bluuebunny/crossref_metadata_2025 huggingface dataset:
Laboratory investigations on the low pathogenic potential of Plesiomonas shigelloides
Authors: Abbott S L , Kokka R P , Janda J M
Published: January 2020

The pathogenic properties of 16 Plesiomonas shigelloides strains recovered from humans with extraintestinal and intestinal illnesses, infected animals, and environmental sources were investigated. Most strains possessed a high cell charge and low surface hydrophobicity analogous to those of Shigella spp.; additionally, serogroup O:17 strains reacted with Shigella group D antisera. However, unlike the shigellae, P. shigelloides strains did not universally bind Congo red, were noninvasive in HEp-2 cell assays, and did not produce a Shiga-like toxin on Vero cells. On HEp-2, Y1, and possibly Vero cells, a low-level cytolysin was consistently produced by all 16 P. shigelloides strains when grown in either Evan Casamino Acids-yeast extract or Penassay broth. The median 50% lethal dose for all 16 P. shigelloides strains in outbred Swiss Webster mice was 3.5 x 10(8) CFU (range, 3.2 x 10(7) to greater than 1 x 10(9) CFU). Animal pathogenicity did not correlate with cytolysin expression, possession of a greater than or equal to 120-MDa plasmid, protein profile, or resistance to complement-mediated lysis. No strain analyzed produced siderophores or a heat-stable enterotoxin. The results suggest that members of the genus Plesiomonas have an overall low pathogenic potential, irrespective of the site of isolation or phenotypic, serologic, or surface properties shared with other traditional enteropathogens.
A single-season epidemic with respiratory syncytial virus subgroup B2 during 10 epidemic years, 1978 to 1988
Authors: Mufson M A , Akerlind-Stopner B , Orvell C , Belshe R B , Norrby E
Published: January 2020

Respiratory syncytial virus strains isolated from infants and children hospitalized with acute respiratory tract disease in Huntington, W. Va., from July 1979 through June 1988 were categorized by subgroup by using two panels of monoclonal antibodies specific for subgroup A and subgroup B. Of 405 strains tested, 319 (78.8%) were subgroup A, 73 (18.0%) were subgroup B1, and 13 (3.2%) were subgroup B2. The subgroup B2 strains occurred as a single-season epidemic during the epidemiologic year July 1980 to June 1981, except for one strain isolated in the epidemiologic year 1978 to 1979. Subgroup B strains were associated with significantly fewer bronchiolitis illnesses than subgroup A strains. The severity of illnesses caused by subgroup B1 and B2 strains was the same.
Comparison of two O-serogrouping systems for mesophilic Aeromonas spp
Authors: Shimada T , Kosako Y
Published: January 2020

Two O-serogrouping systems for mesophilic Aeromonas spp., National Institute of Health, Tokyo, Japan, and National Institute of Public Health and Environmental Hygiene, Bilthoven, The Netherlands, containing 44 and 30 reference strains, respectively, were compared. Nine strains in each system were considered identical. Thirty-four unique strains were identified, and 22 strains of the two systems were in an a,b-a,c type of relationship to each corresponding O group.
Diagnostic value of the polymerase chain reaction for Chlamydia detection as determined in a follow-up study
Authors: Claas H C , Wagenvoort J H , Niesters H G , Tio T T , Van Rijsoort-Vos J H , Quint W G
Published: January 2020

The diagnostic value of the polymerase chain reaction (PCR) for detection of Chlamydia trachomatis in comparison with that of the culture technique was established in a follow-up study of 32 patients (81 samples) who were treated for a C. trachomatis infection. The PCR was performed with two different sets of primers, a genus-specific primer set directed against the rRNA genes and a C. trachomatis-specific set directed against the common endogenous plasmid. After treatment with doxycycline, all patients became culture negative after 1 week. Results for the detection of C. trachomatis by the PCR were in complete agreement with the results by the culture method of detection, except for one culture-negative sample, which was found to be positive by the PCR. The results indicated that 1 week after treatment, no residual chlamydial DNA was found in the samples. Therefore, the PCR can be used for monitoring infections by chlamydiae.
Etiology of childhood diarrhea in Beijing, China
Authors: Kain K C , Barteluk R L , Kelly M T , He X , de Hua G , Ge Y A , Proctor E M , Byrne S , Stiver H G
Published: January 2020

To determine the role of recently recognized enteropathogens in childhood diarrhea in China, 221 children with diarrhea and 108 controls seen at the Beijing Children's Hospital were studied during April and May 1989. Stools were examined for ova, parasites, and rotavirus, cultured for bacterial pathogens, and probed for enterotoxigenic Escherichia coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), and enteropathogenic adherence factor-positive (EAF+) E. coli. Pathogens were identified in 56.5% of children with diarrhea and 43.5% of controls (P = 0.04). Detection of enteropathogens was significantly greater in patients examined within 1 week of symptom onset (65%) than in patients examined later (39%; P = 0.01). ETEC was the most frequently detected pathogen in children with diarrhea, accounting for 20% of the cases. Other agents identified in patients included the following: salmonellae, 12%; rotavirus, 7%; EIEC, 7%; EHEC, 7%; members of the Aeromonas hydrophila group, 6%; EAF+ E. coli, 5%; Ascaris lumbricoides, 3%; shigellae, 3%; campylobacters, 2%; and Vibrio spp., 0.5%. The isolation rates of salmonellae (P = 0.02), EAF+ E. coli (P = 0.04), and mixed pathogens (P = 0.05) were significantly greater for diarrhea patients than for controls. Resistance to multiple antimicrobial agents occurred in 39% of the Salmonella isolates, 22% of the Aeromonas isolates, and 17% of the Shigella isolates. Multiresistant salmonellae (P = 0.05) and shigellae were recovered from diarrheal stools only. Ciprofloxacin, cefotaxime, and imipenem were the only agents tested to which all bacterial isolates were susceptible in vitro. These results suggest that both traditional and newly recognized agents are important causes of childhood diarrhea in Beijing and that therapy may be complicated by indigenous antimicrobial resistance.
Rapid identification of dengue virus serotypes by using polymerase chain reaction
Authors: Morita K , Tanaka M , Igarashi A
Published: January 2020

Four primer pairs were selected on the basis of the published sequence data of four dengue virus serotypes so that each unique target sequence size could be amplified for each serotype by polymerase chain reaction. The procedure consists of (i) RNA preparation, (ii) reverse transcription, and (iii) polymerase chain reaction, all of which could be completed within 2 h in a single tube for each specimen. The amplified sequence size revealed by ethidium bromide-stained agarose gel electrophoresis was unique for each serotype, using infected culture fluid of isolates from dengue fever or dengue hemorrhagic fever patients in Thailand, Indonesia, and the Philippines as well as from prototype viruses, thus facilitating simultaneous identification and typing.
Characterization of risk factors for Helicobacter pylori infection among men attending a sexually transmitted disease clinic: lack of evidence for sexual transmission
Authors: Polish L B , Douglas J M , Davidson A J , Perez-Perez G I , Blaser M J
Published: January 2020

The mechanism of transmission of Helicobacter pylori is unknown. To investigate the role of sexual behavior and demographic factors in the acquisition of H. pylori infection, we evaluated the seroprevalence of antibody to H. pylori in 370 men attending an urban sexually transmitted diseases clinic. Sera from the following three groups were analyzed by enzyme-linked immunosorbent assay for H. pylori-specific immunoglobulin G: 78 human immunodeficiency virus (HIV)-seropositive homosexual men, 102 HIV-seronegative homosexual men, and 190 HIV-seronegative heterosexual men. Overall, the seroprevalence of H. pylori was 100 of 370 men (27%), with rates of 18% in HIV-seropositive homosexual men and 20% in HIV-seronegative homosexual men versus 35% in heterosexual men (P less than 0.005, chi 2 test). By ethnic group, 21 (12%) of 181 Caucasian men, 40 (41%) of 97 black men, and 37 (43%) of 87 Hispanic men were seropositive (P less than 0.001, chi 2 test). Multivariate analysis revealed that race was associated with H. pylori seropositivity independent of HIV status, sexual preference, or age. There was no relationship between H. pylori seropositivity and the number of lifetime sexual partners or previous sexually transmitted diseases. Three HIV-seropositive men with H. pylori immunoglobulin G had essentially identical antibody titers over 8 to 16 months of follow-up. In conclusion, black and Hispanic men have significantly higher H. pylori seroprevalence rates than do Caucasian men, but neither sexual behavior nor HIV infection influences the presence or persistence of H. pylori antibody. Further evaluation of the factors associated with these ethnic differences may lead to a better understanding of H. pylori acquisition and transmission.
16S rRNA sequences of Bartonella bacilliformis and cat scratch disease bacillus reveal phylogenetic relationships with the alpha-2 subgroup of the class Proteobacteria
Authors: O'Connor S P , Dorsch M , Steigerwalt A G , Brenner D J , Stackebrandt E
Published: January 2020

The primary structures of 16S rRNAs of Bartonella bacilliformis, an isolate of the cat scratch disease (CSD) bacillus, and a strain phenotypically similar to the CSD bacillus were determined by reverse transcriptase sequencing. These microorganisms were found to be members of the alpha-2 subgroup of the class Proteobacteria. The sequence from B. bacilliformis was most closely related to the rRNA of Rochalimaea quintana (91.7% homology), the etiologic agent of trench fever. The sequence from the isolate of the CSD bacillus showed the greatest homology with Brucella abortus (89.7%) and, when compared with oligonucleotide catalog data, formed a cluster with Rhodopseudomonas palustris, Pseudomonas carboxidovorans, Nitrobacter species, and Bradyrhizobium species. The 16S rRNA sequence was also determined for the Cleveland Clinic isolate, which was previously shown to be phenotypically similar to and approximately 30% related, by DNA hybridization, to the CSD bacillus. The Cleveland Clinic isolate was isolated from a patient not diagnosed with CSD. The rRNAs from these bacteria exhibited 98.2% homology, confirming that this isolate is a second species in the same genus as the CSD bacillus. Our data suggest that neither B. bacilliformis nor the CSD bacillus is the etiologic agent of bacillary epithelioid angiomatosis.
Amplification of a species-specific DNA fragment of Mycobacterium tuberculosis and its possible use in diagnosis
Authors: Del Portillo P , Murillo L A , Patarroyo M E
Published: January 2020

In recent work, a species-specific Mycobacterium tuberculosis DNA fragment was cloned and sequenced. On the basis of its nucleotide sequence, two oligonucleotides were synthesized and used as primers for polymerase chain reaction (PCR) amplification. A 396-bp fragment was specifically amplified from the M. tuberculosis genome. No amplification was observed from any of 10 different mycobacterial strains, included those belonging to the M. tuberculosis complex. Neither was this fragment amplified from genomes of humans or different species of clinically important bacteria. The PCR product was detected by dot blot hybridization even when as little as 10 fg of purified M. tuberculosis DNA was used. This amplification method was subsequently used to detect and identify bacilli in different clinical samples, such as sputum, urine, and cerebrospinal fluid. A good correlation was observed between the results obtained with the PCR method that we describe and other diagnostic tests currently used. Thus, PCR amplification of this genomic fragment is proposed as a specific, rapid, and sensitive test for the diagnosis of infection with M. tuberculosis.
Development of simian immunodeficiency virus isolation, titration, and neutralization assays which use whole blood from rhesus monkeys and an antigen capture enzyme-linked immunosorbent assay
Authors: Lohman B L , Higgins J , Marthas M L , Marx P A , Pedersen N C
Published: January 2020

Assays that use rhesus macaque whole blood and an antigen capture enzyme-linked immunosorbent assay for the simian immunodeficiency virus (SIV) p27 core protein were developed for the isolation of SIV from the blood of infected animals, the titration of infectivity of SIV inocula, and the quantitation of virus neutralizing antibodies in serum. These assays required small amounts of whole blood, were adaptable to a microtiter format, and used substrates mainly of rhesus macaque origin.
Direct identification of bacterial isolates in blood cultures by using a DNA probe
Authors: Davis T E , Fuller D D
Published: January 2020

This study involved the rapid, direct identification of Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, Enterococcus sp., and Streptococcus agalactiae from positive blood culture bottles (BACTEC, Johnston Laboratories, Inc.) by using the AccuProbe (Gen-Probe, San Diego, Calif.) culture confirmation test. This method uses a chemiluminescent DNA probe that detects the rRNA of the target organisms. The manufacturer's instructions were modified to use a pellet of bacteria made directly from positive blood culture broth rather than a colony from an agar plate. Two separate procedures of selective centrifugation were employed in order to obtain the pellet. The first utilized a routine clinical centrifuge and a large volume of broth (10 to 12 ml) from the blood culture bottle. The second method used a microcentrifuge and less volume (1 to 1.5 ml). A total of 196 clinical specimens taken directly from positive blood culture broths were correctly identified by AccuProbe from pellets made by using the clinical centrifuge technique, while 166 clinical specimens used as negative controls failed to show hybridization. The microcentrifuge technique for obtaining pellets was performed on 105 patient specimens, and all were correctly identified. When combined with the microcentrifuge technique for pellet preparation, the AccuProbe test has several advantages: (i) direct identification of bacteria from blood culture broths, (ii) rapid turn-around time (30 min), (iii) simplicity of the procedure, and (iv) relative low cost.
Evaluation of the L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test
Authors: Leland D S , Kohler R B
Published: January 2020

The purpose of this study was to evaluate the L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test (Access Medical Systems, Inc., Branford, Conn.) for detection of Legionella antigen in urine. A total of 481 frozen urine samples previously tested by an in-house solid-phase radioimmunoassay (RIA) was thawed and retested by using L-CLONE. Included in this sample were 140 RIA-positive samples from culture-positive or serologically confirmed cases of legionellosis and 341 RIA-negative samples from patients with non-Legionella respiratory disease or bacteriuria. The original RIA test result was accepted as the true value. L-CLONE correctly identified 76 of 140 (54%) known positive samples. False-negative results could not be attributed to a low Legionella antigen concentration or to a Legionella antigen subgroup. L-CLONE correctly identified 252 of 341 (74%) known negative samples. False-positive results were experienced in all groups of negative samples, regardless of the patients' underlying diseases. A total of 141 fresh urine samples was tested; all were Legionella antigen negative by RIA. L-CLONE provided 86% specificity. The sensitivity of the L-CLONE in testing fresh urine samples could not be evaluated because of the lack of Legionella antigen RIA-positive samples.
Diagnostic application of polymerase chain reaction for detection of Ehrlichia risticii in equine monocytic ehrlichiosis (Potomac horse fever)
Authors: Biswas B , Mukherjee D , Mattingly-Napier B L , Dutta S K
Published: January 2020

Genomic amplification by the polymerase chain reaction (PCR) was used to identify a unique genomic sequence of Ehrlichia risticii directly in DNA isolated from peripheral-blood buffy coat cells of E. risticii-infected horses (Potomac horse fever) and from infected cell cultures. A specific primer pair, selected from a cloned, species-specific, 1-kb DNA fragment of the E. risticii genome as a template, was used for the amplification of the target DNA of 247 bp. The optimal number of 40 PCR cycles, determined by analyzing an amplification profile obtained with a constant Taq polymerase concentration, was used to achieve maximum amplification of the E. risticii DNA segment. Efficient amplification of target DNA was achieved with specimens processed by either the phenol extraction or rapid lysis method. The specificity of the amplified DNA product was confirmed by the proper size (247 bp) and appropriate restriction enzyme cleavage pattern of the amplified target DNA, as well as by the specific hybridization signal obtained by using a PCR-amplified 185-bp internal DNA probe. A 10(5)- to 10(6)-fold amplification of target DNA, which allowed detection of E. risticii from as few as two to three infected cells in culture and from a very small volume of buffy coat cells from infected horses, was achieved. This PCR amplification procedure was found to be highly specific and sensitive for the detection of E. risticii for the study of Potomac horse fever.
Differences in genomic DNA sequences between pathogenic and nonpathogenic isolates of Entamoeba histolytica identified by polymerase chain reaction
Authors: Tachibana H , Ihara S , Kobayashi S , Kaneda Y , Takeuchi T , Watanabe Y
Published: January 2020

A lambda gt11 cDNA library was constructed from the poly(A)+ RNA of trophozoites of Entamoeba histolytica HM-1:IMSS strain. The library was immunologically screened with monoclonal antibody 4G6, which is specific for the 30,000-Mr antigen of pathogenic isolates. A 0.7-kb clone was isolated, and its nucleotide sequence was determined. To examine whether this gene was specific for pathogenic isolates, a polymerase chain reaction was performed by using four sets of primers and the genomic DNA of pathogenic and nonpathogenic isolates as templates. Amplified DNAs were detected not only in pathogenic isolates but also in nonpathogenic isolates. However, when sequences of amplified DNA of these isolates were compared, minor differences were observed. By considering the presence or absence of recognition sites of some endonucleases, it was possible to distinguish between the pathogenic and nonpathogenic isolates. When various isolates with different zymodemes were examined by polymerase chain reaction and enzyme digestion, the results of typing were entirely in accord with those of zymodeme analysis. These results indicate that there is dimorphism in the genomic DNA coding the 30,000-Mr antigen of E. histolytica and that the combined use of the polymerase chain reaction and enzyme digestion is a useful strategy for identification of species and determination of pathogenicity.
Immunodot detection of Escherichia coli heat-stable enterotoxin b by using enhanced chemiluminescence reaction
Authors: Lortie L A , Harel J , Fairbrother J M , Dubreuil J D
Published: January 2020

An indirect immunodot assay with rabbit antibodies raised against purified heat-stable enterotoxin type b (STb) and with a Western blotting (immunoblotting) detection system (ECL; Amersham International plc, Amersham, United Kingdom) was developed for the detection of STb toxin. Culture supernatants of 62 Escherichia coli isolates from pigs with diarrhea were blotted onto nitrocellulose and incubated with anti-STb serum. The chemiluminescence produced by the action of horseradish peroxidase with luminol and H2O2 was recorded by exposure of X-ray film. Over 90% correlation was observed between the rat or pig intestinal ligated loop assay and a radioactive DNA probe and the ECL immunodot assay for the detection of STb. In addition, using this new and sensitive technique, we could detect STb in the feces of a newborn pig inoculated with an STb-producing E. coli strain. Detection of STb-producing E. coli in pigs with diarrhea will be greatly facilitated by the use of this convenient and rapid diagnostic assay.
Immunomagnetic separation and DNA hybridization for detection of enterotoxigenic Escherichia coli in a piglet model
Authors: Lund A , Wasteson Y , Olsvik O
Published: January 2020

Enterotoxigenic Escherichia coli (ETEC) strains were detected by stool blot hybridization assays using different oligonucleotide probes for the colonization fimbrial antigen F4, heat-stable enterotoxin I (ST I), and heat-labile enterotoxin (LT I) genes. Forty-eight fecal samples and seven samples of intestinal content from ETEC-challenged newborn piglets were processed in two ways: (i) by direct inoculation of bacterial suspension onto nylon membranes overlaying blood agar and (ii) by immunomagnetic enrichment of F4+ ETEC using magnetic beads coated with F4 monoclonal antibodies before inoculation onto nylon membranes. In samples obtained from nondiarrheic piglets pre- and postchallenge, E. coli genes for F4, ST I, and LT I could be detected only after immunomagnetic enrichment. No difference between the two methods in detection of these E. coli genes was observed when stool blots from diarrheic piglets were examined. By using magnetic separation, it was easy to decrease background bacterial flora, intestinal cells, and fecal debris and thus produce purer specimens. The method evaluated in this animal model appeared simple and quick and increased the sensitivity of detection of ETEC strains 100-fold compared with the direct stool blot hybridization assays. Prior bacterial isolation and identification were not necessary.
Comparison of five commercial enzyme-linked immunosorbent assays and Western immunoblotting for human immunodeficiency virus antibody detection in serum samples from Central Africa
Authors: Behets F , Disasi A , Ryder R W , Bishagara K , Piot P , Kashamuka M , Kamenga M , Nzila N , Laga M , Vercauteren G
Published: January 2020

Detection by five different enzyme-linked immunosorbent assays (ELISAs) of antibody to human immunodeficiency virus (HIV) in sera from three Zairian populations consisting of 1,998 individuals with various risks for HIV infection was evaluated. Sera that were reactive by at least one assay and 10% of the nonreactive serum samples were analyzed by Western blot (immunoblot) by using U.S. Public Health Service interpretation criteria. Sera which were positive by ELISA for detection of antibody to HIV-1 and HIV-2 and negative or indeterminate by HIV-1 Western blot were also analyzed by HIV-2 Western blot. Overall, 443 (22.2%) serum specimens were HIV-1 Western blot positive, 390 (19.5%) had indeterminate HIV-1 Western blot patterns, and no samples were HIV-2 Western blot positive. The sensitivity of the ELISAs ranged from 97.5 to 99.8%, and the specificity ranged from 51.7 to 98.4%. By population group, the negative predictive value ranged from 97.1 to 100%, in contrast to the positive predictive value, which varied from 6.6 to 100%. Follow-up results for sera which were indeterminate for antibody to HIV-1 documented only four seroconversions (6.0%) among 67 individuals at high risk for HIV-1 infection and no seroconversions among 202 individuals at relatively low risk for HIV-1 infection. This study demonstrates the importance of evaluating commercial ELISAs with sera from appropriate geographical regions in order to select the most cost-effective and practical assay for use in that region. Furthermore, the high frequency of indeterminate Western blots for African sera emphasizes the continual need for improved confirmatory assays and interpretation criteria.
Serology and leprosy: immunoassays comparing immunoglobulin G antibody responses to 28- and 30-kilodalton proteins purified from Mycobacterium bovis BCG
Authors: Pessolani M C , Peralta J M , Rumjanek F D , Gomes H M , Marques M A , Almeida E C , Saad M H , Sarno E N
Published: January 2020

Two major proteins from Mycobacterium bovis BCG culture filtrates with molecular masses of 28 kDa (P28) and 30 kDa (P30), identified as components of the BCG 85 complex, were purified and used in enzyme-linked immunosorbent assays (ELISAs) for the determination of specific immunoglobulin G (IgG) levels in patients with leprosy or tuberculosis or with exposure to these diseases. High reactivity to both antigens was observed with sera from lepromatous leprosy patients, whereas antibody levels in sera from paucibacillary leprosy patients were not significantly different from those in sera from healthy individuals from an area in which leprosy is endemic. High IgG responses were also found in some contacts of lepromatous leprosy patients. A comparison of the levels of anti-P28 and anti-P30 within the multibacillary leprosy patient group showed much higher IgG reactivity to P28 than to P30, suggesting that the antibody response of lepromatous patients is directed predominantly against the 28-kDa protein. A high degree of correlation in values of ELISAs based on P28 and on the phenolic glycolipid of Mycobacterium leprae was observed in all groups analyzed. The potential use of an assay based on the 28-kDa protein to selectively distinguish individuals destined to develop multibacillary leprosy is discussed, as also is the likelihood that the 28-kDa-30-kDa complex, part of the fibronectin-binding family, is an important component of M. leprae.
Platelia-Toxo IgA, a new kit for early diagnosis of congenital toxoplasmosis by detection of anti-P30 immunoglobulin A antibodies
Authors: Decoster A , Slizewicz B , Simon J , Bazin C , Darcy F , Vittu G , Boulanger C , Champeau Y , Demory J L , Duhamel M
Published: January 2020

With the aim of achieving earlier diagnosis of congenital toxoplasmosis, anti-P30 immunoglobulin A (IgA) antibodies were assayed by using a Platelia-Toxo IgA kit with samples from 72 children born to mothers who seroconverted during pregnancy. A total of 148 serum samples and 1 cerebrospinal fluid samples were from 23 congenitally infected children (2 serum samples were collected from fetuses), and 74 serum samples were from 49 uninfected children. Among the 23 infected children, anti-P30 IgA antibodies were present in all infants either at birth or in the following weeks, whereas anti-P30 IgM antibodies were present in 13 from the 23 infected children either at birth or in the following weeks. Serum samples collected in utero from two infected children were also tested. One of these samples was positive for both anti-P30 IgA and anti-P30 IgM antibodies, whereas both children were negative at birth for these antibodies. Neither anti-P30 IgA nor anti-P30 IgM antibodies were detected in 47 of 49 uninfected children. These results suggest that detection of anti-P30 IgA antibodies by the Platelia-Toxo IgA kit is a very effective method for early diagnosis of congenital toxoplasma infection.
Evaluation of first- and second-generation RIBA kits for detection of antibody to hepatitis C virus
Authors: Chaudhary R K , MacLean C
Published: January 2020

Samples reactive by first-generation recombinant immunoblot assay (RIBA) to detect antibody to hepatitis C virus (anti-HCV) (RIBA-1 [Chiron, Calif.]) remained reactive by a second-generation test (RIBA-2) for HCV antibodies. A total of 75% of specimens indeterminate by RIBA-1 became reactive, 12.5% were nonreactive, and 12.5% remained indeterminate by RIBA-2. Among RIBA-1-nonreactive specimens, 12.0% became positive and 5.1% became indeterminate by RIBA-2. The antigens c33c and c22-3 have increased the sensitivity of RIBA-2.
Identification of related DNA sequences in Borrelia burgdorferi and two strains of Leptospira interrogans by using polymerase chain reaction
Authors: Kron M A , Gupta A , Mackenzie C D
Published: January 2020

The suitability of a polymerase chain reaction assay for Borrelia burgdorferi in epidemiological studies of infected tick populations was evaluated by using 28 strains of Leptospira interrogans and lysates of fixed adult Ixodes tick tissues. Two false positives representing leptospires were differentiated from B. burgdorferi by using an oligonucleotide probe.
Humoral immune response of tuberculous patients against the three components of the Mycobacterium bovis BCG 85 complex separated by isoelectric focusing
Authors: Van Vooren J P , Drowart A , De Cock M , Van Onckelen A , D'Hoop M H , Yernault J C , Valcke C , Huygen K
Published: January 2020

An isoelectric-focusing technique followed by Western blot (immunoblot) analysis was used to investigate the immunoglobulin G response of tuberculous patients against each of the three components of the Mycobacterium bovis BCG antigen 85 complex. The 85A component was stained by the tuberculous as well as the non-tuberculous sera. In contrast, the 85B and the 85C proteins of the complex were not stained by the control sera but were stained by 20 of 28 tuberculous serum samples.
Detection of salmonellae by using Rambach agar and by a C8 esterase spot test
Authors: Freydiere A M , Gille Y
Published: January 2020

In food microbiology, Rambach agar facilitates the differentiation of non-typhi Salmonella through a specific red pigmentation of the colonies. The usefulness of Rambach agar was examined relative to its usefulness to the field of clinical microbiology. Of 170 non-typhi Salmonella strains, 92 and 97% gave bright red colonies after 24 and 48 h of incubation, respectively, while 100% of 112 other members of the family Enterobacteriaceae were of a different color (blue, green, beige, or colorless). Red colonies were also found with five of five Acinetobacter isolates and one of three Pseudomonas isolates. To further detect Salmonella typhi and the rare beige or colorless colonies atypical of Salmonella isolates, a C8 esterase detection spot test was carried out. With UV light, that test revealed fluorescent colonies for all Salmonella isolates tested.
Restriction fragment length polymorphisms in rRNA operons for subtyping Shigella sonnei
Authors: Hinojosa-Ahumada M , Swaminathan B , Hunter S B , Cameron D N , Kiehlbauch J A , Wachsmuth I K , Strockbine N A
Published: January 2020

Shigella sonnei is the most frequent cause of shigellosis in the United States. Epidemiologic studies of this organism have been hampered by the lack of adequate typing procedures. Ribosomal DNA analysis (ribotyping), a method which analyzes restriction fragment length polymorphisms in the chromosomal genes that encode rRNA, has recently been shown to be useful for microbial species identification and subtyping. To determine whether ribotyping could be used to distinguish between S. sonnei isolates, we conducted Southern hybridization studies on isolates from 16 different geographic locations and from four recent outbreaks. S. sonnei genomic DNA fragments generated following digestion with SalI hybridized with Escherichia coli 16S and 23S rRNAs to produce six distinct patterns; strains with patterns 1, 2, and 3 were each further subdivided into two additional patterns by using PvuII, SmaI, and SstI, respectively. Epidemiologically related strains had identical patterns. Ribotyping appears to be a useful tool for epidemiologic studies of shigellosis caused by S. sonnei.
Amplification and characterization of herpesvirus DNA in cerebrospinal fluid from patients with acute encephalitis
Authors: Rozenberg F , Lebon P
Published: January 2020

A single pair of oligonucleotide primers selected within a highly conserved region of the DNA polymerase gene of the herpesviruses was designed to amplify related viral genomes, i.e., herpes simplex virus type 1, herpes simplex virus type 2, Epstein-Barr virus, and cytomegalovirus, by the polymerase chain reaction. A simple restriction enzyme analysis of these amplified products allowed accurate characterization of the herpesvirus type. Ninety-nine cerebrospinal fluid samples from 36 patients (including newborns, children, and adults) with acute encephalitis were tested for the presence and identification of herpesvirus DNA by this approach. High levels of viral DNA, which were readily visualized by simple ethidium bromide staining, were found in all these patients from the first days of the disease and, in some cases, until the third week following the onset of acute encephalitis. The herpesvirus type was rapidly identified by enzymatic digestion in 33 patients' samples and by hybridization and direct sequencing in the last 3 patients' samples. Our results show that the polymerase chain reaction provides a highly sensitive and specific technique for the identification of herpesviruses DNA in cerebrospinal fluid that should be of value for early and rapid diagnosis, therapeutic decisions, prognostic evaluation, and epidemiological studies.
110-kilodalton recombinant protein which is immunoreactive with sera from humans, dogs, and horses with Lyme borreliosis
Authors: Caputa A C , Murtaugh M P , Bey R F , Loken K I
Published: January 2020

EcoRI-digested DNA from Borrelia burgdorferi was ligated into the dephosphorylated vector pWR590 and transformed into Escherichia coli DH5 alpha. When the gene library was screened, 20 clones reacted with pooled dog sera with high titers (immunofluorescent antibody titer, greater than or equal to 1,280) to this spirochete. One clone expressed a 110-kDa antigen that reacted strongly with the high-titered pooled sera from dogs with Lyme borreliosis and serum from goats immunized with B. burgdorferi. The 110-kDa protein was serum from goats immunized with B. burgdorferi. The 110-kDa protein was expressed with and without isopropyl-beta-D-thiogalactosidase, indicating the protein is not a fusion protein with beta-galactosidase. Monospecific antisera to the 110-kDa antigen recognized a 75-kDa Borrelia protein. Of the sera that reacted with B. burgdorferi by immunoblotting; 57, 100, and 83% of human, dog, and horse serum samples, respectively, reacted with the 110-kDa protein. Sera from individuals that tested negative with a B. burgdorferi lysate with immunoblotting showed no reaction with the 110-kDa protein. The 110-kDa antigen appears to be useful for the diagnosis of Lyme borreliosis.
Anti-Salmonella lipopolysaccharide monoclonal antibodies: characterization of Salmonella BO-, CO-, DO-, and EO-specific clones and their diagnostic usefulness
Authors: Luk J M , Lindberg A A
Published: January 2020

To facilitate the identification and serotyping of Salmonella species, we established a wide variety of murine monoclonal antibodies (MAbs) that were reactive with the lipopolysaccharides (LPSs) of Salmonella serogroups B to E. An effective approach for generating LPS-reactive hybridomas was used; this required immunization of mice with LPS-coated bacteria. To screen for diagnostically useful MAbs, the MAbs were tested by enzyme-linked immunosorbent assay against a set of purified LPSs from smooth and rough Salmonella strains. At least four major groups of antibody specificities were identified: Salmonella (i) BO specific, (ii) CO specific, (iii) DO specific, and (iv) EO specific. For a more detailed epitope analysis, a panel of eight different serogroup-specific MAbs which were shown to bind the O-antigenic polysaccharide chains, yielding characteristic ladder patterns in Western blots (immunoblots) against the LPS of Salmonella serogroups B to E, were selected. The availability of various chemically defined LPS structures and Salmonella O-antigen glycoconjugates permitted the definition of O-antigenic polysaccharide epitopes recognized by each MAb that serologically corresponded to factors O3, O4, O5, O6, O7, O8, O9, and O10 on the basis of the Kauffmann-White scheme for Salmonella classification. The diagnostic accuracy of these immunochemically defined O-specific MAbs for Salmonella serotyping was demonstrated by correct identification of all 167 salmonellae (including 72 serotypes from serogroups B to E) among the 294 bacterial strains in a slide agglutination test. No false-positive reactions were detected.
Comparative sequence analysis of human cytomegalovirus strains
Authors: Lehner R , Stamminger T , Mach M
Published: January 2020

Three regions of DNA from five low-passage clinical isolates of human cytomegalovirus were amplified by polymerase chain reaction. The DNA sequences as well as the predicted amino acid sequences were compared with those of the laboratory strains AD169 and Towne. The genomic regions consisted of (i) three regions from the major glycoprotein (gp58/116, unique long [UL]55), (ii) three regions from the integral membrane protein (IMP, UL100), and (iii) a region from the major immediate-early 1 and 2 (IE-1/2) enhancer/promoter. Homologies ranged from 75.8 to 100.0% on the nucleotide level and from 47 to 100% on the amino acid level. The following two patterns were observed. (i) There are regions with a high degree of conservation with few scattered point mutations (mainly in the IE-1/2 enhancer/promoter and in the IMP gene). (ii) There are clusters of highly variable regions (parts of the gp58/116 gene and of the IMP gene). Within the areas of high variability, the strains could be classified into a limited number of subtypes.
Predicting human immunodeficiency virus type 1-positive sera by using two enzyme immunoassay kits in a parallel testing format
Authors: Fonseca K , Anand C M
Published: January 2020

Two algorithms for screening sera for antibody to human immunodeficiency virus type 1 were compared for their efficiency in identifying a true-positive sample in a population with heterogeneous risk factors, using the criteria of specificity and positive predictive value (PPV). In the first algorithm, all sera were screened by using a single enzyme immunoassay (EIA) kit, and a specificity of 98.6% and a PPV of 69.3% was calculated for true-positive sera. The second algorithm employed two different EIA kits in parallel to screen each sample. In the first instance, a specificity and a PPV of 100% was calculated if a positive sample was defined as reactive by both EIA kits; in the second, a specificity of 99.97% and a PPV of 99.4% was obtained if this criterion was extended to include a combination of one reactive and one equivocal result obtained with the two EIA kits.
Biotypes of Haemophilus influenzae that are associated with noninvasive infections
Authors: Harper J J , Tilse M H
Published: January 2020

In this study, we examined the biotypes of Haemophilus influenzae strains associated with noninvasive infections in hospitalized patients. Over an 18-month period, a total of 388 strains were isolated from patients of various ages (neonates to the elderly), and the biotypes of the strains were determined. Strains of biotype II accounted for 48% of the isolates; this was followed by strains of biotypes III and I (26 and 16%, respectively). The remaining 10% of the isolates were made up of strains of biotypes IV, V, VI, and VII. A total of 6% of strains were capsulated. The distribution of biotypes in specimens from the respiratory tract and associated sites was comparable to that obtained in similar investigations, but examination of isolates from neonatal and genital specimens did not support the concept that H. influenzae biotype IV is a major urogenital pathogen. Conflicting results regarding the incidence of certain biotypes in specimens, particularly those from the urogenital tract, may be due to the selection of different subpopulations of patients. Data relating to the specimens were used to evaluate the association between biotype and clinical diagnosis, the presence of other potential bacterial pathogens in the specimens, and the presence of viruses in the specimens. None of the differences in the distribution of biotypes which were examined was statistically significant.
Bacillus piliformis flagellar antigens for serodiagnosis of Tyzzer's disease
Authors: Motzel S L , Riley L K
Published: January 2020

Purified flagella from multiple isolates of Bacillus piliformis were obtained and examined by electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (immunoblot) analyses were used to assess the purity, antigenicity, and cross-reactivity of purified flagellar preparations. SDS-PAGE demonstrated a single, major protein band evident at approximately 53 to 56 kDa in all isolates tested. Results of Western blot analyses indicated a lack of cross-reactivity between flagellar antigens and heterologous isolates. Enzyme-linked immunosorbent assays (ELISAs) were used to compare the efficacies of flagellar preparations from the various isolates as antigens in detecting B. piliformis serum antibodies from several host species. ELISA results indicated that no single flagellar preparation could be relied on to consistently identify serum antibodies in all the host species tested; however, ELISAs that utilized a trivalent flagellar antigen preparation were shown to be specific and sensitive for the detection of antibodies to B. piliformis.
A cloned DNA probe identifies Cowdria ruminantium in Amblyomma variegatum ticks
Authors: Waghela S D , Rurangirwa F R , Mahan S M , Yunker C E , Crawford T B , Barbet A F , Burridge M J , McGuire T C
Published: January 2020

Heartwater, caused by Cowdria ruminantium and transmitted by ticks of the genus Amblyomma, is a constraint to ruminant animal production in sub-Saharan Africa. This rickettsial disease could spread from endemically infected areas of sub-Saharan Africa and certain Caribbean islands to other countries, including the United States, in which Amblyomma ticks exist. To detect C. ruminantium in tick vectors and animals, we made DNA probes from C. ruminantium DNA isolated from endothelial cell cultures. Two clones were evaluated; pCS20 from Crystal Springs (Zimbabwe) strain DNA had a 1,306-bp insert, and pCR9 from Kiswani (Kenya) strain DNA had a 754-bp insert. Both DNA probes detected 1 ng of Crystal Springs DNA; however, the pCS20 probe had a 10-fold-greater ability to discriminate between C. ruminantium DNA and DNA from other organisms. Also, the pCS20 probe did not hybridize to 400 ng (highest amount tested) of DNA from bovine cells, 3 protozoa, 3 rickettsiae, and 12 bacteria. In all experiments, C. ruminantium DNA was detected in midguts from 99 of 160 Amblyomma variegatum nymphs infected as larvae and in midguts from 38 of 80 adult ticks infected as nymphs but not in midguts from control nymphs and adults. The presence of C. ruminantium in nymphs and adults was confirmed by transmission of heartwater to goats. The DNA sequences of both probes were determined; synthetic oligonucleotides from pCS20 are recommended as DNA probes for C. ruminantium.
Detection of high-level aminoglycoside resistance in enterococci other than Enterococcus faecalis
Authors: Sahm D F , Boonlayangoor S , Schulz J E
Published: January 2020

The ability of six screening methods to detect high-level aminoglycoside resistance in enterococcal species other than Enterococcus faecalis was investigated. The 85 Enterococcus isolates, which included 55 E. faecium, 11 E. gallinarum, 9 E. casseliflavus, 5 E. raffinosus, 4 E. avium, and 1 E. mundtii, were tested by using aminoglycoside-supplemented brain heart infusion agar (BHI), Remel EF Synergy Quad plates, high-content aminoglycoside diffusion disks, standard (prepared in-house) microdilution panels, Pasco MIC Gram Positive microdilution panels, and Vitek GPS-TA cards. When tested on BHI, 32 and 35 strains showed resistance to gentamicin and streptomycin, respectively. Resistance profiles obtained with Remel EF Synergy Quad plates were in complete agreement with those obtained on BHI. However, growth on Mueller-Hinton agar-based plates was not as heavy. Some isolates showed only weak growth and required 48 h for resistance to become evident, especially with swab inoculation of quadrants containing 2,000 micrograms of gentamicin per ml. Profiles obtained by use of the agar-based screens were used as the basis for evaluating the other methods. Disk diffusion showed complete agreement. No false resistance occurred by either microdilution method, but 48 h of incubation was needed for detection of some gentamicin-resistant isolates, and 14% of the streptomycin-resistant strains were not detected by standard microdilution. The Vitek GPS-TA card detected 81 and 100% of the gentamicin- and streptomycin-resistant isolates, respectively. In general, most methods used to detect high-level aminoglycoside resistance in E. faecalis appear to be reliable for the testing of the other enterococcal species. However, further investigations with a greater number of resistant E. raffinosus, E. avium, and E. mundtii isolates, when they are available, will be useful for establishing the full range of enterococci that can reliably be tested by the various methods.
Feeding trials of Listeria monocytogenes with a nonhuman primate model
Authors: Farber J M , Daley E , Coates F , Beausoleil N , Fournier J
Published: January 2020

One of the major unanswered questions regarding the presence of Listeria monocytogenes in foods is how many cells must be ingested in order to cause illness. To answer this question, studies were undertaken by using Macaca fascicularis (cynomolgus monkey) as an animal model. Healthy nonhuman primates were dosed with various concentrations of L. monocytogenes suspended in sterile whole milk. Final concentrations of 10(5), 10(7), and 10(9) total cells of the organism were used; a control was also included. Blood samples, as well as fecal and nasal specimens, were taken at various time intervals. Only animals that received 10(9) cells of L. monocytogenes became noticeably ill, with symptoms of septicemia, irritability, loss of appetite, and occasional diarrhea. Monkeys that received 10(7) and 10(9) cells shed L. monocytogenes in the feces for approximately 21 days. In monkeys that received the dose of 10(9) cells, severe lymphopenia and neutrophilia occurred within 48 h. In a separate trial, monkeys received Maalox to reduce the gastric acidity of the stomach. However, no substantial differences were observed between Maalox-treated and control monkeys.
Type F botulism due to neurotoxigenic Clostridium baratii from an unknown source in an adult
Authors: McCroskey L M , Hatheway C L , Woodruff B A , Greenberg J A , Jurgenson P
Published: January 2020

Type F botulism was confirmed in a 54-year-old male with signs compatible with botulism who reported to the emergency unit of a hospital. Botulinal neurotoxin was detected in the patient's serum and fecal specimens, and a neurotoxigenic organism whose physiologic characteristics correspond to those of Clostridium baratii was isolated. The toxin produced by the isolate was neutralized by type F botulinal antitoxin and cross-neutralized with lower efficiency by type E antitoxin. The patient's food history was not suggestive of botulism, and it seems likely that the illness was due to colonization of the gut.
Diagnosis of human cytomegalovirus-induced retinitis in human immunodeficiency virus type 1-infected subjects by using the polymerase chain reaction
Authors: Fenner T E , Garweg J , Hufert F T , Boehnke M , Schmitz H
Published: January 2020

In 13 of 16 AIDS patients with retinitis, a herpesviruslike infection was diagnosed by clinical investigation. In 12 of the 13 patients, human cytomegalovirus (HCMV) DNA was detected in 5 microliters of aqueous humor by using the polymerase chain reaction (PCR). In the aqueous humor of 12 control patients HCMV DNA could not be detected by PCR. PCR may be used to monitor specific antiviral long-term therapy in HCMV retinitis.