bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the BacT/Alert pediatric blood culture system, Pedi-BacT, with conventional culture using the 20-milliliter Becton-Dickinson supplemented peptone broth tube
Authors: Krisher K K , Whyburn D R , Koepnick F E
Published: January 2020

The performance of the Pedi-BacT system, the BacT/Alert (Organon Teknika Corp., Durham, N.C.) pediatric blood culture bottle, was compared with that of a conventional 20-ml supplemented peptone broth tube (Becton-Dickinson Corp., Cockeysville, Md.) (BD system) in matched aerobic cultures. The tubes of the BD system were visually examined daily for 7 days and were subcultured during the first 24 h of incubation. Pedi-BacT cultures were mechanically agitated and continuously monitored for growth by the instrument. Of the 6,628 compliant pairs, 331 (5.0%) were positive in both systems, 220 (3.3%) were positive in the Pedi-BacT system only, and 170 (2.6%) were positive in the BD system only. One (0.02%) false-negative culture and 15 (0.2%) false-positive cultures occurred with the Pedi-BacT system while 20 (0.3%) false-negative cultures and 35 (0.5%) false-positive cultures occurred with the BD system. Of 288 clinically significant organisms detected in matched pairs from which a single isolate was recovered, 176 (61%) were recovered from both systems, 83 (29%) were recovered from the Pedi-BacT system only (P < 0.0001), and 29 (10%) were recovered from the BD system only. Members of the family Enterobacteriaceae (P < 0.01), miscellaneous nonfermenters (P < 0.05), and Candida spp. (P < 0.01) were isolated more frequently in the Pedi-BacT system than in the BD system. No significant difference in recovery of other organisms was found between the systems. The average time to detection for the Pedi-BacT system ranged from 11.5 h for streptococci to 29.7 h for enterococci, while that for the BD system ranged from 20.3 h for streptococci to 66.4 h for some nonfermenters. The BacT/Alert system is a reliable, labor-saving alternative to conventional blood culture methods.
Evaluation of immunoglobulin E-specific antibodies and viral antigens in nasopharyngeal secretions of children with respiratory syncytial virus infections
Authors: Russi J C , Delfraro A , Borthagaray M D , Velazquez B , García-Barreno B , Hortal M
Published: January 2020

Enzyme immunoassays were developed to detect the presence of specific immunoglobulin E (IgE) antibodies and respiratory syncytial (RS) virus structural proteins in nasopharyngeal secretions in order to improve the knowledge on some aspects of the pathogenesis of severe acute lower respiratory tract infections caused by RS virus. These assays were used to analyze clinical specimens from children with RS virus-associated infections (bronchiolitis and pneumonia), and the findings were correlated with the patients' clinical symptoms. The results indicate the presence of specific IgE against the two external glycoproteins (G and F) and the absence of detectable IgE levels for the internal viral antigens. There was a correlation between the levels of IgE-specific antibodies and the amount of viral protein F in the secretions, indicating that the IgE response against the viral glycoproteins might be related to the antigen load. In addition, a correlation was found between higher levels of both viral protein F-specific IgE and F antigen with higher respiratory rates in children with pneumonia. These findings may be relevant because they suggest an association between the virus load and the immune response in the pathogenesis of RS virus infections.
Polymerase chain reaction using 16S rRNA gene sequences distinguishes the two biovars of Ureaplasma urealyticum
Authors: Robertson J A , Vekris A , Bebear C , Stemke G W
Published: January 2020

Several fundamental phenotypic and genotypic differences have separated strains of the genital mycoplasma Ureaplasma urealyticum into two clusters or biovars. However, the lack of an easily performed and unambiguous test to discriminate between them has hampered investigation of the relationship between these biovars and disease. We determined the 16S rRNA nucleotide sequence of U. urealyticum 27, the serovar 3 standard and representative of the parvo biovar (serovars 1, 3, 6, and 14). This sequence was compared with the published sequence of U. urealyticum T960, which is the type strain and the serovar 8 standard and is representative of the T960 biovar which is composed of the 10 intervening serovars. Homology between the two sequences was 98.8%; differences were exploited to provide primers for biovar-specific polymerase chain reactions (PCRs). The results of these reactions placed all 14 serovar standard strains into the correct biovar. The PCRs were also applied to 10 cloned and 8 noncloned isolates that had been serotyped earlier. For 16 of them, we deduced their biovars from the serotyping data and then confirmed them by PCR. One unpredictable isolate and one nonserotypeable isolate were also classified as to biovar. Thus, we have developed a method for biotyping U. urealyticum that is applicable to both laboratory-adapted strains and wild-type isolates and that is appropriate for testing large numbers of clinical isolates. The amplification by the T960 biovar PCR protocol of DNAs from ureaplasmas of animals and certain Mycoplasma species suggested that the parvo biovar has diverged from the mainstream of the evolution of this clade.
Effect of heterosubtypic immunity on infection with attenuated influenza A virus vaccines in young children
Authors: Steinhoff M C , Fries L F , Karron R A , Clements M L , Murphy B R
Published: January 2020

Resistance to infection with an influenza A virus conferred by previous infection with an influenza A virus belonging to another subtype is called heterosubtypic immunity. Heterosubtypic immunity is demonstrable in laboratory animals but is believed to be weak in humans. The present study examined whether heterosubtypic immunity from previous influenza virus infection induced resistance to infection with an attenuated influenza A vaccine virus. Two groups of vaccinees consisting of young infants and children who received either influenza A H1N1 or H3N2 attenuated virus were studied. Influenza A H3N2 virus vaccine recipients were classified by their preexisting H1N1 heterosubtypic antibody level induced by prior infection with wild-type virus, and the H1N1 vaccinees were classified by their history of infection with H3N2 vaccine virus. For both groups of vaccinees, the rates of seroconversion and virus shedding and the level of vaccine virus replication were compared in subjects with and without heterosubtypic immunity. In 48 influenza A H3N2 virus and 39 H1N1 virus vaccinees, heterosubtypic immunity had no demonstrable effect on infectivity, immunogenicity, or replication of attenuated vaccine virus. These observations confirm the weak nature of heterosubtypic immunity in humans and suggest that it will not limit the utility of live attenuated influenza A viruses in young infants and children.
Novel, rapid optical immunoassay technique for detection of group A streptococci from pharyngeal specimens: comparison with standard culture methods
Authors: Harbeck R J , Teague J , Crossen G R , Maul D M , Childers P L
Published: January 2020

A novel immunoassay system based on the changes in the reflection of light, termed an optical immunoassay (OIA), was utilized to directly detect group A streptococcal (GAS) carbohydrate antigen from clinical specimens. In two studies, a total of 1,275 throat swabs were tested for the presence of this antigen with the Strep A OIA rapid detection system and the results were compared with those of standard culture methods. In both studies, the Strep A OIA yielded more positive results than plating of the throat swab onto a selective agar, Trypticase soy agar containing sheep blood, or an enriched broth. In one study, the sensitivity and specificity of Strep A OIA compared with those of the broth-enriched culture were 97.4 and 95.6%, respectively. In a second study a sensitivity of 98.9% and a specificity of 98.6% were achieved. It was also shown that the carbohydrate antigen could be detected in the absence of viable GAS organisms. The Strep A OIA is an easily interpretable method and was shown to be more sensitive than routine culture methods for detecting GAS infections directly from throat swabs.
Restriction endonuclease analysis of the vp7 genes of human and animal rotaviruses
Authors: Gouvea V , Ramirez C , Li B , Santos N , Saif L , Clark H F , Hoshino Y
Published: January 2020

The vp7 genes of 194 strains of group A rotaviruses representing all known G types were analyzed with three restriction enzymes by direct digestion of amplified cDNA copies or by deduction of the restriction patterns from known sequences. Mammalian rotavirus strains were classified into 28 restriction patterns consisting of combinations of the 6 profiles (s1 to s6) obtained by digestion with Sau96I endonuclease, 9 profiles (h1 to h9) obtained with HaeIII, and 15 profiles (b1 to b15) obtained with BstYI. Digestion with Sau96I and HaeIII identified restriction sites common to all, or almost all, rotavirus strains studied, whereas BstYI was the most discriminating among rotavirus strains. A clear correlation between some restriction patterns or individual profiles and G type and/or host species of origin was found. Several discriminatory restriction sites consisted of type-specific nucleic acid sequences that encoded conserved amino acid residues. Although not directly involved in antigenic diversity, these sites appear to indicate the G type of the isolate. The technique permits rapid comparison of a large number of virus isolates directly from fecal specimens and provides useful markers for investigating the evolution of rotavirus vp7 genes and tracing vaccine virus and interspecies transmission.
Characterization and seroepidemiology of a type 5 astrovirus associated with an outbreak of gastroenteritis in Marin County, California
Authors: Midthun K , Greenberg H B , Kurtz J B , Gary G W , Lin F Y , Kapikian A Z
Published: January 2020

The Marin County strain of type 5 astrovirus was associated with two separate outbreaks of nonbacterial gastroenteritis in California in 1978. A safety-tested, bacterium-free filtrate prepared from a stool specimen of an individual who was ill during the original outbreak was given orally to 19 adult volunteers. One volunteer developed a gastrointestinal illness, and nine had serologic responses. Several diarrheal stool specimens from the ill volunteer contained a large number of 27-nm particles. By using immune electron microscopy with acute- and convalescent-phase sera from the original outbreak, these 27-nm particles were shown to be identical to the viral inoculum. The Marin County virus, purified from the stool of the ill volunteer, was shown by immunoprecipitation and polyacrylamide gel electrophoresis to contain a single structural protein with a molecular weight of 30,000. The buoyant density of the virion was 1.39 g/cm3 in cesium chloride. By electron microscopy, approximately 5% of the particles had the characteristic stellate configuration of astrovirus, and serologic studies by immunofluorescence technique confirmed previous classification of the Marin County virus as a type 5 astrovirus. Radioimmunoassay and biotin-avidin immunoassay were used to detect antibody to the Marin County virus in paired acute- and convalescent-phase sera from 32 outbreaks of nonbacterial gastroenteritis, but none of these outbreaks could be attributed to this virus. Prevalence of antibody to this strain of astrovirus was approximately 13% in children 6 months to 3 years of age and increased to 41% in older children and young adults.
Comparison of three enzyme immunoassays, a cytotoxicity assay, and toxigenic culture for diagnosis of Clostridium difficile-associated diarrhea
Authors: Barbut F , Kajzer C , Planas N , Petit J C
Published: January 2020

Enzyme immunoassays (EIAs) based on monoclonal antibodies for the detection of Clostridium difficile toxins have recently been developed for clinical use. The aim of this study was to compare three commercially available EIAs, two for toxin A (Premier C. difficile Toxin A; Meridian, Osi, Elancourt, France; and Vidas C. difficile Toxin A; bioMérieux, Marcy l'Etoile, France) and one for toxins A and B (Cytoclone A + B EIA; Cambridge Biotech Corp., Codiapharm, Evian, France), with a cytotoxicity assay and toxigenic culture for the diagnosis of C. difficile-associated diarrhea (CDAD). The study was performed with 285 fresh stools from 285 patients with suspected CDAD. In case of disagreement, the tests were repeated on a frozen aliquot of the same stool sample, and the patient's chart was reviewed. CDAD diagnosis was established in 55 cases (incidence, 19.3%). The sensitivities and specificities of the methods were, respectively, 92.7 and 100% for the cytotoxicity assay, 96.4 and 99.1% for toxigenic culture, 75.5 and 97.8% for Cytoclone, 65.4 and 99.6% for Premier, and 65.4 and 100% for Vidas. The results were uninterpretable in 3.2% of cases with Cytoclone, 0.3% with Premier, and 2.5% with Vidas. We conclude that the cytotoxicity assay and toxigenic culture remain the best methods for the diagnosis of CDAD even though they lack standardization and require 48 to 96 h to obtain the result. Despite their rapidity and simplicity, EIAs are not sensitive enough to be relied on as the sole laboratory test.
Salmonella typhi O:9,12 polysaccharide-protein conjugates: characterization and immunoreactivity with pooled and individual normal human sera, sera from patients with paratyphoid A and B and typhoid fever, and animal sera
Authors: Aron L , Di Fabio J , Cabello F C
Published: January 2020

Polysaccharide of O:9,12 specificity purified from Salmonella typhi was conjugated to tetanus toxoid or bovine serum albumin in order to obtain defined antigenic material that would contain O chain free of other S. typhi antigens and that would be suitable for characterizing host humoral response to only S. typhi O-chain antigens. These artificial conjugates were strongly reactive in immunodots with 18 pooled and 3 individual serum samples from patients with typhoid fever and with rabbit anti-Salmonella O antiserum (group D, factors 1, 9, and 12). They reacted weakly with one serum sample from one human with paratyphoid A. These results suggest that the periodate oxidation and the reductive amination used in the conjugation conserved the immunogenicity of the O chain and allowed its absorption to nitrocellulose. They also suggest that the bovine serum albumin conjugate could be used in the diagnosis of S. typhi infections as normal sera may react with the protein molecule of the tetanus toxoid conjugate.
Mycobacteriosis due to Mycobacterium genavense in six pet birds
Authors: Hoop R K , Böttger E C , Ossent P , Salfinger M
Published: January 2020

Six cases of mycobacteriosis due to Mycobacterium genavense in three budgerigars (Melopsittacus undulatus), one orange-winged amazon (Amazona amazonica), one flycatcher (Cyanoptila cyanomelana), and one zebra finch (Taeniopygia guttata) are discussed. Gross lesions associated with the infection included a high degree of muscular wasting (five cases), hepatomegaly (four cases), and thickening of the wall of the small intestine (four cases). Granulomas were found in the lung (one case) and the subcutis (one case). Acid-fast bacilli were detected in the liver of all six birds. Only the use of acidic BACTEC mediums consistently led to growth, whereas the egg-based medium failed. These findings point to a possible role of the environment as a reservoir for M. genavense.
DNA fingerprinting of Mycobacterium bovis strains by restriction fragment analysis and hybridization with insertion elements IS1081 and IS6110
Authors: Collins D M , Erasmuson S K , Stephens D M , Yates G F , De Lisle G W
Published: January 2020

Strains of Mycobacterium bovis, the causative organism of bovine tuberculosis, can be clearly distinguished from each other by restriction fragment analysis. This method of DNA fingerprinting has been used for many epidemiological studies in New Zealand, but the technique presents practical difficulties that hinder its widespread use. The insertion element IS6110 is being widely used as a DNA probe for distinguishing restriction fragment polymorphisms among strains of Mycobacterium tuberculosis. Both this element and another recently sequenced element, IS1081, are also present in M. bovis. We assessed the usefulness of these two elements for distinguishing between 160 strains of M. bovis. These strains, most of which were isolated in New Zealand, were selected to be representative of the 95 different types that were identified among 530 strains that were previously typed by restriction fragment analysis. Fifteen IS6110 types were identified, but more than half of the strains representing 46 restriction types had the same IS6110 type. Virtually all M. bovis strains as well as strains of M. tuberculosis and Mycobacterium africanum had the same IS1081 type. The results indicate that for M. bovis, IS1081 cannot be used to type strains, IS6110 can be used to distinguish strains into broad groups, but only restriction fragment analysis is sufficiently sensitive for detailed epidemiological studies. An investigation of the host range of IS1081 revealed that, apart from its presence in species of the tuberculosis complex, it is also present in a strain of Mycobacterium xenopi.
Mitomycin immunoblot colony assay for detection of Shiga-like toxin-producing Escherichia coli in fecal samples: comparison with DNA probes
Authors: Hull A E , Acheson D W , Echeverria P , Donohue-Rolfe A , Keusch G T
Published: January 2020

We developed a direct screening immunoblot assay for the detection of Shiga-like toxin (SLT)-producing organisms in stool samples. The assay takes advantage of the phage-mediated nature of SLT production in Escherichia coli and the phage-inducing effects of mitomycin. The addition of mitomycin significantly enhanced the amount of toxin available for immunologic detection. By using the mitomycin-enhanced immunoblot assay, SLT-producing E. coli could be distinguished from non-toxin-producing E. coli and normal stool flora in ratios of 1:1,000 to 1:5,000. The immunoblot assay was examined in a field setting and compared with direct DNA probing for SLT-I and SLT-II. The assay was able to detect SLT-producing E. coli with a high level of sensitivity and specificity. Specificity was markedly improved by using a monoclonal antibody which cross-reacts with both SLT-I and SLT-II B subunits in place of the polyclonal antitoxin sera. We conclude that the mitomycin-enhanced immunoblot colony assay is a rapid and reliable alternative to DNA probing for the detection of phage-mediated SLT-producing organisms in stool samples, especially when the production and use of nucleic acid probes are not feasible. In addition, it permits isolation of positive colonies for further study and confirmation.
Correlation of serum antibody titers against hepatitis C virus core protein with clinical features by western blot (immunoblot) analysis using a recombinant vaccinia virus expression system
Authors: Nishizono A , Hiraga M , Mifune K , Terao H , Fujioka T , Nasu M , Goto T , Misumi J , Moriyama M , Arakawa Y
Published: January 2020

In order to study the relationships among the clinical features of hepatitis C patients, the presence of hepatitis C virus (HCV) RNA in their blood, and their serum antibody titers against the core protein of virus and to study the antibody levels in asymptomatic HCV carriers, a recombinant vaccinia virus containing a core protein gene was constructed. The recombinant virus expressed a protein with a molecular mass of 22 kDa in RK-13 cells as determined by Western blot (immunoblot) analysis. By using the cell lysate of virus-infected cells and serially diluted serum samples, core antibody titers in the groups of patients in the chronic hepatitis phase and in the convalescent phase as well as in asymptomatic carriers were determined by enhanced chemiluminescence Western blot analysis. Almost all patients in the chronic phase were shown to have high antibody titers of more than 1:500,000 and with no exception had of HCV RNA in their sera. On the other hand, patients who had recovered naturally and were in the convalescent phase were shown to have significantly lower antibody titers, and the antibody was not detected in the lowest serum dilution of 1:500 in 43% of these patients (three of seven total patients). Antibody levels of patients who showed a good response to interferon treatment decreased to intermediate levels between those of patients in the chronic phase and those of patients in convalescent phase. The antibody titers in asymptomatic carriers varied considerably from 1:500,000 to 1:500, and 41% (11 of 27 total individuals) of these carriers showed a high titer equivalent to that of those in the chronic phase. Core antibody was detected consistently in the individuals in whom HCV RNA was detected. This system for core antibody might be useful for identifying the stage of an apparent HCV infection.
Clonal analysis of Escherichia coli of serogroups O9, O20, and O101 isolated from Australian pigs with neonatal diarrhea
Authors: Woodward J M , Connaughton I D , Fahy V A , Lymbery A J , Hampson D J
Published: January 2020

The genetic diversity of 87 isolates of Escherichia coli recovered from Australian pigs with neonatal diarrhea was examined by multilocus enzyme electrophoresis. The isolates were of serogroups O9, O20, and O101, and although most isolates lacked K88(F4), K99(F5), 987P(F6), and F41 fimbriae, they were considered to be involved in the etiology of the diarrhea. The isolates were extremely diverse, considering their origin from a single pathological condition in one country. There were estimated to be 18, 16, and 12 clones of the three respective serogroups in the collection, with serogroup diversities of 0.387, 0.448, and 0.275, respectively. Comparison with the results previously obtained for isolates from piglets with postweaning diarrhea suggested that bacteria from piglets with these two conditions did not come from any particular common genetic background. The overall genetic diversity for the combined collection was the same as that reported by others for representative isolates selected from throughout the species (0.47). The current results indicate that if isolates of these O groups are involved in porcine diarrhea, their pathogenicity is directly linked to their O somatic antigen type and is not simply due to the wide distribution of a small number of virulent clones.
Clonal structure and pathogenicity of Shiga-like toxin-producing, sorbitol-fermenting Escherichia coli O157:H-
Authors: Karch H , Böhm H , Schmidt H , Gunzer F , Aleksic S , Heesemann J
Published: January 2020

We compared a collection of sorbitol-fermenting (SF) Escherichia coli O157:H- strains with SF E. coli O157:H45 and non-SF E. coli O157:H7 and E. coli O157:H- strains by pulsed-field gel electrophoresis. The SF E. coli O157:H- strains had identical or closely related XbaI patterns that differed markedly from those for the other E. coli O157 strains. Plasmid content and the presence of Shiga-like toxin-converting phages were determined for the SF E. coli O157:H- strains, indicating that these strains harbor a single 90-kb plasmid. They are lysogenized by toxin-converting phages and harbor the eae gene. Nonmotile E. coli O157 strains were observed to adhere more efficiently to HEp-2 cells than the motile strains. From their phenotypic and genotypic features, the SF E. coli O157:H- strains may well represent a new clone with non-SF E. coli O157:H7 pathogenic characteristics.
Identification of specific antigens of Pseudomonas pseudomallei and evaluation of their efficacies for diagnosis of melioidosis
Authors: Anuntagool N , Rugdech P , Sirisinha S
Published: January 2020

Current methods for the diagnosis of melioidosis are based on bacteriological culture. A number of serological tests currently available lack specificity and sensitivity. This is largely due to the use of crude antigens which results in a significant cross-reactivity with sera from individuals infected with other bacteria. In this study five different antigens were prepared and evaluated for their potential usefulness in diagnosis of melioidosis. These included a 19.5-kDa antigen which was previously shown to be specific by Western blotting (immunoblotting), a crude cell extract, a veronal extract, a 39.0-kDa antigen, and an immunoaffinity-purified antigen. All antigens were used for detecting antibody in sera from patients with septicemic melioidosis by indirect enzyme-linked immunosorbent assay. The results were compared with those obtained with sera from patients with other bacterial infections and normal sera from areas where the infection is and is not endemic. The 19.5-kDa antigen exhibited the most satisfactory results, with 92% sensitivity, 91% specificity, 81% positive predictive value, and 96% negative predictive value based on a background obtained with normal sera from the area where the infection is nonendemic. These values were 82% sensitivity, 96% specificity, 94% positive predictive value, and 87% negative predictive value based on results with normal sera from the area where the infection is endemic. Results from this study showed that the 19.5-kDa antigen was potentially useful in the diagnosis of melioidosis and deserves further investigation.
Human herpesvirus 6 DNA in peripheral blood cells and saliva from immunocompetent individuals
Authors: Cone R W , Huang M L , Ashley R , Corey L
Published: January 2020

Human herpesvirus 6 (HHV-6) genome equivalents were quantitated in peripheral blood mononuclear cells (PBMCs) and saliva from 20 healthy individuals by the polymerase chain reaction (PCR). Nineteen of 20 subjects (95%) harbored HHV-6 DNA: 18 (90%) had HHV-6 in their PBMCs and 18 had HHV-6 in their saliva. Quantitative PCR revealed HHV-6 DNA levels ranging from negative to 4,000 HHV-6 genome equivalents per 10(6) PBMCs and from negative to 200,000 HHV-6 genome equivalents per ml of saliva. Longitudinal saliva samples from 15 HHV-6-seropositive subjects revealed salivary HHV-6 DNA persistence in 13 subjects. HHV-6 antibodies were detected in 17 of 19 subjects, with titers ranging from 1:400 to 1:51,200 (geometric mean titer, 1:2,500). Antibody titers did not correlate with HHV-6 DNA levels in PBMCs or saliva (P = 0.27 and P = 0.44, respectively). One subject with persistent HHV-6 DNA lacked detectable HHV-6 antibodies. The high prevalence of HHV-6 DNA in PBMCs and saliva supports the concept that HHV-6 exists at these sites in normal individuals.
Direct polymerase chain reaction for detection of human immunodeficiency virus in blood spot residues on filter paper after elution of antibodies: an adjunct to serological surveys for estimating vertical transmission rates among human immunodeficiency virus antibody-positive newborns
Authors: Yourno J
Published: January 2020

Blood spot residues on filter paper circles from which antibodies have been eluted remain suitable for follow-up polymerase chain reaction to detect human immunodeficiency virus (HIV) provirus. A method has been developed for specimen processing and nested polymerase chain reaction amplification with a single tube. HIV-positive specimens can be detected by simple, direct electrophoresis of amplified material to a single copy of provirus. The procedure is particularly suited for survey purposes to estimate rates of vertical transmission of virus among HIV antibody-positive newborns, in whom virus loads may be low.
Mycobacteria in stool specimens: the nonvalue of smears for predicting culture results
Authors: Morris A , Reller L B , Salfinger M , Jackson K , Sievers A , Dwyer B
Published: January 2020

A previous recommendation suggests that stool be cultured for mycobacteria only if the smear is positive. We have correlated smear and culture results of 2,176 stool specimens submitted for mycobacterial culture. The sensitivity, specificity, and positive and negative predictive values for smears to predict culture results are 34, 99, 90, and 87%, respectively. We recommend that the stool smear not be used as a screening technique to decide which specimens from at-risk patients should be cultured because it lacks the necessary sensitivity.
Detection of antibodies to a pathogenic mycoplasma in desert tortoises (Gopherus agassizii) with upper respiratory tract disease
Authors: Schumacher I M , Brown M B , Jacobson E R , Collins B R , Klein P A
Published: January 2020

Mycoplasma agassizii (proposed species novum) is the etiologic agent of an upper respiratory tract disease in the desert tortoise (Gopherus agassizii), which is threatened in most of its range. An enzyme-linked immunosorbent assay (ELISA) for the detection of M. agassizii-specific antibodies in desert tortoises was developed with a monoclonal antibody with specificity for desert tortoise immunoglobulin light chain. Plasma samples from one group of tortoises were tested immediately before and 1 month after challenge either with nasal exudate containing M. agassizii or with a purified preparation of M. agassizii. Plasma samples from a second group of known healthy and sick tortoises were also tested. In the first group, the ELISA detected seroconversion in individual tortoises following challenge with M. agassizii. In the second group, ELISA results were positively correlated with the health status of the tortoises, as determined by clinical and pathologic findings. In addition, the ELISA revealed that tortoise antimycoplasma antibodies were specific for M. agassizii when samples were assayed against M. agassizii, M. pulmonis, M. testudinis, and M. gallisepticum antigens. The observed direct correlation between the presence of nasal mucosal lesions and M. agassizii-specific antibodies proved that the ELISA reliably diagnosed M. agassizii infection in desert tortoises and advocates its use for monitoring M. agassizii-induced upper respiratory tract disease in free-ranging desert tortoises.
Common somatic O and heat-labile serotypes among Campylobacter strains from sporadic infections in the United States
Authors: Patton C M , Nicholson M A , Ostroff S M , Ries A A , Wachsmuth I K , Tauxe R V
Published: January 2020

Somatic O (formerly heat-stable) and heat-labile (HL) serotyping methods are commonly used to type Campylobacter jejuni and Campylobacter coli isolates. Although both systems are effective, the labor and time required for each have limited their application. These systems can be simplified by reducing the number of antisera used. To find an appropriate panel of antisera, we determined the distribution of common serotypes in the United States among a representative sample of 298 Campylobacter isolates. The strains, obtained between July 1989 and June 1990 from persons with sporadic cases of diarrhea, were collected from 19 randomly chosen counties in all geographic (census) regions of the United States. All strains were serotyped by the O and HL systems. By phenotypic methods, 288 C. jejuni, 9 hippurate-negative C. jejuni/C. coli, and 1 Campylobacter lari were identified. Of 57 O antisera, 24 typed 252 (84.6%) strains. Of the 55 HL antisera, 23 serotyped 253 (84.9%) strains. All strains were typeable in the unabsorbed O antisera. In the absorbed HL antisera, four strains were nontypeable and 14 were rough and untypeable. In each geographic region, 9 or more O and HL serotypes were found. Serotypes O:1, O:4, and O:13,16,43,50 and HL 1 were identified in all regions. The combination of both schemes gave greater discrimination than either system alone, but the maintenance of both requires a large resource investment. A serotyping scheme incorporating the 24 most prevalent O and 23 most prevalent HL serotypes could be useful for outbreak support and for surveillance. In the near future, we anticipate using a molecular subtyping method in combination with limited serotyping to distinguish Campylobacter strains.
Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis
Authors: Nachamkin I , Bohachick K , Patton C M
Published: January 2020

We developed and studied a molecular typing approach for Campylobacter spp. with restriction fragment length polymorphism (RFLP) analysis of the flagellin gene flaA in C. jejuni. Using polymerase chain reaction, we amplified the flaA gene from strains comprising different HL:O serotypes by using a primer set directed at the conserved 5' and 3' flaA gene sequence to generate a 1.7-kb amplicon. The amplicon was further digested with the restriction enzyme DdeI, and the fragments generated were analyzed by agarose gel electrophoresis. In 43 non-outbreak strains of six common HL serotypes (HL 1, 2, 4, 5, 9, and 36) in the United States, 18 RFLP patterns were observed. In U.S. outbreak strains previously studied by 10 other typing methods, flaA typing correlated with the HL serotype within each outbreak, and six additional flaA types were identified. Our results suggest that RFLP analysis of the flaA gene from Campylobacter spp. has sufficient discrimination to be useful as a practical typing method for clinical and epidemiologic investigations.
Detection of 2-eicosanol by gas chromatography-mass spectrometry in sputa from patients with pulmonary mycobacterial infections
Authors: Alugupalli S , Olsson B , Larsson L
Published: January 2020

A total of 96 sputum specimens from patients with suspected or known mycobacterial and nonmycobacterial pulmonary infections were analyzed by gas chromatography-mass spectrometry for the presence of 2-eicosanol. This secondary alcohol was detected in all of the 25 sputum specimens culture positive for Mycobacterium tuberculosis, in 7 of the 9 sputum specimens culture positive for M. avium complex, and in all 3 of the studied sputum specimens associated with M. malmoense. The alcohol was not detected in any of the 45 culture-negative sputum specimens or in 14 sputum specimens culture positive for Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenzae, and Streptococcus pneumoniae. The ratio of tuberculostearic acid to 2-eicosanol was much lower in sputum samples culture positive for mycobacteria than in the corresponding in vitro-grown cultures. The present findings indicate that 2-eicosanol may be useful as a chemical marker for rapid diagnosis of pulmonary infections caused by the M. avium complex, M. malmoense, and M. tuberculosis.
Identification of four epitopes in hepatitis C virus core protein
Authors: Ferroni P , Mascolo G , Zaninetti M , Colzani D , Pregliasco F , Pirisi M , Barbone F , Gasparini V
Published: January 2020

We identified four epitopes in hepatitis C virus core protein by using the algorithm of Jameson and Wolf. G15V (amino acids [aa] 31 to 45) appears to be the immunodominant epitope, since it was able to detect antibodies to the core protein in all 40 patients and in 44 of 45 recombinant immunoblot assay-confirmed positive blood donors. This epitope is associated with a low frequency of false-positive results, as found with 522 negative blood donors. A strong reactivity was also observed with the Q15V epitope (aa 7 to 21), although this was associated with low specificity. Occasional reactivity to the R15P (aa 49 to 63) and P15R (aa 99 to 113) epitopes was observed. Q15V and G15V detected antibodies to core protein earlier than the other two epitopes.
Detection of aquareovirus RNA in fish tissues by nucleic acid hybridization with a cloned cDNA probe
Authors: Subramanian K , Lupiani B , Hetrick F M , Samal S K
Published: January 2020

A nucleic acid hybridization assay was developed to rapidly detect small quantities of aquareovirus RNAs in infected cells and organs. Cloned cDNA copies were synthesized from the genomic RNA of the SBR strain of aquareovirus. By using cloned cDNA probes, aquareovirus RNAs were detected in spleen and kidney tissues of experimentally infected fish.
Comparison of the Clearview Chlamydia, the PACE 2 assay, and culture for detection of Chlamydia trachomatis from cervical specimens in a low-prevalence population
Authors: Blanding J , Hirsch L , Stranton N , Wright T , Aarnaes S , de la Maza L , Peterson E M
Published: January 2020

The Clearview Chlamydia assay (Wampole Laboratories, Cranbury, N.J.), the PACE 2 DNA probe assay (GenProbe, San Diego, Calif.), and culture were compared for their abilities to detect Chlamydia trachomatis from cervical specimens in a population with a low prevalence (3.9%) of chlamydial infections. A consensus reference method was used. The consensus reference method defined a positive specimen as one with a positive culture result or positive by both of the two nonculture methods. Of the 940 specimens tested, 37 were positive; 36 were positive by culture, 28 were positive by the PACE 2 assay, and 27 were positive by the Clearview assay, giving sensitivities of 97.3, 75.5, and 72.9%, respectively, and specificities of 100, 97.1, and 98.9%, respectively. There was a direct correlation between the number of inclusion-forming units detected by culture and the ability of the two nonculture methods to detect the positive specimens.
Chondroitin sulfate depolymerase and hyaluronidase activities of viridans streptococci determined by a sensitive spectrophotometric assay
Authors: Homer K A , Denbow L , Whiley R A , Beighton D
Published: January 2020

Sensitive spectrophotometric assays for the detection of bacterial chondroitin sulfate depolymerase and hyaluronidase activities were developed by using Stains-all (1-ethyl-2-[3-(1-ethylnaphtho-[1,2-d]thiazolin-2-ylidene)-2- methylpropenyl]naphtho-[1,2-d]thiazolium bromide). Stains-all interacts with hyaluronic acid to produce a shift in the absorption spectrum with a distinct absorption peak between 620 and 660 nm, while chondroitin sulfate interacts to form a distinct shoulder between 440 and 500 nm. Assays measure undegraded substrate. A collection of 110 strains of viridans streptococci, including representatives of all the currently recognized species, was studied. Streptococcus intermedius and S. constellatus degraded hyaluronic acid, while only strains of S. intermedius, primarily isolated from brain and liver abscesses, produced chondroitin sulfate depolymerase. S. intermedius, of all the viridans streptococci, produces the widest range of glycoprotein- and glycosoaminoglycan-degrading enzymes, which may contribute to the virulence of this species.
Comparative evaluation of detection assays for Chlamydia trachomatis
Authors: Warren R , Dwyer B , Plackett M , Pettit K , Rizvi N , Baker A M
Published: January 2020

The performances of a commercial nucleic acid hybridization test (Gen-Probe Pace 2 Chlamydia trachomatis) and two commercial enzyme immunoassays (EIAs) (Abbott Chlamydiazyme and Pharmacia Chlamydia EIA) were evaluated against cell culture for the detection of C. trachomatis infection, with cervical swabs obtained from 1,037 women visiting a public sexual health center. The positivity rate by cell culture was 4.7%. Sensitivity and specificity for each test were as follows: Gen-Probe, 95.8 and 98.3%; Chlamydiazyme, 80.4 and 99.3%; Pharmacia EIA, 80.8 and 99.1%. Analysis of discrepant results with probe confirmation assay (Gen-Probe) and direct immunofluorescence (Syva Microtrak) revealed 12 cases of C. trachomatis infection for which culture was negative, resulting in the definition of a true-positive case as opposed to a culture positive. The positivity rate by true-positive definition was 5.9%, and sensitivity and specificity for each test were as follows: Gen-Probe, 96.7 and 99.6%; Chlamydiazyme, 77.5 and 100%; Pharmacia EIA, 77.0 and 100%; cell culture, 80.0 and 100%. We conclude that the Gen-Probe Pace 2 C. trachomatis test is a sensitive and specific alternative to cell culture for the detection of C. trachomatis.
Differentiation of Yersinia enterocolitica serotype O:5,27 strains by phenotypic and molecular techniques
Authors: Fukushima H , Gomyoda M , Aleksic S , Tsubokura M
Published: January 2020

Restriction endonuclease analyses of virulence plasmid DNA (REAP) and chromosomal DNA and other phenotypic characteristics were used to study the differentiation of Yersinia enterocolitica serotype O:5,27 strains. There was a close correlation between REAP patterns and the geographical distribution of serotype O:5,27. Human isolates produced only one REAP pattern, which was also found with isolates from pigs and dogs.
Comparison of plasmid- and chromosome-based polymerase chain reaction assays for detecting Chlamydia trachomatis nucleic acids
Authors: Mahony J B , Luinstra K E , Sellors J W , Chernesky M A
Published: January 2020

Several laboratories have demonstrated that the polymerase chain reaction (PCR) is more sensitive than culture or enzyme immunoassay (EIA) for detecting Chlamydia trachomatis in genitourinary tract specimens when various DNA targets are used for amplification, including the cryptic plasmid, major outer membrane protein (MOMP), or rRNA genes. We compared the performances of five different PCR assays, including assays with two plasmid, two MOMP, and one rRNA targets, by amplifying serial dilutions of C. trachomatis DNA and testing genitourinary tract specimens. By using published procedures, two different plasmid primers had sensitivities of 0.1 fg for C. trachomatis plasmid DNA and 10 fg for total cellular DNA. The sensitivities of the assays with the two MOMP primers were 0.1 and 10 pg, and the sensitivity for the assay with the rRNA primers was 1 pg for cellular DNA. Both plasmid-based assays detected 38 of 38 confirmed Chlamydiazyme-positive specimens, whereas the assays with the MOMP and rRNA primers detected 36 of 38 and 29 of 38 confirmed Chlamydiazyme-positive specimens, respectively. Six of 18 Chlamydiazyme-negative specimens collected from individuals whose specimens were positive by culture or immunofluorescence were positive by both plasmid-based PCRs; 4 of these were positive by PCR with the MOMP primers and 3 were positive by PCR with the rRNA primers. The results obtained with both purified DNA and genitourinary tract specimens indicated that the plasmid-based PCRs are more sensitive than bacterial chromosome-based PCRs for detecting C. trachomatis.
Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems
Authors: Clabots C R , Johnson S , Bettin K M , Mathie P A , Mulligan M E , Schaberg D R , Peterson L R , Gerding D N
Published: January 2020

A HindIII restriction endonuclease analysis (REA) typing system for total genomic Clostridium difficile DNA including a rapid and efficient method of DNA extraction and a scheme for organizing unique electrophoretic DNA band patterns was developed. REA typing was performed by two extraction methods for 1,965 C. difficile isolates obtained from patients with symptomatic C. difficile disease, asymptomatic patients who were C. difficile culture positive, and environmental surfaces. This isolate collection yielded 206 unique REA types, which were organized into 75 groups. A reference strain representing each unique REA type was chosen for DNA band pattern comparisons, cytotoxin testing, and plasmid analysis. The DNA band patterns utilizing a guanidine thiocyanate-EDTA-Sarkosyl DNA extraction method were 94% reproducible, while the original and sporadically problematic diethyl pyrocarbonate-sodium dodecyl sulfate DNA extraction method was 98% reproducible when readable patterns were obtained. Reference strains from 43 of the 75 groups were cytotoxin positive, 28 groups were cytotoxin negative, and 4 groups included both toxigenic and nontoxigenic strains. Cytotoxicity of isolates with a particular REA type was always consistent with the toxicity of the reference strain for that type. REA typing was able to discriminate strain differences within types identified by the immunoblot (89 isolates), bacteriophage-bacteriocin (44 isolates), and ribotyping (23 isolates) methods. REA typing is a sensitive, discriminating, reproducible, and rapid method for differentiating C. difficile strains and is suitable for large-scale epidemiologic studies.
Novel Campylobacter-like organism resembling Helicobacter fennelliae isolated from a boy with gastroenteritis and from dogs
Authors: Burnens A P , Stanley J , Schaad U B , Nicolet J
Published: January 2020

We isolated a Campylobacter-like organism resembling Helicobacter fennelliae from a 5 1/2-year-old boy with gastroenteritis. Similar strains had been found previously in fecal specimens from healthy and diarrheic dogs. These isolates could be differentiated from H. fennelliae by a lack of catalase and arylsulfatase activities. This group of organisms seems to be homogeneous by a nonradioactive dot blot DNA hybridization assay.
Sensitivity of culture compared with that of polymerase chain reaction for detection of Helicobacter pylori from antral biopsy samples
Authors: van Zwet A A , Thijs J C , Kooistra-Smid A M , Schirm J , Snijder J A
Published: January 2020

Polymerase chain reaction (PCR) was used to test the sensitivity of standard bacteriological culture of Helicobacter pylori. The tests were concordant in 97% of cases. PCR did not detect more treatment failures after triple treatment than did the standard culture method. In the present study, culture was as sensitive as PCR for the detection of H. pylori.
Metronidazole susceptibility testing for Helicobacter pylori: comparison of disk, broth, and agar dilution methods and their clinical relevance
Authors: DeCross A J , Marshall B J , McCallum R W , Hoffman S R , Barrett L J , Guerrant R L
Published: January 2020

Since the methods for metronidazole susceptibility testing of Helicobacter pylori have not been standardized or validated, we compared three methods that are used to test the metronidazole susceptibilities of 25 isolates of H. pylori. Specifically, we examined the methods of Steer's replicator agar dilution, tube broth microdilution, and modified Kirby-Bauer disk diffusion. The metronidazole disk zone sizes obtained by the disk diffusion method correlated well (r = 0.74) with the MICs obtained by the agar dilution method. Afterward, the disk diffusion method was used to characterize the metronidazole susceptibilities of 44 isolates of H. pylori. Dual therapy (bismuth and metronidazole) proved to be highly effective against metronidazole-susceptible strains (81.6% eradication rate) but fared poorly against resistant strains (16.7% eradication rate; P < 0.01). Using agar dilution testing, we validated the modified Kirby-Bauer disk diffusion method for metronidazole susceptibility testing of H. pylori and conclude that it is practical, accurate, and clinically applicable.
Detection of Ockelbo virus RNA in skin biopsies by polymerase chain reaction
Authors: Hörling J , Vene S , Franzén C , Niklasson B
Published: January 2020

A sensitive assay based on the polymerase chain reaction for the detection of Ockelbo virus RNA was developed. Two primer pairs from the gene coding for the E2 glycoprotein were chosen. By use of a nested strategy for the primers, as few as 1 to 10 PFU could be detected. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The primer pairs allowed amplification of several Ockelbo and Sindbis virus isolates but discriminated between these and other alphaviruses. Ockelbo virus RNA was detected in 4 of 10 skin biopsy specimens collected during the acute stage of the disease. The identities of the amplified products were confirmed by restriction endonuclease cleavage. Acute- and convalescent-phase sera as well as lymphocytes collected during the convalescent phase were negative by the polymerase chain reaction. No infectious virus could be recovered from any of the specimens.