bluuebunny/crossref_metadata_2025 huggingface dataset:
Interpretive criteria and quality control limits for testing susceptibility of Neisseria gonorrhoeae to enoxacin
Authors: Barry A L , Cohen M A , Sesnie J C , Fuchs P C , Washington J A , Murray P R , Baker C
Published: January 2020

For testing the susceptibility of Neisseria gonorrhoeae to enoxacin, a proposed susceptibility category includes strains for which MICs are less than or equal to 0.5 micrograms/ml and zones of inhibition are greater than or equal to 32 mm in diameter. Because of the sparcity of resistant gonococci, a resistance category was not defined, but laboratory-selected resistant mutants were appropriately categorized by the proposed criteria. A review of clinical data confirmed the utility of a single 400-mg oral dose of enoxacin for treating gonorrhea caused by strains judged to be susceptible by the proposed criteria. For quality control purposes, for N. gonorrhoeae ATCC 49226 MICs should be 0.016 to 0.06 micrograms/ml and zones of inhibition should be 43 to 51 mm in diameter.
Ribotyping of coagulase-negative staphylococci with special emphasis on intraspecific typing of Staphylococcus epidermidis
Authors: Izard N C , Hächler H , Grehn M , Kayser F H
Published: January 2020

Coagulase-negative staphylococci (CoNS), particularly Staphylococcus epidermidis, are increasingly being recognized as opportunistic pathogens. They are often multiply antibiotic resistant and can cause nosocomial outbreaks. For clinical and epidemiological reasons, accurate species identification and typing are imperative. Ribotyping, i.e., the generation of characteristic fragment patterns by hybridization of restriction endonuclease fragments of total DNA with labeled standard rRNA from Escherichia coli, has been applied to CoNS for species identification by various investigators. The present study, involving 115 randomly collected clinical isolates of CoNS, provides ambiguous evidence with respect to those findings. Eighty six S. epidermidis strains were ribotyped intraspecifically. Eleven different ribotypes were found after digestion with EcoRI, and 10 were found with HindIII. A combination of the two restriction endonucleases resulted in an increase in the discriminatory power (DP) from 14.3 to 31.6%. A combination of ribotyping with biotyping raised the DP to a maximum of 48.6%. The reproducibility of ribotyping was 100% after greater than 400 generations of growth. No correlation between methicillin resistance and certain ribotypes among the S. epidermidis strains was observed. Ribotyping is considered a useful tool for the intraspecific typing of CoNS for epidemiological purposes. The DP can be increased by the use of additional restriction endonucleases.
Rapid epidemiologic analysis of cytomegalovirus by using polymerase chain reaction amplification of the L-S junction region
Authors: Sokol D M , Demmler G J , Buffone G J
Published: January 2020

A technique based on polymerase chain reaction (PCR) amplification was developed to facilitate the study of the epidemiology of cytomegalovirus (CMV). Consensus oligonucleotide primers from repetitive DNA sequences were designed to amplify interspersed repetitive sequences in an area of heterogeneity within the L-S junction region of the CMV genome, and PCR products were detected by gel electrophoresis. Purified CMV DNAs from 25 CMV isolates, 13 from members of five families in which person-to-person transmission was documented, 9 random clinical isolates of CMV, and 3 laboratory reference strains of CMV (Towne, Davis, and AD169), were analyzed. The gel electrophoretic patterns of DNA bands, or PCR profiles, produced by amplification with the L-S primers were unique for epidemiologically unrelated strains and laboratory reference strains, yet similar patterns were observed for epidemiologically related strains isolated from members of the same family. This method of rapid fingerprinting of CMV DNA within the hypervariable L-S junction region by PCR to produce strain-specific, variably sized PCR products should simplify the molecular epidemiologic analysis of CMV.
Comparison of three nonradioisotopic polymerase chain reaction-based methods for detection of human immunodeficiency virus type 1
Authors: Whetsell A J , Drew J B , Milman G , Hoff R , Dragon E A , Adler K , Hui J , Otto P , Gupta P , Farzadegan H
Published: January 2020

Three nonradioisotopic polymerase chain reaction (PCR)-based detection techniques were evaluated for sensitivity and specificity in detecting human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells. The Roche prototype HIV-1 PCR assay, the Du Pont enzyme-linked oligonucleotide sandwich assay (ELOSA), and the Gen-Probe hybridization protection assay (HPA) were compared with a standard radioisotopic oligonucleotide solution hybridization (OSH) technique. A panel of 111 well-characterized clinical samples that included peripheral blood mononuclear cells from 48 healthy, low-risk, HIV-1 antibody-negative subjects, 24 antibody-positive subjects with stable CD4 counts of less than 200/mm3, and 39 antibody-positive subjects with stable CD4 counts of greater than 800/mm3 were studied. Each method demonstrated good specificity, ranging between 96 and 100%; those of the OSH and ELOSA (Du Pont) were 100%, those of the HPA (Gen-Probe) were 100% with one probe and 96% with the other probe, and that of the HIV-1 PCR assay (Roche) was 96%. Sensitivities ranged from 96 to 100% for the low-CD4-count group, with the OSH, the HIV-1 PCR assay (Roche), and the HPA (Gen-Probe) all attaining a sensitivity of 100%. For the high-CD4-count group, sensitivities ranged from 69 to 97%, with the OSH attaining a sensitivity of 97% and the HPA attaining sensitivities of 97% with one probe and 95% with the other probe. These data indicate that the nonradioisotopic techniques are sensitive and specific for the detection of HIV-1 proviral DNA in clinical samples.
Serologic confirmation of simian T-lymphotropic virus type I infection by using immunoassays developed for human T-lymphotropic virus antibody detection
Authors: Rudolph D L , Yee J , Mone J , Foung S K , Lipka J J , Reyes G R , Hadlock K , Chan L , Villinger F , Lairmore M D
Published: January 2020

Serum specimens from diverse species of Old World monkeys, categorized as seropositive (n = 97) or seronegative (n = 23) for human T-lymphotropic virus (HTLV) infection, were tested by using recombinant env-spiked Western immunoblot assays and synthetic peptide assays for simultaneous detection and discrimination of simian T-lymphotropic virus (STLV) infection. Of the 97 seropositive specimens, 93 reacted with the recombinant transmembrane (r21env) protein and 90 reacted with a recombinant, MTA-1, derived from the central region of the external glycoprotein of HTLV-I (rgp46env), thus yielding test sensitivities of 96 and 93%, respectively. While 1 of the 23 negative monkey specimens reacted with r21env, none reacted with rgp46env, for overall specificities of 96 and 100%, respectively. Analysis of synthetic peptide-based immunoassays demonstrated that while 85 of 97 (88%) seropositive specimens reacted with HTLV-I-specific epitope (p19gag), none of the specimens reacted with HTLV-II-specific epitope (gp52env). These results show that recombinant envelope-spiked Western blots provide a simple means for serologic confirmation of STLV-I infection and that type-specific synthetic peptides can be used to confirm the virus type in seropositive monkey specimens.
Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts
Authors: Kielstein P , Rapp-Gabrielson V J
Published: January 2020

Previous independent investigations of the serotyping of Haemophilus parasuis strains have led to the designation of serovars A to D, 1 to 7, Jena 6 to Jena 12, and ND1 to ND5. Heat-stable antigen preparations from the reference strains for these serovars were tested by immunodiffusion with rabbit hyperimmune antisera. The existence of 15 distinct serologic groups was apparent, for which we propose the designations serovars 1 to 15. Examination of 290 field isolates from swine in the former German Democratic Republic indicated a prevalence of serovars 4 and 5, which together accounted for 41% of the isolates examined. However, 26.2% of the isolates were nontypeable with this test procedure and available antisera. Intraperitoneal inoculation of specific-pathogen-free pigs with cells representing the 15 serovars indicated differences in virulence which may be serovar related. Cells of strains representing serovars 1, 5, 10, 12, 13, and 14 were the most virulent, causing death or moribundity in inoculated pigs. Cells of serovars 2, 4, 8, and 15 caused polyserositis, but not death, in inoculated pigs. However, inoculation of cells of strains representing serovars 3, 6, 7, 9, and 11 resulted in no clinical symptoms or lesions indicative of H. parasuis infection.
Direct detection of influenza virus antigen in nasopharyngeal specimens by direct enzyme immunoassay in comparison with quantitating virus shedding
Authors: Döller G , Schuy W , Tjhen K Y , Stekeler B , Gerth H J
Published: January 2020

We developed a direct enzyme immunoassay [EIA; Enzygnost Influenza A(Ag) and Enzygnost Influenza B(Ag)] for the direct detection of influenza A and B virus antigens in nasopharyngeal secretion specimens (NPS). The test is performed without sonification of specimens, and results are obtained within 4 h. A direct comparison between direct EIA and quantitation of virus shedding for influenza A and B virus antigen detection was carried out. A total of 210 NPS and 98 nasopharyngeal wash specimens (NPW) were investigated. We isolated influenza A viruses from 79 (37.6%) of 210 NPS; of these 79 cell-culture-positive NPS, 70 (88.6%) were also positive by direct EIA. Of 29 (13.8%) NPS from which influenza B virus was isolated, 24 (82.8%) NPS were positive by direct EIA. Virus shedding was determined quantitatively in 48 NPS from patients with influenza A and in 24 NPS from patients with influenza B. Only a crude correlation between optical density values and virus concentrations was observed. Detection of influenza virus antigens in NPS by direct EIA showed sensitivities of 89.7% for influenza A virus and 87.9% for influenza B virus and specificities of 99.3% for influenza A virus and 100% for influenza B virus. With direct EIA, all NPW were negative for influenza A virus, although virus was isolated from 21 (21.4%) NPW. Of 15 NPW from which influenza B virus was isolated, 7 showed positive results in direct EIA. In addition, direct EIA is suitable for detecting influenza A and B viruses in cell cultures before the appearance of any cytopathic effects and can be used as a cell culture confirmation test.
Isolation and characterization of a species-specific DNA fragment for detection of Candida albicans by polymerase chain reaction
Authors: Miyakawa Y , Mabuchi T , Kagaya K , Fukazawa Y
Published: January 2020

A 2-kbp DNA fragment, EO3, that was present in multiple copies in the Candida albicans genome was isolated for use in developing a detection method for C. albicans by polymerase chain reaction (PCR). Dot blot hybridization revealed that EO3 was specific for the 40 isolates of C. albicans serotypes A and B used. Using a set of primers (20-mer each) derived from the nucleotide sequence of EO3, we performed specific amplification of a 1.8-kbp DNA fragment within EO3 by PCR. All 40 isolates belonging to C. albicans serotypes A and B contained amplifiable 1.8-bkp fragments, although the DNA of the amplified products exhibited small variations in size, yielding three different fragment groups. Southern blot hybridization probed with EO3 showed that these 1.8-kbp fragments were derived from the EO3 region. Conversely, the 1.8-kbp fragment was not amplified from 38 isolates belonging to seven other medically important Candida species or from isolates of Cryptococcus neoformans, Saccharomyces cerevisiae, various bacteria, and a human cell line. The detection limit of the PCR assay for C. albicans with the EO3 fragment was shown to be approximately 2 to 10 cells and 100 cells in saline and human urine, respectively, by ethidium bromide staining and 2 and 10 cells, respectively, by Southern blot analysis. In addition, EO3 was assumed to originate from mitochondrial DNA on the basis of the results of its characterizations. These results indicate that the PCR system using the 1.8-kbp fragment as a target is a reliable method for identifying C. albicans isolates, thereby suggesting its potentials for specific and sensitive detection of C. albicans in samples from patients with candidiasis.
Neutralizing serum antibodies to serotype 6 human rotaviruses PA151 and PA169 in Ecuadorian and German children
Authors: Brüssow H , Gerna G , Sidoti J , Sarasini A
Published: January 2020

Serum samples from 726 Ecuadorian children who underwent natural rotavirus (RV) exposure were tested for neutralizing serum antibodies against two serotype 6 (ST6) human RV (HRV) isolates from Italy, PA151 and PA169, and two ST6 bovine RV (BRV) isolates, NCDV and UK. Gene 4 was distinct in all four ST6 strains. Ninety-one, 56, 67, and 65 serum samples neutralized HRV PA151 (13%), HRV PA169 (8%), BRV NCDV (9%), and BRV UK (9%), respectively. A total of 44 of the 91 serum samples which neutralized HRV PA151 did not neutralize the other three ST6 RV strains. In addition, we identified three serum samples that neutralized HRV PA151 but none of the six human or four animal RV STs. However, we failed to identify serum samples that neutralized HRV PA169 without neutralizing at least one of the major HRV STs. With a hospital-based serum collection from German children (excluding gastroenteritis patients), we identified 3 out of 197 serum samples tested that neutralized HRV PA151 specifically, whereas none neutralized HRV PA169 exclusively. None of the 71 German infants hospitalized with primary RV gastroenteritis showed a PA151- or a PA169-specific antibody response.
Typing of Enterococcus species by DNA restriction fragment analysis
Authors: Hall L M , Duke B , Guiney M , Williams R
Published: January 2020

Enterococci are a frequent cause of hospital-acquired infection, being associated with urinary tract infections, wound sepsis, bacteremia, and endocarditis. The source of infection is usually thought to be endogenous, but some evidence points to cross-infection between patients. A better understanding of the epidemiology of enterococci has been limited by the lack of a good discriminatory typing system. This report describes the application of two DNA-based typing methods to Enterococcus faecalis and Enterococcus faecium: comparison of restriction fragments from total DNA by conventional electrophoresis and comparison of restriction fragments hybridizing to an rRNA gene probe (ribotyping). Comparison of restriction fragments (from SstI digestion) by conventional electrophoresis was simple and highly discriminatory. The results of analysis of blood culture isolates and of repeat isolates from individual patients are reported. Ribotyping (with BscI digestion) was more applicable at the level of species discrimination.
Development of DNA probes for early diagnosis and epidemiological study of cryptococcosis in AIDS patients
Authors: Polacheck I , Lebens G , Hicks J B
Published: January 2020

We report the isolation of middle-repetitive DNA sequences from Cryptococcus neoformans that are species and variety specific. These probes were used for assessing strain relatedness among cryptococcal isolates from patients with and without AIDS who were from Zaire and the United States. Five distinct hybridization patterns were observed for the 60 isolates examined, regardless of the restriction enzyme used for digestion. The most common pattern among the isolates from the patients without AIDS was also the most common among the isolates from the patients with AIDS who were from the United States and was the only pattern observed for all isolates tested from patients with AIDS who were from Zaire. On the basis of the high specificity and sensitivity of the signals observed by hybridization, we suggest that these sequences provide a means for both biotyping and early diagnosis of C. neoformans.
Molecular cloning of a highly repeated DNA element from Mycobacterium tuberculosis and its use as an epidemiological tool
Authors: Ross B C , Raios K , Jackson K , Dwyer B
Published: January 2020

In order to develop a technique for distinguishing between isolates of Mycobacterium tuberculosis, we cloned two hypervariable DNA fragments from NdeII-digested genomic DNA. The cloned DNA fragments of 3.8 and 4.7 kb were found to contain the same repetitive element, which was different from previously characterized repetitive elements. It is present in at least 30 copies per genome and is distributed among mycobacterial species other than those of the tuberculosis complex, including M. kansaii, M. gastri, and M. szulgai. When used as a probe on restriction enzyme-digested DNA, it can distinguish between strains from unrelated cases of tuberculosis while demonstrating identical banding patterns for isolates from epidemiologically related cases.
DNA fingerprinting by pulsed-field gel electrophoresis is more effective than ribotyping in distinguishing among methicillin-resistant Staphylococcus aureus isolates
Authors: Prevost G , Jaulhac B , Piemont Y
Published: January 2020

Pulsed-field gel electrophoresis (PFGE) after SmaI restriction of DNA from 239 methicillin-resistant Staphylococcus aureus isolates (from 142 patients) produced 26 different fingerprints. The deduced chromosome sizes ranged from 2,200 to 3,100 kb (+/- 100 kb). A total of 81 isolates taken from 65 patients were then typed by PFGE and ribotyping with ClaI, EcoRI, and HindIII. Ribotypes were less discriminating than PFGE. Ribotyping did not discriminate isolates from a given PFGE fingerprint into different subsets. PFGE may be a more effective epidemiological tool than ribotyping for the typing of methicillin-resistant S. aureus strains.
Evaluation of the standardized disk diffusion and agar dilution antibiotic susceptibility test methods by using strains of Neisseria gonorrhoeae from the United States and Southeast Asia
Authors: Putnam S D , Lavin B S , Stone J R , Oldfield E C , Hooper D G
Published: January 2020

Presently, most Neisseria gonorrhoeae testing is based on beta-lactamase tests and agar dilution with common therapeutic agents. The National Committee for Clinical Laboratory Standards (NCCLS) recently described a disk diffusion test that produced results similar to the reference agar dilution method for the antibiotic susceptibility of N. gonorrhoeae. We obtained 71 gonococcal isolates from active-duty males aboard a United States Navy vessel while deployed in the Western Pacific during 1989. In addition, 47 isolates of N. gonorrhoeae were obtained from sexually transmitted disease clinics within the branch clinic operations of the Naval Hospital, San Diego (SD), and tested. Antibiotic susceptibility tests by using the NCCLS agar dilution and disk diffusion techniques were compared. Among the Southeast Asia (SEA) isolates, 47% were beta-lactamase producers compared with 10.5% of the SD isolates. The mean MICs (SEA/SD) in micrograms per milliliter for both groups were as follows: penicillin, 88/15; tetracycline, 2.2/0.95; erythromycin, 1.2/0.49; ceftriaxone, 0.016/0.012; cefotaxime, 0.034/0.03; cefuroxime, 0.44/0.17; cefoxitin, 1.3/0.97; spectinomycin, 150/131; ciprofloxacin, 0.07/0.034; norfloxacin, 0.77/0.29; lomefloxacin, 0.15/0.0.056; and ofloxacin, 0.07/0.036. The established NCCLS interpretive criteria for both susceptibility methods appear applicable to domestic gonococcal strains. However, modifications may be necessary for the more antimicrobial agent-resistant SEA isolates on the basis of the clinical success and cure rates following the indicated single-dose regimens for the geographic region.
Comparison of different approaches to measuring influenza A virus-specific hemagglutination inhibition antibodies in the presence of serum inhibitors
Authors: Subbarao E K , Kawaoka Y , Ryan-Poirier K , Clements M L , Murphy B R
Published: January 2020

The A/Los Angeles/2/87 (H3N2) (A/LA/2/87) virus is sensitive to inhibitors of hemagglutination present in certain human sera. It was found that the effect of these inhibitors could be removed by treating sera with high-concentration receptor-destroying enzyme or trypsin-periodate or by using inhibitor-resistant viruses in the hemagglutination inhibition (HAI) test. Inhibitor-resistant viruses were not effective for detecting rises in antibody titers in the sera of volunteers infected with the A/LA/2/87 wild-type virus, while rises in antibody titer were readily detected in sera treated with trypsin-periodate and tested against A/LA/2/87 wild-type virus in an HAI test. It is therefore suggested that chemical or enzymatic methods be used to inactivate serum inhibitors and that standard virus be used in the HAI test for the currently circulating H3N2 viruses.
Time course of anti-SL-IV immunoglobulin G antibodies in patients with tuberculosis and tuberculosis-associated AIDS
Authors: Martin-Casabona N , Gonzalez Fuente T , Papa F , Rosselló Urgell J , Vidal Plá R , Codina Grau G , Ruiz Camps I
Published: January 2020

Immunoglobulin G (IgG) and IgM antibodies against the SL-IV antigen of Mycobacterium tuberculosis in the sera of patients with tuberculosis with negative serology for human immunodeficiency virus (HIV) infection (TB group; n = 97), patients with tuberculosis with positive serology for HIV infection (TB-HIV group; n = 59), and healthy controls (n = 289) were determined by enzyme-linked immunosorbent assay. All sera were obtained at the onset of tuberculosis, i.e., when clinical symptoms appeared. Clinical specimens were collected and cultured for the isolation of M. tuberculosis, and treatment with antituberculous drugs was started. Sera were also obtained from patients in the TB group at fixed intervals during treatment; sera were available from 13 patients in the TB-HIV group before the onset of tuberculosis. The best specificity and positive predictive values were obtained with the IgG assays. In the IgG assays at specificities above 96.0%, the sensitivities of the tests were 45.3 and 72.8% for the TB and TB-HIV groups, respectively, and the sensitivity was 51.9% when data from both groups were combined for analysis. For the TB group, results of this study indicated that the levels of IgG antibodies remain high during treatment. Thus, repetitive serological assays may not be useful for treatment follow-up. In the TB-HIV group, 12 of 13 patients had IgG-specific antibodies against the SL-IV antigen between 1 and 30 months before the onset of tuberculosis, so we suggest that the IgG antibody assay against SL-IV may be helpful for identifying tuberculosis in patients infected with HIV.
Use of serum antibody and lysozyme levels for diagnosis of leprosy and tuberculosis
Authors: Near K A , Lefford M J
Published: January 2020

Active tuberculosis (TB) and leprosy are difficult to diagnose early because there are few organisms to detect and the specific immune response does not distinguish between active and inactive disease. We developed an immunoassay for lysozyme to see whether serum lysozyme levels could be used to identify individuals with clinical leprosy or TB. The immunoassay for lysozyme proved superior to standard enzyme assays that were less sensitive and reliable. The lysozyme assay was compared with assays for antibodies to Mycobacterium tuberculosis lipoarabinomannan (LAM) and M. leprae phenolic glycolipid-1. The sera tested were from Ethiopian leprosy (paucibacillary and multibacillary) and TB patients and from healthy Ethiopian and U.S. controls. The lysozyme assay was able to detect more of the individuals with TB (sensitivity, 100% for 19 patients) or leprosy (sensitivity, 86% for 36 patients) than either antibody assay. In particular, lysozyme levels were raised in a higher proportion of the paucibacillary leprosy patients (83% of 17), for whom the antibody assays were less sensitive; the LAM IgG and the phenolic glycolipid-1 IgM levels were raised in only 62 and 44% of 16 patients, respectively. The data suggest that lysozyme measurements may be useful in the diagnosis of mycobacterial infections and other chronic infectious granulomatoses.
New assays for quantitative determination of viral markers in management of chronic hepatitis B virus infection
Authors: Zoulim F , Mimms L , Floreani M , Pichoud C , Chemin I , Kay A , Vitvitski L , Trepo C
Published: January 2020

We performed a quantitative study of serum hepatitis B virus (HBV) markers, including new parameters such as pre-S1 antigen (Ag), pre-S2 Ag, and anti-HBx, in 88 chronic hepatitis B surface antigen (HBsAg) carriers. New IMx assays for HBsAg and immunoglobulin M (IgM) anti-HBc detection were also used. The population studied was composed of 65 chronic hepatitis cases (40 positive for hepatitis B antigen [HBeAg] and 25 positive for anti-HBe) and 23 anti-HBe-positive, asymptomatic HBsAg carriers. Serum HBsAg levels detected by IMx were higher in HBeAg-positive than in anti-HBe-positive HBsAg carriers (all patient subgroups included) and correlated with the serum HBV DNA level (P = 0.0001). Both pre-S1 and pre-S2 Ags were detected by enzyme immunoassays in almost all HBsAg carriers. Both pre-S1 and pre-S2 Ag titers correlated positively with the serum HBsAg concentration (P = 0.0001), but only the pre-S1 Ag titer correlated with the level of serum HBV DNA (P = 0.02). The detection of low levels of IgM anti-hepatitis B core (anti-HBc) antibodies by IMx was associated with the presence of liver disease (P = 0.05) but not with the level of viral replication. The prevalence of anti-HBx antibodies detected by the enzyme immunoassay was slightly, although not significantly, higher in patients with high levels of HBV DNA (greater than 100 pg/ml) than in patients without detectable HBV DNA (P = 0.16). In anti-HBe-positive chronic HBsAg carriers, the quantitative detection of serum HBV DNA, pre-S Ag titers, and IgM anti HBc allowed us to predict which patients suffered from chronic liver disease and/or supported viral replication (P < 0.05). In a follow-up study of eight patients undergoing antiviral therapy, the clearance of both pre-S1 Ag and HBV DNA was associated with a subsequent clearance of HBV. Therefore, the quantitative determination of HBV DNA, pre-S Ags, IgM anti-HBc may prove useful for the decision to use and the monitoring of antiviral therapy, especially in anti-HBe-positive HBsAg carriers.
Identification of subgroups of bovine respiratory syncytial virus
Authors: Baker J C , Wilson E G , McKay G L , Stanek R J , Underwood W J , Velicer L F , Mufson M A
Published: January 2020

The occurrence of antigenic variation among nine isolates of bovine respiratory syncytial virus (BRSV) was determined by examining their reaction patterns to human respiratory syncytial virus (HRSV) subgroup A and B monoclonal antibodies (MAbs) by enzyme immunoassay and radioimmunoprecipitation with fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using MAbs and polyclonal antisera to HRSV and BRSV. Shared epitopes were demonstrated on four of five structural proteins between BRSV and both subgroups A and B of HRSV. The nine isolates of BRSV showed different patterns of reactivity in enzyme immunoassays with panels of MAbs to HRSV subgroups A and B. Major variations in the molecular weights of the P (phosphoprotein) and F (fusion protein) proteins were demonstrated among the BRSV isolates tested. These results suggest that BRSV belongs to a different antigenic grouping than HRSV and that BRSV is composed of two distinct subgroups.
Astrovirus-associated diarrhea among Guatemalan ambulatory rural children
Authors: Cruz J R , Bartlett A V , Herrmann J E , Cáceres P , Blacklow N R , Cano F
Published: January 2020

Fecal excretion of astroviruses was monitored in 321 children, 0 to 3 years old, living in the rural highlands of Guatemala. During the longitudinal study, from February 1987 to February 1989, we examined 5,000 stool specimens, including 1,805 collected during 1,369 episodes of diarrhea, 830 collected during the convalescent week, and 216 and 244 collected 2 weeks and 1 week, respectively, before the onset of diarrhea. Routine specimens were taken once a month from every child who had been free from diarrhea for at least three consecutive weeks. Of the children, 124 (38.6%) excreted astroviruses during the study. In total, we identified 184 infections by astroviruses. Of the samples collected 2 weeks and 1 week before the initiation of symptoms, 0.9 and 4.9%, respectively, were positive, while 7.3% of the diarrhea episodes were associated with astroviruses. Of the convalescent specimens, 3.4% were shown to be positive; 2.4% of the 1,905 specimens taken in diarrhea-free periods contained astroviruses. Infections by other potential enteropathogens were documented in 54 and 65% of the asymptomatic and symptomatic astrovirus infections, respectively. Diarrhea associated with astroviruses alone had a median duration of 5 days and was associated with vomiting in 8.6%, with fever in 17.1%, with dehydration in 5.7%, and with loss of appetite in 34.3% of the episodes. Diarrhea due to astroviruses was accompanied by negative changes in weight gain. Astrovirus diarrhea contributes to the high morbidity observed in young children living under poor conditions and has a deleterious effect on their nutritional status.
Evaluation of culture and the Gen-Probe PACE 2 assay for detection of Neisseria gonorrhoeae and Chlamydia trachomatis in endocervical specimens transported to a state health laboratory
Authors: Limberger R J , Biega R , Evancoe A , McCarthy L , Slivienski L , Kirkwood M
Published: January 2020

The Gen-Probe PACE 2 assay (Gen-Probe Inc., San Diego, Calif.) was compared with culture for the detection of Neisseria gonorrhoeae in endocervical specimens that were mailed to the laboratory. During mail transport, the specimens were exposed to extremes of hot and cold weather for several days before arriving in the laboratory. Specimens on culture plates deteriorated during transport, as evidenced by many dead gonococcus-like colonies. The manufacturer's recommendation for reporting PACE 2 assay-positive results was modified to create a suspicious category for samples with relative light units near the positive cutoff value. Of a total of 4,869 specimens tested, 30 were positive by both methods and 102 were positive only by the PACE 2 assay. These additional 102 positive specimens were likely to be true positives, as indicated by several lines of indirect evidence, including detailed probe competition analysis, patient history, and the lack of false-positive results in hand-delivered specimens. Although Gen-Probe Inc. indicates that specimens are stable for up to 7 days, N. gonorrhoeae was easily detectable by the PACE 2 assay after 1 month of incubation at room temperature in the PACE 2 transport buffer. We also compared the Gen-Probe PACE 2 assay for Chlamydia trachomatis with culture on endocervical specimens delivered by same-day courier. Of 398 endocervical specimens tested, the PACE 2 assay detected 19 of 20 culture-positive samples. Although the assay failed to detect one culture-positive sample, it was able to detect two very weak culture-suspicious samples. Finally, PACE 2 assays for N. gonorrhoeae and C. trachomatis performed on the same samples indicated that the coinfection rate was 40% for women attending five family planning clinics. We concluded that the Gen-Probe PACE 2 assay system should be considered for use in testing those specimens that are transported to the laboratory through the mail.
Interpretive criteria for susceptibility testing of CI-960 (PD127391, AM-1091), fleroxacin, lomefloxacin, and temafloxacin against Neisseria gonorrhoeae, including drug stability in GC agar medium
Authors: Erwin M E , Jones R N
Published: January 2020

CI-960, fleroxacin, lomefloxacin, and temafloxacin were tested against over 100 strains of Neisseria gonorrhoeae. Each organism was tested in triplicate by using agar dilution and disk diffusion methods recommended by the National Committee for Clinical Laboratory Standards. CI-960 was the most potent compound, with a MIC against 90% of the strains tested of 0.008 microgram/ml, and the least active was fleroxacin (MIC against 90% of strains, 0.12 microgram/ml). Only the susceptible interpretive category was recommended for the CI-960 tests as follows: 5-micrograms disk, greater than or equal to 39 mm (MIC correlate, less than or equal to 0.12 microgram/ml). Three interpretive categories were proposed for the other fluoroquinolones as follows: fleroxacin, 5-micrograms disk susceptible at greater than or equal to 33 mm (MIC correlate, less than or equal to 0.25 microgram/ml), intermediate at 28 to 32 mm (MIC correlate, 0.5 microgram/ml), and resistant at less than or equal to 27 mm (MIC correlate, greater than 0.5 microgram/ml); lomefloxacin, 10-micrograms disk susceptible at greater than or equal to 35 mm (MIC correlate, less than or equal to 0.12 microgram/ml), intermediate at 28 to 34 mm (MIC correlates, 0.25 to 0.5 microgram/ml), and resistant at less than or equal to 27 mm (MIC correlate, greater than 0.5 microgram/ml); and temafloxacin, 5-micrograms disk susceptible at greater than or equal to 36 mm (MIC correlate, less than or equal to 0.06 microgram/ml), intermediate at 28 to 35 mm (MIC correlates 0.12 to 0.25 microgram/ml), and resistant at less than or equal to 27 mm (greater than 0.25 microgram/ml). Interpretive agreement between disk diffusion results and the MICs was 100% for each agent, with the exception of lomefloxacin, which had a 0.9% minor error. All drugs were stable in GC agar medium for at least 21 days when stored at 2 to 5 degrees C.
Novel immunoenzymatic assay for identification of coagulase- and protein A-negative Staphylococcus aureus strains
Authors: Guzmàn C A , Guardati M C , Fenoglio D , Coratza G , Pruzzo C , Satta G
Published: January 2020

A purified monoclonal antibody (MAb) which specifically reacts with Staphylococcus aureus glucosaminidase was obtained. This MAb was utilized to develop an immunoenzymatic assay for the identification of S. aureus strains. The sensitivity of this assay, based on the simultaneous detection of S. aureus glucosaminidase and protein A, was evaluated by analyzing a total of 196 strains, 26 of which did not exhibit one or more of the following properties: protein A, clumping factor, and staphylocoagulase. All strains yielded positive results by the MAb-based immunoenzymatic test. The assay's ability to differentiate between S. aureus and other staphylococci was then analyzed by testing a total of 277 non-S. aureus strains that yielded negative results. Our data demonstrate that this immunoenzymatic assay can be used as a single S. aureus identification criterion, particularly useful for those strains negative for clumping factor, staphylocoagulase, or protein A.
Enzyme immunoassay for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae
Authors: Uldum S A , Jensen J S , Søndergård-Andersen J , Lind K
Published: January 2020

An enzyme immunoassay (EIA) for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae was developed. The EIA was evaluated on the basis of results in the M. pneumoniae complement fixation (MPCF) test and the cold agglutinin test. Serum samples from 430 patients with respiratory infections of known or unknown etiology, from 91 healthy children and adults and from 20 patients with rheumatoid factor, were investigated. By the criteria chosen for positive diagnostic EIA values, we found that the combined measurement of specific IgM and IgG gave a specificity of 99.7% and a sensitivity of 97.8%. If only IgM antibodies were measured, the specificity was 100% and the sensitivity was 88%. For IgG alone the specificity was 99.7%, but the sensitivity was only 46% because of the high EIA cutoff value chosen for IgG. We found no false positives among serum samples from patients with non-M. pneumoniae respiratory infection of known etiology, and there were no false IgM positives due to rheumatoid factor. In some cases the IgM EIA results became positive earlier in the course of illness than the MPCF titer. While children and teenagers responded predominantly with IgM antibodies, patients older than 40 years often had an IgG response only (56% of cases), probably because of reinfection. We conclude that this EIA is a good alternative to the combined MPCF and cold agglutinin tests in the diagnosis of M. pneumoniae infection.
Evaluation of the abilities of three diagnostic tests based on the polymerase chain reaction to detect Mycobacterium paratuberculosis in cattle: application in a control program
Authors: van der Giessen J W , Haring R M , Vauclare E , Eger A , Haagsma J , van der Zeijst B A
Published: January 2020

Three assays for the specific detection of Mycobacterium paratuberculosis by dot spot hybridization of polymerase chain reaction products were applied to fecal samples of dairy cattle. The first two tests used polymerase chain reaction primers and a DNA probe derived from M. paratuberculosis-specific sequences of the 16S rRNA gene and insertion element IS900, respectively. These two tests were carried out on spiked fecal samples to determine the detection limits. The 16S rRNA test was able to detect 10(7) bacteria per g of feces, and the IS900 test detected 10(4) to 10(5) per g of feces. Next, we studied the usefulness of these tests in a control program for paratuberculosis. Therefore, the tests and a third, commercially available, test (IDEXX Corp.) were used twice with an interval of 3 months on fecal samples of 87 cows from two dairy herds with a history of Johne's disease. We compared the results of these tests with those of culturing. This showed that the tests are specific but that the sensitivity ranged from 3 to 23%. Further improvement of the sensitivity is needed before the tests can be used in a control program to eradicate Johne's disease.
DNA amplification and reverse dot blot hybridization for detection and identification of mycobacteria to the species level in the clinical laboratory
Authors: Fiss E H , Chehab F F , Brooks G F
Published: January 2020

A method incorporating DNA amplification and reverse dot blot hybridization for the detection and identification of mycobacteria to the species level is described. The amplification procedure allowed for the incorporation of digoxigenin-labeled UTP, which was detected by chemiluminescence, removing the need for radioactivity. Using a set of primers and probes from the gene for the 65-kDa heat shock protein of mycobacteria, previously reported in the literature, the reverse dot blot method correctly identified 12 of the 12 M. tuberculosis isolates and 45 of the 50 M. avium complex isolates. Two of the nonhybridizing M. avium complex isolates were reidentified as M. xenopi. The other three nonhybridizing M. avium complex isolates, which were identified as M. intracellulare, hybridized with the probe for M. tuberculosis, as did two ATCC strains of M. intracellulare. The amplified DNA of M. intracellulare was sequenced, and the sequence was compared with the sequence from M. tuberculosis. The sequence for M. avium differed from M. tuberculosis by 5 of 20 bases. The sequence for M. intracellulare differed from M. tuberculosis by 2 of 20 bases, but this difference did not result in sufficient thermal instability to affect hybridization. The use of chemiluminescence allowed as few as 10(2) CFU to be detected. The format of the assay is readily applicable for implementation in the clinical laboratory.
Large restriction fragment patterns of genomic Mycobacterium fortuitum DNA as strain-specific markers and their use in epidemiologic investigation of four nosocomial outbreaks
Authors: Hector J S , Pang Y , Mazurek G H , Zhang Y , Brown B A , Wallace R J
Published: January 2020

Pulsed-field gel electrophoresis and restriction endonucleases with rare recognition sites were used to generate large restriction fragment (LRF) patterns of genomic DNA from 48 isolates of Mycobacterium fortuitum biovariant fortuitum. Epidemiologically unrelated isolates gave highly diverse patterns when AsnI, HpaI, AflII, DraI, NdeI, XbaI, SpeI, or SspI was used. Epidemiologically related isolates produced identical or minimally different LRF patterns. Minor variations in LRF patterns were seen in two epidemic isolates digested with XbaI, suggesting that genetic alteration had occurred. LRF patterns were used to study three cardiac surgery wound infection outbreaks and one respiratory disease nosocomial outbreak. In two outbreaks, LRF patterns confirmed the reported clustering of isolates on the basis of multiple phenotyping methods. In the remaining two outbreaks, isolates which could not be separated by prior typing methods were easily distinguished by LRF pattern analysis. Environmental water isolates from two outbreaks had LRF patterns identical to those of the disease-producing strains, confirming that the local environment was the source of infection. Pulsed-field gel electrophoresis of LRFs of genomic DNA offers great promise as an epidemiologic tool for the study of M. fortuitum.
Emergence of a new clone of serogroup C Neisseria meningitidis in São Paulo, Brazil
Authors: Sacchi C T , Zanella R C , Caugant D A , Frasch C E , Hidalgo N T , Milagres L G , Pessoa L L , Ramos S R , Camargo M C , Melles C E
Published: January 2020

Serogroup C isolates of Neisseria meningitidis recovered from 121 patients with meningitis or septicemia in Greater São Paulo, Brazil, between 1976 and 1990 were analyzed with respect to serotype and multilocus enzyme genotype. The distribution of serotypes has changed since 1989 when serotype 2b started to replace serotype 2a. There were 48 distinct multilocus genotypes (electrophoretic types [ETs]) and 13 distinct complexes. Among the 41 serotype C:2b:- strains analyzed, 38 (93%) were found in complex 11. The percentage of complex 11 increased from 8% in 1988 to 50 and 66% in 1989 and 1990, respectively. Although we have been in an epidemic situation due to serogroup B:4:P1.15 ET-5 complex since 1988, the appearance and increase of a new unrelated strain, C:2b:- of ET-11 complex, in 1989 and 1990 provide enough data to conclude that the presence of two different complexes, ET-5 and -11, of N. meningitidis were responsible for the high levels of meningococcal disease in Greater São Paulo during this period.
High correlation in antibody titers between the Sabin-Feldman dye test and an enzyme-linked immunosorbent assay detecting immunoglobulin G antibodies to the nucleoside triphosphate hydrolase of Toxoplasma gondii
Authors: Asai T , Mizuno F , Kojima S , Takeuchi T , Kobayashi A , Suzuki Y
Published: January 2020

An enzyme-linked immunosorbent assay to detect immunoglobulin G antibodies against nucleoside triphosphate hydrolase, which is a specific and dominant antigen of Toxoplasma gondii, was developed, and the sensitivity and specificity of the test were compared with those of the Sabin-Feldman dye test. One hundred percent agreement was observed in comparative study between those tests on 37 positive and 50 negative human sera. Antibody titers in the enzyme-linked immunosorbent assay test, which were expressed as the reciprocal of the highest positive dilution of serum, were just 100 times those in the dye test on 81% (30 of 37) of the positive sera.
Changing clonal patterns of Salmonella enteritidis in Maryland: evaluation of strains isolated between 1985 and 1990
Authors: Morris J G , Dwyer D M , Hoge C W , Stubbs A D , Tilghman D , Groves C , Israel E , Libonati J P
Published: January 2020

We examined isolates from 203 sporadic and outbreak-associated Salmonella enteritidis cases occurring in Maryland between 1985 and 1990. Plasmid profiles were determined for all isolates; 52 isolates were phage typed. Ten plasmid profiles were identified. A single profile (consisting of a single ca. 55-kb plasmid) emerged as the predominant profile in Maryland during the study period. This profile (which was closely associated with phage type 8) accounted for 86% of a group of isolates from sporadic cases in 1988 and 1989, compared with 43% of the 1985 isolates. Strains with this profile were identified in four of nine outbreaks, including one of three outbreaks in which eggs were implicated as a vehicle. While plasmid profiles and phage typing appear to provide complementary means of identifying specific strains of S. enteritidis, the emergence of what appears to be a single predominant clone has reduced the discriminant ability of both typing systems. The factors be a single predominant clone has reduced the discriminant ability of both typing systems. The factors contributing to the emergence of this one clone are still not well understood.
Traveller's coccidioidomycosis: case report of pulmonary infection diagnosed in Israel
Authors: Lefler E , Weiler-Ravell D , Merzbach D , Ben-Izhak O , Best L A
Published: January 2020

A 60-year-old temporary Israeli resident travelled to Arizona, developed an influenzalike infection, and returned with a space-occupying lesion in the lung. Since the patient was a heavy smoker, lung cancer was suspected and he was operated on. A granuloma was reported on frozen sections, and Coccidioides immitis was revealed on stained preparations and by microbiological investigation. Coccidioidomycosis is unusual in Israel; therefore, it is important to be aware of this mycosis in patients who have a history of recent visits to areas of endemicity in North America, Central America, and South America.
Detection of human group C rotaviruses by an enzyme-linked immunosorbent assay using monoclonal antibodies
Authors: Fujii R , Kuzuya M , Hamano M , Yamada M , Yamazaki S
Published: January 2020

An enzyme-linked immunosorbent assay using monoclonal antibodies was established for the detection of human group C rotaviruses. Seventeen clinical samples which were found to contain group C rotaviruses were all strongly positive, whereas 9 samples containing group A rotaviruses and 51 samples lacking rotaviruses were all negative with this test.
Subtyping of Streptococcus uberis by DNA amplification fingerprinting
Authors: Jayarao B M , Bassam B J , Caetano-Anollés G , Gresshoff P M , Oliver S P
Published: January 2020

Total DNA of Streptococcus uberis from cows with mastitis was analyzed by DNA amplification fingerprinting (DAF) and compared with restriction endonuclease fingerprinting (REF). DAF grouped 22 strains into 15 distinct patterns, while REF grouped them into 12 patterns. These results suggest that DAF is a useful technique for subtyping strains of S. uberis.
Pseudoepidemic of Nocardia asteroides associated with a mycobacterial culture system
Authors: Patterson J E , Chapin-Robertson K , Waycott S , Farrel P , McGeer A , McNeil M M , Edberg S C
Published: January 2020

Nocardia isolations increased from 0.7 to 11.7/1,000 acid-fast bacillus and mycological cultures (P less than 0.000001). Only three isolations from one patient represented infection. Pseudoepidemic strain identity was confirmed by DNA fingerprinting; the isolate causing infection was distinct. The end of the pseudoepidemic was associated with changing the needle sterilizer and prolonging needle sterilization time on the BACTEC 460 machine. To our knowledge, this is the first reported Nocardia asteroides pseudoepidemic.