bluuebunny/crossref_metadata_2025 huggingface dataset:
A new method for identification of Trichomonas vaginalis by fluorescent DNA in situ hybridization
Authors: Muresu R , Rubino S , Rizzu P , Baldini A , Colombo M , Cappuccinelli P
Published: January 2020

The protozoan flagellate Trichomonas vaginalis is responsible for human trichomoniasis, one of the most widespread sexually transmitted diseases in the world. Several methods are currently used for laboratory diagnosis, including direct microscopic observation, cell culture, immunological techniques, and more recently, DNA probing and gene amplification. This report describes an in situ hybridization technique with specific DNA probes labeled with either biotin, rhodamine, or fluorescein for detection of T. vaginalis with fluorescence microscopy. Repetitive DNA sequences were evident in the nuclei of the protozoa as intensively fluorescent regions, giving a spotted pattern. No cross-reactivity between the probes and the DNAs of mammalian cells, yeasts, or bacteria was noted. This technique is potentially useful for the diagnosis of human trichomoniasis in clinical samples.
Molecular epidemiology of catheter-associated bacteriuria in nursing home patients
Authors: Rahav G , Pinco E , Silbaq F , Bercovier H
Published: January 2020

Urine samples from 19 nursing home patients with long-term urinary catheters were cultured every 3 months for 18 months. Providencia stuartii, present in 74% of the elderly and in 59% of urine specimens, was the most frequently isolated bacteria. The persistence of P. stuartii was significantly higher among females than among males. In order to study the epidemiology of bacteriuria in this nursing home, bacteria were characterized by biochemical tests, antibiotic susceptibility pattern, and restriction fragment length polymorphism (RFLP) analysis. The antibiotic susceptibility pattern indicated that each patient had two to three different strains of P. stuartii during the 18 months of follow-up. In contrast, the RFLP analysis revealed that a specific strain had persisted in the urinary tract of the patient during the entire follow-up period. According to the biochemical profile, 74% of the patients had the same bacteria in urine cultures, pointing to a common source of transmission. RFLP analysis, however, demonstrated different patterns of RFLP, suggesting concomitant multiple sources of infection.
Prevalence of Cryptosporidium species in elementary school children
Authors: Nimri L F , Batchoun R
Published: January 2020

The project described here was conducted to study the prevalence of various parasites in elementary school children in northern Jordan. A single stool specimen was collected from each of 1,000 students in the 6- to 14-year-old age group. A questionnaire covering demographic information, health status, and other relevant information was filled out by one of the parents of each student. Fresh stool specimens were processed by using wet mount preparations, formalin-ether, and Sheather's sugar flotation techniques. Permanently stained slides were prepared by acid-fast, Giemsa, and trichrome staining. Cryptosporidium species was found in 40 specimens (4%); however, only 15 specimens had Cryptosporidium species alone, and these 15 specimens were from symptomatic children with diarrheic stools. The symptoms reported most often were abdominal pain, cramps, malaise, nausea, and headache. The number of cases of infection was higher in villages, where contact with animals was evident and where contaminated drinking water could have been a major source of the infections.
Bacteremia by multidrug-resistant Capnocytophaga sputigena
Authors: Gomez-Garces J L , Alos J I , Sanchez J , Cogollos R
Published: January 2020

A case of bacteremia caused by a multiresistant strain of Capnocytophaga sputigena in a patient with hematological malignancy is described. The strain presented with a pattern of marked resistance to beta-lactams, with MICs of > 256 mg/liter for ampicillin, ticarcillin, piperacillin, cefazolin, and cefuroxime, 64 mg/liter for cefotaxime, and 32 mg/liter for ceftazidime. In addition, the MIC of ciprofloxacin was 16 mg/liter. Both of these groups of antimicrobial agents are frequently used in the empiric treatment of infections in immunocompromised patients. The appearance of resistant strains suggests the need for antimicrobial susceptibility studies in all patients with severe infections caused by Capnocytophaga spp. or other capnophilic organisms present in the oral microflora of these patients.
Preliminary interpretive susceptibility testing criteria for FK-037 with 30-microgram disks
Authors: Jones R N
Published: January 2020

FK-037, a new parenteral cephalosporin, was tested against 483 organisms from clinical infections to establish preliminary susceptibility testing criteria with 30-micrograms disks. The proposed breakpoint zone diameters were > or = 17 mm (MIC correlate, < or = 8 micrograms/ml) for the susceptible category and < or = 13 mm (MIC correlate, > or = 32 micrograms/ml) for the resistant category. These interpretive guidelines produced no very major (false-susceptible) errors, 2.9% major (false-resistant) errors, and 6.2% minor errors for a total absolute agreement between methods of 90.9%.
Chemiluminescent hybridization assay for hepatitis delta virus RNA in serum
Authors: Gallagher M , Morris T T , Fields H A
Published: January 2020

A nonradioactive hybridization assay for the detection of hepatitis delta virus RNA in serum is described. This assay utilizes a digoxigenin-labeled RNA hybridization probe and chemiluminescent immunodetection. The probe can detect as little as 0.4 pg of cDNA with a 60-min exposure. Results obtained are in agreement with serological indicators of hepatitis delta virus infection and are comparable to those obtained by hybridization assays employing radioactively labeled RNA.
Fluconazole-resistant recurrent oral candidiasis in human immunodeficiency virus-positive patients: persistence of Candida albicans strains with the same genotype
Authors: Millon L , Manteaux A , Reboux G , Drobacheff C , Monod M , Barale T , Michel-Briand Y
Published: January 2020

Thirty human immunodeficiency virus-positive patients carrying Candida albicans in their oropharynx were treated with fluconazole and were monitored for 90 to 570 days. Fluconazole-resistant C. albicans (MIC, > 32 micrograms/ml) appeared only in seven patients and only after 90 days of treatment corresponding to a total dose of more than 10 g. Resistance was not associated with resistance to other azole derivatives. Susceptible and resistant strains from each patient had the same genotype (as defined by electrophoretic karyotype and restriction fragment length polymorphism). Thus, the resistant strains were selected by the antimycotic treatment from the susceptible strain present in each case.
Effect of delayed processing of blood samples on performance of cytomegalovirus antigenemia assay
Authors: Niubò J , Pérez J L , Carvajal A , Ardanuy C , Martín R
Published: January 2020

This prospective, parallel, and blind study of 120 blood samples from immunocompromised patients examined the influence of delayed sample processing on cytomegalovirus antigenemia assay. Cytomegalovirus was detected in 49 samples (40.8%): 41 were processed within 2 to 4 h, and 40 were reprocessed the following day. Results were discrepant in 17 of the samples with the lowest positive cell counts. Differences between the two days did not reach statistical significance.
Diagnosis and epidemiological association of Listeria monocytogenes strains in two outbreaks of listerial encephalitis in small ruminants
Authors: Wiedmann M , Czajka J , Bsat N , Bodis M , Smith M C , Divers T J , Batt C A
Published: January 2020

Two outbreaks of epizootic listerial encephalitis, one in sheep and one in goats, were investigated through pathology, microbiology, and DNA amplification-based techniques. Efforts were made to survey the diversity of Listeria monocytogenes strains in the silage consumed by affected animals and to verify the causal relationship between silage and disease outbreak. In both outbreaks, L. monocytogenes was isolated from silage and brain tissue samples. Random amplified polymorphic DNA patterns revealed two distinct L. monocytogenes strains, one of which was identical to the sheep brain isolate, in the silage associated with the outbreak in sheep. Three brain isolates and one silage isolate, all of which had different random amplified polymorphic DNA patterns, were found in the outbreak involving goats. All isolates from both outbreaks were indistinguishable in an in vitro assay for cell-to-cell spread and growth in macrophages. All brain isolates from the goat outbreak had identical intracellular ActA patterns, which were different from the pattern for the silage isolate. While the sheep brain isolate had an ActA pattern different from that of the corresponding silage isolate, the patterns for the brain isolates from the two outbreaks were not identical. This survey demonstrates the diversity of L. monocytogenes in silage and suggests the existence of one or more selective processes by which certain strains are more prone to give rise to disease.
Intracellular Campylobacter-like organism from ferrets and hamsters with proliferative bowel disease is a Desulfovibrio sp
Authors: Fox J G , Dewhirst F E , Fraser G J , Paster B J , Shames B , Murphy J C
Published: January 2020

Proliferative bowel disease is an intestinal disorder of a variety of domestic animals associated with the presence of an intracellular Campylobacter-like organism (ICLO). We have identified the ICLO obtained from a ferret with proliferative colitis by 16S rRNA sequence analysis. In this ferret, proliferative bowel tissue containing the ICLO had translocated to the mesenteric lymph nodes, omentum, and liver. The 16S rRNA genes of the ICLO were amplified from an infected fragment of extraintestinal tissue by using universal prokaryotic primers. Approximately 1,480 bases of the amplified 16S rRNA gene were sequenced by cycle sequencing. Comparison of the sequence of the ICLO with those of over 400 bacteria in our data base indicated that the sequence of the ICLO was most closely related to that of Desulfovibrio desulfuricans (87.5% similarity). Phylogenetic analysis with 12 Desulfovibrio species and 20 species from related genera placed the ICLO in a subcluster within the genus Desulfovibrio with D. desulfuricans and 5 other Desulfovibrio species. We will refer to this organism as the intracellular Desulfovibrio organism (IDO). Specific primers were produced for PCR amplification of a 550-base fragment of the 16S rRNA gene of the IDO in proliferative intestinal tissue samples. This unique 550-base segment was amplified from samples of frozen intestinal tissue from nine ferrets and three hamsters with ICLO-associated disease but not in four intestinal tissue samples from animals without the ICLO-associated disease. The 550-base amplified products from the bowel tissues of one hamster and one ferret were fully sequenced. The ferret IDO partial sequence was identical to the previously determined 16S rRNA sequence over its length, and the hamster IDO sequence differed by a single base. The same intracellular organism has been identified in proliferative intestinal tissues of swine and that the organism has been successfully maintained in tissue culture. The availability of specific primers for PCR-based detection of this intracellular Desulfovibrio organism will aid in the determination of its role in the pathogenesis of proliferative bowel disease in a variety of infected hosts.
Ureaplasma urealyticum biovar specificity and diversity are encoded in multiple-banded antigen gene
Authors: Teng L J , Zheng X , Glass J I , Watson H L , Tsai J , Cassell G H
Published: January 2020

Ureaplasma urealyticum is a commensal organism of the lower genital tract of females, but in a subpopulation of individuals, it can invade the upper genital tract. It is a significant cause of chorioamnionitis and neonatal morbidity and mortality. There are 14 recognized serovars of U. urealyticum; these can be divided into two distinct clusters or biovars. Biovar 1 is composed of serovars 1, 3, 6, and 14, Biovar 2 is composed of serovars 2, 4, 5, 7, 8, 9, 10, 11, 12, and 13. We previously identified a surface antigen, the multiple-banded (MB) antigen, which contains both serovar-specific and cross-reactive epitopes. Genotypic characterization of the C-terminal region of the MB antigen of serovar 3 indicates that serovar specificity and MB antigen size variation reside in that domain. In the present study, we used PCR analysis with primers derived from the serovar 3 MB antigen gene DNA sequence to determine if the MB antigen gene was present in the remaining 13 reference serovars as well as in invasive clinical isolates. The results indicated that not only was the MB antigen gene present in all serovars but that the genes' 5' regions were markers of biovar specificity and diversity. Further analysis of this region should reveal the phylogenetic relationship among serovars of U. urealyticum and, possibly, their invasive potential.
Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR
Authors: Elder R O , Duhamel G E , Schafer R W , Mathiesen M R , Ramanathan M
Published: January 2020

A PCR assay for the detection of Serpulina hyodysenteriae in diagnostic specimens was developed on the basis of sequence analysis of a recombinant clone designated pRED3C6. Clone pRED3C6, which contained a 2.3-kb DNA fragment unique to S. hyodysenteriae, was identified by screening a plasmid library of S. hyodysenteriae isolate B204 genomic DNA in Escherichia coli by colony immunoblot with the mouse monoclonal antibody 10G6/G10, which was produced against cell-free supernatant antigens from the same isolate. Southern blot analysis of HindIII-digested genomic DNA of S. hyodysenteriae serotypes 1 through 7 and of four weakly beta-hemolytic intestinal spirochetes, including Serpulina innocens, with the 2.3-kb DNA fragment of pRED3C6 indicated that the cloned sequence was present exclusively in the seven serotypes of S. hyodysenteriae. An oligonucleotide primer pair for PCR amplification of a 1.55-kb fragment and an internal oligonucleotide probe were designed and synthesized on the basis of sequence analysis of the 2.3-kb DNA fragment of pRED3C6. Purified genomic DNAs from reference isolates of S. hyodysenteriae serotypes 1 through 9, S. innocens, weakly beta-hemolytic intestinal spirochetes belonging to genotypic groups distinct from those of reference Serpulina spp., other cultivable reference isolates of the order Spirochaetales, and enteric bacteria including Escherichia coli, Salmonella spp., Campylobacter spp., and Bacteroides vulgatus were amplified with the oligonucleotide primer pair in a hot-start PCR. The 1.55-kb products were obtained only in the presence of genomic DNA from each of the nine serotypes of S. hyodysenteriae. The specificity of the 1.55-kb products for S. hyodysenteriae was confirmed on the basis of production of a restriction endonuclease pattern of the PCR products identical to the predicted restriction map analysis of pRED3C6 and positive hybridization signal with the S. hyodysenteriae-specific internal oligonucleotide probe. By using total DNA obtained from normal swine feces inoculated with decreasing concentrations of S. hyodysenteriae cells, the sensitivity of the PCR assay was calculated to be between 1 and 10 organisms per 0.1 g of feces. The PCR assay was 1,000 times more sensitive than conventional culture of dysenteric feces on selective medium. There was complete agreement between the results of PCR assays and anaerobic culture on selective agar medium with diagnostic specimen (n = 9) obtained from six farms on which there were cases with clinical signs suggestive of swine dysentery. Detection of S. hyodysenteriae by PCR amplification of DNA has great potential for rapid identification of S. hyodysenteriae in diagnostic specimens.
Serological follow-up after treatment of patients with erythema migrans and neuroborreliosis
Authors: Hammers-Berggren S , Lebech A M , Karlsson M , Svenungsson B , Hansen K , Stiernstedt G
Published: January 2020

To investigate the duration and kinetics of immunoglobulin M (IgM) and IgG antibodies against Borrelia burgdorferi in serum after treatment of Lyme borreliosis, consecutive serum samples from 30 seropositive patients with erythema migrans and 91 seropositive patients with neuroborreliosis were analyzed with a capture IgM enzyme-linked immunosorbent assay (ELISA) and an indirect IgG ELISA, both using B. burgdorferi flagella as the antigen. All the patients improved after treatment: 97 patients had a complete clinical recovery, while 24 patients had sequelae. The results showed that patients with erythema migrans and early neuroborreliosis more often initially had highly elevated IgM optical density (OD) values and low IgG OD values against B. burgdorferi, while the opposite was found in patients with late neuroborreliosis. During follow-up, the majority of patients had developed negative or significantly declining IgM ODs after 1 to 1.5 years but persistently positive IgM ODs were found up to 17 months after treatment of erythema migrans and 3 years after treatment of neuroborreliosis. IgG antibody levels declined more slowly and remained elevated to a larger extent, but more than half of the patients had developed negative IgG ODs within 5 years after therapy. However, positive IgG OD values were found after 9 to 10 years for patients treated for neuroborreliosis as well as erythema migrans. Both IgM and IgG antibodies against B. burgdorferi may persist for months to years after successful treatment of Lyme borreliosis. Consequently, a single serum sample with antibodies against B. burgdorferi must always be carefully evaluated and correlated to clinical symptoms.
Antimicrobial susceptibilities of mycobacteria as determined by differential light scattering and correlation with results from multiple reference laboratories
Authors: Conville P S , Witebsky F G , MacLowry J D
Published: January 2020

The DAWN Model B laser light scattering instrument (Wyatt Technology Corporation, Santa Barbara, Calif.) was evaluated to assess its potential to provide rapid mycobacterial antimicrobial susceptibility test results. For Mycobacterium tuberculosis there was a clear separation between susceptible and resistant results with the isolates tested, and there was excellent correlation with reference laboratory results. For Mycobacterium avium there was no obvious breakpoint between susceptible and resistant results with the isolates tested, and correlation with reference laboratory results was less good than for M. tuberculosis. However, for M. avium there was also less agreement among reference laboratory results than for M. tuberculosis. Significant instrument design and software program changes would be required for the instrument to become a useful tool for mycobacterial susceptibility testing in the diagnostic laboratory.
Immunohistochemical detection of a novel 22- to 25-kilodalton glycoprotein of Paracoccidioides brasiliensis in biopsy material and partial characterization by using species-specific monoclonal antibodies
Authors: Figueroa J I , Hamilton A , Allen M , Hay R
Published: January 2020

Two murine monoclonal antibodies (MAbs) specific to Paracoccidioides brasiliensis (as determined by enzyme-linked immunosorbent assay [ELISA] and Western blot [immunoblot]) were produced by using a modification of standard hybridization protocols, with cyclophosphamide included as an immunomodulator to abolish responses to highly cross-reactive immunodominant epitopes. MAbs PS14 and PS15 are two different clones which exhibit similar characteristics by ELISA and Western blot. They are directed against a 22- to 25-kDa antigen which is present in P. brasiliensis and which could not be identified in other dimorphic fungi by ELISA or Western blot. Partial purification of the antigen was accomplished by isoelectric focusing, and deglycosylation studies suggested that the 22- to 25-kDa antigen is a glycoprotein with a pI of between 4.5 and 5 and that O-linked sugars may be part of the recognized epitope. The MAbs stained the cytoplasm of P. brasiliensis yeast and hyphal cells in cryostat sections of fresh cultures of the fungus. In addition, the MAbs stained the wall of paracoccidioidomycotic granulomas, as well as the cytoplasm of the fungus, as determined by the use of immunofluorescence, immunoperoxidase, and immuno-alkaline phosphatase staining techniques in paraffin-embedded sections of human biopsy material, and they failed to stain granulomas resulting from other clinical conditions. These findings suggest that these MAbs have potential use in the immunohistochemical identification of P. brasiliensis.
Development and evaluation of a rapid and simple procedure for detection of Pneumocystis carinii by PCR
Authors: Cartwright C P , Nelson N A , Gill V J
Published: January 2020

We report the development of a simplified PCR-based assay for the detection of Pneumocystis carinii DNA in clinical specimens. The adoption of a rapid DNA extraction procedure and the introduction of a type of enzyme-linked immunosorbent assay for PCR product detection enabled this procedure to be carried out in a single working day in a clinical microbiology laboratory. The PCR assay was prospectively compared with an immunofluorescent-antibody (FA) staining method for the detection of P. carinii in induced sputum and bronchoalveolar lavage (BAL) specimens. The results of the study showed that, for induced sputum specimens, FA staining had a sensitivity of 78% (32 of 41 specimens) and a specificity of 100% (166 of 166 specimens); PCR was 100% (41 of 41 specimens) sensitive and 98% (162 of 166 specimens) specific. For BAL specimens, FA staining was 100% sensitive (21 of 21 specimens) and 100% specific (133 of 133 specimens), and PCR had a sensitivity of 100% (21 of 21 specimens) and a specificity of 99% (132 of 133 specimens). These results strongly suggest that use of our PCR-based assay could effect clinically useful improvements in the sensitivity of induced sputum specimens for the detection of P. carinii.
Selection of candidate quality control isolates and tentative quality control ranges for in vitro susceptibility testing of yeast isolates by National Committee for Clinical Laboratory Standards proposed standard methods
Authors: Pfaller M A , Bale M , Buschelman B , Lancaster M , Espinel-Ingroff A , Rex J H , Rinaldi M G
Published: January 2020

The National Committee for Clinical Laboratory Standards has developed a proposed standard method for in vitro antifungal susceptibility testing of yeast isolates (National Committee for Clinical Laboratory Standards, document M27-P, 1992). In order for antifungal testing by the M27-P method to be accepted, reliable quality control (QC) performance criteria must be developed. In the present study, five laboratories tested 10 candidate QC strains 20 times each against three antifungal agents: amphotericin B, fluconazole, and 5-fluorocytosine. All sites conformed to the M27-P standards and used a common lot of tube dilution reagents and RPMI 1640 broth medium. Overall, 98% of MIC results with amphotericin B, 95% with fluconazole, and 92% with 5-fluorocytosine fell within the desired 3-log2 dilution range (mode +/- 1 log2 dilution). Excellent performance with all three antifungal agents was observed for six strains: Candida albicans ATCC 90028, Candida parapsilosis ATCC 90018, C. parapsilosis ATCC 22019, Candida krusei ATCC 6258, Candida tropicalis ATCC 750, and Saccharomyces cerevisiae ATCC 9763. With these strains, 3-log2 dilution ranges encompassing 94 to 100% of MICs for all three drugs were established. Additional studies with multiple lots of RPMI 1640 test medium will be required to establish definitive QC ranges.
Ability of PCR assay to identify Mycobacterium tuberculosis in BACTEC 12B vials
Authors: Forbes B A , Hicks K E
Published: January 2020

Introduction of PCR to directly detect Mycobacterium tuberculosis in clinical specimens has shown promise; however, interfering substances in clinical material have contributed to lowered assay sensitivities. We evaluated the ability of a PCR assay to detect M. tuberculosis in BACTEC 12B broth cultures. Clinical specimens were processed and inoculated into BACTEC 12B vials. Evaluation was approached in two phases, starting with an initial evaluation in which an aliquot of 12B broth was removed when the growth index (GI) was > or = 10 and stored at 4 degrees C until assayed by PCR. Of the 290 specimens initially assayed, 129 were culture negative for mycobacteria as well as PCR negative for M. tuberculosis. Except for one, cultures (n = 102) which grew mycobacteria other than M. tuberculosis were all PCR negative. The remaining 59 broths were all culture and PCR positive for M. tuberculosis; 39% (n = 23) of these cultures when assayed by PCR had GIs of < or = 50. Following initial evaluation, 200 12B BACTEC vials with GIs of > or = 10 were assayed in a similar manner except that specimens were amplified twice weekly to determine PCR's impact on the length of time to identification of M. tuberculosis as compared with standard laboratory practices. Utilization of PCR resulted in a mean time to detection of M. tuberculosis of 14 days, compared with 29 days by using commercially available nucleic acid probes to identify M. tuberculosis complex from growth of BACTEC 12B subcultures on solid media. In light of an overall sensitivity and specificity of 100 and 99.7%, respectively, coupled with the ability to identify M. tuberculosis days or weeks before other methods can be applied, we conclude that PCR might prove to be a rapid alternative for identification of M. tuberculosis in culture and allow for earlier setup of susceptibility testing.
Use of recombinant OspC from Borrelia burgdorferi for serodiagnosis of early Lyme disease
Authors: Padula S J , Dias F , Sampieri A , Craven R B , Ryan R W
Published: January 2020

Infection with Borrelia burgdorferi, the etiologic agent of Lyme disease, is associated with an early and dominant humoral response to the spirochete's 23-kDa outer surface protein C (OspC). We have cloned and expressed OspC as a fusion protein in Escherichia coli and have shown that patient serum samples react with it in an enzyme-linked immunosorbent assay (ELISA) (S. J. Padula, A. Sampieri, F. Dias, A. Szczepanski, and R. W. Ryan, Infect. Immun. 61:5097-5105, 1993). Now we have compared the detection of B. burgdorferi-specific immunoglobulin M antibodies in 74 individuals with culture-positive erythema migrans by a whole-cell ELISA, immunoblot, and the recombinant OspC (rOspC) ELISA. Seventy-six negative controls were also studied. With all of the tests, there was a statistically significant association between the duration of disease and the frequency of a positive result. With the rOspC ELISA, the predictive value of a positive test was 100% and the predictive value of a negative test was 74%. Similar results were obtained with the whole-cell ELISA and with the immunoblot using as the source of test antigen a strain of B. burgdorferi which expresses abundant levels of OspC. We conclude that the use of rOspC in an ELISA is a convenient, readily automated, and easily standardized test for the serodiagnosis of early Lyme disease.
Detection of Salmonella serovars from clinical samples by enrichment broth cultivation-PCR procedure
Authors: Stone G G , Oberst R D , Hays M P , McVey S , Chengappa M M
Published: January 2020

To overcome problems associated with application of PCR to clinical samples, we have combined a short cultivation procedure with a Salmonella-specific PCR-hybridization assay to specifically identify Salmonella serovars from clinical samples of various animal species. The technique was investigated by using fecal samples seeded with known numbers of Salmonella organisms and cultivated for different lengths of time in assorted selective and nonselective enrichment media. The ability of PCR to amplify a Salmonella-specific DNA product (457-bp sequence covering the Salmonella invE and invA genes) was examined in Southern hybridizations with an internal oligonucleotide probe. Forty-seven Salmonella isolates representing 32 serovars were evaluated, and all Salmonella isolates resulted in a 457-bp product that hybridized with the oligonucleotide probe, whereas no hybridizations were evident with 53 non-Salmonella organisms. The assay detected as few as 9 CFU of Salmonella organisms in pure culture and as little as 300 fg of purified chromosomal DNA. Rappaport-Vassiliadis and tetrathionate broths were inhibitory to PCR, whereas brain heart infusion and selenite-cystine broths were not. The PCR-hybridization assay coupled with a brain heart infusion enrichment culture incubated for 2 h detected as few as 80 CFU of Salmonella organisms in seeded feces. We have successfully identified Salmonella serovars in clinical samples from swine, horses, and cattle more rapidly than with conventional culture techniques. The sensitivity and specificity of this assay were both 100% compared with culture results. These results indicate that a combined cultivation-PCR-hybridization assay could be applicable and advantageous in the rapid identification of Salmonella serovars in routine diagnostic situations.
Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory
Authors: Geha D J , Uhl J R , Gustaferro C A , Persing D H
Published: January 2020

A multiplex PCR assay for detection of the staphylococcal mecA gene (the structural gene for penicillin-binding protein 2a) was compared with agar dilution and disk diffusion susceptibility test methods for identifying methicillin resistance. The multiplex PCR assay combined two primer sets (mecA and 16S rRNA) in a single reaction. A total of 500 staphylococcal isolates (228 isolates of Staphylococcus aureus and 272 isolates of coagulase-negative staphylococci) from clinical specimens were studied. For S. aureus, 40 of 40 mecA-positive isolates and 4 of 188 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 100 and 98%, respectively). In 3 of 4 discordant isolates, resistance was due to hyperproduction of beta-lactamase. For coagulase-negative staphylococci, 148 of 159 mecA-positive isolates and 0 of 113 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 93 and 100%, respectively). Twenty-six isolates were categorized as indeterminate because of the absence of a detectable 16S rRNA product. Four of these 26 isolates contained mecA when retested. The assay is designed to be incorporated into the work flow of the clinical microbiology laboratory and allows for the identification of intrinsic resistance in a timely and reliable manner.
Evaluation of rapid commercial enzyme immunoassay for detection of Giardia lamblia in formalin-preserved stool specimens
Authors: Scheffler E H , Van Etta L L
Published: January 2020

Two hundred twenty-three formalin-preserved stool specimens were evaluated by using ProSpecT Giardia Rapid Assay (membrane bound) (Alexon, Inc., Sunnyvale, Calif.). Enzyme immunoassay (EIA) results were compared with those by conventional microscopic examination. Two hundred four specimens were negative by both methods, and 13 (6.3%) were positive. Five specimens were negative by initial microscopic exam and positive by EIA; three of these specimens were found to be positive upon extensive microscopic reexamination. The remaining two specimens were from patients who previously tested positive and who had recurrent symptoms of or responded to therapy for giardia. Therefore, we consider both cases to be true positives. One specimen exhibited a single cyst by microscopic exam and was negative by EIA Resolved results yielded a relative sensitivity of 95%, a relative specificity of 100%, a positive predictive value of 99.6%, and a negative predictive value of 100%, compared with a sensitivity of 74% for conventional microscopy.
Detection of leptospires in urine by PCR for early diagnosis of leptospirosis
Authors: Bal A E , Gravekamp C , Hartskeerl R A , De Meza-Brewster J , Korver H , Terpstra W J
Published: January 2020

We tested urine samples from patients at different stages of current leptospirosis and thereafter to determine whether use of the PCR for detection of leptospires in urine can be a valuable alternative to culturing. The procedure of DNA extraction and subsequent PCR applied to 15 freshly voided urine samples proved to be twice as sensitive as culturing. Overall, we were able to detect leptospires in approximately 90% (26 of 29) of the urine samples. Urine and serum samples were obtained from seven patients, before the eighth day of illness. Although it is generally assumed that leptospiruria starts approximately in the second week of illness, we were able to detect leptospires in all of these early urine samples. In contrast, only two of seven corresponding serum samples gave positive PCR results, which suggests that PCR analysis of urine can be more successful for early diagnosis of leptospirosis than PCR analysis of serum. Urine samples from six patients who had been treated with antibiotics at the time of illness were positive by PCR, implying that the patients were still shedding leptospires in their urine despite treatment. Some of these samples were even taken years after the infection, indicating that shedding of leptospires in urine may last much longer than is generally assumed. We conclude that detection of leptospires in urine with PCR is a promising approach for early diagnosis of leptospirosis and may also be useful in studying long-term shedding.
Gas-liquid chromatography of cellular fatty acids for identification of staphylococci
Authors: Stoakes L , John M A , Lannigan R , Schieven B C , Ramos M , Harley D , Hussain Z
Published: January 2020

A commercially available, computer-assisted microbial identification system (MIS) employs gas-liquid chromatographic analyses of bacterial fatty acids. The MIS was used to identify 470 isolates of Staphylococcus species. The accuracy of the MIS was compared with the accuracies of conventional methods. There was a complete agreement between the MIS and conventional methods in the identification of 413 (87.8%) strains. For 36 of 45 misidentified strains, the correct identification was listed by the MIS as a choice but not as the first choice. Twelve strains could not be matched. All strains of Staphylococcus cohnii, S. epidermidis, S. intermedius, S. lugdunensis, S. schleiferi, S. sciuri, S. simulans, and S. xylosus were correctly identified. Two species, S. hominis and S. saprophyticus, accounted for 52.6% (30 of 57) of the misidentifications. Seventy-eight organisms were retested. Identification of 73 organisms remained unchanged, and for five organisms, the second choice became first and vice versa. The overall performance of the MIS is acceptable, and the system can be used as an alternate identification method for staphylococci.
Enzyme-linked immunosorbent assay for measuring ileal symbiont intracellularis-specific immunoglobulin G response in sera of pigs
Authors: Holyoake P K , Cutler R S , Caple I W , Monckton R P
Published: January 2020

Proliferative enteritis (PE) is a common intestinal disease on pig farms. The disease is caused by ileal symbiont (IS) intracellularis (Campylobacter-like organisms) bacteria. An enzyme-linked immunosorbent assay (ELISA) was developed to measure IS intracellularis-specific immunoglobulin G (IgG) response in the sera of pigs. The antigen used in the ELISA was filtered, percoll gradient-purified IS intracellularis extracted from the intestines of pigs affected with proliferative hemorrhagic enteropathy. The antibody responses of pigs challenged with intestinal homogenates from pigs affected with proliferative hemorrhagic enteropathy containing IS intracellularis or percoll-gradient purified IS intracellularis were low and variable. The low IgG titers measured in challenged pigs support previous findings that IgG plays a minor role in the immune response of pigs to IS intracellularis. On a farm in which infection was endemic, pigs seroconverted at between 7 and 24 weeks of age. High IgG titers, indicative of maternally acquired antibody, were present in 3-week-old pigs. The IgG titers in piglets were lowest at 6 weeks of age, which approximates the age of onset of clinical disease. These results suggest that IgG plays a role in determining the susceptibilities of pigs to natural infection. Measurements of seroconversion by the ELISA might aid in epidemiological investigations of PE in naturally infected herds. However, the variable antibody responses in experimentally challenged pigs would seem to limit its usefulness as an antemortem diagnostic test for PE.
Application to immunoglobulin M capture hemadherence assays of hemagglutination of monkey erythrocytes by native and recombinant human parvovirus B19 antigens
Authors: Cubel R C , Alferes A C , Cohen B J , Nascimento J P
Published: January 2020

Human parvovirus B19 recently was shown to agglutinate baboon and human erythrocytes. We have now demonstrated that both recombinant and native B19 antigens agglutinate rhesus, cynomolgus, and Saimiri monkey erythrocytes. Using cynomolgus erythrocytes and the recombinant antigen, we developed an immunoglobulin M (IgM) antibody capture hemadherence test (MACHAT) for the detection of specific B19 IgM antibodies in human sera. The results obtained with MACHAT were compared with those obtained with an IgM capture enzyme immunoassay (MACEIA) employing the native antigen routinely used in our laboratory. For 229 patient serum samples, we found 96% agreement between the results of the two assays. There was some evidence that MACHAT was slightly more sensitive than MACEIA. Our results add to the range of erythrocytes that can be agglutinated by B19 virus and show that native as well as recombinant antigens may be used in MACHAT.
Comparison of methods for detection of hepatitis B virus DNA
Authors: Zaaijer H L , ter Borg F , Cuypers H T , Hermus M C , Lelie P N
Published: January 2020

We compared the performance of four assays for detection of hepatitis B virus (HBV) DNA: the PCR; the branched DNA hybridization assay (Chiron); and two hybridization assays that use liquid hybridization (Abbott) or direct membrane hybridization (MH). Testing 109 random hepatitis B surface antigen-positive patient samples, the percentages found to be HBV DNA positive among 30 hepatitis B e antigen (HBeAg)-positive samples and 79 HBeAg-negative samples were as follows: PCR, 100 and 90%; Chiron, 73 and 25%; Abbott, 67 and 13%; and MH, 40 and 8%. In six hepatitis B surface antigen-positive, HBeAg-negative samples, all three hybridization assays detected HBV DNA. Testing dilutions prepared from the Eurohep HBV DNA standards, the detection limits of the assays appeared to be the following: PCR, 2.5 x 10(2) HBV genomes per ml; Chiron, 2.5 x 10(6) genomes per ml; and Abbott and MH, 2.5 x 10(7) genomes per ml. HBV DNA levels in the dilution series, as reported by the Chiron and MH assays, were, on average, 2 times higher than calculated; the Abbott results were, on average, 19 times lower than calculated. We concluded that high levels of HBV DNA and the presence of HBeAg do not necessarily coincide, that the application of hybridization assays is limited to the monitoring of relatively high levels of HBV DNA, and that further standardization of quantitative HBV DNA assays is necessary to facilitate comparison of HBV DNA levels.
Elimination of false-positive serum reactivity in latex agglutination test for cryptococcal antigen in human immunodeficiency virus-infected population
Authors: Whittier S , Hopfer R L , Gilligan P
Published: January 2020

We recently tested serum from a human immunodeficiency virus-infected patient for the presence of cryptococcal antigen using the Meridian latex agglutination (LA) test (Cryptococcal Antigen Latex Agglutination System). Two pronase-treated serum specimens from the patient had LA titers of 80 and 160, but the patient had no evidence of cryptococcal disease. The serum was negative for rheumatoid factor, a well-documented cause of false-positive LA reactions. Seven blood culture supernatants from the patient were also LA positive, but were culture negative for cryptococcus. When the sera and blood culture supernatants were treated with 0.01 M 2-beta-mercaptoethanol (2-ME), the agglutinating activity was ablated. Similar results were seen when the sera were tested by two other commercial LA assays. Serum and cerebrospinal fluid specimens from patients with confirmed cryptococcal disease were treated with 2-ME, and the results were compared with those obtained after pronase (sera) or heat (cerebrospinal fluid) inactivation. The titers were identical (n = 56) or within 1 dilution (n = 3). One hundred serum specimens from human immunodeficiency virus-seropositive patients with no known history of cryptococcal disease were examined to determine the frequency of false-positive reactivity in this patient population. Of this group, three were positive following pronase treatment. One remained positive after 2-ME treatment; the remaining two were negative. These data indicate that 2-ME can be used to eliminate nonspecific reactivity in the LA test without affecting true-positive results.
Rapid discrimination of Mycobacterium tuberculosis strains by random amplified polymorphic DNA analysis
Authors: Linton C J , Jalal H , Leeming J P , Millar M R
Published: January 2020

Investigations of the epidemiology of tuberculosis have been hampered by the lack of strain-specific markers that can be used to differentiate isolates of Mycobacterium tuberculosis. We report the development of a rapid protocol for random amplified polymorphic DNA analysis which included the use of a commercially available DNA extraction kit (GeneReleaser). This was applied to 14 strains of M. tuberculosis, including strains associated with temporal and geographical clusters of tuberculosis in the United Kingdom and those from India, Africa, and Saudi Arabia. Strains of M. tuberculosis could be discriminated in about 8 h by this method, which is therefore a rapid and simple alternative to restriction fragment length polymorphism analysis.
A simple, specific, and highly sensitive blocking enzyme-linked immunosorbent assay for detection of antibodies to bovine herpesvirus 1
Authors: Kramps J A , Magdalena J , Quak J , Weerdmeester K , Kaashoek M J , Maris-Veldhuis M A , Rijsewijk F A , Keil G , van Oirschot J T
Published: January 2020

By using a monoclonal antibody directed against an epitope located on glycoprotein B of bovine herpesvirus 1 (BHV1), a simple, convenient blocking enzyme-linked immunosorbent assay (ELISA) which combines a high sensitivity with a low false-positive rate has been developed. The test can be performed at low variance on undiluted bovine serum samples. The epitope on glycoprotein B appears to be conserved, because it could be detected by immunostaining in all of 160 BHV1 isolates originating from 10 countries. In testing 215 anti-BHV1 antibody-negative and 179 anti-BHV1 antibody-positive serum samples, specificity and sensitivity were 0.96 and 0.99, respectively. This blocking ELISA is superior to a commercially available indirect ELISA and to the 24-h virus neutralization test in detecting low antibody levels in serum. In addition, this blocking ELISA is able to detect specific antibodies in serum as early as 7 days postinfection. To minimize any risk of introducing latent BHV1 carriers among noninfected cattle, this blocking ELISA would be, in our opinion, the test of choice.
Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples
Authors: Vilcek S , Elvander M , Ballagi-Pordány A , Belák S
Published: January 2020

Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme ScaI, which specifically cleaved, products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the method of choice in the analysis of clinical specimens of BRSV.
Early detection of anti-HCc antibody in acute hepatitis C virus (HCV) by western blot (immunoblot) using a recombinant HCV core protein fragment
Authors: Yeh C T , Han C M , Lo S Y , Ou J H , Fan K D , Sheen I S , Chu C M , Liaw Y F
Published: January 2020

Crude extract from Escherichia coli which expressed a recombinant protein containing amino acids 2 to 127 of the hepatitis C virus (HCV) core protein was used to detect the antibody against HCV core protein (anti-HCc). After electrophoretic separation of proteins from the extract, Western blot (immunoblot) analysis was performed with the serum samples. This method was compared with a commercially available second-generation enzyme immunoassay (EIA) which employed synthetic peptides corresponding to highly antigenic segments of both structural and nonstructural portions of HCV. Also, reverse transcription PCR for HCV RNA was used for comparison. Seventy-two serum samples from three groups of patients were tested. Groups I and II represented healthy subjects and subjects with acute hepatitis A or B, respectively. Group III included patients with newly acquired acute hepatitis C. By Western blot analysis, 31 of 31 (100%) samples from group I were negative for anti-HCc antibody, whereas 4 of 22 (18%) samples from group II were positive for anti-HCc. One of these four samples was also positive for anti-HCV antibody by the second-generation EIA (1 of 22 [4.5%]). Among 19 patients diagnosed with newly acquired acute hepatitis C, 4 (21%) were positive for anti-HCV by the second-generation EIA, whereas 12 of 19 (63%) were positive for anti-HCc by Western blot analysis. Of EIA-positive subjects, 4 of 4 (100%) were also positive for anti-HCc by Western blot analysis, whereas among EIA-negative subjects, 8 of 15 (53%) were positive. For HCV RNA detected by reverse transcription PCR, 15 of 19 (80%) of this group of samples were positive. Strikingly, the peak bilirubin level for patients with EIA-negative and Western blot-positive results is significantly higher than that for patients with consistent EIA and Western blot results (22.7 versus 7.2 mg/dl). A series of serum samples from a patient with concurrent hepatitis B and C viral infection was also studied by both tests. Although anti-HCc persisted throughout the course of infection, anti-HCV by EIA converted from negative to positive 20 days after admission and then converted back to negative 30 days later.