bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic similarity of intestinal spirochetes from humans and various animal species
Authors: Koopman M B , Käsbohrer A , Beckmann G , van der Zeijst B A , Kusters J G
Published: January 2020

The chromosomal DNA of spirochetes isolated from human, swine, dog, mouse, rat, and chicken intestine or feces was subjected to restriction enzyme analysis and hybridization with three different DNA probes, derived from a flagellin gene, a hemolysin gene, and the 16S rDNA sequence of the pathogenic swine intestinal spirochete Serpulina hyodysenteriae. This genetic analysis showed that intestinal spirochetes represent a heterogeneous but related population of bacteria. In general, unique genotypes were distinguished among isolates from the same host species; they were not present among isolates from other host species. This suggests the host specificity of some strains. An exception to this are isolates from humans and dogs suffering from gastrointestinal disorders; these isolates showed highly similar or even identical genotypes. None of them resembled any of the genotypes of isolates found in other host species without apparent disease.
Humoral immune response to human cytomegalovirus DNA polymerase
Authors: Landini M P , Lazzarotto T , Ertl P F
Published: January 2020

In this work, we show that antibodies to baculovirus recombinant DNA polymerase of human cytomegalovirus are present in human sera during natural infection. Specific antibody was found to be of the immunoglobulin G class, produced at low titers only for a short period of time at the beginning of acute infection and not detectable in sera from patients with a convalescent or a latent phase of infection. These results were compared with those obtained with procaryotically expressed p52, the polymerase accessory protein.
Two cases of Campylobacter mucosalis enteritis in children
Authors: Figura N , Guglielmetti P , Zanchi A , Partini N , Armellini D , Bayeli P F , Bugnoli M , Verdiani S
Published: January 2020

Two cases of Campylobacter mucosalis enteritis in children are reported. The patients recovered without antimicrobial therapy. Strains were isolated only by the feces filtration technique. In one child, bactericidal antibodies to the homologous strain were detected in a convalescent-phase serum sample. C. mucosalis should be considered a primary intestinal pathogen.
Characterization of Centers for Disease Control group NO-1, a fastidious, nonoxidative, gram-negative organism associated with dog and cat bites
Authors: Hollis D G , Moss C W , Daneshvar M I , Meadows L , Jordan J , Hill B
Published: January 2020

Seventeen strains of fastidious, nonoxidative, gram-negative rods, isolated from human wounds resulting primarily from dog or cat bites, formed a distinct group, which was designated Centers for Disease Control (CDC) group nonoxidizer 1 (NO-1). The phenotypic characteristics of CDC group NO-1 were most similar to non-acid-producing Acinetobacter species, with the major difference being a negative reaction in the transformation assay test for Acinetobacter spp. The cellular fatty acid composition of CDC group NO-1 was different from those of Acinetobacter species and all other bacteria tested to date. The isolates were susceptible to a variety of antimicrobial agents including the aminoglycosides, beta-lactam antibiotics, tetracyclines, quinolones, and sulfonamides. Fifty percent of the isolates were resistant to trimethoprim. Ubiquinone-8 was present as the major isoprenoid quinone in CDC group NO-1.
Susceptibility of Haemophilus influenzae to piperacillin-tazobactam combinations: interpretive criteria and quality control limits for standardized tests
Authors: Barry A L , Fuchs P C , Jorgensen J H , Tenover F C , Allen S D , Hardy D J , McLaughlin J C
Published: January 2020

In vitro studies evaluated methods for testing the susceptibility of Haemophilus influenzae to piperacillin-tazobactam combinations. Ampicillin-resistant beta-lactamase-nonproducing strains of H. influenzae may be presumed to be relatively resistant to combinations of piperacillin-tazobactam, even though they frequently appear to be susceptible by disk diffusion methods. Other ampicillin-resistant or -susceptible strains were predictably susceptible; i.e., 130 such strains gave zones of inhibition > or = 26 mm in diameter, and MICs for these strains were < or = 0.125/4.0 micrograms/ml (< or = 1.0/0.12 micrograms/ml when an 8:1 ratio was tested). A resistant category has yet to be defined. For quality control purposes, H. influenzae ATCC 49247 should give zones of inhibition 32 to 38 mm in diameter, and broth microdilution MICs should be 0.12/4.0 to 0.5/4.0 micrograms/ml.
Nucleotide sequence variation in Pneumocystis carinii strains that infect humans
Authors: Lee C H , Lu J J , Bartlett M S , Durkin M M , Liu T H , Wang J , Jiang B , Smith J W
Published: January 2020

The nucleotide sequences of a portion of the large-subunit mitochondrial rRNA gene of Pneumocystis carinii derived from 12 patients were examined. Five sequences were found to be identical to the prototype sequence reported by Sinclair et al. (K. Sinclair, A. E. Wakefield, S. Banerji, and J. M. Hopkin, Mol. Biochem. Parasitol. 45:183-184, 1991). Six sequences differed from the prototype sequence at one to three positions. The remaining sequence was markedly different from the prototype sequence and appeared to be a hybrid of the human and rat P. carinii sequences. The results of this study indicate the existence of multiple P. carinii strains infecting humans and suggest that coinfections of animal and human P. carinii strains in the same host are possible.
Properties and type antigen patterns of group B streptococcal isolates from pigs and nutrias
Authors: Wibawan I W , Lämmler C , Smola J
Published: January 2020

All 59 group B streptococcal cultures isolated from pigs and nutrias reacted with group B-specific antiserum and gave a positive CAMP reaction in the zone of staphylococcal beta-lysin. Most of the cultures were pigmented; all cultures hydrolyzed Na hippurate and utilized salicin, maltose, and saccharose but not esculin, mannitol, or inulin. Fifty-three percent of the group B streptococci from pigs and none of those from nutrias were lactose positive. Serotyping revealed that most of the group B streptococci from pigs were of serotype III and most of those from nutrias were of type Ia/c. Protein c was present as c beta antigen. All group B streptococci were susceptible to penicillin and bacitracin (10 U), and most of the porcine cultures were resistant to tetracycline. According to these results, group B streptococci from pigs and nutrias differ from bovine and human group B streptococci and seem to play no role in cross-infections between animals or between animals and humans.
Comparison of the Seradyn Color Vue passive agglutination test and complement fixation for detection of Mycoplasma pneumoniae antibodies
Authors: Leland D S , Barth K A , Cunningham E B
Published: January 2020

We compared traditional complement fixation (CF) with a new passive agglutination method, the Seradyn Color Vue (SCV) test (Seradyn, Indianapolis, Ind.), for detection of Mycoplasma pneumoniae antibodies in 170 stored serum samples. The SCV test was 90% sensitive in identifying as positive 27 of 30 CF high-titer (> or = 1:64) serum samples and 100% specific in identifying as negative 134 of 134 CF low-titer (< or = 1:32) or negative (< 1:8) serum samples. The SCV test was technically undemanding, and it required no expensive equipment.
Prevalence of antibodies to Norwalk virus in England: detection by enzyme-linked immunosorbent assay using baculovirus-expressed Norwalk virus capsid antigen
Authors: Gray J J , Jiang X , Morgan-Capner P , Desselberger U , Estes M K
Published: January 2020

A total of 3,250 serum specimens collected in England in 1991 and 1992 were tested by an indirect enzyme-linked immunosorbent assay for antibody to Norwalk virus using baculovirus-expressed capsid antigen, and 2,382 (73.3%) were positive. The prevalence of Norwalk virus antibody differed regionally. It was lowest (24.6%) in 6- to 11-month-old infants and increased to 89.7% in persons over 60 years old.
Progress toward a simplified polymerase chain reaction and its application to diagnosis of tuberculosis
Authors: Wilson S M , McNerney R , Nye P M , Godfrey-Faussett P D , Stoker N G , Voller A
Published: January 2020

The complexity, expense, and susceptibility to contamination of the polymerase chain reaction (PCR) are all issues which need to be overcome if PCR is to be used outside of research laboratories. We addressed these problems with respect to the diagnosis of tuberculosis. First, we simplified the procedure for extracting Mycobacterium tuberculosis DNA from sputum samples. Two methods of sample preparation were compared: the chaotrope-silica method and a novel, more simple chloroform method. Second, we developed a colorimetric method for product detection. This method was as sensitive and specific as agarose gel electrophoresis for detection of PCR product. By using a one-tube nested protocol, 5 to 50 genome equivalents of M. tuberculosis DNA were detected. The simplified colorimetric PCR was compared with microscopy and culture for detection of M. tuberculosis in clinical specimens of sputum. A total of 171 sputum samples were investigated from 108 patients, 12 of whom were subsequently found to have tuberculosis by culture and/or microscopy. PCR of samples prepared by the chaotrope-silica method had a sensitivity of 75% and a specificity of 100% whereas PCR of samples prepared by the chloroform method had a sensitivity of 92% and a specificity of 99% when compared with the sensitivities and specificities of the combined classical microbiological methods for the diagnosis of tuberculosis. The simplified colorimetric PCR in combination with the chloroform sample preparation method was at least as sensitive as microscopy but had a greater specificity because samples with atypical mycobacteria were not detected by PCR. The sensitivity of the method for detection of smear-negative and extrapulmonary tuberculosis remains to be investigated.
Comparison of phage typing and DNA fingerprinting by polymerase chain reaction for discrimination of methicillin-resistant Staphylococcus aureus strains
Authors: van Belkum A , Bax R , Peerbooms P , Goessens W H , van Leeuwen N , Quint W G
Published: January 2020

A typing procedure for methicillin-resistant Staphylococcus aureus (MRSA) based on the polymerase chain reaction (PCR) amplification of both mecA sequences and variable DNA sequences as present in the prokaryotic genome has been developed. Two primers based on the sequences of DNA repeats as discovered in gram-negative members of the family Enterobacteriaceae allow detection of variable regions in the genome of a gram-positive bacterium such as S. aureus, as does a newly described arbitrary primer. This procedure, enabling the detection of 23 different genotypes in a collection of 48 MRSA isolates, was validated by comparisons with phage typing studies. It appeared that within the same group of isolates only 13 different phagovars could be identified. Combination of the results from both phage typing and genotyping allowed the discrimination of 34 of 48 isolates. However, depending on the primer-variable complexity of the PCR fingerprints, which could also be modulated by combination of PCR primers, clear homologies between the groups defined by either phage typing or fingerprinting were observed. An analysis of an MRSA outbreak in a geriatric institution showed a collection of genetically homogeneous isolates. In agreement with phage typing, PCR fingerprinting revealed the identical natures of the MRSA strains isolated from all patients.
Role of beta-hemolytic group C streptococci in pharyngitis: incidence and biochemical characteristics of Streptococcus equisimilis and Streptococcus anginosus in patients and healthy controls
Authors: Fox K , Turner J , Fox A
Published: January 2020

The biochemical characteristics and the isolation rates of the two Lancefield group C streptococcal species (S. equisimilis and S. anginosus) from patients with pharyngitis and asymptomatic controls were compared. Some 239 strains of beta-hemolytic group C streptococci were isolated from 1,480 patients (209 strains) with pharyngitis and 227 controls (30 strains). A total of 44 strains displayed broad hemolysis, were Voges-Proskauer test negative, and produced glucuronidase. Some 72.7% of these strains also fermented ribose. A second group of 159 strains was Voges-Proskauer test positive and glucuronidase negative; 98.8% also displayed minute hemolysis and only 3.7% fermented ribose. These two groups represent typical large-colony S. equisimilis and small-colony S. anginosus, respectively. A small number of strains (36 in total) exhibited intermediate characteristics; morphologically, all 36 strains resembled S. anginosus, but 6 strains biochemically resembled S. equisimilis. No strains of S. equisimilis fermented sorbitol (unlike S. zooepidemicus) and all fermented trehalose (unlike S. equi). The structural carbohydrate profiles of S. equisimilis and S. anginosus both included galactosamine (consistent with their being group C organisms), but the profiles were not distinguishable. In total, 78.5% of strains from controls and 83.3% of strains from patients were determined to be the species S. anginosus. S. equisimilis was isolated from 3.0% of patients and 2.2% of controls, and S. anginosus was isolated from 11.1% of patients and 11.0% of controls. Thus, S. equisimilis and S. anginosus are both members of the normal flora of asymptomatic individuals. The incidence and biochemical characteristics of these two species are similar in patients who are healthy and those who have disease. However, a companion article provides clinical evidence associating S. equisimilis (but not s. anginosus) with pharyngitis.
Evaluation of commercially available acridinium ester-labeled chemiluminescent DNA probes for culture identification of Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, and Histoplasma capsulatum
Authors: Stockman L , Clark K A , Hunt J M , Roberts G D
Published: January 2020

Four commercially available acridinium ester-labeled DNA probes directed against rRNA were evaluated for their ability to identify Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, and Cryptococcus neoformans in culture. rRNA was extracted by sonication of 1- to 2-mm2 portions of cultures of fungi in two chaotropic reagents with glass beads. Following a heat inactivation step, the extracts were hybridized in solution with probes specific for each pathogen. The acridinium ester reporter moiety of nonhybridized probe was selectively hydrolyzed, and chemiluminescence of specific DNA:RNA hybrids was quantitated in relative light units with a luminometer. A positive identification required a relative light unit value of > or = 50,000. Sensitivity and specificity of the probes were determined by probing cultures of the respective pathogenic fungi (target) and nontarget fungi. Both mycelial and yeast forms of the dimorphic fungi (B. dermatitidis and H. capsulatum) were tested. For B. dermatitidis, sensitivity and specificity were 87.8 and 100%, respectively (74 target and 219 nontarget fungi tested). For C. immitis, sensitivity and specificity were 99.2 and 100%, respectively (122 target and 164 nontarget fungi tested). For H. capsulatum, sensitivity and specificity were 100 and 100%, respectively (86 target and 154 nontarget fungi tested). For C. neoformans, sensitivity and specificity were 97 and 100%, respectively (100 target and 230 nontarget fungi tested). For B. dermatitidis, C. immitis, and C. neoformans, repeat testing increased the respective sensitivities to 97.3, 100, and 100%. The high sensitivities and specificities of the probes, the relatively short time (less than 1 h) required to perform the assay, and the availability of standardized reagent kits make the acridinium ester-labeled DNA probes well suited to laboratories in need of a rapid method to identify these fungal pathogens. Further, use of the probes to identify pathogenic fungi as soon as colonies appear on primary recovery media significantly shortens the time to reporting.
Computerized surveillance in clinical microbiology with time series analysis
Authors: Dessau R B , Steenberg P
Published: January 2020

An automatic surveillance system to detect changes in the incidences of microorganisms diagnosed in the department of clinical microbiology has been developed. The program is incorporated into the laboratory computer system and gives a weekly list of microorganisms whose isolation rates compared with those of a previous period exceed a chosen limit. The system uses time series analysis with moving weighted averages, and the detection limit is based on the distribution of the residuals. Output from the system included information about potential outbreaks of gastroenteritis, nosocomial infection with Corynebacterium jeikeium, and a seasonal epidemic of respiratory syncytial virus. The system also listed organisms not commonly isolated in the laboratory and detected incorrect reports. We conclude that continuous surveillance of laboratory data with time series analysis is a valuable tool for epidemiologic surveillance and quality control. Large quantities of data may be screened.
Detection of human papillomavirus 16 and 18 DNA in epithelial lesions of the lower genital tract by in situ hybridization and polymerase chain reaction: cervical scrapes are not substitutes for biopsies
Authors: Margall N , Matias-Guiu X , Chillon M , Coll P , Alejo M , Nunes V , Quilez M , Rabella N , Prats G , Prat J
Published: January 2020

Human papillomavirus (HPV) types 16 and 18 in 66 women with histologically documented lesions of the genital tract and 64 control cohorts were investigated. The efficacies of in situ hybridization and polymerase chain reaction (PCR) in detecting HPV 16 and 18 DNA were analyzed. In order to assess the usefulness of replacing biopsies with cervical scrapes, the two samples were compared by PCR. The prevalence rates of HPV infection by PCR were 59.1 and 10.9% in patients and controls, respectively. PCR was three times more sensitive than in situ hybridization (52.6 versus 17.8%). However, the need to improve PCR sensitivity by subsequent dot blot hybridization reduced one of the main advantages of PCR, i.e., expeditious diagnosis. Cervical scrapes were less sensitive than biopsies (13.6 versus 53%), although with four (6.1%) patients with intraepithelial neoplasias, HPV DNA was identified only by means of cervical scraping. We conclude that obtaining biopsy specimens and cervical scraping are complementary sampling procedures.
Cloning and expression of portions of the 34-kilodalton-protein gene of Mycobacterium paratuberculosis: its application to serological analysis of Johne's disease
Authors: De Kesel M , Gilot P , Misonne M C , Coene M , Cocito C
Published: January 2020

Paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle that is caused by Mycobacterium paratuberculosis, is characterized by incoercible diarrhea and fecal shedding of bacteria. The present work aimed at developing a specific serological test for this disease. We have recently shown that a 34-kDa protein belonging to the major antigen complex A36 of M. paratuberculosis is immunodominant and contains epitopes specific with respect to all mycobacteria tested, including Mycobacterium bovis and the closely related species Mycobacterium avium. From a lambda gt11 genomic library of M. paratuberculosis, three portions of the gene coding for this 34-kDa protein have been isolated. Two of them expressed cross-reacting mycobacterial epitopes. One portion (in clone a362) expressed a polypeptide which cross-reacted with all tested M. paratuberculosis strains but not with 20 other bacteria tested, including many strains of the M. avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum group. The occurrence at the M. paratuberculosis surface of epitopes corresponding to the a362 polypeptide was shown by immune electron microscopy. The recombinant a362 polypeptide was used as reagent for an enzyme-linked immunoassay for paratuberculosis. This assay correctly diagnosed all the tested blood samples from infected cattle at all stages of the disease.
Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to bovine leukemia virus in serum and milk
Authors: Nguyen V K , Maes R F
Published: January 2020

The results of examination of 375 bovine serum and 150 bovine milk samples for detection of bovine leukemia virus infection by the immunodiffusion technique were compared with those of an enzyme-linked immunosorbent assay (ELISA). It was concluded that the ELISA is another useful method for the detection of antibodies to bovine leukemia virus in serum and milk. The ELISA provides a quantitative result and has the advantage of being more sensitive and less time-consuming than the conventional immunodiffusion technique.
Comparison of nucleic acid hybridization and nucleic acid amplification using conserved sequences from the 5' noncoding region for detection of bovine viral diarrhea virus
Authors: Ridpath J F , Bolin S R , Katz J
Published: January 2020

Primers and probes derived from conserved sequences located in the 5' noncoding region of pestiviruses were evaluated for detection of bovine viral diarrhea virus. With these reagents, hybridization and polymerase chain reaction tests detected 62 of 90 and 90 of 90 bovine viral diarrhea virus isolates, respectively. A quick lysis method for preparing RNA for use in polymerase chain reaction amplification also was evaluated.
Arbitrarily primed polymerase chain reaction provides rapid differentiation of Proteus mirabilis isolates from a pediatric hospital
Authors: Bingen E , Boissinot C , Desjardins P , Cave H , Brahimi N , Lambert-Zechovsky N , Denamur E , Blot P , Elion J
Published: January 2020

During a systematic survey, maternal carriage of Proteus mirabilis was found over a 25-day period in 18 pregnant women admitted to the delivery ward of our hospital maternity. Five neonates born to these mothers were found to be colonized with P. mirabilis. We report here on the use of DNA fingerprinting by the arbitrarily primed polymerase chain reaction technique (AP-PCR) for the epidemiological investigation of this sudden outbreak. This approach was compared with the analysis of restriction fragment length polymorphisms of ribosomal DNA regions (ribotyping). Results of the AP-PCR and of ribotyping were in complete agreement in showing the genetic unrelatedness of the isolates obtained from each mother. Moreover, the results showed mother-to-infant vertical transmission of P. mirabilis in the neonates. AP-PCR is a rapid and discriminative method which seems particularly well suited to the epidemiological study of P. mirabilis.
Direct detection and genotyping of Chlamydia trachomatis in cervical scrapes by using polymerase chain reaction and restriction fragment length polymorphism analysis
Authors: Lan J , Walboomers J M , Roosendaal R , van Doornum G J , MacLaren D M , Meijer C J , van den Brule A J
Published: January 2020

Detection and genotyping of Chlamydia trachomatis were optimized by using a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis performed directly with crude cells of cervical scrapes. Different PCR pretreatment methods were evaluated on samples which were positive for C. trachomatis by cell culture. In comparison with DNA extraction and different proteolytic digestion methods, a simple pretreatment of 10 min of boiling appeared to be optimal for PCR amplification. Crude samples (n = 209) were first screened for C. trachomatis by both cell culture and plasmid PCR. Subsequently, positive samples found by plasmid PCR were subjected to a direct omp1 PCR-based RFLP analysis to differentiate C. trachomatis serovars A to K, Ba, Da, and L1 to L3 and serovariant D-. All cervical scrapes that were found positive for C. trachomatis by cell culture (n = 30) were also positive by plasmid PCR and omp1 PCR and could be easily genotyped. In addition, of the culture-negative group, eight samples were found positive by plasmid PCR. Five of these eight samples were also positive by omp1 PCR; of these five, two were positive by a nested omp1 PCR. Genotyping by RFLP analysis of the 35 omp1 PCR-positive samples showed that serovars D, E, and F are the most prevalent types found in cervical scrapes, while serovariant D- was also detected. This study shows that direct PCR and PCR-based RFLP analysis are feasible for detection and genotyping of C. trachomatis in cervical scrapes and are more sensitive than culture-based serotyping.
Genotyping by multiplex polymerase chain reaction for detection of endemic hepatitis B virus transmission
Authors: Repp R , Rhiel S , Heermann K H , Schaefer S , Keller C , Ndumbe P , Lampert F , Gerlich W H
Published: January 2020

A nested polymerase chain reaction (PCR) protocol was developed for rapid genotyping of hepatitis B virus (HBV). During the first PCR round, a universal HBV primer pair was used to amplify the entire pre-S region of the HBV genome. Within the pre-S region, many nucleotide exchanges are observed. These are partly correlated to the serological hepatitis B surface antigen subtypes. Five additional subtype-specific primers were selected from that region which, together with two universal non-group-specific primers, generated specific combinations of two to four DNA fragments of defined sizes. By this approach, 55 hepatitis B surface antigen-positive patients from a pediatric oncology unit in Germany were analyzed. Fifty-four patients who had been infected within 2 years had an identical pattern in the multiplex PCR, suggesting a common source of infection and person-to-person transmission within the unit. One child who was infected 5 years later had a different PCR pattern and, therefore, must have been infected from a different source. Furthermore, 109 serum samples taken from pregnant Cameroonian women and 25 serum samples from their babies taken 6 months after birth were analyzed. In one case, mother-to-infant transmission of the virus was demonstrated. Apart from its role in epidemiological studies on HBV, multiplex PCR may also be a useful tool for rapid genetic analysis in other fields if there is a moderate degree of sequence variation which enables the design of specific primers.
Intracellular bacteria of porcine proliferative enteropathy: cultivation and maintenance in vitro
Authors: Lawson G H , McOrist S , Jasni S , Mackie R A
Published: January 2020

An obligate intracellular bacterium was isolated from the intestines of all 10 cases of porcine proliferative enteropathy from four different pig farms. The organism grew in a rat enterocyte cell line (IEC-18) and was maintained over 20 passages. The growth of the bacteria was assessed by immunostaining of cells exposed to infection. Infection was not associated with morphological cell change, and growth was confined to cells infected at the time of each transfer of infection and the progeny of these cells. The bacterium is a microaerophilic, cell dependent, curved or rod-shaped, gram-negative bacillus that multiplies freely in the enterocyte cytoplasm. Cell cultures containing the intracellular bacteria appear to be free of other microorganisms, including chlamydiae and viruses.
Molecular epidemiology of a variant of coxsackievirus A24 in Taiwan: two epidemics caused by phylogenetically distinct viruses from 1985 to 1989
Authors: Lin K H , Wang H L , Sheu M M , Huang W L , Chen C W , Yang C S , Takeda N , Kato N , Miyamura K , Yamazaki S
Published: January 2020

In order to know the phylogenetic relationship and the route of transmission of a variant of coxsackievirus A24 (CA24v), an agent that caused four sequential outbreaks of acute hemorrhagic conjunctivitis from 1985 to 1989 in Taiwan, the nucleotide sequence variations in the virus-encoded proteinase 3C region (549 nucleotides) were studied with 19 isolates. The prototype strain (EH24/70), four isolates from Japan, and two isolates from Hong Kong were used for comparison. The nucleotide sequences of the Taiwan strains from the 1985-1986 and 1988-1989 epidemics were closely related within each epidemic, while they were more distantly related between strains from two epidemics. Phylogenetic analysis by the unweighted pairwise grouping method of the arithmetic average revealed that the 19 Taiwan isolates had diverged into two groups, 1985-1986 and 1988-1989 groups. The time at which these two groups diverged was estimated to be around May 1982, more than 3 years prior to the first appearance of the CA24v epidemic in Taiwan. On each occasion, the viruses caused a 2-year epidemic and then disappeared. The Taiwan isolates from 1985 to 1986 were closely related to the Japan isolates from 1985 to 1986 and the Taiwan isolates from 1988 to 1989 were phylogenetically close to the 1989 Japan isolates, indicating that Taiwan and Japan had two common-source outbreaks. However, none of the 1988 Taiwan isolates were phylogenetically close to the 1988 Japan or Hong Kong isolates. The evidence revealed that Taiwan has had two repeated but discontinuous introductions of CA24v since its first appearance in Taiwan in 1985. None of the other CA24v strains have been detected so far.
Prevalence and significance of hepatitis C virus (HCV) viremia in HCV antibody-positive subjects from various populations
Authors: François M , Dubois F , Brand D , Bacq Y , Guerois C , Mouchet C , Tichet J , Goudeau A , Barin F
Published: January 2020

Hepatitis C virus (HCV) infection is currently assessed by detection of antibodies to HCV with immunoassays. However, in the absence of an in vitro system to isolate the virus, or an immunoassay to identify HCV antigen in blood, an ongoing acute or chronic HCV infection can be diagnosed only by detection of HCV RNA by polymerase chain reaction. We used a reverse transcription-nested polymerase chain reaction to detect an HCV 5' noncoding viral RNA sequence in serum specimens collected from anti-HCV-positive individuals belonging to different risk groups and compared the results with those obtained with a prototype recombinant immunoblot assay (Chiron HCV SIA prototype recombinant immunoblot assay [RIBA]) containing four different viral peptides (c22, c33c, c100, and NS5). The prevalence of HCV viremia ranged from 25.9% in HCV antibody-positive blood donors to 92% in HCV antibody-positive hemophiliacs. Elevated alanine aminotransferase values in HCV antibody-positive patients were clearly associated with viremia. Ninety-six percent of HCV RNA-positive patients reacted to two viral antigens or more, compared with only 64% of HCV RNA-negative patients. Contrary to previous reports, HCV viremia was not associated with either the presence or the absence of a particular antibody specificity. The newly introduced NS5 peptide did not improve the sensitivity or specificity of the RIBA. Although 20% of the patients in our study whose sera reacted to all of the antigens were HCV RNA negative, the positive predictive value of a RIBA considered positive by the manufacturer (two or more bands), was rather high (78%) and may allow suspicion of viremia in EIA2 enzyme-linked immunosorbent assay-positive patients.
Identifying bovine respiratory syncytial virus by reverse transcription-polymerase chain reaction and oligonucleotide hybridizations
Authors: Oberst R D , Hays M P , Hennessy K J , Stine L C , Evermann J F , Kelling C L
Published: January 2020

An assay to identify tissue culture cells infected with bovine respiratory syncytial virus (BRSV) that utilizes reverse transcription (RT), the polymerase chain reaction (PCR), and a synthetic oligonucleotide hybridization probe has been developed. The RT-PCR assay uses a BRSV-specific negative-sense oligonucleotide primer to synthesize cDNA from a BRSV fusion protein mRNA template and another BRSV-specific oligonucleotide primer (positive sense) upstream from the negative-sense primer for PCR amplification. In the presence of mRNA templates of BRSV isolates originating from locations throughout the United States, the BRSV RT-PCR assay resulted in amplified products (381 bp) that were specific to BRSV, as demonstrated in hybridizations with a positive-sense oligonucleotide probe complementary to internal sequences and in sequence comparisons with the F protein of BRSV 391-2. In analysis of the BRSV RT-PCR assay with prototype strains of human RSV subgroups A and B, amplification of a similar 381-bp RT-PCR product was not evident, and no RT-PCR product hybridized with the internal probe. We conclude that the specific ability to amplify DNA sequences of BRSV F protein mRNA by RT-PCR and then to demonstrate the presence of the amplified product with a BRSV-specific oligonucleotide probe will greatly add to the speed, sensitivity, and specificity of BRSV diagnostics.
Testing of Streptococcus pneumoniae for resistance to penicillin
Authors: Marshall K J , Musher D M , Watson D , Mason E O
Published: January 2020

The increasing prevalence of penicillin-resistant Streptococcus pneumoniae requires antibiotic susceptibility tests that can be done with greater ease and reliability. We measured the MIC of penicillin for pneumococci by the tube macrodilution method with Mueller-Hinton broth (MHB), Haemophilus Test Medium (HTM), Todd-Hewitt broth with 0.5% yeast extract (THY), and MHB with 3% lysed horse blood (LHB). Eight (19%) and 6 (14%) of 42 pneumococcal isolates failed to generate turbid growth in MHB and HTM, respectively, whereas all pneumococcal isolates did so in THY and LHB. For those strains that replicated to turbidity, the mean MICs of penicillin were lower in MHB and HTM than in THY and LHB, with differences being significant (P < 0.05) for comparisons with LHB. Four isolates appeared to be penicillin susceptible in HTM but were actually moderately resistant in THY and LHB, and two isolates appeared to be moderately resistant but were resistant. A similar failure to detect resistance was seen with MHB. S. pneumoniae ATCC 49619, a moderately penicillin-resistant strain that has been proposed for quality control testing, gave variable results in MHB or THM and appeared to be susceptible to penicillin in some assays, whereas the MICs for S. pneumoniae ATCC 49619 in THY or LHB fell within a twofold dilution range, with geometric means of 0.16 and 0.18 micrograms/ml, respectively. Pneumococcal isolates thus may appear falsely susceptible to penicillin when tested in MHB or HTM. LHB remains the standard medium; however, because THY is an easily prepared clear medium that can be used in automated systems and appears to yield results similar to those obtained with LHB, THY deserves consideration for routine use.
Epidemiologic analysis and genotypic characterization of a nosocomial outbreak of vancomycin-resistant enterococci
Authors: Boyle J F , Soumakis S A , Rendo A , Herrington J A , Gianarkis D G , Thurberg B E , Painter B G
Published: January 2020

We are reporting on a nosocomial outbreak of 213 cases of vancomycin-resistant enterococcus infection involving 2,812 enterococcal isolates from patients over a period of 36 months. In 1990, the Enterococcus faecium vancomycin susceptibility rate was found to be 85.7% (36 of 42 cases), and an incidence of 10.9% (42 of 383) was noted. The 1991 data showed E. faecium with a vancomycin susceptibility rate of 61.8% (110 of 178) and an incidence of 26.0% (178 of 684). Subsequently, in 1992, the incidence of E. faecium increased to 34.0% (599 of 1,745), with a decreased vancomycin susceptibility rate of 25.8% (155 of 599). The E. faecalis vancomycin susceptibility rate remained near 97% (1,768 of 1,823) over the 36-month period. Of 115 vancomycin-resistant enterococcus (VRE) clinical isolates identified by the MicroScan MIC Combo-6 panels (Baxter Healthcare, Sacramento, Calif.), the agar dilution method indicated the resistance rate to be 92.3% (106 of 115) (high level), 3.5% (4 of 115) midlevel, and 3.5% (4 of 115) (low level). Genotypic characterization of 32 different VRE isolates by field-inversion gel electrophoresis demonstrated 19 dissimilar restriction endonuclease patterns, with 9 patterns associated with VRE quinolone resistance. Statistical analysis of case-control data for 32 patients with VRE infections indicated a positive association with intrabdominal surgical procedures (odds ratio, 24.12), multidrug therapy (odds ratio, 37.80), preexposure to vancomycin (odds ratio, 20.21), and death (odds ratio, 17.50).
Lipopolysaccharide profile typing as a technique for comparative typing of gram-negative bacteria
Authors: Aucken H M , Pitt T L
Published: January 2020

We have applied the technique of lipopolysaccharide (LPS) profiling in sodium dodecyl sulfate-polyacrylamide gel electrophoresis to the typing of 124 isolates of 12 gram-negative species from suspected outbreaks of infection. LPS was prepared by proteinase K digestion or micro-phenol-water extraction. A total of 11 of the 12 species gave clear ladder band profiles, the exception being Acinetobacter baumannii. When compared with conventional typing for Enterobacter cloacae, Pseudomonas aeruginosa, and Serratia marcescens, LPS profile type alone was sufficient to allow relatedness or distinguishability of isolates to be established, and this was corroborated by serotype and phage type data. Serologically nontypeable isolates invariably lacked O repeating units and thus could not be classified by their silver stain profile. We conclude that LPS profiling is useful for the epidemiological investigation of small clusters of isolates in order to determine whether or not cross-infection between patients has occurred.
Epidemic of diarrhea caused by Vibrio cholerae non-O1 that produced heat-stable toxin among Khmers in a camp in Thailand
Authors: Bagchi K , Echeverria P , Arthur J D , Sethabutr O , Serichantalergs O , Hoge C W
Published: January 2020

An epidemic of a cholera-like disease occurred among Khmers in a camp in Aranyaprathet, Thailand, in May 1990. Of 215 patients with diarrhea, Vibrio cholerae O1 was isolated from 25 (12%) and V. cholerae non-O1 was isolated from 15 (7%). Five of 15 (33%) non-O1 V. cholerae isolates hybridized with two different oligonucleotide probes previously used to detect V. cholerae non-O1 that produces a heat-stable toxin. This is the first description of an epidemic of diarrhea caused by V. cholerae non-O1 that produces heat-stable toxin.
Evaluation of five enzyme immunoassays for detection of immunoglobulin M antibodies to Epstein-Barr virus viral capsid antigens
Authors: Wiedbrauk D L , Bassin S
Published: January 2020

Five enzyme immunoassays for the detection of Epstein-Barr virus immunoglobulin M were evaluated versus indirect immunofluorescence assays with 128 serum specimens. The sensitivities and specificities, respectively, of these kits were as follows: Gull, 92 and 100%; Incstar, 53 and 100%; Ortho, 89 and 100%; Sigma, 78 and 86%; and BioWhittaker, 94 and 70%. Indeterminate results were produced by 2, 2, 0, 8, and 14 specimens with the Gull, Incstar, Ortho, Sigma, and BioWhittaker tests, respectively. The Gull and Sigma tests had the best day-to-day reproducibilities, and the Gull test was the easiest to use. No correlation between immunofluorescence titers and enzyme immunoassay values was observed.
Evaluation of intraspecies genetic variation within the 16S rRNA gene of Mycoplasma hominis and detection by polymerase chain reaction
Authors: Blanchard A , Yáñez A , Dybvig K , Watson H L , Griffiths G , Cassell G H
Published: January 2020

Mycoplasma hominis is a heterogeneous species with DNA-DNA hybridization values ranging from 51 to 100%. We report here the sequencing of the 16S rRNA gene of a strain (183) that greatly differs from the type strain (PG21) of this species. Comparison of 16S rDNA sequences from these two strains showed limited differences, indicating that the two strains belong to the same rRNA species complex. Using these nucleotide sequence data, we established a rapid method for the detection of M. hominis by using polymerase chain reaction. This method was shown to be sensitive and specific when tested with reference strains and clinical isolates.
Evaluation of the RapID NH system for identification of Haemophilus somnus, Pasteurella multocida, Pasteurella haemolytica, and Actinobacillus pleuropneumoniae isolated from cattle and pigs with respiratory disease
Authors: Salmon S A , Watts J L , Yancey R J
Published: January 2020

Haemophilus somnus, Pasteurella haemolytica, Pasteurella multocida, and Actinobacillus pleuropneumoniae from cattle and pigs with respiratory disease were used to evaluate the RapID NH system (Innovative Diagnostics, Atlanta, Ga.). Minor modifications of the RapID NH system to include animal source and growth requirements would permit the identification of all isolates tested.
Isolation of Shigella dysenteriae serotypes 11, 12, and 13 from patients with diarrhea in Bangladesh
Authors: Ansaruzzaman M , Kibriya A K , Mitra A K , Sack R B , Albert M J
Published: January 2020

Nine isolates of bacteria biochemically resembling Shigella dysenteriae but not belonging to the 10 recognized serotypes were isolated from patients with diarrhea in Bangladesh. Further studies suggested that two, one, and six isolates belonged to the recently recognized S. dysenteriae serotypes 11, 12, and 13, respectively.
Enzyme-linked oligosorbent assay for detection of polymerase chain reaction-amplified human immunodeficiency virus type 1
Authors: Mallet F , Hebrard C , Brand D , Chapuis E , Cros P , Allibert P , Besnier J M , Barin F , Mandrand B
Published: January 2020

An enzyme-linked oligosorbent assay (ELOSA) was developed for the detection on microtiter plates of polymerase chain reaction (PCR)-amplified human immunodeficiency virus type 1 (HIV-1) DNA. The denatured PCR product was hybridized with a passively adsorbed oligonucleotide capture probe and a horseradish peroxidase-labeled oligonucleotide detection probe. The sensitivity and specificity of the PCR-ELOSA technique depended to some extent on the nucleotide sequences of the oligonucleotide primer and probe quartet used in the amplification and detection. We evaluated five oligonucleotide quartets located in the gag, pol, vpr, env, and nef regions of HIV-1. DNAs from 39 HIV-1-seropositive individuals and 27 healthy HIV-1-seronegative controls were amplified by the PCR procedure, and the products were detected by ELOSA. Ten copies of HIV-1 DNA against a background of 1 microgram of human DNA were specifically detected by PCR-ELOSA. Specificities and sensitivities were, respectively, 100 and 95% for the gag system, 100 and 97% for the pol system, 100 and 85% for the vpr system, 96 and 95% for the env system, and 100 and 95% for the nef system. The simplicity of ELOSA makes it suitable for automation and applicable to genetic testing and detection of viral and bacterial DNAs or RNAs in most routine laboratories.
Discrimination between human T-cell lymphotropic virus type I and II (HTLV-I and HTLV-II) infections by using synthetic peptides representing an immunodominant region of the core protein (p19) of HTLV-I and HTLV-II
Authors: Bonis J , Baillou A , Barin F , Verdier M , Janvier B , Denis F
Published: January 2020

We describe enzyme immunoassays that use synthetic oligopeptides to discriminate serologically between human T-cell lymphotropic virus type I and II (HTLV-I and HTLV-II) infections. The peptides represented 20-amino acid segments between residues 111 and 130 (MA1) and residues 116 and 135 (MA2) of the p19 gag proteins of HTLV-I and HTLV-II, respectively. The assays were sensitive since 69 of 74 HTLV-positive sera were reactive to at least one of the two matrix (MA) peptides (sensitivity, 93.2%). By using the ratio of the optical density of MA1 to the optical density of MA2, which represents for every serum sample the ratio between the absorbance value obtained in the MA1 assay and the absorbance value obtained in the MA2 assay, 59 of the 69 reactive serum samples were clearly and easily typed as positive for either antibody to HTLV-I or antibody to HTLV-II. Eight of the 10 remaining reactive serum samples were analyzed further by an inhibition procedure, and their type specificities were then clearly identifiable. Therefore, the results indicate that all MA-reactive sera were serologically distinguished by our peptide assays.
Comparison of a whole-virus enzyme immunoassay (EIA) with a peptide-based EIA for detecting rubella virus immunoglobulin G antibodies following rubella vaccination
Authors: Zrein M , Joncas J H , Pedneault L , Robillard L , Dwyer R J , Lacroix M
Published: January 2020

A total of 250 human serum samples were tested for rubella virus immunoglobulin G antibodies by two enzyme immunoassays (EIAs), one using whole rubella virus antigen and the other based on the use of synthetic peptide antigen. The samples were taken from 125 volunteers before and after their immunization with the RA 27/3 rubella vaccine. This study indicates that a synthetic peptide-based EIA can favorably replace current viral lysate-based EIAs to detect rubella virus antibodies following immunization. Because the synthetic peptide used in this newly developed EIA represents a putative neutralization epitope of the rubella virus, it could also be instrumental in determining rubella immune status and in assessing vaccine program efficiency.
Development of species-specific DNA probes for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari by polymerase chain reaction fingerprinting
Authors: Giesendorf B A , van Belkum A , Koeken A , Stegeman H , Henkens M H , van der Plas J , Goossens H , Niesters H G , Quint W G
Published: January 2020

The application of polymerase chain reaction (PCR) fingerprinting assays enables discrimination between species and strains of microorganisms. PCR primers aiming at arbitrary sequences in combination with primers directed against the repetitive extragenic palindrome (REP) or enterobacterial repetitive intergenic consensus (ERIC) motifs generate isolate-specific DNA banding patterns. Analysis of these PCR fingerprints obtained for 33 isolates of Campylobacter jejuni, 30 isolates of Campylobacter coli, and 8 isolates of Campylobacter lari revealed that besides generation of isolate-specific fragments, species-specific DNA fragments of identical size were synthesized. It appeared that these DNA fragments could be used as species-specific probes, since they are unique for the pattern which they are deriving from. The probes do not cross-react with amplified DNA originating from a large panel of nonrelated microorganisms. Moreover, these probes displayed species specificity, as they reacted with a single restriction fragment on Southern blots containing DNA from C. jejuni, C. coli, and C. lari and other Campylobacter species. This combination of PCR fingerprinting and probe hybridization results in a highly specific identification assay and provides an example of specific test development without the prior need for DNA sequence information. The principle of the procedure holds great promise for the rapid isolation of DNA probes which, in combination with a general PCR assay, may lead to efficient typing and detection procedures for a multitude of medically important nonviral microorganisms.