were infected with inocula of 10(4) and 10(5) or more, respectively. Passive transfer of undiluted and 10-fold-diluted anti-297 rabbit serum and active immunization of 50 to 100 micrograms of lyophilized whole cells completely protected mice from infection with B. burgdorferi. These results suggest that the outbred mouse is a convenient model for experimental infection with B. burgdorferi.
Use of the E test to predict high-level resistance to aminoglycosides among enterococci
Authors: Sanchez M L , Barrett M S , Jones R N
Published: January 2020

The E test and the reference agar dilution methods were compared for detecting high-level aminoglycoside resistance (HLAR) among 71 selected clinical isolates, including 62 Enterococcus faecalis and 9 Enterococcus faecium isolates. High-level gentamicin resistance alone was found in 11% (5 E. faecalis and 3 E. faecium strains) and high-level streptomycin resistance was found in 42% (28 E. faecalis, 2 E. faecium strains) of the strains tested, and 31% of the strains demonstrated high-level resistance to both antimicrobial agents (21 E. faecalis and 1 E. faecium strains). The E test detected all HLAR populations, but the streptomycin strip may require recalibration to achieve absolute MIC comparisons with the reference value (twofold less) or the use of an alternative interpretive resistance breakpoint, e.g., > 1,000 micrograms/ml. By the E test, MIC results indicate that ampicillin, imipenem, penicillin, piperacillin, and vancomycin remain active against the HLAR E. faecalis isolates; however, these tested drugs were less effective on the HLAR E. faecium isolates (< 50%).
Western immunoblot analysis and serologic characterization of Blastomyces dermatitidis yeast form extracellular antigens
Authors: Hurst S F , Kaufman L
Published: January 2020

A major 98-kDa extracellular protein antigen of Blastomyces dermatitidis was shown in Western blot (immunoblot) analysis to be highly reactive with serum antibodies from patients with blastomycosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of yeast form B. dermatitidis culture filtrates and cell extracts demonstrated over 50 proteins, only 24 of which were immunoreactive. Of these, a 98-kDa protein was found to be the most specific and was isolated. This protein was found in both broth culture filtrates and extracts of yeast forms. Western blot tests with this antigen detected serum antibodies in 91% of 32 patients with clinically proven blastomycosis, in contrast to an enzyme-linked immunosorbent assay (ELISA) with DEAE-purified antigen, which detected 88% of the cases. The Western blot test also exhibited lower reactivity with a panel of sera from patients with heterologous fungal infections that were cross-reactive in the ELISA with DEAE-purified B. dermatitidis antigen. The 98-kDa protein electroeluted from polyacrylamide gels was identical to the diagnostic A antigen used in the blastomycosis immunodiffusion test. Comparison of the 98-kDa antigen with a previously described 120-kDa yeast form cell wall protein antigen of B. dermatitidis showed that these two antigens are almost identical.
Mycobacteria in Crohn's disease: DNA probes identify the wood pigeon strain of Mycobacterium avium and Mycobacterium paratuberculosis from human tissue
Authors: McFadden J , Collins J , Beaman B , Arthur M , Gitnick G
Published: January 2020

Mycobacterium paratuberculosis is known to cause Johne's disease, a granulomatous ileitis in ruminants, and may be involved in some cases of Crohn's disease. Like M. paratuberculosis, the wood pigeon strain of Mycobacterium avium may also show mycobactin dependence on primary isolation that is attenuated on further subculturing. A wood pigeon strain, M. avium restriction fragment length polymorphism (RFLP) type A/I, is also capable of causing granulomatous ileitis in experimental animal models but is not known to cause disease in humans. M. avium RFLP type A is associated with disease in immunocompromised hosts. Three DNA probes, pMB22 and the two subclones pMB22/S4 and pMB/S12, were found to be capable of distinguishing among M. paratuberculosis, M. avium type A, and M. avium type A/I (wood pigeon strain) on the basis of RFLPs. These DNA probes were used to identify two mycobacterial isolates (M. paratuberculosis and M. avium type A/I, wood pigeon strain) derived from the intestinal tissues of two patients with Crohn's disease. In addition, the wood pigeon strain of M. avium was identified from a patient with ulcerative colitis, and M. avium RFLP type A was identified from a patient with colonic carcinoma. This is the first time that M. avium A/I (wood pigeon strain) is known to have been isolated from human tissue. There are too few isolates to speculate about the etiological significance of mycobacteria and inflammatory bowel disease, but it is reasonable to conjecture that M. paratuberculosis may be responsible for some cases of Crohn's disease and that the wood pigeon strain of M. avium may also be an inflammatory bowel disease pathogen in humans.
Functional and serological analysis of type II immunoglobulin G-binding proteins expressed by pathogenic group A streptococci
Authors: Raeder R , Otten R A , Chamberlin L , Boyle M D
Published: January 2020

Bacterial immunoglobulin-binding proteins expressed on the surface of group A streptococci represent a heterogeneous family of functionally related proteins. In this report, we describe efficient methods for extracting immunoglobulin-binding proteins and classifying them functionally and antigenically. A common characteristic of immunoglobulin-binding proteins expressed by group A streptococci appears to be the absence of internal methionine residues in the binding protein. This has enabled development of a rapid, efficient, cyanogen bromide-based extraction procedure for solubilizing these molecules from intact bacteria. Studies carried out with a series of monospecific polyclonal antibodies prepared in chickens have identified two major antigenic classes of immunoglobulin-binding proteins. The methods described in this report facilitate a rapid functional and serological screening of immunoglobulin-binding proteins that should now enable detailed epidemiological studies of the importance of these molecules in group A streptococcal infections and their relationship to other surface proteins, in particular, the antiphagocytic M protein.
Preliminary evaluation of the ligase chain reaction for specific detection of Neisseria gonorrhoeae
Authors: Birkenmeyer L , Armstrong A S
Published: January 2020

Rapid identification of Neisseria gonorrhoeae in clinical specimens is essential for effective control. Traditional culture requires a minimum of 24 h, and for some specimens harboring gonococci, the gonococci fail to grow or are misidentified. The recently described ligase chain reaction (LCR) is a highly specific and sensitive DNA amplification technique which was evaluated as an alternative to routine culture. Three LCR probe sets were used. Two of the probe sets were directed against the multi-copy Opa genes (Omp-II), while the third set was targeted against the multicopy Pilin genes. Each LCR probe set was evaluated with 260 microorganisms including 136 global isolates of N. gonorrhoeae, 41 isolates of N. meningitidis, and 10 isolates of N. lactamica; 26 nonpathogenic Neisseria strains; and 47 isolates of non-Neisseria species that may reside in clinical specimens. Amplification products were detected by using the IMx LCR format (Abbott Laboratories, Abbott Park, Ill.). Strains of N. gonorrhoeae were assayed at 270 cells per LCR (approximately 6.7 x 10(4) CFU/ml) with the Opa and Pilin probes, producing signals at least 21 and 15 times above background, respectively. In contrast, only background values were observed when testing the probe sets with 124 nongonococcal strains at 1.3 x 10(6) cells per LCR (approximately 3.2 x 10(8) CFU/ml). One hundred urogenital specimens were assayed by LCR, and compared with culture, the three probes were 100% sensitive (8 of 8) and 97.8% specific (90 of 92), resulting in an agreement of 98% (98 of 100). On the basis of the results of these preliminary studies, LCR has the potential to be an accurate and rapid DNA probe assay for the detection of N. gonorrhoeae in clinical specimens.
Effects of fixation on polymerase chain reaction detection of Mycobacterium leprae
Authors: Fiallo P , Williams D L , Chan G P , Gillis T P
Published: January 2020

The effects of standard fixatives (10% neutral buffered formalin, ethanol and mercury based) on the detection of Mycobacterium leprae DNA by the polymerase chain reaction (PCR) were studied. Mercury-based fixatives (Zenker's and Carnoy-Lebrun's fluids) strongly inhibited PCR amplification of M. leprae DNA. Ten percent neutral buffered formalin was inhibitory, but significant inhibition was observed only when fixation times exceeded 24 h. Ethanol-based fixatives provided the best medium for holding specimens for subsequent PCR with both free bacilli and skin biopsy specimens containing M. leprae. The M. leprae-specific, 360-bp region of the 18-kDa protein gene could be amplified from paraffin-embedded sections of formalin-fixed skin biopsy specimens from patients with either multibacillary or paucibacillary infections when proper fixation conditions were used. Results of the study demonstrate that tissues properly fixed with two standard fixatives (10% neutral buffered formalin and 50 or 70% ethanol) can be analyzed by PCR for the presence of M. leprae with no loss in specificity and only minimal diminution in sensitivity compared with the specificities and sensitivities obtained by use of freshly prepared, unfixed specimens.
Comparative evaluation of a chemiluminescent DNA probe and an exoantigen test for rapid identification of Histoplasma capsulatum
Authors: Padhye A A , Smith G , McLaughlin D , Standard P G , Kaufman L
Published: January 2020

A chemiluminescent DNA probe (Accuprobe) assay developed by Gen Probe, Inc., for the rapid identification of Histoplasma capsulatum was evaluated and compared with the exoantigen test by using 162 coded cultures including Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Histoplasma capsulatum var. farciminosum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and morphologically related saprobic fungi. Each test uses a chemiluminescent, acridinium ester-labeled, single-stranded DNA probe that is complementary to the rRNA of the target organism. Lysates of the test cultures were prepared by sonication with glass beads and heat treated. After the rRNA was released from the target organism, the labeled DNA probe combined with the target H. capsulatum rRNA to form a stable DNA-RNA hybrid. A hybridization protection assay was used, and the chemiluminescence of hybrids was measured initially with a Leader 1 luminometer as relative light units and later during the investigation with a probe assay luminometer as probe light units. Of the 162 coded mycelial cultures tested by the Accuprobe assay, 105 were identified as H. capsulatum. The test could be performed with an inoculum of a few square millimeters (1 to 2 mm2) of growth. In the primary evaluation, the Accuprobe identified 103 of the 105 cultures as H. capsulatum within 2 h. The remaining two cultures, contaminated with bacteria, had to be purified before the Accuprobe assay identified them correctly as H. capsulatum. Since each coded culture was concurrently tested for H. capsulatum, B. dermatitidis, and C. immitis exoantigens, the identification of all three dimorphic pathogens was provided simultaneously. Of the 162 coded cultures tested, 105 were identified by the exoantigen test as H. capsulatum, 12 were identified as B. dermatitidis, 13 were identified as C. immitis, and 32 were negative for H. capsulatum, B. dermatitidis, and C. immitis. The bacterial contamination in two isolates did not interfere with the exoantigen testing. The exoantigen test required 7- to 10-day-old colonies and required 48 to 72 h of incubation before definitive identification was obtained.
Evaluation of the RapID ANA II and API ZYM systems for identification of Actinomyces species from clinical specimens
Authors: Brander M A , Jousimies-Somer H R
Published: January 2020

Classification and identification of fermentative actinomycetes are labor-intensive and problematic. In this study, we evaluated the applicability and reliability of the RapID ANA II system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) and the discriminatory value of the API ZYM system (Societes Analytab Products Inc., La Balme Les Grottes, France) in the identification of Actinomyces-like bacteria by using conventional methods as a reference. Eighty-five strains, including 71 isolates from mixed anaerobic infections and 14 reference strains, were tested. The RapID ANA II system correctly identified all Actinomyces odontolyticus strains and 65% of Actinomyces israelii strains. All Arcanobacterium haemolyticum strains were misidentified as Actinomyces pyogenes. The most common isolates in the study were Actinomyces meyeri-like organisms, 84% of which, however, were aerotolerant. The identification of these aerotolerant strains thus remains unresolved and warrants further studies. New characteristics and changes to the conventional API ZYM enzyme profiles are suggested. The API ZYM enzyme profiles of A. odontolyticus and A. israelii were very similar, the only discriminating enzyme being alpha-fucosidase. In differentiation between A. pyogenes and Arcanobacterium haemolyticum, the production of beta-glucuronidase by the former and the production of acid phosphatase by the latter are suggested as new helpful characteristics for use in clinical laboratories. In summary, the RapID ANA II and API ZYM systems can be used as rapid preliminary methods in the identification of Actinomyces species but accurate identification requires supplementary conventional tests and gas-liquid chromatography.
Detection of Norwalk virus in stool specimens by reverse transcriptase-polymerase chain reaction and nonradioactive oligoprobes
Authors: De Leon R , Matsui S M , Baric R S , Herrmann J E , Blacklow N R , Greenberg H B , Sobsey M D
Published: January 2020

A reverse transcriptase (RT)-polymerase chain reaction (PCR)-oligoprobe (OP), or RT-PCR-OP, method was developed for the detection of the Norwalk virus, which causes acute, epidemic gastroenteritis, in stool specimens. The Norwalk virus genome regions encoding the following two proteins were amplified by RT-PCR: the RNA polymerase (260-bp product) and a putative immunogenic protein (224-bp product). The resulting DNA fragments (amplicons) were hybridized to a digoxigenin-labeled internal OP specific to each amplicon. The detection limit of Norwalk virus, as determined by the endpoint of RT-PCR amplification for serially diluted, positive stool specimens, was similar to the actual virion titer as estimated by electron microscopy and at least 100-fold greater than the titer determined by radioimmunoassay (RIA). The RT-PCR-OP assay was specific for Norwalk virus and negative for other enteric viruses, including human and animal caliciviruses, hepatitis E virus, Snow Mountain agent, astroviruses, 16 human enteroviruses, and 5 human rotaviruses. Components of fecal specimens that interfere with RT-PCR were removed successfully by Sephadex G-200 gel chromatography. Of 20 stool specimens from human volunteers that were positive for Norwalk virus by RIA, a specific RT-PCR-OP result was obtained in 95% (19 of 20) of the samples by using the immunogenic protein primers and 75% (15 of 20) by using the polymerase primers. Twenty-six stool specimens from asymptomatic children and adults were negative by the Norwalk virus RT-PCR-OP. RT-PCR-OP detected Norwalk virus in the 4 of 21 coded fecal specimens that were also positive by enzyme immunoassay. Two samples that were positive by RIA or enzyme immunoassay were negative by RT-PCR, perhaps because viral RNA was not present or RT-PCR inhibitors were not adequately removed.
Sensitive and specific serodiagnosis of invasive amebiasis by using a recombinant surface protein of pathogenic Entamoeba histolytica
Authors: Lotter H , Mannweiler E , Schreiber M , Tannich E
Published: January 2020

A recombinantly expressed protein, recEh-P1, representing part of an immunodominant surface antigen of pathogenic Entamoeba histolytica, was used for serodiagnosis of invasive amebiasis. Expression was performed under the control of a T7-RNA promoter by using a modified procaryotic expression vector, designated pHisT7. This vector allowed high-yield expression of recEh-P1 fused to a stretch of sequence containing eight histidine residues, which facilitated purification by metal chelate affinity chromatography on Ni2+ columns under highly denatured conditions. Purified recEh-P1 was found to be water soluble after prolonged dialysis and was used as the antigen for the detection of antiamebic serum antibodies by immunoblotting and enzyme-linked immunosorbent assay. In both tests all sera of patients with invasive amebiasis reacted to recEh-P1 whereas none of those collected from healthy controls, including individuals with noninvasive amebiasis, or from patients suffering from bacterial or protozoan infections unrelated to E. histolytica did so.
Antibody response to Brucella outer membrane proteins in bovine brucellosis: immunoblot analysis and competitive enzyme-linked immunosorbent assay using monoclonal antibodies
Authors: Cloeckaert A , Kerkhofs P , Limet J N
Published: January 2020

Sera from Brucella-infected bovines were analyzed by immunoblotting by using sonicated cell extracts of B. melitensis or B. abortus and a competitive enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies against outer membrane proteins (OMPs) with molecular masses of 10, 16.5, 19, 25 to 27, 36 to 38, and 89 kDa. Antibody responses against OMPs were compared with antibody responses against smooth lipopolysaccharide. Immunoblot analysis indicated that the antibody response in infected animals was largely different from one animal to another. The antigens of concern were OMPs with molecular masses of 10, 16.5, 19, 25 to 27, 36 to 38, and 89 kDa and other proteins with molecular masses of between 40 and 80 kDa. According to the specificity of the competitive ELISA, OMPs useful for the detection of infected animals are the OMPs of 10, 16.5, 19, 25 to 27, and 36 to 38 kDa. A competitive ELISA with the anti-89 kDa monoclonal antibody was not specific. Results of the competitive ELISA confirmed the individual variability of the humoral immune response against OMPs. It therefore seems that a combination of several protein antigens is necessary for the development of an immunoassay with a sensitivity comparable to that of the smooth lipopolysaccharide ELISA.
Evaluation of methods for differentiation of coagulase-positive staphylococci
Authors: Roberson J R , Fox L K , Hancock D D , Besser T E
Published: January 2020

The purpose of this study was to determine the minimum number of tests that could be used to differentiate between the coagulase-positive strains of staphylococcus: Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus intermedius. Eighty coagulase-positive strains of each of the three species were examined. The five tests conducted were growth on modified Baird-Parker agar, growth on P agar supplemented with acriflavin, production of acetoin, anaerobic fermentation of mannitol, and presence of beta-galactosidase. Positive test percentages for S. aureus were 100% for growth on modified Baird-Parker agar, 100% for growth on P agar supplemented with acriflavin, 94% for production of acetoin, 99% for anaerobic fermentation of mannitol, and 0% for presence of beta-galactosidase. Positive test percentages for S. intermedius were 0% for growth on modified Baird-Parker agar, 0% for growth on P agar supplemented with acriflavin, 1% for production of acetoin, 0% for anaerobic fermentation of mannitol, and 100% for presence of beta-galactosidase. S. hyicus isolates were negative in all five tests. Results from the 240 coagulase-positive staphylococcus strains tested would suggest correct identification of coagulase-positive staphylococci with P agar supplemented with acriflavin and the beta-galactosidase test. These two tests are simple to conduct and result in quick and easy differentiation of the three coagulase-positive staphylococcal species.
Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus
Authors: Cuypers H T , Bresters D , Winkel I N , Reesink H W , Weiner A J , Houghton M , van der Poel C L , Lelie P N
Published: January 2020

We have noticed that suboptimal specimen processing and storage conditions may cause false-negative results in the detection of hepatitis C virus (HCV) RNA in plasma or serum. To establish the influence of specimen handling in a serological laboratory on the rate of detection of HCV RNA by the cDNA polymerase chain reaction (cDNA-PCR), we tested routine serum samples and fresh-frozen plasma samples from the same bleeding from confirmed anti-HCV-positive blood donors. When primers from the NS3/NS4 region were used, HCV RNA was detected in fresh-frozen plasma from 67% of the donors, whereas positive results were obtained with only 50% of the serum samples that had been subjected to routine serological procedures. Analysis of the same samples with primers from the highly conserved 5'-terminal region (5'-TR) revealed an HCV RNA detection rate of 92% for both the routine and the fresh-frozen samples. However, the yield of the amplification product in routine samples was strongly reduced compared with that in fresh-frozen plasma. Comparison of both primer sets for cDNA-PCR showed that the 5'-TR primer set was 10- to 100-fold more effective in detecting HCV RNA. We also analyzed the effect of storage of whole EDTA-blood and serum at room temperature and at 4 degrees C on the yield of the amplification product. A rapid decline in detectable HCV RNA of 3 to 4 log units was observed within 14 days when whole blood and serum were stored at room temperature. By contrast, no perceptible reduction in the cDNA-PCR signal was found in freshly prepared serum stored at 4 degrees C.
Rotavirus diarrhea in Bangladeshi children: correlation of disease severity with serotypes
Authors: Bern C , Unicomb L , Gentsch J R , Banul N , Yunus M , Sack R B , Glass R I
Published: January 2020

To improve the understanding of the relative importance of serotypes of rotavirus in dehydrating diarrhea, we examined the correlation of clinical characteristics and disease severity with serotype in 2,441 diarrheal episodes among children younger than 2 years of age in rural Bangladesh. Of 764 rotavirus-associated episodes, a single G type (serotype 1, 2, 3, or 4) was determined by oligonucleotide probe in 485 (63%), while 233 episodes were nontypeable. Episodes with G types 2 and 3 were associated with more-severe dehydration than episodes associated with G type 1 or 4 or with nontypeable rotavirus. Episodes did not differ by G type in prevalence of vomiting, copious diarrhea, fever, abdominal pain, or length of treatment center stay. Rotavirus reinfections were detected in seven children, with homologous reinfection (G type 2) in one. Twelve children with diarrhea who died had rotavirus detected in stool specimens within 30 days of death. Children who died were more likely to be malnourished than were surviving children with rotavirus diarrhea. Of 40 specimens tested by polymerase chain reaction, 29 (72.5%) were P type 1, 9 (22.5%) were P type 2, 1 (2.5%) was P type 3, and 1 (2.5%) was nontypeable. One severely symptomatic diarrheal episode was associated with P type 3 rotavirus, a serotype usually found in asymptomatic nursery infections. Although G types 2 and 3 were associated with more-severe dehydration than other serotypes, the differences do not appear to be of major clinical importance. Effective vaccines should protect against all four major G types.
Evaluation of a new monoclonal antibody combination reagent for direct fluorescence detection of Giardia cysts and Cryptosporidium oocysts in human fecal specimens
Authors: Garcia L S , Shum A C , Bruckner D A
Published: January 2020

Giardia lamblia and Cryptosporidium parvum can cause severe symptoms in humans, particularly in the immunologically compromised. Monoclonal antibody reagents offer increased sensitivity and an excellent alternative to conventional staining methods. These reagents are helpful when screening large numbers of patients or those with minimal symptoms. Problems of false-positive and false-negative results with routine staining methods for stool parasites can be eliminated with monoclonal antibody reagents. Known positive formalinized specimens [Giardia sp. (n = 60), Cryptosporidium sp. (n = 55), and mixed Giardia-Cryptosporidium spp. (n = 10)] and negative formalinized specimens (n = 105), of which 46 contained other yeast or human cells or protozoa), were tested by the MERIFLUOR Cryptosporidium-Giardia direct immunofluorescence detection procedure. The MERIFLUOR reagent exhibited +/- to 4+ (majority, 2+ to 3+) on all Giardia cysts and 2+ to 4+ (majority, 3+ to 4+) on all Cryptosporidium oocysts. The cysts were generally oval (11 to 15 microns), while the oocysts were round (4 to 6 microns); both showed apple-green fluorescence against a background free of nonspecific fluorescence. All specimens positive for Giardia sp. and/or Cryptosporidium sp. showed fluorescence, and all specimens negative for the two organisms showed no fluorescence. There were eight specimens previously negative by the ova and parasite examination which were positive by the direct fluorescence method; four contained Giardia sp., and four contained Cryptosporidium sp. These positive results were confirmed after the examination of additional trichrome and modified acid-fast smears. The MERIFLUOR reagent was very easy to use, and even with a lower fluorescence intensity for Giardia sp. cysts, no false-negative or false-positive results among the specimens tested for either organism were found.
Peripheral intramonocytic leishmanias in an AIDS patient
Authors: Fillola G , Corberand J X , Laharrague P F , Levenes H , Massip P , Recco P
Published: January 2020

Intramonocytic leishmanias were unexpectedly observed in blood smears of a Spanish AIDS patient. In immunodepressed patients from exposed countries, careful microscopic examination of blood smears should be requested by the clinician in cases of prolonged fever, and biologists must be informed that leishmanias may be fortuitously observed in the peripheral blood.
Aspergillus quadrilineatus, a new causative agent of fungal sinusitis
Authors: Polacheck I , Nagler A , Okon E , Drakos P , Plaskowitz J , Kwon-Chung K J
Published: January 2020

Aspergillus quadrilineatus was found to be the etiologic agent of pansinusitis in a patient suffering from acute nonlymphoblastic leukemia and who had undergone allogeneic bone marrow transplantation. A. quadrilineatus was cultured from biopsy specimens of the maxillary sinus, and tissue sections with fungal stains showed a necrotic area containing dichotomously branching septate hyphae, which is morphologically consistent with Aspergillus species. The patient was successfully treated with a combination of surgical debridement, granulocyte transfusions, and intravenous administration of amphotericin B-cholesterol sulfate colloidal dispersion. This is the first report of an infection caused by A. quadrilineatus.
Clinical significance of Staphylococcus warneri bacteremia
Authors: Kamath U , Singer C , Isenberg H D
Published: January 2020

Twenty-seven episodes of bacteremia caused by Staphylococcus warneri were identified at Long Island Jewish Medical Center in New York between 1984 and 1989. Fourteen of these were thought to represent true bacteremias and 13 to represent contaminants. Of the 14 true bacteremias, 5 were in pediatric and 9 were in adult patients. Eight of 14 patients (57%) had catheter-related bacteremia and 5 of 14 had bacteremia of unknown source. There was one case of fulminant native valve S. warneri endocarditis. All cases of catheter-related bacteremia, except one, were nosocomially acquired, and 75% of these patients had an underlying immunosuppressive condition. Only 40% of patients with bacteremias of unknown source were immunocompromised, and S. warneri appeared to be noninvasive in this group. Interestingly, all five of the pediatric isolates were oxacillin susceptible, although four of five were resistant to penicillin, despite the fact these patients were hospitalized an average of 29 days. In contrast, seven of nine adult isolates were resistant to both oxacillin and penicillin. The only case of native valve S. warneri endocarditis occurred in a patient who had no known underlying valvular heart disease, but had an underlying immunosuppressive condition. Identification to species level of coagulase-negative staphylococci may lead to appreciation of the importance of bacteria such as S. warneri as human pathogens.
Characterization of a novel Rochalimaea species, R. henselae sp. nov., isolated from blood of a febrile, human immunodeficiency virus-positive patient
Authors: Regnery R L , Anderson B E , Clarridge J E , Rodriguez-Barradas M C , Jones D C , Carr J H
Published: January 2020

Isolation of a Rochalimaea-like organism from a febrile patient infected with human immunodeficiency virus was confirmed. Analysis of 16S rRNA gene sequences, together with polymerase chain reaction and restriction endonuclease length polymorphism analysis of a portion of the citrate synthase gene, demonstrated that the agent is closely related to members of the genus Rochalimaea and that the isolate is genotypically identical to the presumptive etiologic agent of bacillary angiomatosis. However, the same genotypic analyses readily differentiated the new isolate from isolates of other recognized Rochalimaea species as well as other genera of bacteria previously suggested as putative etiologic agents of bacillary angiomatosis and related syndromes. We propose that the novel species be referred to as Rochalimaea henselae sp. now.
Sensitivity and specificity of a recombinant transmembrane glycoprotein (rgp21)-spiked western immunoblot for serological confirmation of human T-cell lymphotropic virus type I and type II infections
Authors: Lal R B , Brodine S , Kazura J , Mbidde-Katonga E , Yanagihara R , Roberts C
Published: January 2020

Serum specimens (n = 2,712) obtained from individuals residing in diverse geographic regions and categorized as seropositive (n = 122), seroindeterminate (n = 523), or seronegative (n = 2,067) for human T-cell lymphotropic virus (HTLV) infection in accordance with U.S. Public Health Service guidelines were retested by recombinant transmembrane protein (rgp21)-spiked Western immunoblotting. Of the 122 HTLV-positive specimens, those from 85 of 85 (100%) U.S. blood donors, 2 of 2 (100%) Brazilians, 1 of 2 (50%) Indonesians, 14 of 14 (100%) Solomon Islanders, and 18 of 19 (95%) Papua New Guineans reacted with rgp21, yielding an overall sensitivity of 98% (120 of 122). Specimens from individuals whose infections were confirmed to be HTLV type I or HTLV type II by the polymerase chain reaction assay reacted equally well with rgp21. Of the 523 HTLV-indeterminate specimens, those from 21 of 379 (5.5%) U.S. blood donors, 3 of 6 (50%) Brazilians, 10 of 23 (44%) Ugandans, 8 of 49 (16%) Indonesians, 4 of 36 (11%) Solomon Islanders, and 5 of 30 (17%) Papua New Guineans reacted with rgp21. None of these 51 specimens reacted with native gp46 and/or gp61/68 in a radioimmunoprecipitation assay, suggesting a false-positive reaction (9.75%). Of the 2,067 HTLV-negative specimens, 12 reacted with rgp21, yielding a false-positivity rate of 0.6%. These data indicate that while detection of rgp21 is highly sensitive, it can yield false-positive results. Thus, specimens exhibiting reactivity with rgp21 in the absence of reactivity with native gp46 and/or gp61/68 by Western blot should be tested further by a radioimmunoprecipitation assay to verify HTLV type I or type II infection.
Vibriophage VcA-3 as an epidemic strain marker for the U.S. Gulf Coast Vibrio cholerae O1 clone
Authors: Almeida R J , Cameron D N , Cook W L , Wachsmuth I K
Published: January 2020

Toxigenic and nontoxigenic Vibrio cholerae O1, El Tor biotype strains, which are endemic to the U.S. Gulf Coast, can be lysogenic for bacteriophage VcA-3. To evaluate the presence of VcA-3 as an indicator of toxigenicity and as an epidemic strain marker, phage production and the presence of phage and cholera toxin genes were assayed in 98 strains of V. cholerae O1 (35 U.S. and 63 foreign strains). By using a HindIII chromosomal digest for Southern blot analysis, 39 of the study strains hybridized with the VcA-3 probe in 10 banding patterns. The 15 toxigenic and 6 of the 20 nontoxigenic U.S. isolates gave four VcA-3-related patterns. Among the foreign isolates, 12 of 12 toxigenic classical biotype strains, 1 of 43 toxigenic El Tor biotype strains, and 3 of 8 nontoxigenic atypical strains gave six patterns that were clearly distinct from that of VcA-3. Compared with Southern blot analysis, the phage production assay had a sensitivity of 1.0 and a specificity of 0.48, while the colony hybridization assay had a sensitivity of 1.0 and a specificity of 0.77 for identification of VcA-3. Neither assay reliably identified the toxigenic Gulf Coast clone. The presence of VcA-3, as defined by Southern blot analysis, always separated toxigenic U.S. from foreign isolates and often from nontoxigenic U.S. isolates of V. cholerae O1.
Quantitation of pneumococcal C polysaccharide in sputum samples from patients with presumptive pneumococcal pneumonia by enzyme immunoassay
Authors: Parkinson A J , Rabiego M E , Sepulveda C , Davidson M , Johnson C
Published: January 2020

Although the Gram stain and culture of expectorated sputum are considered standard methods for the diagnosis of presumptive pneumococcal pneumonia, these methods remain relatively insensitive and nonspecific. We developed an enzyme immunoassay (EIA) for the quantitation of pneumococcal C polysaccharide (PnC) in the sputum of patients with presumptive pneumococcal pneumonia. Of 34 patient sputum samples collected within 24 h of the first radiographic report of pneumonia, 12 grew Streptococcus pneumoniae on culture. By using a cutoff point of 0.5 micrograms of PnC per ml of sputum, all 12 specimens were positive (sensitivity, 100%) by EIA. PnC levels ranged from 1.43 to 57.53 micrograms/ml. Blood samples from 18 of the 34 patients were cultured. S. pneumoniae grew in the culture of a blood sample from one patient, whose sputum also had the highest PnC level. Of 22 sputum samples from patients with pneumonia that did not grow S. pneumonia, two were positive by EIA (specificity, 90.1%). Sputa from both patients had low levels of PnC (2.7 and 4.5 micrograms/ml), and both patients had received antibiotics before sputum collection. The positive predictive value of the quantitative EIA was 85.7%. Quantitation of PnC has the potential for improving the accuracy of sputum examination for S. pneumoniae, monitoring disease severity and the effectiveness of antibiotic therapy, and differentiating between those patients with invasive pneumococcal disease and those who are carriers of S. pneumoniae.
Combination of immunofluorescence and immunoperoxidase techniques for serotyping mixtures of Mycoplasma species
Authors: Bencina D , Bradbury J M
Published: January 2020

We describe a method for the simultaneous identification of up to three Mycoplasma species by the use of contrast-labeled fluorescent antibodies against two species and peroxidase-labeled antibody against a third species. The procedure enabled the rapid identification of colonies of three artificially mixed avian Mycoplasma species on agar blocks and also mixtures of species in cultures from naturally infected chickens. Furthermore, it was possible to quantitate the components of a mixture of Mycoplasma gallisepticum, M. synoviae, and M. gallinarum. This new procedure offers an improvement over existing methods in terms of both speed and analytical capability.