bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of specific DNA fragments of Mycobacterium avium and their possible use in diagnosis
Authors: Thierry D , Vincent V , Clément F , Guesdon J L
Published: January 2020

We cloned and sequenced two DNA fragments (DT1 and DT6) from Mycobacterium avium serotype 2 for use in the identification of members of the M. avium-M. intracellulare complex (MAC). Reference strains of MAC belonging to serovars 1 to 28 were examined by using these DNA fragments as probes. The study revealed that the DT6 probe hybridized with DNAs from M. avium strains (serovars 1 to 6, 8 to 11, and 21), while the DT1 probe hybridized with DNAs from serovars 2, 3, 7, 12 to 20, and 23 to 25. DT1- and DT6-derived oligonucleotides were selected for use as primers in a polymerase chain reaction test. Amplification of the DT1 and DT6 sequences may provide the basis for a rapid and reliable assay for the detection of mycobacteria belonging to MAC.
Standardized nested polymerase chain reaction-based assay for detection of human immunodeficiency virus type 1 DNA in whole blood lysates
Authors: Sauvaigo S , Barlet V , Guettari N , Innocenti P , Parmentier F , Bastard C , Seigneurin J M , Chermann J C , Teoule R , Marchand J
Published: January 2020

The routine detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in clinical samples requires a standardized, simple, and sensitive test. To identify the HIV-1 proviral DNA in blood, we used a solid-phase assay based on the affinity capture and the gamma counting of the amplified product after a nested polymerase chain reaction (AMPLICIS test). In order to simplify the general process, whole-blood lysates rather than peripheral blood mononuclear cell lysates were used for the amplifications. The solid-phase capture and counting of the final amplified products allowed us to define precise interpretive criteria to determine the positivity level of the test. Three new primer sets located in the gag and pol structural genes and in the tat regulatory gene of HIV-1 were studied. The results obtained in 54 seropositive and 120 seronegative individuals demonstrated the ability of the AMPLICIS test to be used for HIV-1 provirus detection: 53 of 54 of the seropositive specimens were found to be positive with at least two primer sets. We also assessed the usefulness of this test for the estimation of the HIV-1 DNA load by the end point dilution method with serial dilutions of blood lysates from 26 HIV-1-seropositive patients.
Monoclonal antibody-based blocking enzyme-linked immunosorbent assay for specific detection and titration of peste-des-petits-ruminants virus antibody in caprine and ovine sera
Authors: Saliki J T , Libeau G , House J A , Mebus C A , Dubovi E J
Published: January 2020

A blocking enzyme-linked immunosorbent assay (B-ELISA), using two neutralizing monoclonal antibodies (MAbs), was established and compared with the virus neutralization test (VNT) for detecting specific peste-des-petits-ruminants virus (PPRV) antibody in caprine and ovine sera. This technique was developed because VNT, the only available specific serological test for PPRV and the cross-reactive rinderpest virus (RPV), is time-consuming and unaffordable for most laboratories in regions where both peste des petits ruminants and rinderpest occur. The test depends on the blocking of the binding of the MAb to a specific epitope in the presence of positive serum. Test conditions were optimized by using peste-des-petits-ruminants and rinderpest sera that were known to be VNT positive and negative. A blocking format, in which serum is preincubated with a solid-phase PPRV antigen and then incubated with the MAb, yielded levels of sensitivity and specificity superior to those of a competitive format, in which the two reagents are added simultaneously. A threshold value of 45% inhibition, representing the mean for a negative population (n = 277) plus 2.7 standard deviations, was adopted for routine screening. A total of 605 serum samples were screened by B-ELISA and the VNT. The sensitivity and specificity of B-ELISA relative to the VNT were 90.4 and 98.9%, respectively. Of 264 field serum samples tested, 11 (4.2%) could not be assayed by the VNT because of contamination or cytotoxicity; the overall agreement quotient between results of the two tests (n = 253) was 0.91. A high correlation (r>/=0.98) was observed between B-ELISA and the VNT for endpoint titration of sera (n=57). Because B-ELISA proved to be nearlyas sensitive and specific as the VNT while being simpler and more rapid, it would be an adequate substitute for the VNT for assessing herd immune status and for epidemiologic surveillance.
Detection of Mycoplasma pneumoniae by polymerase chain reaction and nonradioactive hybridization in microtiter plates
Authors: Lüneberg E , Jensen J S , Frosch M
Published: January 2020

In order to improve the diagnosis of a Mycoplasma pneumoniae infection, we developed a polymerase chain reaction (PCR)-based assay. The gene encoding elongation factor Tu (tuf) was selected as the target sequence. Oligonucleotides derived from variable stretches of the tuf gene were able to prime the amplification of a 950-bp fragment exclusively when M. pneumoniae DNA was used as the template. The sensitivity of the assay was increased 10-fold when the amplification products were hybridized with an internal M. pneumoniae-specific oligonucleotide. The use of three to four genome copies for PCR was sufficient for obtaining a hybridization signal. In addition, we substituted radioactive filter hybridization with a microtiter plate assay. Via a biotin moiety of one PCR primer, the amplification products were immobilized on streptavidin-coated microtiter plates. Subsequent hybridization with a digoxigenin-labeled oligonucleotide resulted in the same sensitivity and specificity as those obtained by filter hybridization. Clinical application of the assay was performed on 102 throat swab specimens from patients with respiratory tract infections. Of 21 culture-positive samples, 19 were confirmed to be positive in the PCR-based assay (sensitivity, 90%). Furthermore, 14 of 19 seropositive but culture-negative samples gave a positive hybridization signal. Of 62 culture-negative and seronegative specimens, 60 gave a negative result in our assay (specificity, 97%). Of the 33 samples that were positive in our PCR-based assay, 5 samples initially gave false-negative results because of the presence of inhibitory substances in those specimens. Inhibition of Taq polymerase in these five cases was prevented by an additional step of phenol extraction and subsequent ethanol precipitation.
Chlamydia trachomatis infection in a high-risk population: comparison of polymerase chain reaction and cell culture for diagnosis and follow-up
Authors: Vogels W H , van Voorst Vader P C , Schröder F P
Published: January 2020

A study to compare the polymerase chain reaction (PCR) test with the cell culture method in diagnosing urogenital Chlamydia trachomatis infections was performed. From 497 patients (212 women, 285 men) attending an outpatient clinic for sexually transmitted diseases, a total of 814 samples (female patients, cervix and urethra; male patients, urethra) were collected. This total included follow-up samples from 35 women and 35 men positive for C. trachomatis by cell culture and/or PCR test, which were collected 2 weeks after treatment with doxycycline (two 100-mg doses per day for 7 days). The PCR test was performed directly on clinical samples without performing phenol-chloroform extraction and ethanol precipitation of DNA. The prevalence of C. trachomatis as measured by positive cell culture was 64 of 497 (12.9%) for all patients, 31 of 212 (14.6%) for women, and 33 of 285 (11.6%) for men. The prevalences as measured by positive PCR test were 71 of 497 (14.3%), 36 of 212 (17.0%), and 35 of 285 (12.3%), respectively. The sensitivities of the cell culture and the PCR test compared with that of true-positive samples were 77.5 to 78.4% and 99.0 to 100.0%, respectively. Discrepancies between cell culture and the PCR test were found for 23 of 497 patients (4.9%), 19 of 212 females (9.0%), and 4 of 285 males (1.4%). Nineteen pretreatment samples from 19 patients (4 female endocervical, 13 female urethral, and 2 male urethral samples) were cell culture negative and PCR test positive, while 1 pretreatment female endocervical sample was cell culture positive and PCR test negative. The posttreatment samples from all patients were cell culture negative, but the PCR test remained positive for 3 of 70 patients (1 female endocervical and 2 male urethral samples). One of these samples became spontaneously negative in three more weeks. The medical history of the individual patient and the negative PCR tests after treatment for nearly all patients support our hypothesis that the positive PCR test results were clinically relevant for the cell culture-negative but PCR test-positive but PCR test-positive patients of the population studied.
Application of polymerase chain reaction to fingerprinting Aspergillus fumigatus by random amplification of polymorphic DNA
Authors: Loudon K W , Burnie J P , Coke A P , Matthews R C
Published: January 2020

A new method for fingerprinting Aspergillus fumigatus by random amplification of polymorphic DNA (RAPD) by using single primers with arbitrary sequences is described. Five primers were examined with 19 isolates from six patients with aspergilloma as well as with A. fumigatus NCPF 2109. Two of the primers (GCT GGT GG and GCG CAC GG, 5' to 3') gave adequate discrimination between isolates, generating five and six types, respectively. Combination of the results obtained with each of these two primers generated 12 types. This compares very favorably with immunoblot fingerprinting and XbaI-generated restriction fragment length polymorphisms on the same isolates. Typeability and reproducibility were good with RAPD, and RAPD was less labor-intensive than immunoblot fingerprinting. RAPD typing results suggested that aspergillomas sometimes contain isolates of more than one type.
Detection of hog cholera virus and differentiation from other pestiviruses by polymerase chain reaction
Authors: Wirz B , Tratschin J D , Müller H K , Mitchell D B
Published: January 2020

Reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the detection and differentiation of pestiviruses. For this purpose, one primer pair was selected from a highly conserved region of the genome of pestiviruses. Using these primers (PEST 1-PEST 2), DNA fragments of between 72 and 74 bp could be amplified from all pestivirus isolates tested. In order to differentiate hog cholera virus (HCV) from bovine viral diarrhea virus (BVDV) and border disease virus (BDV), we selected a primer pair from a conserved region in the genome of HCV strains that differed from that sequenced in the genome of BVDV strains. By using these primers (HCV 1-HCV 2), a DNA fragment of 478 bp could be specifically amplified from HCV isolates. By these means, viral RNA was detected in extracts of lymph node, spleen, tonsil, and lung. Such extracts were used directly for RT-PCR without prior RNA isolation. We also performed multiplex PCR by using both the PEST 1-PEST 2 and HCV 1-HCV 2 primer pairs in a single reaction. This allowed the differentiation of HCV from BVDV and BDV in one step. To assess the sensitivity of the method, RT-PCR was compared with virus propagation in tissue culture and subsequent detection by immunofluorescence staining. The results show that RT-PCR is useful for the rapid detection and differentiation of pestiviruses.
A novel mu-capture enzyme-linked immunosorbent assay based on recombinant proteins for sensitive and specific diagnosis of hemorrhagic fever with renal syndrome
Authors: Zöller L G , Yang S , Gött P , Bautz E K , Darai G
Published: January 2020

Hantavirus nucleocapsid protein has recently been identified as a major antigen inducing an early and long-lasting humoral immune response in patients with hemorrhagic fever with renal syndrome. A mu-capture enzyme-linked immunosorbent assay utilizing recombinant nucleocapsid proteins of Hantavirus strains Hantaan 76-118 (Hantaan serotype) and CG 18-20 (Puumala serotype) as diagnostic antigens and specific monoclonal antibodies as the detection system has been developed. Histidine-tailed recombinant proteins were expressed in Escherichia coli and purified in a single step by affinity chromatography on a nickel-chelate resin. The assay was evaluated with a panel of sera from patients with hemorrhagic fever with renal syndrome originating from various geographic regions. The overall sensitivity of the mu-capture enzyme-linked immunosorbent assay (both recombinant antigens) was 100%, and its specificity was also found to be 100%. Immunoglobulin M antibodies were detected as early as on day 3, and maximum titers were obtained between days 8 and 25 after onset of the disease. The assay was regularly found to be positive within 3 to 4 months but in some cases up to 2 years after the acute phase of the disease.
Direct detection of Chlamydia trachomatis in urine specimens from symptomatic and asymptomatic men by using a rapid polymerase chain reaction assay
Authors: Jaschek G , Gaydos C A , Welsh L E , Quinn T C
Published: January 2020

Screening for Chlamydia trachomatis infection in men has traditionally been limited to men who present with urethral symptoms, thereby limiting the detection of asymptomatic chlamydia infection in men. In order to effectively screen both symptomatic and asymptomatic men, we evaluated a newly developed polymerase chain reaction (PCR) assay, Amplicor C. trachomatis, from Roche Molecular Systems for the detection of C. trachomatis in urine specimens in comparison with urethral culture. A total of 530 male urine specimens were collected from 322 symptomatic and 208 asymptomatic men attending two sexually transmitted disease clinics in Baltimore, Md. The prevalence of C. trachomatis by culture was 9.8% (10.6% in symptomatic men and 8.2% in asymptomatic men). Compared with culture, the sensitivity of the PCR was 92.8%, the specificity was 94.7%, the positive predictive value was 68.4%, and the negative predictive value was 99.1%. Discrepant results between culture and PCR were further analyzed by direct fluorescent-antibody staining of elementary bodies in urine sediment and in culture transport vials and by major outer membrane protein PCR of transport media for specimens with negative culture. The revised sensitivity and specificity of PCR for urine were 95.0 and 99.8%, respectively, and the positive and negative predictive values were 98.7 and 99.1%, respectively. The sensitivity of culture compared with PCR and/or direct fluorescent-antibody staining was 68.4%. These results indicate that the PCR assay is a highly sensitive and specific assay for the detection of C. trachomatis in male urine specimens and provides a noninvasive technique for routine screening of chlamydia infection in both symptomatic and asymptomatic men.
Evidence for two genetic variants of Pneumocystis carinii coinfecting laboratory rats
Authors: Cushion M T , Zhang J , Kaselis M , Giuntoli D , Stringer S L , Stringer J R
Published: January 2020

Pneumocystis carinii pneumonia is an oftentimes fatal infection for hosts in an immunocompromised state. The disease occurs in a wide variety of mammals, but the etiologic agent of this disease has been referred to as P. carinii regardless of the host species. However, even within a single host species, such as laboratory rats, distinct varieties of P. carinii have been identified from differences in the electrophoretic migration of chromosomes in agarose gels. Here we present evidence indicating that some laboratory rats can contain two different genetic variants of P. carinii that differ not only in electrophoretic karyotype but also in the presence of a particular repeated DNA sequence, in the presence of an intron in the 18S ribosomal RNA gene, and in the sequence of part of the 18S rRNA gene. Most of the rat colonies studied were infected with P. carinii that contained the repeated DNA and the 18S rRNA gene intron. The other type of rat-derived P. carinii, which lacked the repeated DNA and the intron in the 18S rRNA gene, was found as a coinfection with the first. Parasite populations from different coinfected rats contained the two variants in different proportions.
Identification of spheroplast-like agents isolated from tissues of patients with Crohn's disease and control tissues by polymerase chain reaction
Authors: Wall S , Kunze Z M , Saboor S , Soufleri I , Seechurn P , Chiodini R , McFadden J J
Published: January 2020

Mycobacterium paratuberculosis has been isolated from tissue taken from patients with Crohn's disease and has been implicated in the etiology of this disease. On culture, the organisms appear initially as cell wall-deficient, spheroplast-like forms that are difficult to identify by conventional techniques. Here we examine 30 unidentified cultures by the polymerase chain reaction using primers specific for M. paratuberculosis, Mycobacterium tuberculosis, and Mycobacterium avium restriction fragment length polymorphism type A/I and also by a non-species-specific mycobacterial polymerase chain reaction. Six of these cultures, all from Crohn's disease, were shown to contain DNA from M. paratuberculosis. Cultures from both Crohn's disease and controls were found to contain mycobacterial DNA of unknown specific origin.
Standardization of medium for culturing Lyme disease spirochetes
Authors: Pollack R J , Telford S R , Spielman A
Published: January 2020

To standardize the procedure for isolating and culturing Lyme disease spirochetes, we modified the composition of the medium generally used for this purpose (BSK-II) and developed a system for its distribution. This medium contains no gelatin or agarose, and various components are used in proportions that differ from those in BSK-II. Each of the major proteinacious components was screened by substitution in samples of the complete product. The final medium was evaluated for the capacity to grow related spirochetes including Borrelia burgdorferi N40, Guilford, and JD-1 as well as strains of Borrelia hermsii (HS-1) and of Borrelia coriaceae (CO53). Each isolate developed from inocula containing as few as one to five organisms. Doubling time of B. burgdorferi during log-phase growth at 37 degrees C was 10 to 12 h. Lyme disease spirochetes were isolated in this medium from ear punch biopsies and dermal aspirates from naturally infected mice and rabbits, from dermal biopsies from a human patient, and by sampling field-collected deer ticks (Ixodes dammini). Cultured spirochetes remained infective to mice and to ticks. The medium can be stored at -20 degrees C or lower temperatures for at least 8 months without effect on its ability to support growth of small inocula to densities exceeding 10(8) spirochetes per ml. Lyme disease spirochetes remained infective to mice after being stored at -80 degrees C in this medium for at least 8 months. We anticipate that the availability of this standardized medium (Sigma Chemical Co.), supplemented with prescreened rabbit serum, will facilitate comparison of research results between laboratories and may eventually permit definitive clinical diagnosis of Lyme disease based on demonstration of the pathogen. The standardized medium is designated BSK-H.
Determination of the optimal cutoff value for a serological assay: an example using the Johne's Absorbed EIA
Authors: Ridge S E , Vizard A L
Published: January 2020

Traditionally, in order to improve diagnostic accuracy, existing tests have been replaced with newly developed diagnostic tests with superior sensitivity and specificity. However, it is possible to improve existing tests by altering the cutoff value chosen to distinguish infected individuals from uninfected individuals. This paper uses data obtained from an investigation of the operating characteristics of the Johne's Absorbed EIA to demonstrate a method of determining a preferred cutoff value from several potentially useful cutoff settings. A method of determining the financial gain from using the preferred rather than the current cutoff value and a decision analysis method to assist in determining the optimal cutoff value when critical population parameters are not known with certainty are demonstrated. The results of this study indicate that the currently recommended cutoff value for the Johne's Absorbed EIA is only close to optimal when the disease prevalence is very low and false-positive test results are deemed to be very costly. In other situations, there were considerable financial advantages to using cutoff values calculated to maximize the benefit of testing. It is probable that the current cutoff values for other diagnostic tests may not be the most appropriate for every testing situation. This paper offers methods for identifying the cutoff value that maximizes the benefit of medical and veterinary diagnostic tests.
Detection and characterization of the eae gene of Shiga-like toxin-producing Escherichia coli using polymerase chain reaction
Authors: Gannon V P , Rashed M , King R K , Thomas E J
Published: January 2020

In this study, the polymerase chain reaction (PCR) was used in the detection of the attaching and effacing (eae) gene of Shiga-like toxin-producing Escherichia coli (SLT-EC). Oligonucleotide primers, complementary to the 5' portion of the eae gene of the enteropathogenic E. coli E2348/69 (O127:H6) and of SLT-EC CL8 and EDL933 (O157:H7), generated PCR products of the predicted sizes with DNA from the majority of human clinical SLT-EC strains tested from O serogroups 5, 26, 103, 111, 121, 128, 145, and 157; all SLT-EC strains of O serogroups 5, 26, and 111 from cattle; and a minority of porcine SLT-EC strains (one strain each from O serogroups 107 and 130 and one rough strain). Five HaeIII digestion profiles were obtained for PCR products generated by amplification of a 2.3-kb DNA fragment from the 5' end of eae. The HaeIII profiles for SLT-EC O serogroups, such as 26, 103, and 157, differed from each other but were consistent among strains within these O serogroups. Oligonucleotide primer pairs complementary to the 3' end of either the O127:H6 E. coli or the O157:H7 eae nucleotide sequence only amplified DNA from E. coli strains from a few of the SLT-EC O serogroups examined. One primer pair with homology to the 3' nucleotide sequence of eae from E. coli O157:H7 appeared to be relatively specific for this O serogroup by PCR. No PCR products were obtained in amplification experiments with the eae primers using DNA from human SLT-EC of O serogroups 38 (1 0f 1) and 91 (3 or 3), 15 of 15 SLT-EC strains from edema disease, or 29 of 29 non-SLT-EC strains from pigs and calves with diarrhea.
Accuracy of reporting of methicillin-resistant Staphylococcus aureus in a provincial quality control program: a 9-year study
Authors: Mackenzie A M , Richardson H , Missett P , Wood D E , Groves D J
Published: January 2020

We report the results of a province-wide quality control program in which five methicillin-resistant Staphylococcus aureus strains were circulated to all Ontario laboratories (hospital, private, and public health laboratories) on nine occasions between 1980 and 1989. The level of expression of methicillin resistance in each of the isolates was determined by performing viable colony counts on serial dilutions of methicillin in agar, and each isolate was assigned to an expression class according to previous published criteria (A. Tomasz, S. Nachman, and H. Leaf, Antimicrob. Agents Chemother. 35:124-129, 1991). Over this time there was an improvement in the performance of laboratories in the recognition of three strains that were relatively easy to detect (strains B, C, and E). These strains were of expression class II, and 98% of laboratories reported correct identifications in 1986. Performance in identifying two strains (strains A and D) of expression class I remained poor. Strain A was circulated in two surveys in 1987 and 1989, and laboratories were sent a questionnaire requesting details of the methods used in those two surveys. The methods used by the laboratories were classified into three categories: disk diffusion, single-plate screening by agar incorporation, and automated methods, which included premanufactured MIC panels. Between the 1987 and 1989 surveys, there was no change in the performance of the disk diffusion test (60% correct on both occasions), but there was improvement in the sensitivity of the agar incorporation test (36% correct in 1987 and 84% correct in 1989) and in automated methods (43% correct in 1987 and 79% correct in 1989). Over a decade, there was overall improvement in the performance of laboratories in detecting easy-to-detect strains, but there were difficulties in detecting organisms of low expression class, and an organism of very low expression class should be designated as a control organism for routine testing of methicillin-resistant s. aureus isolates.
Innovative index system for reporting microbiology laboratory results
Authors: Kelble K M , Kehm G , Roe M H , Todd J K
Published: January 2020

A new computerized format for presenting microbiology laboratory results has been designed and evaluated. This new system uses an index format providing a concise summary of the status of each test ordered and the results for each test organized by the source of the specimen on the first page of the report. This is followed by complete, detailed microbiology results sorted in reverse chronological order. This new system was significantly (P < 0.05) faster and easier to use than a standard source-category reporting system used in many hospitals. In addition, the index format was overwhelmingly preferred by the individuals evaluating both systems.
Mixed-linker polymerase chain reaction: a new method for rapid fingerprinting of isolates of the Mycobacterium tuberculosis complex
Authors: Haas W H , Butler W R , Woodley C L , Crawford J T
Published: January 2020

Rapid recognition of multidrug-resistant strains of Mycobacterium tuberculosis is a desirable goal for treatment of patients and protection of health care workers. DNA fingerprints produced with the insertion sequence IS6110 generate restriction fragment length polymorphism (RFLP) patterns that reliably identify M. tuberculosis complex strains. This report describes a rapid technique for RFLP typing using the polymerase chain reaction. The method uses one primer specific for IS6110 and a second primer complementary to a linker ligated to the restricted genomic DNA. In one strand the linker contains uracil in place of thymidine, and specific amplification is obtained by elimination of this strand with uracil N-glycosylase. Mixed-linker fingerprinting clearly differentiated multidrug-resistant isolates from 12 outbreaks and unambiguously assigned them to 26 RFLP groups.
Solid-phase antibody capture hemadsorption assay for detection of hepatitis A virus immunoglobulin M antibodies
Authors: Summers P L , Dubois D R , Cohen W H , Macarthy P O , Binn L N , Sjogren M H , Snitbhan R , Innis B L , Eckels K H
Published: January 2020

A solid-phase antibody capture hemadsorption (SPACH) assay was developed to detect hepatitis A virus (HAV)-specific immunoglobulin M (IgM) antibodies in sera from humans recently infected with hepatitis. The assay is performed with microtiter plates coated with anti-human IgM antibodies to capture IgM antibodies from the test sera. HAV-specific IgM antibody is detected by the addition of HAV hemagglutinating antigen and goose erythrocytes. Hemadsorption of erythrocytes to antigen-antibody complexes attached to the solid phase indicate the presence of IgM antibodies. The SPACH assay was compared to a commercial radioimmunoassay and was found to be equally or more sensitive and specific for the detection of HAV IgM antibodies. The SPACH assay is an alternative, rapid assay that doesn't require hazardous substrates or radioactivity for the detection of HAV-specific antibodies.
Rapid detection of human group C rotaviruses by reverse passive hemagglutination and latex agglutination tests using monoclonal antibodies
Authors: Kuzuya M , Fujii R , Hamano M , Nagabayashi T , Tsunemitsu H , Yamada M , Nii S , Mori T
Published: January 2020

Reverse passive hemagglutination (RPHA) tests and a latex agglutination test with monoclonal antibodies (MAbs) were developed for the rapid detection of noncultivatable human group C rotaviruses. For RPHA tests, two MAbs, MAb 5A12 recognizing the outer capsid and MAb 13A3 recognizing the inner capsid, were separately used for the coating of sheep erythrocytes (SRBCs). Forty-six fecal samples were examined to confirm the practicality of the tests. As a result, there was concordance between the RPHA test with SRBCs coated with MAb 5A12 and polyacrylamide gel electrophoresis of viral RNA (RNA-PAGE) in 44 (95.6%) of 46 samples, while the diagnoses by the RPHA test with SRBCs coated with MAb 13A3 were in complete agreement with those by RNA-PAGE. Furthermore, a latex agglutination test with MAb 13A3 was also developed, and this test was fast enough and sensitive enough to successfully detect the viruses from most fecal samples within 2 min. The present procedures would be useful for the diagnosis of human group C rotavirus infections in clinical laboratories which are not well equipped.
Identification of a novel group of Serpulina hyodysenteriae isolates by using a lipopolysaccharide-specific monoclonal antibody
Authors: Alderton M R , Smith S C , Coloe P J
Published: January 2020

A monoclonal antibody to Serpulina hyodysenteriae 8930 was produced and was used to probe pronase-treated cell lysates of S. hyodysenteriae isolates in immunblots. The results showed that the monoclonal antibody was specific for only five closely related S. hyodysenteriae isolates: 8930, 5380, 70A, RMIT 88, and RMIT 97.
Categorization of clinical isolates of Helicobacter pylori on the basis of restriction digest analyses of polymerase chain reaction-amplified ureC genes
Authors: Moore R A , Kureishi A , Wong S , Bryan L E
Published: January 2020

Restriction endonuclease analyses of a 1.1-kb polymerase chain reaction-amplified portion of the ureC gene from Helicobacter pylori were used to group or to differentiate 21 clinical isolates. Isolates were placed into 4 groups after HindIII digestion alone, and placement was expanded into 15 groups after isolates were digested with AluI and PvuI.
Comparison of the VIDAS RSV assay and the Abbott Testpack RSV with direct immunofluorescence for detection of respiratory syncytial virus in nasopharyngeal aspirates
Authors: Miller H , Milk R , Diaz-Mitoma F
Published: January 2020

The sensitivity and accuracy of the VIDAS RSV assay in testing fresh specimens were 82.7 and 87.1%, respectively, whereas specimens previously frozen at -70 degrees C gave a sensitivity of 96.2% and an accuracy of 95.4%. The sensitivity and accuracy of Abbott Testpack RSV were 92.6 and 91.3% for fresh specimens and 86.8 and 88.1% for frozen specimens. The advantages and drawbacks of the two assays are discussed.
New latex reagent using monoclonal antibodies to capsular polysaccharide for reliable identification of both oxacillin-susceptible and oxacillin-resistant Staphylococcus aureus
Authors: Fournier J M , Bouvet A , Mathieu D , Nato F , Boutonnier A , Gerbal R , Brunengo P , Saulnier C , Sagot N , Slizewicz B
Published: January 2020

A new latex agglutination test (Pastorex Staph-Plus, Sanofi Diagnostics Pasteur), consisting of a mixture of latex particles coated with fibrinogen and immunoglobulin G for the detection of clumping factor and protein A and latex particles sensitized with monoclonal antibodies directed to Staphylococcus aureus serotype 5 and 8 capsular polysaccharides, was compared with three commercially available rapid agglutination methods for the identification of 220 isolates of S. aureus (61 oxacillin resistant) and 128 isolates of coagulase-negative staphylococci. The sensitivity for identification of S. aureus was high with the Pastorex Staph-Plus test (98.6%) compared with those of the other tests, which ranged from 91.8 to 84.5%. Test sensitivities for the identification of oxacillin-resistant S. aureus were as follows: Pastorex Staph-Plus, 95.1%; Pastorex Staph, 73.8%; Staphyslide, 72.1%; and StaphAurex, 49.2%.
Zygomycotic necrotizing fasciitis caused by Apophysomyces elegans
Authors: Lakshmi V , Rani T S , Sharma S , Mohan V S , Sundaram C , Rao R R , Satyanarayana G
Published: January 2020

A case of necrotizing fasciitis of the anterior abdominal wall caused by the zygomycete Apophysomyces elegans in a healthy male following inguinal herniorrhaphy is reported. The portal of entry of the fungus into the incised skin and subcutaneous tissues was probably through either contaminated surgical sutures or postoperative surgical dressings. Broad, aseptate fungal hyphae were seen in the necrosed tissues with an associated necrotizing vasculitis. Extensive tissue debridements and a low dose of amphotericin B were not successful in controlling the rapid invasion of the tissues by the fungus.
Two-site comparison of broth microdilution and semisolid agar dilution methods for susceptibility testing of Cryptococcus neoformans in three media
Authors: Anaissie E , Shawar R , Paetznick V , Ensign L G , Witte Z , LaRocco M
Published: January 2020

This study evaluated the inter- and intralaboratory agreement between results of the semisolid agar dilution and broth microdilution methods of antifungal susceptibility testing of Cryptococcus neoformans. Three media were tested in two laboratories. The drugs tested were amphotericin B, flucytosine, itraconazole, fluconazole, and Schering 39304. Analysis by kappa statistics revealed good agreement between the laboratories for the two methods. The highest level of inter- and intralaboratory agreement was observed in RPMI 1640 with L-glutamine followed by Eagle's minimum essential medium and yeast nitrogen broth. The broth microdilution method appears more suitable than the semisolid agar dilution method for testing cryptococci because of its ease in performance, cost, and simplicity.
Evaluation of a 2-minute anti-human immunodeficiency virus (HIV) test using the autologous erythrocyte agglutination technique with populations differing in HIV prevalence
Authors: Sirivichayakul S , Phanuphak P , Tanprasert S , Thanomchat S , Uneklabh C , Phutiprawan T , Mungklavirat C , Panjurai Y
Published: January 2020

A total of 1,800 blood specimens (1,000 from healthy blood donors, 300 from patients with sexually transmitted disease, and 500 from intravenous drug users) were simultaneously tested with anti-human immunodeficiency virus enzyme-linked immunosorbent assay (ELISA) kits and a newly developed 2-min test for anti-human immunodeficiency virus based on the principle of autologous erythrocyte agglutination (AGEN Biomedical Limited). We found that AGEN's rapid test was as sensitive and specific as the other ELISA kits.
Conventional identification characteristics, mycolate and fatty acid composition, and clinical significance of MAIX AccuProbe-positive isolates of Mycobacterium avium complex
Authors: Viljanen M K , Olkkonen L , Katila M L
Published: January 2020

A total of 145 isolates belonging to the Mycobacterium avium complex (MAC) were tested with commercial acridinium ester-labeled DNA probes (AccuProbe, Gen-Probe). M. avium and M. intracellulare probes reacted with 102 and 36 isolates, respectively. The remaining seven isolates were clearly positive with the new probe, designated MAIX. Thus, the combined sensitivity of M. avium and M. intracellulare probes was 95.2%. The MAIX probe improved the sensitivity up to 100%. The MAIX probe also reacted with all M. avium (n = 20) and M. intracellulare (n = 20) isolates tested. Three of the seven MAIX-positive isolates were considered clinically significant. We conclude that the new MAIX probe should be used, in addition to M. avium and M. intracellulare probes, for the identification of MAC isolates. Our results also suggest that the probe may be used alone to cover the whole MAC when species differentiation is not required.
Hydroxy-fatty acid profiles of Legionella species: diagnostic usefulness assessed by principal component analysis
Authors: Jantzen E , Sonesson A , Tangen T , Eng J
Published: January 2020

Twenty-nine species (76 strains) of members of the genus Legionella were analyzed for their cellular hydroxylated fatty acids (OH-FAs). The individual patterns were unusually complex and included both monohydroxylated and dihydroxylated chains of unbranched or branched (iso and anteiso) types. Comparison of the strain profiles by SIMCA (Soft Independent Modelling of Class Analogy) principal component analysis revealed four main groups. Group 1 included Legionella pneumophila plus L. israelensis strains, and group 2 included L. micdadei and L. maceacherneii strains. These two closely related groups were characterized by the occurrence of di-OH-FAs and differed mainly in the amounts of 3-OH-a21:0, 3-OH-n21:0, 3-OH-n22:0, and 3-OH-a23:0. Group 3 (13 species) was distinguished by i14:0 at less than 3%, 3-OH-3-OH-n14:0 at greater than 5%, 3-OH-n15:0 at greater than 2%, and minute amounts of OH-FAs with chains longer than 21:0. Group 4 (12 species) was heterogeneous. Its main characteristics were the presence of 3-OH-n12:0 and 3-OH-n13:0, 3-OH-i14:0 at greater than 5%, as well as significant amounts of 3-OH-a21:0 and 3-OH-n21:0. The groupings obtained by OH-FA profiles were found to reflect DNA-DNA homology groupings reasonably well, and the profiles appear to be useful for differentiation of Legionella species.
Restriction endonuclease analysis of clinical Pseudomonas aeruginosa strains: useful epidemiologic data from a simple and rapid method
Authors: Maher W E , Kobe M , Fass R J
Published: January 2020

Newer genetic techniques have replaced phenotypic methods of subtyping Pseudomonas aeruginosa strains. Widespread application of newer methodologies, however, may be limited by technologic complexity and the cost of equipment. We conducted restriction endonuclease analysis (REA) of sheared genomic DNAs from 48 clinical P. aeruginosa strains using the enzyme SalI and electrophoresis in horizontal, low-concentration (0.3 to 0.6%) agarose gels. Each REA profile consisted of a smear of lower-molecular-mass bands as well as a countable number of well-resolved bands in the 8.3- to 48.5-kbp range which could easily be compared when isolates were run side-by-side on the same gel. In general, the REA patterns of strains recovered from different patients differed by at least seven bands, and those of serial isolates from individual patients were identical or differed by, at most, two bands over this 8.3- to 48.5-kbp range. REA of strains already subtyped by field inversion gel electrophoresis revealed that the two techniques generally paralleled each other. Overall, some unrelated strains had similar REA profiles, but the relative simplicity and low cost of the approach coupled with the ability to demonstrate differences between most unrelated strains should make this type of REA an attractive first step in the investigation of institutional P. aeruginosa problems.
Typing of group B streptococci: comparison of pulsed-field gel electrophoresis and conventional electrophoresis
Authors: Gordillo M E , Singh K V , Baker C J , Murray B E
Published: January 2020

The SmaI restriction endonuclease digestion patterns of chromosomal DNAs from 35 group B streptococci were analyzed by pulsed-field gel electrophoresis (PFGE). Nineteen different patterns and four possible variants were identified. Twenty-four isolates were previously analyzed by conventional electrophoresis of HindIII-digested and/or BglII plus EcoRI double-digested chromosomal DNA. Although interpretations by both methods were essentially the same, PFGE identified as variants two isolates that were previously classified as the same isolate. More importantly, PFGE of the chromosomal DNA of group B streptococci digested with SmaI generated more easily defined patterns, since fewer and better separated bands were obtained, whereas digestion with HindIII or EcoRI plus BglII typically generated 100 or more bands. SalI digestion also yielded easily evaluable results, although the SalI fragments were somewhat smaller than those generated by SmaI. In our hands, PFGE patterns were more easily discerned and interpreted than were patterns previously generated by conventional electrophoresis.
Detection of Mycobacterium tuberculosis in sputum samples by polymerase chain reaction using a simplified procedure
Authors: Kocagöz T , Yilmaz E , Ozkara S , Kocagöz S , Hayran M , Sachedeva M , Chambers H F
Published: January 2020

A repetitive sequence of Mycobacterium tuberculosis DNA was amplified by polymerase chain reaction (PCR), from sputum samples, for the diagnosis of pulmonary tuberculosis. The method of heating the sample in a boiling water bath to break down the bacterial cell wall and to release the DNA was compared with that of enzymatic lysis of bacteria and then phenol-chloroform extraction of DNA. Heating the sample was the better method with a sensitivity of approximately 10 microorganisms. A total of 78 sputum specimens prepared by heating were examined by PCR, and the results were compared with the results of acid-fast stained smears, cultures, and clinical data. M. tuberculosis was detected by PCR in all smear- and culture-positive and smear-negative, culture-positive cases. Additionally, PCR was capable of detecting four of nine cases which were smear and culture negative but clinically suspected of tuberculosis. DNA amplification by PCR is a sensitive and specific method for the diagnosis of tuberculosis, and with this simplified DNA isolation procedure it can be used in routine clinical practice.
Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction
Authors: Song J H , Cho H , Park M Y , Na D S , Moon H B , Pai C H
Published: January 2020

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella typhi in the blood specimens from patients with typhoid fever. Two pairs of oligonucleotide primers were designed to amplify a 343-bp fragment of the flagellin gene of S. typhi. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization by using a 32P-labeled 40-base probe internal to the amplified DNA. The nested PCR with two pairs of primers could detect 10 organisms of S. typhi as determined by serial dilutions of DNA from S. typhi. The peripheral mononuclear cells from 11 of 12 patients with typhoid fever confirmed by blood culture were positive for DNA fragment of the flagellin gene of S. typhi, whereas 10 blood specimens of patients with other febrile diseases were negative. With the nested PCR, S. typhi DNAs were detected from blood specimens of four patients with suspected typhoid fever on the basis of clinical features but with negative cultures. We suggest that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases.
Diagnosis of Helicobacter pylori infection by using pyloriset EIA-G and EIA-A for detection of serum immunoglobulin G (IgG) and IgA antibodies
Authors: Granberg C , Mansikka A , Lehtonen O P , Kujari H , Grönfors R , Nurmi H , Räihä I , Ståhlberg M R , Leino R
Published: January 2020

We evaluated the performance of new enzyme immunoassay (EIA) kits (Pyloriset; Orion Corporation, Orion Diagnostica, Espoo, Finland) for the detection of immunoglobulin G (IgG) and IgA antibodies to Helicobacter pylori in serum. Serum samples from 195 patients with upper abdominal complaints were collected. Biopsy specimens of the gastric mucosae were taken for histological analysis and bacterial culture. The sensitivity, specificity, and positive and negative predictive values, and efficacy of the Pyloriset EIA-G in detecting IgG antibodies to H. pylori were 92, 84, 88, 90, and 89%, respectively, when compared with those of the reference methods used. The corresponding data for detection of IgA antibodies were 80, 89, 89, 79, and 84%, respectively. The overall prevalence of defined H. pylori positivity was 54%. Moreover, the antibody tests showed a very good correlation with the biopsy findings. IgG antibodies were found in 93% of sera from patients with documented gastritis and H. pylori positivity, whereas only 4% of the sera from patients with documented gastritis and H. pylori-negative patients was positive. The results obtained for IgA antibodies were 81 and 6%, respectively. We conclude that the Pyloriset EIA-G, the test for IgG antibodies, is a good and reliable test for the detection of antibodies to H. pylori and as an indication of H. pylori infection. The determination of IgA antibodies may be used as a test that complements the IgG antibody assay.
Characterization and partial nucleotide sequence of the DNA fingerprinting probe Ca3 of Candida albicans
Authors: Anderson J , Srikantha T , Morrow B , Miyasaki S H , White T C , Agabian N , Schmid J , Soll D R
Published: January 2020

The moderately repetitive Ca3 fragment of Candida albicans has been used as an effective DNA fingerprinting probe in epidemiological studies. EcoRI digestion of Ca3 DNA results in seven fragments of 4.2 kb (A), 2.98 kb (B), 2.85 kb (C), 0.77 kb (D1), 0.77 kb (D2), 0.38 kb (E), and 0.30 kb (F). Five of these EcoRI fragments have been mapped in the 5'-3' order C B D1 A D2. The intact Ca3 probe and the three largest EcoRI fragments, A, B, and C, were individually used to probe Southern blots of EcoRI-digested DNA of a set of test strains, transverse alternating field electrophoresis-separated chromosomes of strain 3153A, and Northern (RNA) blots of test strain 3153A. Fragments, A, B, and C each generate a different Southern blot hybridization pattern with EcoRI-digested whole-cell DNA; Ca3 sequences are present in at least five of seven separable chromosomes and a minichromosome of strain 3153A; fragments A, B, and C are distributed differently on chromosomes; and fragments A, B, and C do not cross-hybridize. Ca3 hybridizes to three major transcripts of 2.8, 2.3, and 1.5 kb. Fragment A hybridizes intensely to the 1.5-kb transcript, while fragments B and C both hybridize intensely to the 2.8- and 2.3-kb transcripts. The B fragment, which contains 2,980 bp and contributes to the major portion of the Ca3 pattern, was sequenced. Both direct and inverted repeat sequence motifs were identified. These results provide us with initial insights into the evolution of the Ca3 pattern and the nature of the probe.
Mapping of serotype-specific, immunodominant epitopes in the NS-4 region of hepatitis C virus (HCV): use of type-specific peptides to serologically differentiate infections with HCV types 1, 2, and 3
Authors: Simmonds P , Rose K A , Graham S , Chan S W , McOmish F , Dow B C , Follett E A , Yap P L , Marsden H
Published: January 2020

The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenicity of the NS-4 protein was investigated by epitope mapping and by enzyme-linked immunosorbent assay with branched oligopeptides. Epitope mapping of the region between amino acid residues 1679 and 1768 in the HCV polyprotein revealed two major antigenic regions (1961 to 1708 and 1710 to 1728) that were recognized by antibody elicited upon natural infection of HCV. The antigenic regions were highly variable between variants of HCV, with only 50 to 60% amino acid sequence similarity between types 1, 2, and 3. Although limited serological cross-reactivity between HCV types was detected between peptides, particularly in the first antigenic region of NS-4, type-specific reactivity formed the principal component of the natural humoral immune response to NS-4. Type-specific antibody to particular HCV types was detected in 89% of the samples from anti-HCV-positive blood donors and correlated almost exactly with genotypic analysis of HCV sequences amplified from the samples by polymerase chain reaction. Whereas almost all blood donors appeared to be infected with a single virus type (97%), a higher proportion of samples (40%) from hemophiliacs infected from transfusion of non-heat-inactivated clotting factor contained antibody to two or even all three HCV types, providing evidence that long-term exposure may lead to multiple infection with different variants of HCV.