bluuebunny/crossref_metadata_2025 huggingface dataset:
Serologic surveillance for the Lyme disease spirochete, Borrelia burgdorferi, in Minnesota by using white-tailed deer as sentinel animals
Authors: Gill J S , McLean R G , Shriner R B , Johnson R C
Published: January 2020

To determine the effectiveness of white-tailed deer as sentinel animals in serologic surveillance programs for Borrelia burgdorferi, we performed enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting analyses on 467 deer serum samples. The seropositivity rate in the ELISA was 5% for the 150 samples collected at the three sites in which the tick Ixodes scapularis was absent. The three sites with established I. scapularis populations had a seropositivity rate of 80% for 317 samples. Results were similar for two closely situated sites, one with an established I. scapularis population and one without; these sites were only 15 km apart. Rates of seropositivity were significantly higher in yearling and adult deer than in fawns. The mean numbers of bands seen on Western immunoblots were 3.0 for samples negative in the ELISA and 13.8 for samples positive in the ELISA; all of these samples were collected from sites in which I. scapularis was established. At sites in which I. scapularis was absent, the mean numbers of bands seen were 1.6 for samples negative in the ELISA and 8.2 for samples positive in the ELISA. There were 14 different B. burgdorferi antigens that reacted with more than 50% of the ELISA-positive samples from areas with I. scapularis. A 19.5-kDa antigen reacted with 94% of the ELISA-positive samples. Reactivity against OspA and OspB was weak a infrequent (2%). Serologic analysis of white-tailed deer sera appears to be an accurate and sensitive surveillance method for determining whether B. burgdorferi is present in specific geographic locations.
Purification of Chlamydia trachomatis strains in mixed infection by monoclonal antibody neutralization
Authors: Lampe M F , Stamm W E
Published: January 2020

A Chlamydia trachomatis D* serovariant strain was found to be mixed with an F serovar strain in a clinical specimen. By using a monoclonal antibody which neutralizes serovar F infectivity in hamster kidney cells, the D* variant strain was enriched until it could be cloned by limiting dilution. This newly described neutralization enrichment procedure can be used to purify a C. trachomatis serovar present in small numbers in a mixed culture or, potentially, to identify nonneutralizable mutants.
Effects of blood medium supplements on activities of newer cephalosporins tested against enterococci
Authors: Buschelman B J , Jones R N , Bale M J
Published: January 2020

This comparative study determined the effect of blood on the antienterococcal activities of the newer cephalosporins. Standardized disk diffusion susceptibility tests were performed with 57 strains of enterococci (30 Enterococcus faecalis strains) on Mueller-Hinton agar with and without 5% sheep blood supplementation. Twelve cephalosporins representing five different structural groups (based on the 7-alpha position substitution) were tested. The greatest frequency of activity enhancement by blood was observed with cefdaloxime and cefdinir (7-alpha hydroxyimino group) against E. faecalis. Cephalosporins with a 7-alpha methoxyimino group (cefpodoxime, cefepime, and cefpirome) had marked increases in zone diameters (3 to > 9 mm) when tested with the blood supplement. Cephems with 7-alpha amino or carboxy substitutions did not demonstrate any enhanced activity. Awareness of this phenomenon is important for the interpretation and accuracy of cephalosporin susceptibility testing.
Nosocomial septicemia caused by Serratia plymuthica
Authors: Domingo D , Limia A , Alarcón T , Sanz J C , Del Rey M C , López-Brea M
Published: January 2020

We report a case of nosocomial septicemia in a 79-year-old patient caused by Serratia plymuthica with no evident focus of infection. The patient was treated with gentamicin (40 mg every 8 h) during 10 days; clinical resolution of the infection was obtained after the 10-day treatment period.
Dilution assay statistics
Authors: Myers L E , McQuay L J , Hollinger F B
Published: January 2020

A parametric method of statistical analysis for dilution assays is developed in detail from first principles of probability and statistics. The method is based on a simple product binomial model for the experiment and produces an estimate for the concentration of target entities, a confidence interval for this concentration, and an indicator of the quality of the assay called the p value for goodness of fit. The procedure is illustrated with data from a virologic quantitative micrococulture assay used to quantify free human immunodeficiency virus in clinical trials. The merits of the procedure versus those of nonparametric methods of estimating the dilution inducing a 50% response rate are discussed. Advantages of the proposed approach include plausibility of the underlying assumptions, ability to assess plausibility of specific experimental outcomes through their likelihood, and plausibility of confidence intervals.
Isolation of Borrelia burgdorferi from saliva of the tick vector, Ixodes scapularis
Authors: Ewing C , Scorpio A , Nelson D R , Mather T N
Published: January 2020

A method for cultivating and isolating Lyme disease spirochetes, Borrelia burgdorferi, from the saliva of vector ticks, Ixodes scapularis (formerly known as Ixodes dammini), is described. Saliva was collected from partially engorged ticks after application of pilocarpine to induce salivation. B. burgdorferi was isolated from 8 of 14 (57%) of the saliva samples derived from ticks infected with the bacteria, as determined by direct immunofluorescent-antibody assay of tick hemolymph. A comparison of the protein profiles of the salivary isolates and a highly passaged strain (B31) showed that the salivary isolates all lacked a 22-kDa protein known to increase with continuous passage, but exhibited larger amounts of the OspA and OspB proteins than did the highly passaged B31 strain.
Investigation of an outbreak of Clostridium difficile infection in a general hospital by numerical analysis of protein patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Authors: Costas M , Holmes B , On S L , Ganner M , Kelly M C , Nath S K
Published: January 2020

One hundred forty-five cultures of Clostridium difficile, including strains from an apparent nosocomial outbreak of infection, were characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins. Each protein pattern was characterized by the presence of one to three dense bands which were highly reproducible. The first 100 strains (in chronological order) were used as the basis for a numerical analysis which divided the strains into 17 phenons (EP types 1 to 17). The protein patterns of the remaining 45 strains were identified to type by comparing their individual patterns against a data base made up of the protein patterns of the first 100 strains. EP type 1 was the most common, with 70 of 139 (50%) patient isolates having this pattern type, and it accounted for 26 of 35 strains (74%) from patients in a medical teaching ward from which the outbreak was believed to have originated. This type was also found as a high proportion of isolations in a number of other medical and oncology wards, but the majority of these isolates occurred subsequent to the isolations on the initial outbreak ward. This technique can therefore provide a method for tracing the possible spread of epidemic strains in hospitals and other institutions and may contribute to a better understanding of the epidemiology of C. difficile.
Detection of anti-human immunodeficiency virus type 1 (HIV-1) immunoglobulin G in urine by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) with recombinant reverse transcriptase as an antigen
Authors: Hashinaka K , Hashida S , Hirota K , Saitoh A , Nakata A , Shinagawa H , Oka S , Shimada K , Ishikawa E
Published: January 2020

Anti-human immunodeficiency virus type 1 immunoglobulin G in urine was detected by an immunoassay with reverse transcriptase as the antigen and beta-D-galactosidase as the label; this immunoassay was 30-fold more sensitive than the previous immunoassay with peroxidase as the label. The sensitivity and specificity were both 100%. The lowest signal for asymptomatic carriers was 20-fold higher than the highest signal for seronegative subjects.
Utility of paired blood cultures and smears in diagnosis of disseminated Mycobacterium avium complex infections in AIDS patients
Authors: Stone B L , Cohn D L , Kane M S , Hildred M V , Wilson M L , Reves R R
Published: January 2020

For 273 patients evaluated for disseminated Mycobacterium avium complex infection, a total of 1,047 mycobacterial blood cultures (MBCs) were submitted; the M. avium complex was recovered from 140 (13%) of the specimens. Results for the paired MBCs were highly concordant: in 392 of 462 (85%) culture sets, both MBCs were negative, in 53 of 462 (11%) sets, both MBCs were positive, and in only 17 of 462 (4%) sets was one culture positive and the other negative. Acid-fast smears were done on sediments from 671 specimens; smears were positive for 4 of 98 (4%) cultures that grew the M. avium complex. A single MBC should be obtained and then repeated if negative and disseminated M. avium complex infection is still clinically suspected. Use of direct acid-fast smears of sediments is not a reliable means of detecting mycobacteremia.
Rapid detection of Vibrio cholerae O1 in stools of Peruvian cholera patients by using monoclonal immunodiagnostic kits. Loyaza Cholera Working Group in Peru
Authors: Carillo L , Gilman R H , Mantle R E , Nunez N , Watanabe J , Moron J , Quispe V , Ramirez-Ramos A
Published: January 2020

We compared stool culture with two commercial Vibrio cholerae O1 rapid diagnostic kits which detect antigen in 100 adults with cholera in Peru. Serum vibriocidal-antibody titer was used as an external reference. Both rapid diagnostic kits appeared to detect cholera more frequently than did culture and were highly specific.
Use of a hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin and part of the 83-kDa protein as antigen for serodiagnosis of Lyme disease
Authors: Rasiah C , Rauer S , Gassmann G S , Vogt A
Published: January 2020

A hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin (an 18-kDa fragment) and a 59-kDa fragment (lacking the N-terminal part) of the 83-kDa protein has been constructed by genetic engineering. It was expressed as a nonfusion protein of an apparent molecular weight of 77,000 in Escherichia coli. The suitability of this new antigen for the diagnosis of Lyme disease was tested by immunoblotting; for comparison, the recombinant variable region of the flagellin, the 18-kDa fragment (p18), and the whole recombinant 83-kDa protein (p83), both expressed in E. coli, were used. A total of 120 serum samples from various stages of Lyme disease, which were positive in two serological assays, a passive hemagglutination assay and an indirect immunofluorescence assay, were tested. By indirect immunofluorescence, 74 samples were positive for immunoglobulin G (IgG) antibodies and 72 were positive for IgM antibodies. Of these serum samples, 69 of 74 (93%) contained IgG antibodies against p18 and/or p83, and IgG antibodies were detected by the hybrid protein in 67 (90%) samples. IgM antibodies against p18 and/or p83 were detected in 60 of 72 (83%) serum samples, and 57 (79%) serum samples were reactive with the hybrid protein. Twenty serum samples of patients with a history of syphilis and 40 serum samples, negative in routine B. burgdorferi serology, were tested as controls. The hybrid protein, made up of specific epitopes of an early (p18) and late (p83) antigen, is recognized by almost the same number of patient serum samples as the individual antigens.
Detection of leptospiral DNA by PCR
Authors: Kee S H , Kim I S , Choi M S , Chang W H
Published: January 2020

An EcoRI fragment (1.2 kb) which is highly conserved among Leptospira interrogans isolated in Korea was cloned into pBluescript vector from L. interrogans serovar lai WH20. The EcoRI fragment was sequenced, and a pair of primers (LP1 and LP2) was designed for PCR assay. PCR amplification of target DNA obtained from cultured L. interrogans showed that 274 bp could be detected when as little as 100 fg of leptospiral genomic DNA was used in the reaction mixture. No amplification of DNA was detected from DNA of Leptospira biflexa serovars patoc and sau paulo, Borrelia burgdorferi, Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium. Amplification of 274-bp target DNA could be detected in DNA samples purified from 500 microliters of blood collected from experimentally infected gerbils 2 days after infection, while antibodies to L. interrogans could be detected by the microscopic agglutination test 7 days after infection. The specificity and high sensitivity of the test provided valuable tools for the early diagnosis of leptospirosis.
Molecular analysis of rRNA and cholera toxin genes carried by the new epidemic strain of toxigenic Vibrio cholerae O139 synonym Bengal
Authors: Faruque S M , Abdul Alim A R , Roy S K , Khan F , Nair G B , Sack R B , Albert M J
Published: January 2020

Vibrio cholerae O139 synonym Bengal recently caused large epidemics of cholera-like disease in Bangladesh and India. We compared the restriction fragment length polymorphisms of ctxA and rRNA genes (ribotypes) in 27 isolates of V. cholerae O139 from patients in Bangladesh and India with those of 48 isolates of V. cholerae O1 from patients and 21 V. cholerae isolates from surface waters in Bangladesh, which included 2 O139 and 19 other non-O1 isolates. Ribotyping of the isolates with BglI revealed that all 29 isolates of O139 vibrios belonged to a single ribotype, suggesting a clonal nature of the infection. However, the O139 vibrios comprised two ctxA genotypes and carried three or more copies of the ctxA gene, and the chromosomal locations of these copies were unlike those of the El Tor or classical vibrios. Analysis of the restriction fragment length polymorphisms of the rRNA genes suggested that V. cholerae O139 isolates are more closely related to El Tor strains of V. cholerae O1 than were 19 other non-O1 vibrios and 33 classical V. cholerae O1 isolates that were studied. However, further studies are needed to determine whether V. cholerae O139 originated from mutations and genetic changes in a V. cholerae O1 strain or was due to the acquisition of virulence genes by a previously unknown V. cholerae non-O1 strain.
Modified technique for efficient detection of microsporidia
Authors: Kokoskin E , Gyorkos T W , Camus A , Cedilotte L , Purtill T , Ward B
Published: January 2020

Changes in temperature (from room temperature to 50 degrees C) and staining time (from 90 to 10 min) were evaluated as a means of improving the detection of microsporidia from stool specimens. A blinded and independent comparison of 50 known positive matched-specimen pairs by three technologists resulted in consistently easier microscopic detection. The background is clearer, and spores stain more intensely. Staining time is reduced by 80 min.
Comparison of novel synthetic peptide-based DETECT-RUBELLA enzyme immunoassays with Enzygnost and IMx for detection of rubella-specific immunoglobulin G
Authors: Pedneault L , Zrein M , Robillard L , Landry F , Lacroix M , Joncas J
Published: January 2020

Enzyme immunoassays (EIAs) using synthetic peptides SP-E1 and SP-E1E2 (DETECT-RUBELLA [Bio-Chem]) were compared with two viral lysate-based EIAs (Enzygnost [Behring] and IMx [Abbott]) for the detection of rubella virus-specific immunoglobulin G antibodies. Sensitivities of 94.7, 100, 98.6, and 100% and specificities of 100, 97.4, 100, and 73.7% were found for the SP-E1, SP-E1E2, Enzygnost, and IMx EIAs, respectively.
Method for differentiating Klebsiella planticola and Klebsiella terrigena from other Klebsiella species
Authors: Monnet D , Freney J
Published: January 2020

Conventional methods usually fail to identify Klebsiella planticola and Klebsiella terrigena, which represent up to 19% of clinical Klebsiella isolates. By combining four carbon substrate assimilation tests and two conventional tests, the method identified these species with a specificity and a sensitivity of 100%. Overall, sensitivity for Klebsiella identification was 94.7%.
Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats
Authors: Díaz-Aparicio E , Marín C , Alonso-Urmeneta B , Aragón V , Pérez-Ortiz S , Pardo M , Blasco J M , Díaz R , Moriyón I
Published: January 2020

Five serological assays were evaluated for the diagnosis of brucellosis in goats: the rose bengal test (RBT), complement fixation test (CFT), radial immunodiffusion (RID) with Brucella and Yersinia enterocolitica O:9 polysaccharides, counterimmunoelectrophoresis (CIEP) with cytosol, and enzyme-linked immunosorbent assay (ELISA) with polyclonal and protein G conjugates and smooth lipopolysaccharide (S-LPS), native hapten polysaccharide (NH), or cytosol antigens. For optimal sensitivity, RBT had to be used with sera-antigen at a 3:1 dilution. In the RID test, Brucella melitensis biotype 1 NH could not be replaced by Brucella abortus biotype 1 or Y. enterocolitica 0:9 polysaccharides. In the ELISA, S-LPS and NH gave similar results and the protein G conjugate increased the specificity. With the sera from 55 B. melitensis culture-positive goats, the sensitivity was 100% for RBT, CFT (titer > or = 4), and ELISA with S-LPS or NH; 94% for RID; and 93% for CIEP. All tests were negative (100% specific) when testing the sera from 127 brucella-free goats. Larger discrepancies among the results of the serological tests were obtained with sera from goats of areas where brucellosis is endemic. When the sera of 20 young goats vaccinated subcutaneously (10(9) CFU of B. melitensis Rev 1) and bled 6 months later were examined, the specificities were as follows: NH ELISA, 60%; CFT and S-LPS ELISA, 75%; RBT, 80%; CIEP, 90%; and RID, 94%. With the sera from 10 young goats vaccinated conjunctivally (10(9) CFU of B. melitensis Rev 1) all tests were 100% specific 4 months after vaccination. The proportion of goats giving a positive reaction after vaccination decreased faster in RID than in other tests.
VP4 typing of bovine and porcine group A rotaviruses by PCR
Authors: Gouvea V , Santos N , Timenetsky M do C
Published: January 2020

A PCR typing assay was developed to identify rotavirus P types (VP4 specificity) of bovine NCDV, UK, and B223 strains and porcine OSU and Gottfried strains. Thirty-nine human and animal strains representing all known, and some undefined, rotavirus P types were used to develop and evaluate the specificity of the method. No cross-amplification was observed. The PCR results agreed with previous characterizations by monoclonal antibodies, sequence analysis, and hybridization assays, except for the Gottfried strain, which showed a P type distinct from the human asymptomatic strains. Analysis of a small number of field specimens suggested a high level of VP4 polymorphism among porcine strains. The assay should be of value in typing field isolates and tracing interspecies infections.
Identification of bovine and porcine rotavirus G types by PCR
Authors: Gouvea V , Santos N , Timenetsky M do C
Published: January 2020

A new seminested PCR typing assay has been extended to identify the important veterinary rotavirus serotypes G5, G6, G10, and G11, as well as the rare human serotype G8. The specificity of the method was evaluated with 30 standard laboratory strains of the G1 to G6 and G8 to G11 types. Rotavirus strain types G6 and G8, not previously recognized in pigs, were identified in field specimens of porcine origin.
Comparison of monoclonal antibody-based sandwich enzyme-linked immunosorbent assay and virus isolation for detection of peste des petits ruminants virus in goat tissues and secretions
Authors: Saliki J T , House J A , Mebus C A , Dubovi E J
Published: January 2020

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (S-ELISA) was developed for specific detection of peste des petits ruminants virus. Compared with virus isolation in Vero cell cultures using 89 paired tissue and secretion samples from six experimentally infected goats, S-ELISA was significantly more sensitive (71.9% versus 65.2%; P < 0.05). The S-ELISA is a suitable alternative to virus isolation.
Mycobacterium smegmatis strain for detection of Mycobacterium tuberculosis by PCR used as internal control for inhibition of amplification and for quantification of bacteria
Authors: Kolk A H , Noordhoek G T , de Leeuw O , Kuijper S , van Embden J D
Published: January 2020

For the detection of Mycobacterium tuberculosis by PCR, the IS6110 sequence was used. A modified target was constructed by insertion of 56 nucleotides in the IS6110 insertion element of Mycobacterium bovis BCG. This modified insertion sequence was integrated into the genome of Mycobacterium smegmatis, a mycobacterium species which does not contain the IS6110 element. When DNA from the modified M. smegmatis 1008 strain was amplified with IS6110-specific primers INS1 and INS2, a band of 301 bp was seen on agarose gel, whereas the PCR product of M. tuberculosis complex DNA was a 245-bp fragment with these primers. The addition of a small number of M. smegmatis 1008 cells to clinical samples before DNA purification enables the detection of problems which may be due to the loss of DNA in the isolation procedure or to the presence of inhibitors. The presence of inhibitors of the amplification reaction can be confirmed by the addition of M. smegmatis 1008 DNA after the DNA isolation procedure. Furthermore, competition between the different target DNAs of M. smegmatis 1008 DNA and M. tuberculosis complex DNA enables the estimation of the number of IS6110 elements in the clinical sample.
Restriction endonuclease analysis of virulence plasmids for molecular epidemiology of Yersinia pseudotuberculosis infections
Authors: Fukushima H , Gomyoda M , Kaneko S , Tsubokura M , Takeda N , Hongo T , Shubin F N
Published: January 2020

Restriction endonuclease analysis of virulence plasmid DNA was used to study the epidemiology of Yersinia pseudotuberculosis infections. The origin of Y. pseudotuberculosis could be divided into two focus areas: Eastern Asia and Europe. Wild animals were an important reservoir for the Y. pseudotuberculosis seen in infections in humans.
Evaluation of commercial amplification kit for detection of Legionella pneumophila in clinical specimens
Authors: Matsiota-Bernard P , Pitsouni E , Legakis N , Nauciel C
Published: January 2020

A commercial kit (EnviroAmp) designed to detect the DNA of Legionella species in environmental water samples using PCR and reverse dot hybridization was applied to clinical specimens. Results correlated well with culture for bronchoalveolar lavages. In addition, this test was easy to perform and showed good sensitivity.
Direct and rapid detection of Erysipelothrix rhusiopathiae DNA in animals by PCR
Authors: Makino S , Okada Y , Maruyama T , Ishikawa K , Takahashi T , Nakamura M , Ezaki T , Morita H
Published: January 2020

Erysipelothrix rhusiopathiae is a gram-positive rod capable of causing erysipelas in swine. To establish a method for specifically detecting E. rhusiopathiae for practical applications, such as for the inspection of slaughterhouses, the feasibility of using primers derived from the DNA sequence coding for 16S rRNA in a PCR-specific detection system was investigated. Oligonucleotide primers were designed to amplify a 407-bp DNA fragment by PCR. The amplification was specific to the Erysipelothrix DNA but not to that of other bacterial genera tested. This PCR-based method efficiently and specifically detected the Erysipelothrix DNA sequence in joint and spleen samples from mice within 6 h, and application of the 407-bp DNA segment from samples containing very low numbers of bacteria (< 20 bacteria per spleen from mice) was possible. Although this PCR amplification is specific for the Erysipelothrix genus, which contains at least two species, E. rhusiopathiae and E. tonsillarum, it can be concluded that all Erysipelothrix strains detected by this PCR system in diseased pigs are E. rhusiopathiae because only E. rhusiopathiae is virulent for pigs. These results show that this PCR amplification system using the DNA sequence coding for 16S rRNA is very rapid and reliable and avoids cumbersome and lengthy cultivation steps, demonstrating that this system could be used for practical applications.
Multiplex PCR assay specific for the multidrug-resistant strain W of Mycobacterium tuberculosis
Authors: Plikaytis B B , Marden J L , Crawford J T , Woodley C L , Butler W R , Shinnick T M
Published: January 2020

In 1991, a multidrug-resistant strain of Mycobacterium tuberculosis was isolated from eight people with tuberculosis at a state correctional facility in New York. This strain, which is designated strain W (IS6110 restriction fragment length polymorphism type 212072), was resistant to isoniazid, rifampin, ethambutol, streptomycin, kanamycin, ethionamide, and rifabutin. Since that outbreak, the W strain has been associated with outbreaks in five hospitals in the New York City area and is a continuing public health problem in the area. To be able to identify this strain rapidly, we developed a multiplex PCR assay which targets a direct repeat of IS6110 with a 556-bp intervening sequence (NTF-1). The amplification generates two amplicons from strain W, which indicate the presence and orientation of the NTF-1 sequence between the direct repeat of IS6110, and a third amplicon, which serves as an internal PCR control. The assay was evaluated with 193 isolates of M. tuberculosis, and all 48 strain W isolates among those 193 isolates were correctly identified.
Evaluation of temperature sensitivity of human papillomavirus type 11 by using the human xenograft severe combined immunodeficiency mouse model
Authors: Bonnez W , Rose R C , Borkhuis C , Da Rin C , Reichman R C
Published: January 2020

The temperature sensitivity of human papillomavirus type 11 was evaluated by using a human xenograft severe combined immunodeficiency mouse model. Incubation of the virus for 1 h at a temperature higher than 56 degrees C but lower than 72 degrees C was sufficient to inhibit the virally induced growth of infected human tissue. However, 100 degrees C was necessary to completely inactivate HPV type 11 genome expression.
Serotypes of Streptococcus pneumoniae blood culture isolates from adults in Franklin County, Ohio
Authors: Plouffe J F , Moore S K , Davis R , Facklam R R
Published: January 2020

Isolates of Streptococcus pneumoniae in cultures of blood from 258 adults seen in 10 Franklin County, Ohio, hospitals from 1991 and 1992 were serotyped. Most strains (230 [89.2%]) belonged to serotypes that are included in the current pneumococcal vaccine. An additional 16 isolates (6.2%) were immunologically related to strains with serotypes that are included in the vaccine. Only 12 isolates (4.6%) were not covered by the vaccine. The rate of mortality from pneumococcal bacteremia in adults remains high (20%). While recent studies have documented the efficacy of the pneumococcal vaccine for preventing pneumococcal bacteremia (56 to 70%), use of the pneumococcal vaccine in susceptible patients by physicians remains low (19% in Franklin County). Additional efforts need to be expended to increase the use of the pneumococcal vaccine.
Reisolation of Ehrlichia canis from blood and tissues of dogs after doxycycline treatment
Authors: Iqbal Z , Rikihisa Y
Published: January 2020

We present evidence that supports the carrier status of dogs experimentally infected with Ehrlichia canis after treatment with doxycycline. Canine ehrlichiosis was induced in five dogs by intravenous inoculation with E. canis-infected DH82 cells. All animals developed mild clinical signs of transient fever, body weight loss, thrombocytopenia, and increased gamma globulin levels in plasma. An indirect fluorescent-antibody test (IFA) revealed that all dogs had seroconverted (titer, 5,120) by day 10 postinoculation (p.i.). E. canis was reisolated from blood samples collected at intervals throughout the 2-month period p.i. Doxycycline was administered orally once daily at 10 mg/kg of body weight per day for 1 week starting at 2 months p.i. Following treatment, gamma globulin levels in plasma were decreased. At necropsy on days 54 to 59 after the start of treatment, spleen, liver, kidney, and lymph nodes were collected for E. canis culture and histopathologic examination. Although the dogs did not show significant clinical signs during or after treatment with the antibiotic, E. canis was reisolated from the blood and tissue samples of three of five dogs. A 16-fold reduction in IFA titer was noted in two dogs which were negative for E. canis reisolation at day 49 after the start of treatment, whereas a zero- to fourfold reduction in IFA titer was seen in the remaining three dogs. Western immunoblot reactions to higher-molecular-size E. canis antigens in the sera of two dogs which were negative for E. canis on blood culture decreased, whereas they remained continuously high or only transiently decreased for the duration of the study for antigens in the sera of three dogs from which E. canis was reisolated. Histopathologically, prominent plasmacytosis in the kidney cortex was present in three dogs from which E. canis was reisolated, whereas the kidney cortices of two dogs had moderate to minor plasmacytosis. These findings pose questions regarding the efficacy, dosage and duration of doxycycline treatment in dogs with E. canis infection. In addition, it was shown that IFA and Western immunoblotting may aid in assessing the efficacy of antibiotic therapy when definitive reisolation procedures are not readily available.
Comparison of commercial kits for detection of cryptococcal antigen
Authors: Tanner D C , Weinstein M P , Fedorciw B , Joho K L , Thorpe J J , Reller L
Published: January 2020

Although kits to detect cryptococcal antigen are used widely to diagnose cryptococcal infection, the comparative performance of commercially available assays has not been evaluated in the past decade. Therefore, we compared the sensitives and specificities of five commercially available kits for detecting cryptococcal antigen (four latex agglutination test kits--Calas [Meridian Diagnostics])--Crypto-LA [International Biological Labs], Myco-Immune [MicroScan], and Immy [Immunomycologics]--and an enzyme immunoassay kit, Premier [Meridian Diagnostics]) with culture for the diagnosis of cryptococcal meningitis and fungemia. Of 182 cerebrospinal fluid (CSF) and 90 serum samples submitted for cryptococcal antigen and fungal culture, 49 (19 and 30 samples, respectively) from 20 patients had a culture positive for Cryptococcus neoformans. For CSF specimens, the sensitivities and specificities of all kits were comparable (sensitivity, 93 to 100%; specificity, 93 to 98%). There was a significant difference in sensitivities of the kits when serum samples were tested with the International Biological Labs and MicroScan kits, which do not pretreat serum with pronase. These kits were less sensitive (sensitivity, 83%) than the Immy and Meridian latex kits (sensitivity, 97%), which do pretreat with pronase. The sensitivity of the Meridian enzyme immunoassay kit was comparable to that of the pronase-containing latex kits. These kits were of equivalent specificities (93 to 100%) when testing serum. Some of the currently available kits have limitations that need to be recognized for proper interpretation of results. Specifically, the use of pronase on serum samples reduces the number of false-positive results, and a titer of < or = 1:4 can be a false-positive result when CSF samples are being tested.
Polyclonal infections due to Mycobacterium avium complex in patients with AIDS detected by pulsed-field gel electrophoresis of sequential clinical isolates
Authors: Slutsky A M , Arbeit R D , Barber T W , Rich J , von Reyn C F , Pieciak W , Barlow M A , Maslow J N
Published: January 2020

Invasive infection with organisms of the Mycobacterium avium complex (MAC) is common among patients with advanced human immunodeficiency virus infection. In previous studies, we analyzed multiple individual colonies of MAC isolated from specimens obtained at the same time and observed that 14 to 20% of patients are simultaneously infected with more than one strain. In this study, we examined sequential isolates from 12 patients with AIDS who had two or more MAC isolates available from clinical specimens collected more than 1 week apart; the intervals between the first and last specimens ranged from 8 to 192 (median, 46) days. For each isolate, restriction digests of genomic DNA were analyzed by pulsed-field gel electrophoresis; DNA was prepared by using a protocol, described here in detail, which had been optimized for conditions of bacterial growth and lysis. The pulsed-field gel electrophoresis analysis identified four patients (33%) infected with two different MAC strains. Both M. avium and M. intracellulare were cultured from blood specimens from two patients. In each of the four patients, the second strain was identified from a culture taken within 14 days of the initial study isolate, and in three of these patients, the first strain was detected again in a subsequent culture. These observations suggest that the presence of two different strains among isolates from sequential cultures may reflect ongoing polyclonal infection. We conclude that polyclonal infection with MAC is common among patients with AIDS. The identification of such infections may be critical in the development of effective treatments.
Tentative evidence of AIDS-associated biotype of Mycobacterium kansasii
Authors: Tortoli E , Simonetti M T , Lacchini C , Penati V , Urbano P
Published: January 2020

Previous studies revealed heterogeneous behavior within the species Mycobacterium kansasii against commercially available DNA probes (Accuprobe M. kansasii culture identification test; Gen-Probe); several isolates, conventionally identified as M. kansasii, failed in fact to hybridize. Looking for a possible association with phenotypic features, we tested a fully characterized panel of 69 clinical isolates of M. kansasii (19 of which were Accuprobe negative) with a semiquantitative micromethod which tests for 19 enzymatic activities (Api Zym; BioMérieux). The strains were from 25 hospitals in 18 Italian towns; 20 isolates came from human immunodeficiency virus type 1-positive patients who fulfilled the Centers for Disease Control criteria for AIDS diagnosis. On the basis of the whole set of phenotypic traits, our strains clustered in two groups, allowing the differentiation of biotypes within the species. There was a perfect association between biotype 2 and hybridization failures with Accuprobe and a very significant association between this novel biotype 2 and AIDS status, which suggests that it differs in virulence.
Three new isolates of porcine respiratory coronavirus with various pathogenicities and spike (S) gene deletions
Authors: Vaughn E M , Halbur P G , Paul P S
Published: January 2020

Three new isolates of porcine respiratory coronavirus (PRCV) were isolated and partially characterized. These PRCV isolates showed a selective tropism for respiratory tissue and were antigenically related to transmissible gastroenteritis virus. PCR amplification of the 5' half of the spike (S) genes of the three PRCV isolates indicated that a large deletion, characteristic of PRCV, was present. By using cDNA probes specific for the transmissible gastroenteritis virus S gene, the PCR products were shown to be specific in a Southern blot. The three new PRCV isolates were shown to vary in S gene deletion size. In a separate study, these isolates have also been shown to vary in pathogenicity. These new PRCV isolates should serve as important tools in gaining a better understanding of the pathogenesis of coronavirus infections.
Biochemical identification of citrobacteria in the clinical laboratory
Authors: Janda J M , Abbott S L , Cheung W K , Hanson D F
Published: January 2020

We biochemically identified 235 Citrobacter strains to the species level on the basis of the recently proposed taxonomic changes of Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993). Citrobacter isolates were initially identified as C. koseri or as members of the C. freundii complex or C. amalonaticus group on the basis of indole production, formation of H2S, malonate utilization, and acid production from D-arabitol and adonitol. On the basis of the results of these tests, 68% of the Citrobacter strains were identified as members of the C. freundii complex, 25% were C. koseri, and 8% were members of the C. amalonaticus group. By using a 15-test system recently proposed by Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993) to help identify new species in the C. freundii complex and C. amalonaticus group, 81% of the C. freundii complex strains and 100% of the C. amalonaticus strains could be definitively assigned to one of the previously established or recently designated species or hybridization groups of the genus Citrobacter. Within the C. freundii complex, C. freundii predominated overall (37%), followed by C. youngae (24%), C. braakii (13%), and C. werkmanii (6%). Only one strain each of C. sedlakii and Citrobacter DNA group 11 was identified in this study. Among C. amalonaticus complex members, all were identified as C. amalonaticus with the singular exception of one fecal isolate of C. farmeri. C. freundii and C. koseri were the two Citrobacter species most commonly (80 of 93 [86%]) isolated from extraintestinal sources (genitourinary tract, wounds, blood).
Characterization of Leptospira isolates from serovar hardjo by ribotyping, arbitrarily primed PCR, and mapped restriction site polymorphisms
Authors: Perolat P , Merien F , Ellis W A , Baranton G
Published: January 2020

Leptospira serovar hardjo isolates of the hardjoprajitno and hardjobovis genotypes were characterized by ribotyping, arbitrarily primed PCR (AP-PCR) fingerprinting, and the study of mapped restriction site polymorphisms (MRSPs) in rrs and rrl genes. After restriction of chromosomal DNA with BglII, EcoRI, or HindIII, each genotype was individualized with a distinct ribotype. The fingerprints produced by AP-PCR with seven primers clearly separated the two groups; primers KF and RSP produced species-specific products which assigned hardjoprajitno and hardjobovis isolates to the species L. interrogans sensu stricto and L. borgpetersenii, respectively. Furthermore, AP-PCR fingerprints gave evidence of a considerable genomic heterogeneity at the strain level among the hardjobovis group. Conversely, the hardjoprajitno group was homogeneous. MRSP profiles in ribosomal genes indicated that hardjoprajitno and hardjobovis isolates belonged to L. interrogans MRSP group B and L. borgpetersenii group C, respectively. AP-PCR and determination of MRSPs in ribosomal genes proved to be quick and reliable methods for typing Leptospira strains and for studying intraspecific population structures.
Evaluation of Biolog system for identification of some gram-negative bacteria of clinical importance
Authors: Holmes B , Costas M , Ganner M , On S L , Stevens M
Published: January 2020

The Biolog system (Biolog, Inc., Hayward, Calif.) was evaluated for the identification of 55 gram-negative taxa (789 strains) likely to be encountered commonly in clinical laboratories. The Biolog system performed best with oxidase-positive fermenters and biochemically active nonfermenters and had the most problems with unreactive nonfermenters. It gave significantly better results when the MicroPlates were read manually rather than when they were read by the automated reader. Plates read manually gave the following performances: oxidase-positive fermenters, five taxa, 64 strains, 92% correct, 3% not identified, and 5% incorrect; biochemically active nonfermenters, eight taxa, 122 strains, 88% correct, 6% not identified, and 6% incorrect; members of the family Enterobacteriaceae, 35 taxa, 511 strains, 77% correct, 8% not identified, and 15% incorrect; unreactive nonfermenters, seven taxa, 92 strains, 38% correct, 24% not identified, and 38% incorrect. We found the system easy to use, but while for 39 of 55 of the taxa an identification rate of > 70% was achieved, problems were encountered, particularly with identification of capsulated strains of some Enterobacter and Klebsiella taxa, as well as the least biochemically active Moraxella and Neisseria strains.
Evaluation of microagglutination test for differentiation between Serpulina (Treponema) hyodysenteriae and S. innocens and serotyping of S. hyodysenteriae
Authors: Diarra A T , Mittal K R , Achacha M
Published: January 2020

Swine dysentery is a mucohemorrhagic diarrheal disease caused by the anaerobic spirochete Serpulina hyodysenteriae. At present, the serotyping is done by immunodiffusion testing with lipopolysaccharide (LPS) extract as antigen and rabbit hyperimmune sera produced against different serotypes of S. hyodysenteriae. Since the preparation of LPS is time-consuming and requires a large quantity of bacteria, it is desirable to use a serotyping method which does not require the extraction of LPS. In the present investigation, microagglutination was evaluated by using both formalinized whole- and boiled-cell suspensions as antigens and rabbit hyperimmune sera produced against formalinized whole-cell suspensions of reference strains of S. hyodysenteriae and S. innocens B256. Use of boiled cell suspension as antigen permitted the differentiation between isolates of S. hyodysenteriae and S. innocens as well as serotyping of S. hyodysenteriae strains accurately. A total of 18 isolates were identified as S. hyodysenteriae, and 3 isolates were identified as S. innocens. The microagglutination test was found specific, sensitive, and easy to perform; thus, it was judged suitable for routine identification and serotyping of S. hyodysenteriae isolates.
Evaluation of a novel colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates
Authors: Pfaller M A , Barry A L
Published: January 2020

A comparative evaluation of two broth microdilution methods for antifungal susceptibility testing of 600 clinical yeast isolates (Candida spp., Torulopsis glabrata, and Cryptococcus neoformans) against amphotericin B, fluconazole, and flucytosine (5FC) was conducted. Microdilution testing was performed according to National Committee for Clinical Laboratory Standards (NCCLS) recommendations (NCCLS document M27-P). By using the growth control for comparison, reference microdilution MIC endpoints for amphotericin B were scored as the lowest concentration at which a score of 0 (complete absence of growth) was observed, and those for 5FC and fluconazole were scored at the lowest concentration at which a score of 2 (prominent decrease in turbidity) (MIC-2) was observed. The second microdilution method employed a colorimetric endpoint using an oxidation-reduction indicator (Alamar Biosciences, Inc., Sacramento, Calif.) and was assessed independently of the reference microdilution MICs. The MICs for the two microdilution test systems were read after 24 and 48 h of incubation. Excellent agreement between the reference and colorimetric microdilution MICs was observed. Overall agreement was > or = 95% for all three drugs at 24 h. At 48 h, agreement was > or = 98% for amphotericin B and 5FC but dropped to 84% for fluconazole. Given these results it appears that the colorimetric microdilution approach to antifungal susceptibility testing may be viable alternative to the NCCLS reference method for testing yeasts.
Effects of enhancing agents on detection of cytomegalovirus in clinical specimens
Authors: St George K , Rinaldo C R
Published: January 2020

Dimethyl sulfoxide, dexamethasone, and calcium were tested in combination for their enhancing effects on cytomegalovirus detection in shell vial cultures on 1,579 clinical specimens obtained primarily from adult solid-organ transplant recipients. Fluorescent-focus counts were elevated for the cytomegalovirus-positive urine specimens (P < 0.01) and throat washings (P < 0.05) but not for the tissue biopsy or blood samples. Epidermal growth factor also increased focus counts but provided no additional benefit when used in combination with the other agents. The triple-combination treatment did not increase the number of positive specimens identified.
Clinical comparison of isolator and thiol broth with ESP aerobic and anaerobic bottles for recovery of pathogens from blood
Authors: Kellogg J A , Bankert D A , Manzella J P , Parsey K S , Scott S L , Cavanaugh S H
Published: January 2020

The recovery of pathogens and the speed of their detection were determined for our conventional blood culture system (an Isolator [Wampole] and a 100-ml Thiol bottle [Difco]) compared with automated ESP aerobic and anaerobic bottles (80 ml each; Difco). Each of the four culture devices was inoculated with approximately 10 ml of blood from symptomatic patients weighing more than 80 lb (ca. 36 kg). From 7,070 sets of cultures for 2,841 patients, 607 clinically significant isolates were recovered: 456 (75.1%) from the Isolator, 353 (58.2%) from Thiol, 377 (62.1%) from ESP aerobic bottles, and 346 (57.0%) from ESP anaerobic bottles. Of the 607 isolates, 149 (24.5%) were detected only with the conventional system (Isolator and/or Thiol), and 65 (10.7%) were detected only with the ESP two-bottle system (P < 0.001). Our conventional system allowed for detection of significantly more isolates of members of the family Enterobacteriaceae (P < 0.001), Staphylococcus aureus (P < 0.01), Staphylococcus spp. (coagulase-negative) (P < 0.01), and Enterococcus spp. (P < 0.05), and ESP facilitated detection of significantly more isolates of S. pneumoniae (P < 0.01). When all four devices in a culture set were positive for the same isolate, no microbial species or group was detected significantly earlier ( > or = 24 h) by either blood culture system. The Isolator contamination rate (4.8%) was > or = 6 times the rate for any of the bottles. Of pathogens detected by the Isolator, 50% were recovered in counts of < or = 1.0 CFU/ml and 18% were recovered only as a single colony. The ESP system offered an automated, less labor-intensive blood culture system for which routine subcultures were not required, but the important considerations of culturing large volumes of blood and of obtaining at least two sets from each patient in our population were reemphasized.
Application of typing by pulsed-field gel electrophoresis to the study of Clostridium difficile in a neonatal intensive care unit
Authors: Kato H , Kato N , Watanabe K , Ueno K , Ushijima H , Hashira S , Abe T
Published: January 2020

Pulsed-field gel electrophoresis (PFGE) analysis of restriction pattern polymorphism was applied to type Clostridium difficile isolated from neonates hospitalized in a neonatal intensive care unit, and the results were compared with those of immunoblot analysis. C. difficile was isolated from fecal specimens of 41 (61%) of 67 neonates during a 5-month investigation. All of these neonates were asymptomatic. Fifty-five C. difficile isolates from 32 patients were analyzed by PFGE after digestion with SmaI and SacII endonucleases and by immunoblotting with 10 different antisera. Fifty-three of 55 isolates from 30 patients were identical by PFGE analysis after SmaI and SacII digestion and immunoblot analysis. Two isolates were different from each other and from the epidemic group by both PFGE and immunoblot analysis. All 53 epidemic isolates were nontoxigenic, while the two remaining isolates were toxigenic. These results suggest that nosocomial spread of nontoxigenic C. difficile infection in the neonatal intensive care unit and suggest that both PFGE and immunoblot are powerful typing tools for the epidemiological study of C. difficile.