bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid microbial detection and enumeration using gel microdroplets and colorimetric or fluorescence indicator systems
Authors: Williams G B , Weaver J C , Demain A L
Published: January 2020

A new micromethod employing gel microdroplets (GMDs) and optical measurements can be used for rapid detection and enumeration of viable microorganisms (J. C. Weaver, G. B. Williams, A. M. Klibanov, and A. L. Demain, Bio/Technology 6:1084-1089, 1988) and has several potential applications in clinical microbiology. This method involves entrapping microorganisms in GMDs (10 to 100 microns in diameter) which are surrounded by a hydrophobic (low dielectric) fluid, subsequently distinguishing occupied and unoccupied GMDs with colorimetric or fluorescence indicators, counting both occupied and unoccupied GMDs, and applying Poisson statistical analysis. Acid-producing microorganisms were used to compare colorimetric and fluorescence pH indicator systems. Fluorescence systems were generally superior, particularly for detection before microbial growth occurred. Although colorimetric detection was reasonably fast for fast-growing microorganisms, significantly longer times were needed for slow-growing microorganisms. We investigated the dependence of the detection time on microbial division time, GMD size, and buffering capacity of the medium within GMDs. It was found possible to use a single preparation of GMDs, containing a range of GMD sizes, to simultaneously provide a viable enumeration of growing and nongrowing (e.g., stressed) cells. This was possible because small GMDs responded rapidly to both growing and nongrowing cells, while large GMDs, although slower, responded much more rapidly to growing cells than to nongrowing cells. Separate analysis of small and large GMDs in the same preparation yielded two enumerations, one of nongrowing cells and the other of growing cells. GMDs can also be used with conventional light microscopy to detect and enumerate fast-growing acid-producing bacteria much more quickly than conventional plating methods.
Identification of Campylobacter cinaedi isolated from blood and feces of children and adult females
Authors: Vandamme P , Falsen E , Pot B , Kersters K , De Ley J
Published: January 2020

Five Campylobacter-like organisms isolated from blood and feces were identified by numerical analysis of gel electrophoretic protein profiles and immunotyping as Campylobacter cinaedi. Two of these strains were isolated from adult females; the remaining three strains were isolated from children, two of whom were girls. C. cinaedi has hitherto been isolated only from rectal swabs and blood of homosexual and bisexual males with gastrointestinal symptoms. The results presented extend our knowledge of the features and the habitat of C. cinaedi.
Evaluation of five methods for respiratory syncytial virus detection
Authors: Halstead D C , Todd S , Fritch G
Published: January 2020

A total of 117 nasal aspirates were cultured for respiratory syncytial virus (RSV) and tested for RSV antigen by a direct fluorescent-antibody (DFA) test (Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.), the Directigen enzyme immunoassay (EIA; Becton Dickinson Microbiology Systems, Cockeysville, Md.), the TestPack EIA (Abbott Laboratories, North Chicago, Ill.), and RSV EIA (Abbott). Agreement of two of five methods or a positive RSV culture were required to validate a result. A total of 57 of 117 (48.7%) specimens were culture positive in HEp-2 cells, A549 cells, or both. A total of 5 of 117 (4.3%) additional specimens met the criteria of a positive specimen; i.e., 62 of 117 (53.0%) specimens were positive. Results obtained from 77 of 117 (65.8%) specimens were concordant for all five methods. The sensitivities, specificities, and positive and negative predictive values for the culture and DFA methods were 91.9, 100, 100, and 91.7% and 91.9, 96.4, 96.6, and 91.4%, respectively. The sensitivities, specificities, and positive and negative predictive values for the three EIA procedures, Directigen, TestPack, and RSV EIA, were 75.8, 80.0, 81.0, and 74.6%; 93.6, 100, 100, and 93.2%; and 71.0, 100, 100, and 75.3%, respectively. New self-contained EIA configurations and the DFA method offer attractive alternatives to the culture method. Technical simplicity, rapid turnaround time, performance, and cost must all be considered when selecting a system for RSV detection.
Detection of Epstein-Barr virus genomes in normal human lacrimal glands
Authors: Crouse C A , Pflugfelder S C , Cleary T , Demick S M , Atherton S S
Published: January 2020

Epstein-Barr virus (EBV) has been implicated in several ocular diseases; however, detection of the EBV genome in ocular tissues has not been documented. We report the detection of amplified EBV genomic sequences in 11 of 26 normal lacrimal gland DNA samples by using the polymerase chain reaction. Serum was available for 19 of the lacrimal gland donors. All 19 were EBV seropositive, although of the 19 lacrimal gland-seropositive patients, EBV sequences were detected in only 10 of the samples. Further, amplified EBV sequences were not detected in circulating lymphocyte DNA from normal seropositive volunteers, most likely because of the low frequency of circulating EBV-infected B cells. Amplification of EBV from cadaver lacrimal gland DNA was possible with minute quantities of DNA, whereas peripheral blood mononuclear cell DNA from normal volunteers did not amplify EBV sequences. Interestingly, the peripheral blood mononuclear cell polymerase chain reactions contained approximately 100 times more DNA than the lacrimal gland polymerase chain reactions. We conclude that the lacrimal gland may be a site for EBV persistence and that positive EBV serology is not an indicator of which individuals may have EBV harbored within their lacrimal glands.
Isolation, identification, and characterization in Israel of Brucella melitensis biovar 1 atypical strains susceptible to dyes and penicillin, indicating the evolution of a new variant
Authors: Banai M , Mayer I , Cohen A
Published: January 2020

During characterization by conventional biotyping tests of numerous Brucella melitensis isolates obtained in Israel in the last 2 years, we identified several strains of the biovar 1 serotype that are atypically susceptible to dyes and penicillin. Their coisolation from two brothers in a family that rears sheep and from the milk of one of their sheep and the prevalence of such strains in two distinct geographical zones in Israel provide epidemiological support for the notion that a new variant has been identified.
Unsaturated fatty acids and viability of Helicobacter (Campylobacter) pylori
Authors: Hazell S L , Graham D Y
Published: January 2020

Helicobacter (Campylobacter) pylori was found to be sensitive to the toxic effects of an unsaturated fatty acid (arachidonic acid). Data are presented that support the hypothesis that exogenous catalase added to basal media enhances the growth of H. pylori by preventing the formation of toxic peroxidation products from long-chain unsaturated fatty acids.
Simple, efficient purification of filamentous hemagglutinin and pertussis toxin from Bordetella pertussis by hydrophobic and affinity interaction
Authors: Skelton S K , Wong K H
Published: January 2020

Two major antigens of Bordetella pertussis, filamentous hemagglutinin (FHA) and pertussis toxin (PT), were efficiently purified from culture filtrate by exploiting their relative hydrophobicities and differences in affinity to sialic acid-containing protein. High yields of FHA (40 to 80 mg/liter) and PT (8 to 16 mg/liter) were first produced by growing the bacteria in the modified CL medium. The FHA and PT in the culture filtrate were adsorbed onto butyl-Sepharose by hydrophobic interaction at appropriately high ionic strength. Elution of the antigens was effected by decreasing their hydrophobicities with a buffer of low ionic strength. FHA was then separated from PT with an affinity column of fetuin-Sepharose. The fraction passing through the column contained purified FHA, and the fetuin-bound PT was eluted with buffered MgCl2. The FHA and PT purified by these steps were electrophoretically and serologically identical to the reference purified FHA and PT preparations. Approximately 16 to 32 mg of purified FHA and 4 to 8 mg of purified PT were obtained from 1 liter of culture filtrate. The described procedure for making FHA and PT antigens from B. pertussis for serologic and immunologic use is very simple, efficient, and reproducible.
Modified method for testing the quality of albumin-containing enrichments used in growth media for mycobacteria
Authors: Butler W R , Warren N G , Kubica G P , Kilburn J O
Published: January 2020

Many commercially available media for cultivation of mycobacteria have failed to support the growth of these organisms. This is especially true of media prepared with albumin-containing enrichments. Earlier, we developed a method for rapid identification of good albumin enrichments for agar-based media used to test the susceptibility of tubercle bacilli to pyrazinamide. The method was modified to make testing of the acceptability of albumin enrichments for primary isolation media for mycobacteria possible. We describe here a simple turbidimetric test using a specific Bacillus subtilis strain to assay quickly (24 h) different lots of albumin-containing enrichments that may be used in the preparation of growth media for mycobacteria.
Recovery of an unusual Flavobacterium group IIb-like isolate from a hand infection following pig bite
Authors: Goldstein E J , Citron D M , Merkin T E , Pickett M J
Published: January 2020

An unusual gram-negative rod (RMA 1571) was isolated from a hand infection following a pig bite. This unclassified isolate was characterized by growth requirements, microscopic examination, biochemical characteristics, antimicrobial susceptibility tests, and cellular fatty acid analysis. It was indole positive and produced yellow-pigmented growth, which placed it in the genus Flavobacterium, but its other features, including cellular fatty acid analysis, did not appear to be those of a named species.
Evaluation of the CMV-CUBE assay for detection of cytomegalovirus serologic status in marrow transplant patients and marrow donors
Authors: Gleaves C A , Wendt S F , Dobbs D R , Meyers J D
Published: January 2020

We evaluated a new membrane dot immunobinding assay (CMV-CUBE; Difco Laboratories) for the detection of cytomegalovirus (CMV) antibody in marrow transplant patients and donors. The CMV-CUBE assay was compared with a commercially available enzyme immunoassay (EIA; CMV STAT) and a latex agglutination (LA; CMVScan) test. Serum samples were collected from 311 transplant patients and donors prior to transplantation. A total of 164 serum specimens were positive for CMV antibody by one or more of the three assays, with 153 of 164 samples (93.3%) positive by all three tests. A total of 147 serum specimens were CMV antibody negative. CMV-CUBE detected 154 of 164 (94%) of the positive samples, EIA detected 160 of 164 (97.5%), and LA detected 157 of 164 (95.7%) CMV-positive samples. Compared with EIA, CMV-CUBE had a sensitivity of 95.6% and a specificity of 99.3%. Compared with LA, CMV-CUBE had a sensitivity of 97.5% and a specificity of 99.4%. CMV-CUBE is a simple and rapid visual assay which can be used for the qualitative detection of antibody to CMV in patient serum.
Molecular detection and identification of enteroviruses using enzymatic amplification and nucleic acid hybridization
Authors: Chapman N M , Tracy S , Gauntt C J , Fortmueller U
Published: January 2020

Analysis of enteroviral genomes has revealed that the 5' nontranslated region is highly conserved, providing consensus sequences for the design of oligonucleotides which should anneal to most, if not all, human enteroviral RNAs. We designed and used a pair of such generic primers to enzymatically amplify cDNA from coxsackievirus group B types 1 through 6, poliovirus types 1 through 3, 4 coxsackievirus A types, and 29 echoviruses. The polymerase chain reaction (PCR) products generated with these enteroviral primers were analyzed by agarose gel electrophoresis, Southern blotting, or slot blot hybridization. A genotype-specific PCR was used to detect coxsackievirus B3, to the exclusion of other enteroviruses, by using a coxsackievirus B3 genome-specific primer pair that was derived from sequences coding for part of a capsid protein. A technique is demonstrated by which individual genotypes, for which no sequence information is known, can be identified by high-criterion hybridization analysis following amplification with generic enterovirus PCR primers.
Comparison of a dot immunobinding assay, latex agglutination, and cytotoxin assay for laboratory diagnosis of Clostridium difficile-associated diarrhea
Authors: Woods G L , Iwen P C
Published: January 2020

C. diff-CUBE, a dot immunobinding assay (DIA) (Difco Laboratories, Ann Arbor, Mich.) for detection of Clostridium difficile toxin A in stool specimens, was compared with latex agglutination (LA) (Marion Laboratories, Kansas City, Mo.) and cytotoxin assay (CTA) for the laboratory diagnosis of C. difficile-associated diarrhea. A total of 200 stool specimens collected from 169 patients with suspected C. difficile diarrhea were tested. Of the 198 specimens evaluated by all three methods, 36 (18%) from 36 patients were positive by one or more of the tests. Twenty-five, 26, and 23 specimens were positive by CTA, DIA, and LA, respectively; 14 were positive by all three methods. Eight specimens yielded nonspecific LA test results; all eight were negative by CTA, and one was positive by DIA. DIA results agreed with CTA results in 183 (92%) cases and with LA results in 175 (88%) cases. CTA and LA results agreed in 179 (90%) cases. Freezing of the specimen did not appear to adversely affect either the DIA or LA test. These preliminary results suggest that C. diff-CUBE may be useful as a rapid screen for the diagnosis of C. difficile-associated diarrhea. However, for optimum laboratory diagnosis, further testing of all stools that are negative by DIA is warranted.
Vibrio cholerae serogroup O1 in northeast Thailand
Authors: Kuyyakanond T , Nakamura S , Manmontri W , Iwanaga M
Published: January 2020

Strains of Vibrio cholerae serogroup O1 biotype El Tor that are susceptible to Mukerjee cholera bacteriophage group IV (S. Mukerjee, Bull. W.H.O. 28:333-336, 1963) were found. Cholera vibrios isolated from epidemics in northeast Thailand were characterized, and 57 of 60 strains isolated in 1986 were susceptible to cholera phage IV. However, all 113 strains isolated in 1988 were not susceptible to the phage. All isolates in both epidemics revealed behaviors typical of El Tor vibrios, except phage IV susceptibility in the 57 strains. Although the plaques of phage IV were generally translucent, plaques on some isolates looked transparent, just like those on classical vibrios. The organisms grown in the plaques were lysogenized. If this kind of strain is frequently isolated, the biotype of V. cholerae O1 should be reconsidered.
Comparison of two assay methods for patterns of adherence to HEp-2 cells of Escherichia coli from patients with diarrhea
Authors: Vial P A , Mathewson J J , DuPont H L , Guers L , Levine M M
Published: January 2020

To determine whether methodological differences in the HEp-2 adherence assay could explain conflicting results of field studies, 244 strains of Escherichia coli from Mexican children with diarrhea were tested for patterns of adherence by the method used at the Center for Vaccine Development, University of Maryland (CVD), and at the Center for Infectious Diseases, University of Texas Medical School and School of Public Health (UTH). The CVD assay differentiated three phenotypes of adherent E. coli, including localized, diffuse, or aggregative adherence (LA, DA, or AA, respectively). There was agreement on pattern of adherence in 241 of the 244 strains (98.8%) tested by the CVD method in both Baltimore and Houston, and AA+ was the most common phenotype (28.5% of isolates). Among these isolates, the UTH assay detected only two adherent phenotypes (LA and DA), since it did not distinguish the AA pattern. The LA+ strains detected by each assay were compared for positivity with the enteropathogenic E. coli adherence factor (EAF) gene probe. Of the 16 strains LA+ by the CVD method, 100% were EAF+; in contrast, only 11 of 22 strains LA+ by the UTH method were EAF+ (P = 0.00074). These results help explain why in pediatric field studies in Mexico where isolates were tested by the UTH method (J. J. Mathewson, R. A. Oberhelman, H. L. Dupont, F. J. de la Cabada, and E. V. Garibay, J. Clin. Microbiol. 25:1917-1919, 1987) LA+ strains often did not belong to enteropathogenic E. coli O serogroups and why the AA pattern was not observed; the opposite was found in studies of pediatric diarrhea in Chile in which the CVD assay was used (M. M. Levine, V. Prado, R. M. Robins-Browne, H. Lior, J. B. Kaper, S. Moseley, K. Gicquelais, J. P. Nataro, P. Vial, and B. Tall, J. Infect. Dis. 158:224-228, 1988). Since it appears that both assays identify E. coli strains associated with diarrheal illness, the genetic relationships among these strains should be examined in future studies.
Laboratory observations on Plesiomonas shigelloides strains isolated from children with diarrhea in Peru
Authors: Olsvik O , Wachsmuth K , Kay B , Birkness K A , Yi A , Sack B
Published: January 2020

Eleven strains of Plesiomonas shigelloides isolated from 10 Peruvian children with diarrhea were examined. All the strains were resistant to two or more antibiotics, most commonly ampicillin, gentamicin, erythromycin, kanamycin, and streptomycin. The strains were all negative in the Sereny and cell culture assays used to test for enteroinvasiveness. One strain showed cytotoxic activity on Vero cells. The strains showed no antigenic relationship with Shigella organisms. Both bioassays and enzyme-linked immunosorbent assays used for detection of Escherichia coli enterotoxins were negative. Nucleic acid probes for such toxins likewise gave negative results. The strains all possessed a large (approximately 200-megadalton) plasmid in addition to one or more other plasmids. Several different plasmid profiles were observed among these 11 P. shigelloides strains, indicating that the isolates were not acquired from a common source or from a single bacterial clone.
Chromosomal DNA, iron-transport systems, outer membrane proteins, and enterotoxin (heat labile) production in Salmonella typhi strains
Authors: Faundez G , Aron L , Cabello F C
Published: January 2020

We examined a representative collection of Salmonella typhi strains from Chile, Peru, Mexico, India, and England for the presence of several properties. All strains had a conserved pattern of outer membrane proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The electrophoresis profiles of chromosomal DNA digested with EcoRI and PstI restriction enzymes were similar for all the strains. A conserved pattern of hybridization was observed when digested chromosomal DNA was hybridized with DNA probes for the 36-kilodalton porin, enterobactin synthesis, and enterobactin receptor genes. All the strains produced enterobactin but not aerobactin in bioassays. None of the strains produced heat-labile toxin, as measured by an enzyme-linked immunosorbent assay. Colony and Southern hybridizations with DNA probes for aerobactin synthesis and its receptor and heat-labile toxin genes were negative. These results indicate that S. typhi strains from different origins have similar phenotypic and genetic properties and, as has been suggested, constitute a clone.
Immunoassay for detection of feline immunodeficiency virus core antigen
Authors: Tilton G K , O'Connor T P , Seymour C L , Lawrence K L , Cohen N D , Andersen P R , Tonelli Q J
Published: January 2020

The feline immunodeficiency virus (FIV) is a recently identified feline lentivirus that has been found at significant levels in domestic cat populations worldwide. A microdilution plate format, monoclonal antibody-based enzyme-linked immunosorbent assay was developed for the detection of the FIV group-associated antigen (gag) designated p24. Assays of serially diluted samples containing disrupted virus showed that the assay had a sensitivity limit of approximately 0.2 ng/ml for FIV p24. The assay was approximately eightfold more sensitive than the assay for viral reverse transcriptase activity when it was tested with diluted tissue culture samples. A qualitative confirmation assay by standard antibody inhibition techniques was coupled to the screening test methodology. The test was used to detect and confirm the presence of virus in cultured feline lymphocytes from infected animals.
Geographic distribution, frequency, and specimen source of Mycobacterium avium complex serotypes isolated from patients with acquired immunodeficiency syndrome
Authors: Yakrus M A , Good R C
Published: January 2020

Isolates of Mycobacterium avium complex from 727 patients with acquired immunodeficiency syndrome (AIDS) were submitted by medical centers across the United States to the Centers for Disease Control for serotyping. We were able to type 630 (87%) of these isolates by our seroagglutination procedure. Almost all typeable isolates were M. avium (serotypes 1 to 6 and 8 to 11). Blood was the major specimen source for both M. avium and the nontypeable isolates. M. intracellulare serotypes made up only 3% of all isolates from AIDS patients, with sputum being the major specimen source. More than 50% of the isolates originated from either New York or California, with serotype 4 being isolated most frequently in New York and serotype 8 appearing most frequently in California. AIDS patients in Los Angeles had a significantly higher isolation frequency for serotype 8 and a significantly lower one for serotype 4 in comparison with patients in either San Francisco or New York City.
Unusual fatty acid substitution in lipids and lipopolysaccharides of Helicobacter pylori
Authors: Geis G , Leying H , Suerbaum S , Opferkuch W
Published: January 2020

Cellular fatty acids, phospholipid fatty acids, and lipopolysaccharide fatty acids of four strains of Helicobacter pylori were analyzed by gas-liquid chromatography. The presence of myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, 19-carbon cyclopropane fatty acid, beta-hydroxypalmitic acid, and beta-hydroxystearic acid was confirmed. In phospholipids, myristic acid and 19-carbon cyclopropane fatty acid were the major fatty acids. Hydroxy fatty acids and unsaturated fatty acids were not detected or occurred only in small amounts. The major fatty acids of lipopolysaccharides were stearic acid, beta-hydroxypalmitic acid, and beta-hydroxystearic acid. Unsaturated fatty acids and 19-carbon cyclopropane fatty acid were not found. The unusual compositions of H. pylori phospholipid and lipopolysaccharide fatty acids may have important implications for the taxonomy, physicochemical membrane properties, and biological activity of lipopolysaccharides.
Swab-wash method for quantitation of cutaneous microflora
Authors: Keyworth N , Millar M R , Holland K T
Published: January 2020

We describe a comparison of the scrub-wash method of Williamson and Kligman and a swab-wash method for the enumeration of cutaneous microflora. The swab-wash method provides a less traumatic alternative to the scrub-wash method and can be used to sample the cutaneous microflora of premature neonates.
Growth of Trichomonas vaginalis in commercial culture media
Authors: Gelbart S M , Thomason J L , Osypowski P J , Kellett A V , James J A , Broekhuizen F F
Published: January 2020

There are only two commercially available, ready-to-use culture media which are approved by the Food and Drug Administration for clinical diagnosis of vaginal trichomoniasis: Kupferberg's STS and Diamond's medium (modified). Diamond's medium (Klaas modification), recommended by the Centers for Disease Control for the isolation of Trichomonas vaginalis, was compared in vitro to Kupferberg's (STS) medium. Growth studies using six fresh clinical isolates, all from different patients, showed that while generation time was about 6 h in both STS and Diamond's, the period of exponential growth was longer in Diamond's. More important, in STS there was a 4-h lag period during which the population significantly decreased prior to exponential growth. This did not occur in Diamond's medium. Three hundred organisms inoculated into Diamond's reached a population of over 10(5) organisms in 72 h. In STS, the same inoculum could multiply to only 6 x 10(3) organisms. The fact that there is a lag phase in STS which is not seen in Diamond's could explain why low numbers of T. vaginalis do not multiply in STS but do multiply and can be detected in Diamond's. We conclude that because Diamond's medium (modified) allows more prolific growth over a shorter period of time, it is more suitable than Kupferberg's (STS) for detecting T. vaginalis in patients with vaginitis.
Enhanced recovery of cytomegalovirus in conventional tube cultures with a spin-amplified adsorption
Authors: Oefinger P E , Shawar R M , Loo S H , Tsai L T , Arnett J K
Published: January 2020

Low-speed centrifugation-mediated adsorption was evaluated as an enhancement of infectivity of clinical and laboratory strains of cytomegalovirus (CMV) occurring with cells grown in conventional culture tubes. The time required for reporting of primary isolates of CMV from urine specimens adsorbed onto monolayers of WI-38 cells in culture tubes was calculated. Of 668 specimens adsorbed by the stationary phase (SP) method, 98 were positive by cytopathic effect (CPE) that required an average of 16.8 days for recovery in culture. However, the appearance of CPE required a shorter average time of 11.9 days for 70 CMV strains isolated from 283 specimens adsorbed in tube cultures by the spin-amplified (SA) method. In another phase of clinical CMV recovery, urine specimens were adsorbed by the SA method onto cell cultures grown in both shell vials and test tubes. Of 594 specimens inoculated, a total of 74 were positive by either CPE in test tubes or immunostaining-localized early antigen in shell vials. Approximately one-third of these CMV isolates were recovered only by CPE from specimens adsorbed by the SA method in test-tube cultures. In a related study to further evaluate differences between adsorption methods, the AD-169 laboratory strain of CMV was adsorbed by SP and SA methods onto MRC-5 cells grown in both culture vessels. Early antigen detection by immunomicroscopy was found in the infected cells at least 2 to 4 days prior to the appearance of CPE, regardless of adsorption procedure. In both vessels, the replication of AD-169 virus in cultures adsorbed by the SA method consistently exceeded that of virus adsorbed by the SP procedure. CPE occurred 24 to 48 h earlier and progressed two to four times more extensively; early antigen was expressed two- to fourfold greater within 24 to 48 h postinfection; and foci of infected cells containing late antigen were two to four times greater in number at 1, 2, and 5 days postinfection. Overall, the replication and enhancement of infectivity of laboratory and clinical strains of CMV as determined by CPE and early and late antigen expression occurred most efficiently with specimens adsorbed by the SA method onto cultures grown in conventional tubes or shell vials.
Detection by polymerase chain reaction amplification of human herpesvirus 6 DNA in peripheral blood of patients with exanthem subitum
Authors: Kondo K , Hayakawa Y , Mori H , Sato S , Kondo T , Takahashi K , Minamishima Y , Takahashi M , Yamanishi K
Published: January 2020

The polymerase chain reaction was used to detect human herpesvirus 6 (HHV-6) DNA in peripheral blood mononuclear cells from patients with exanthem subitum. Amplified products were detected by agarose gel electrophoresis and dot blot hybridization with a cloned DNA probe. No cross-hybridization with DNAs of five other human herpesviruses was observed in this system, and all HHV-6 strains gave positive reactions when the primer pairs were used. Immunoglobulin M antibody appeared about 5 days after the onset of disease, reaching a maximum after about 2 to 3 weeks and then decreasing to less than 1:10 1 month after the onset of disease in almost all patients. Mononuclear cells from seven patients with exanthem subitum all gave positive reactions in this system, and viral DNA was found in samples even during the convalescent phase of the disease. We conclude that this test, using polymerase chain reaction amplification, which takes only 1 to 2 days, is useful for the diagnosis of HHV-6 infection.
Rapid diagnosis of tuberculous meningitis by frequency-pulsed electron-capture gas-liquid chromatography detection of carboxylic acids in cerebrospinal fluid
Authors: Brooks J B , Daneshvar M I , Haberberger R L , Mikhail I A
Published: January 2020

The frequency-pulsed electron-capture gas-liquid chromatography technique described previously by Brooks et al. was modified and applied to the studies of coded and routine clinical specimens. Uncentrifuged cerebrospinal fluid (2 ml) was extracted under acidic conditions, derivatized, and analyzed by frequency-pulsed electron-capture gas-liquid chromatography on large-bore fused silica polar and nonpolar capillary columns. The frequency-pulsed electron-capture gas-liquid chromatography profile of carboxylic acids (C2 through C22) along with identification of tuberculostearic acid, established by retention time comparison of derivatized tuberculostearic acid and derivatized sample extract, strongly suggests the presence of Mycobacterium tuberculosis in patients with lymphocytic meningitis. Results from 41 coded cases and 75 clinical cases showed that the frequency-pulsed electron-capture gas-liquid chromatography test had a specificity of 91% and a sensitivity of 95%.
Detection of Borrelia burgdorferi using the polymerase chain reaction
Authors: Malloy D C , Nauman R K , Paxton H
Published: January 2020

Segments of the ospA gene of Borrelia burgdorferi were amplified by the polymerase chain reaction (PCR). Oligonucleotide primers used in the reaction flank a 309-base-pair segment within the ospA gene. After 35 cycles of amplification, the product could be detected by agarose gel electrophoresis or dot hybridization with a 32P-labeled probe. This segment was amplified in all strains of B. burgdorferi tested, but it was not detected in other bacterial species. An additional primer pair which has a broad specificity for conserved 16S rRNA sequences that are present in eubacteria amplified a 215-base-pair fragment in all organisms tested. The sensitivity of PCR for the detection of B. burgdorferi in clinical samples was evaluated by seeding blood and urine specimens with B. burgdorferi and subjecting them to amplification. We were able to detect 10 organisms per ml of blood or urine, using PCR with dot hybridization detection. DNA extraction is not required for sample preparation. Blood and urine specimens were obtained from canines with clinical and serologic evidence of Lyme disease and subjected to PCR analysis. Of 17 clinical specimens from 15 animals, one blood specimen showed reactivity in the PCR.
Amino acid requirements for growth of Chlamydia pneumoniae in cell cultures: growth enhancement by lysine or methionine depletion
Authors: Kuo C C , Grayston J T
Published: January 2020

Amino acid requirements for growth of two isolates of Chlamydia pneumoniae were studied and compared with those for one strain of Chlamydia trachomatis in a HeLa 229 cell culture. It was shown that among 13 amino acids in Eagle minimum essential medium, C. pneumoniae required all amino acids except lysine. A true requirement for arginine, isoleucine, leucine, threonine, and valine could not be determined because depletion of 100% of these amino acids caused cell detachment. Consequently, a requirement for these amino acids was based on 90% depletion. C. trachomatis biovar trachoma required all amino acids except threonine, which was indeterminable. Depletion of 100 and 90% of the lysine and 90 to 70% of the methionine was shown to enhance the growth of C. pneumoniae. This phenomenon was shown to be a property of C. pneumoniae because the effect of lysine and methionine reduction was also demonstrated in another human line, HL cells, and a mouse line, McCoy cells.
Serological evaluation of Escherichia coli-expressed human T-cell leukemia virus type I env, gag p24, and tax proteins
Authors: Coates S R , Harris A J , Parkes D L , Smith C M , Liu H L , Akita R W , Ferrer M M , Sampson E K , Brandis J W , Sliwkowski M X
Published: January 2020

Three proteins (env, gag, and tax) encoded by the human T-cell leukemia virus type I (HTLV-I) genome were cloned and expressed in Escherichia coli. The env protein contained a substantial part of the gp46 domain and a majority of the p21e domain. The gag protein contained all of p24 and portions of p19 and p15. In addition to these two structural proteins, a full-length tax (p40X) construct was obtained. All three recombinant proteins were purified to near homogeneity. When used in an immunoblot assay, the three recombinant proteins detected antibodies in more HTLV-I antibody-positive patient sera than did the corresponding native proteins. Antibodies to at least two of these three different gene products were detected in 98.4% of adult T-cell leukemia patients, 100% of HTLV-I-associated myelopathy patients, 97.4% of asymptomatic carriers, and 94% of uncharacterized HTLV-I-positive patients. Antibody to recombinant tax was found in 4.9% of adult T-cell leukemia patients, whereas antibody to recombinant env could not be detected. These recombinant proteins from three different gene products may be useful in detecting or confirming the presence of antibodies to HTLV-I.
Rapid detection of respiratory syncytial virus and influenza A virus in cell cultures by immunoperoxidase staining with monoclonal antibodies
Authors: Waris M , Ziegler T , Kivivirta M , Ruuskanen O
Published: January 2020

Peroxidase-labeled monoclonal antibodies against respiratory syncytial virus (RSV) and influenza A virus were used for immunoperoxidase staining (IPS) of cell cultures inoculated with nasopharyngeal aspirates. Cells were grown in 24-well plates, and specimens were inoculated by low-speed centrifugation. Cultures were incubated for 2 days at 37 degrees C and then fixed, stained, and observed by light microscopy. IPS was compared with standard virus isolation by using cultures of human diploid fibroblasts and Vero, HEp-2, and HeLa cell lines for RSV and Madin-Darby canine kidney cells for influenza A virus; these cultures were inoculated with specimens that were previously stored at -70 degrees C. Of 40 known RSV-positive specimens, 30 were found to be positive on reinoculation by both methods, and an additional 5 specimens were found to be positive by IPS only. Of 190 specimens tested for influenza A virus, 14 were positive by IPS and in tubes, and a further 8 specimens were positive by IPS only. IPS was also compared with direct detection of viral antigens in nasopharyngeal aspirates by a time-resolved fluoroimmunoassay (TR-FIA). Fresh nasopharyngeal aspirates were inoculated into human diploid fibroblasts and Madin-Darby canine kidney cells and tested for RSV and influenza A virus, respectively, by IPS. Of 110 specimens tested for RSV, 37 were positive in total, 32 were positive by IPS, and 33 were positive by TR-FIA. Of 150 specimens tested for influenza A virus, 39 were positive in total, 35 were positive by IPS, and 34 were positive by TR-FIA. IPS of cultures inoculated by centrifugation and incubated for 2 days is a sensitive method for the diagnosis of respiratory virus infections, and 24-well plates allow for the easy processing of a large number of specimens.
Antibody-independent inhibition of Plasmodium falciparum in vitro cultures
Authors: Perlaza B L , Herrera M A , Villegas A , Carrasquilla G , Herrera S
Published: January 2020

The sera of 100 Colombian individuals of African origin living in a malaria-endemic area of the Pacific coast were studied with regard to their capacity to inhibit Plasmodium falciparum cultures in vitro. Antimalarial antibody levels determined by indirect immunofluorescence were higher in the group of infected individuals than in the noninfected individuals, and inhibitory activity assessed by the inhibition of parasite incorporation of 3H-hypoxanthine in vitro was present in the sera of both the infected and noninfected patients. We believe that the noninfected patients were probably immune. The sera of some of the infected patients had high inhibitory capacities for the P. falciparum FCB-1 isolate. When the inhibitory effects of some of the sera were tested by using four parasite isolates from different regions of the world, striking differences among them were found.
Species-specific monoclonal antibodies to Rickettsia japonica, a newly identified spotted fever group rickettsia
Authors: Uchiyama T , Uchida T , Walker D H
Published: January 2020

A total of 192 hybridomas were developed from mice immunized with Rickettsia japonica, a newly identified spotted fever group rickettsia pathogenic for humans. Of these hybridomas, 101 were species specific, 37 were spotted fever group reactive, and the other 54 were also reactive with one or more of the other pathogenic species of spotted fever group rickettsiae, Rickettsia akari, Rickettsia australis, Rickettsia conorii, Rickettsia rickettsii, and Rickettsia sibrica. Seven of the species-specific monoclonal antibodies were characterized. These monoclonal antibodies all belong to the immunoglobulin G class and react with all five strains of R. japonica at the same immunofluorescence titers, indicating that the five strains all belong to a single species. The species-specific epitopes reactive with these monoclonal antibodies are located on the surface proteins of the organisms demonstrated as 145- and 120-kilodalton bands on Western immunoblots. These two antigenic bands were shown to be proteins, because treatment with proteinase K completely destroyed the reactivity of the bands with the monoclonal antibodies.
Antibody to cytotoxin in infection by Helicobacter pylori
Authors: Leunk R D , Ferguson M A , Morgan D R , Low D E , Simor A E
Published: January 2020

Gastrointestinal disease and colonization by Helicobacter pylori were determined in 36 asymptomatic volunteers and 30 symptomatic individuals undergoing endoscopy and biopsy. Serum antibody immunoglobulin G (IgG) and IgA to H. pylori were measured by enzyme-linked immunosorbent assay. Serum antibody to a cytotoxin produced by H. pylori was detected with a neutralization assay. Serum IgG was 95% predictive of infection by H. pylori, and serum IgA was 88% predictive. Antibody to the cytotoxin was detected in 12 of 18 infected individuals. Antibody to the cytotoxin was a highly specific (96%), but not a very sensitive (67%), indicator of infection by H. pylori. The neutralization assay was 87% predictive of infection. These data confirm the diagnostic value of serum antibody to H. pylori for the detection of infection. The toxin-neutralizing activity of sera from individuals infected with H. pylori suggests that the cytotoxin is produced in vivo. It may therefore contribute to disease associated with H. pylori.
Proposal of a new serovar and altered nomenclature for Haemophilus paragallinarum in the Kume hemagglutinin scheme
Authors: Blackall P J , Eaves L E , Rogers D G
Published: January 2020

By using the Kume hemagglutinin serotyping scheme, 13 Australian isolates of Haemophilus paragallinarum were shown to constitute a new serovar within the presently termed serogroup II. Because of the likelihood that new serovars will continue to be established, we propose a rationalization of the nomenclature of the Kume scheme. Under this altered scheme, the three recognized serogroups I, II, and III are renamed A, C, and B, respectively. Within each of the serogroups, the serovars are numbered sequentially, allowing new serovars to be added in numerical order. Thus, the nine currently recognized Kume serovars are termed A-1, A-2, A-3, A-4, B-1, C-1, C-2, C-3, and C-4.
Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction
Authors: Hermans P W , Schuitema A R , Van Soolingen D , Verstynen C P , Bik E M , Thole J E , Kolk A H , van Embden J D
Published: January 2020

During the screening of a Mycobacterium tuberculosis lambda gt-11 gene library with monoclonal antibodies, we detected a recombinant clone, lambda PH7311, which contained a mycobacterial DNA insert that hybridized specifically with DNA of M. tuberculosis complex strains. Part of this insert was sequenced and used for the development of an M. tuberculosis complex-specific polymerase chain reaction (PCR). Only strains belonging to species of the M. tuberculosis complex group contained an amplifiable fragment of 158 base pairs (bp). This fragment was absent in all strains tested belonging to 15 other mycobacterial species. After amplification by PCR and dot blot hybridization with a digoxigenin-labeled oligonucleotide, the limit of detection of purified genomic M. tuberculosis DNA amounted to a quantity corresponding to 20 bacterial cells. By this technique about 10(3) M. tuberculosis bacteria were detectable in sputum. Using PCR, we were also able to detect M. tuberculosis cells in clinical material such as pleural fluid, bronchial washings, and biopsies, and these results were comparable with those obtained by classical bacterial culture. Of 34 M. tuberculosis strains, 5 did not carry the amplifiable 158-bp fragment, which occurs usually as a single copy in the chromosome. Evidence is presented that the 158-bp fragment is located near a repeated sequence in the chromosome. We presume that strains which did not carry the 158-bp fragment have lost a chromosomal segment by a genetic rearrangement induced by the repetitive DNA element.
Enzyme-linked immunosorbent assay for immunoglobulin M specific antibody for the diagnosis of melioidosis
Authors: Kunakorn M , Boonma P , Khupulsup K , Petchclai B
Published: January 2020

Indirect hemagglutination (IHA) is commonly used for serodiagnosis of melioidosis. However, in endemic areas, high background titers in normal populations and occasional low titers in patients with septicemic melioidosis prompted a search for a more sensitive and more specific method of serodiagnosis. An indirect fluorescent-antibody test for immunoglobulin M (IgM) specific antibody to Pseudomonas pseudomallei was more sensitive and more specific, but fluorescence microscopes are rarely available in the endemic areas. An enzyme-linked immunosorbent assay (ELISA) for IgM antibody is an attractive alternative. An indirect ELISA for IgM antibody (IgM ELISA) and an IgM antibody capture ELISA for melioidosis were developed. Both tests, together with IHA, were evaluated for 153 serum specimens from blood donors and 16 serum specimens from 16 melioidosis patients. It was found that IHA, the IgM ELISA, and the IgM antibody capture ELISA had sensitivities of 88, 88, and 75%, respectively, with specificities of 97.4, 92.2, and 91.5%, respectively. When IHA was combined with IgM ELISA, a sensitivity of 100% and a specificity of 95.4% were obtained. The IgM ELISA and IHA should be used in combination for serodiagnosis of melioidosis.
Choice of reference assay for the detection of rotavirus in fecal specimens: electron microscopy versus enzyme immunoassay
Authors: Dennehy P H , Gauntlett D R , Spangenberger S E
Published: January 2020

Two previously demonstrated sensitive and specific enzyme immunoassays (EIAs) for rotavirus, one using polyclonal and monoclonal antisera (TestPack Rotavirus [TPK]; Abbott Laboratories) and the other using only monoclonal anti-rotavirus antibodies (Rotaclone [RTC]; Cambridge BioScience Corporation), were evaluated as potential reference assays for rotavirus testing in comparison with direct negative-staining electron microscopy (EM), the current laboratory standard. Two hundred and seven stool samples collected consecutively during the winter of 1989 from children with acute diarrhea admitted to a ward for infants from 0 to 2 years of age were tested by the EIAs and by EM. TPK specimens were read visually; RTC results were read spectrophotometrically. Specimens with discordant EIA and EM results were further evaluated by a fluorescent focus assay. Specimens positive by EM and those negative by EM but positive by fluorescent focus assay were considered to be positive for rotavirus. Of the 207 stools tested, 35 (17%) were positive for rotavirus by these criteria. EM had a sensitivity of only 80%. Specificities were 100% for RTC and EM and 89% for TPK. These findings indicate that EM, although very specific, is relatively insensitive compared with a highly sensitive monoclonal antibody-based EIA. An EIA with high sensitivity and specificity, such as RTC, is a more appropriate reference standard for rotavirus testing.
Rapid focus reduction neutralization test of influenza A and B viruses in microtiter system
Authors: Okuno Y , Tanaka K , Baba K , Maeda A , Kunita N , Ueda S
Published: January 2020

A rapid neutralization test for influenza A and B viruses was developed. In this method, a 96-well tissue culture plate was used for the preparation of cell monolayers and the peroxidase-antiperoxidase staining technique was used for the visualization of foci infected with these viruses. In the presence of trypsin and tragacanth gum, clear foci developed 1 day after infection. A linear relationship between virus dilutions and numbers of foci was observed. When neutralizing antibodies in some test sera were assayed, a good correlation was observed between the titers obtained by the focus method and those obtained by the ordinary plaque method. In addition, many serum specimens were investigated by the neutralization test, and it was demonstrated that the test is useful for serological studies of influenza.