bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of cytomegalovirus in blood donors by PCR using the digene SHARP signal system assay: effects of sample preparation and detection methodology
Authors: Krajden M , Shankaran P , Bourke C , Lau W
Published: January 2020

Cytomegalovirus (CMV) is an important cause of transfusion-associated morbidity and mortality; however, only 0.4 to 12% of the blood products obtained from seropositive blood donors transmit infection. The effects of three commercially available whole-blood sample preparation kits on the detection of CMV PCR products by a semiquantitative adaptation of the Digene SHARP Signal System Assay (DSSSA) in samples from volunteer blood donors was assessed. Of 101 samples from seropositive blood donors, CMV was detected in 0 (0%) of the samples extracted with a QIAamp blood kit (QIAGEN), 1 (1%) of the samples extracted with an Amplicor whole-blood specimen preparation kit (Roche), and 8 (8%) of the samples extracted with an Isoquick nucleic acid extraction kit (modified by the addition of carrier tRNA) (Microprobe). CMV DNA was not detected in samples from seronegative blood donors (n = 13). Nested PCR of selected samples confirmed the detection of CMV in the sane eight samples extracted with the modified Isoquick nucleic acid extraction kit and detected an additional nine CMV-positive samples (n = 50). Samples from volunteer blood donors contain low copy numbers of CMV DNA. PCR amplification of such specimens can result in analytical sampling errors, giving results similar to the variations in titers recognized during determinations of the 50% tissue culture infective dose. The detection of CMV in blood samples from volunteer blood donors by PCR is a function of sample preparation, amplification conditions, and detection methodology. Accurate assessments of the clinical utility of CMV DNA detection by nucleic acid amplification for blood product screening and patients will require highly standardized and quantitative methodology.
Discrimination of respiratory syncytial virus subgroups A and B by reverse transcription-PCR
Authors: Gottschalk J , Zbinden R , Kaempf L , Heinzer I
Published: January 2020

Reverse transcription (RT)-PCR with shared primers differentiating respiratory syncytial virus (RSV) subgroups A and B was developed for subtyping of RSV isolates. Results of RT-PCR were compared with those of an indirect immunofluorescence test using monoclonal antibodies. Viral RNA isolated from cell cultures infected with RSV served as a template for cDNA synthesis with random primers. For PCR, we used three synthetic oligonucleotides corresponding to the G protein mRNA sequence of subgroup A (bases 248 to 267; 3'ATGCAACAAGCCAGATCAAG), subgroup B (bases 314 to 333; 3'ACTCATCCAAACAACCCACA), or both (bases 511 to 530; 3'GGWACAAARTTGAACACTTC). PCR products of RSV subgroups A and B had molecular sizes of 283 and 217 bp, respectively. Specific cutting sites for RSV A and B in amplified cDNA were demonstrated by restriction fragment analysis with four restriction endonucleases. Our RT-PCR assay divided 68 RSV isolates into 47 strains of subgroup A and 21 strains of subgroup B in full agreement with subtyping by monoclonal antibodies. RT-PCR seems to be a good alternative to subtyping of RSV with monoclonal antibodies.
Comparison of infectivities of six tick-derived isolates of Borrelia burgdorferi for rodents and ticks
Authors: Peavey C A , Lane R S
Published: January 2020

The infectivity and dissemination to the skin of six isolates of Borrelia burgdorferi were evaluated by inoculating them into groups of deer mice (Peromyscus maniculatus), hamsters, and Swiss Webster mice. Rodent infection was assayed by culture of ear punch biopsy specimens taken at 4, 8, and 12 weeks postinoculation (p.i.). Spirochetes were detected in biopsy specimens from individuals of all three host species that had been inoculated with four isolates (CA3, CA4, CA7, and CA8). Ear punch biopsy specimens taken from Swiss Webster mice at 12 weeks p.i. yielded an additional reisolate (CA2), even though these animals did not seroconvert. The remaining isolate (CA9) was not recovered from any host. However, two deer mice and all hamsters and Swiss Webster mice inoculated with CA9 seroconverted. All six isolates were of low infectivity to ticks when inoculated intramuscularly into hosts. Only 4 (1.6%) of 250 Ixodes pacificus larvae acquired and transstadially maintained infection from hosts inoculated intramuscularly. Infectivity of three isolates for ticks also was tested in Swiss Webster mice injected intradermally. The mean prevalences of infection in xenodiagnostic ticks fed on these mice at 4 weeks p.i. were 47.9, 1.2, and 2.2% for isolates CA4, CA7, and CA8, respectively. The mean prevalences of infection for ticks fed on the same mice at 12 weeks p.i. were 36.4, 11.8, and 20.4%, respectively. Such differences in the infectivity and rate of dissemination of individual isolates of B. burgdorferi should be considered during studies of reservoir and vector competence.
Significance of indeterminate third-generation hepatitis C virus recombinant immunoblot assay
Authors: Pawlotsky J M , Bastie A , Pellet C , Remire J , Darthuy F , Wolfe L , Sayada C , Duval J , Dhumeaux D
Published: January 2020

Indeterminate hepatitis C virus (HCV) third-generation recombinant immunoblot assay (RIBA3.0; Ortho Diagnostic Systems) patterns were arbitrarily defined by the manufacturer as the detection of only one antibody out of the four that were sought, namely, c100 (NS4 encoded), c22 (core encoded), c33c (NS3 encoded), and NS5 (NS5 encoded). The aims of the present study were (i) to determine the prevalence of indeterminate RIBA3.0 patterns in patients consecutively tested for anti-HCV antibodies in a university hospital; (ii) to evaluate the significance of these patterns in terms of viral replication, liver disease, and risk factors for HCV; and (iii) to get an insight into the mechanism underlying this peculiar immune response. Among 3,074 serum samples consecutively tested for anti-HCV antibodies, 588 were found to be positive by screening assays. Fifty-nine of them (10%) were RIBA3.0 indeterminate and were compared with 59 RIBA3.0-positive ones. Thirty-one RIBA3.0-indeterminate and 53 RIBA3.0-positive serum samples were HCV RNA positive by PCR (53 versus 90%; P < 10(-6). RIBA3.0-indeterminate and RIBA-3.0-positive patients with positive PCR results were not significantly different for the prevalence of risk factors for HCV infection and elevated serum alanine aminotransferase activities. Immunosuppression, attributable to coexisting human immunodeficiency virus infection, organ transplantation, or the administration of immunosuppressive drugs, was significantly more frequent in PCR-positive, RIBA3.0-indeterminate patients than in PCR-negative, RIBA3.0 indeterminate patients (P < 0.001) and PCR-positive patients with a positive RIBA3.0 result (P < 0.01). The distribution of HCV genotypes did not differ significantly between HCV RNA-positive patients with indeterminate or positive RIBA3.0 results. In conclusion, the prevalence of indeterminate RIBA3.0 patterns in virology laboratories is about 10%; in about half of these patients HCV replication is detected by PCR; the main factor responsible for indeterminate RIBA3.0 patterns could be immunosuppression, whereas HCV genotypes do not seem to play major role.
Evaluation of the Microbial Identification System for identification of clinically isolated yeasts
Authors: Crist A E , Johnson L M , Burke P J
Published: January 2020

The Microbial Identification System (MIS; Microbial ID, Inc., Newark, Del.) was evaluated for the identification of 550 clinically isolated yeasts. The organisms evaluated were fresh clinical isolates identified by methods routinely used in our laboratory (API 20C and conventional methods) and included Candida albicans (n = 294), C. glabrata (n = 145), C. tropicalis (n = 58), C. parapsilosis (n = 33), and other yeasts (n = 20). In preparation for fatty acid analysis, yeasts were inoculated onto Sabouraud dextrose agar and incubated at 28 degrees C for 24 h. Yeasts were harvested, saponified, derivatized, and extracted, and fatty acid analysis was performed according to the manufacturer's instructions. Fatty acid profiles were analyzed, and computer identifications were made with the Yeast Clinical Library (database version 3.8). Of the 550 isolates tested, 374 (68.0%) were correctly identified to the species level, with 87 (15.8%) being incorrectly identified and 89 (16.2%) giving no identification. Repeat testing of isolates giving no identification resulted in an additional 18 isolates being correctly identified. This gave the MIS an overall identification rate of 71.3%. The most frequently misidentified yeast was C. glabrata, which was identified as Saccharomyces cerevisiae 32.4% of the time. On the basis of these results, the MIS, with its current database, does not appear suitable for the routine identification of clinically important yeasts.
Serodiagnosis of Porphyromonas gingivalis infection by immunoblot analysis with recombinant collagenase
Authors: Wittstock M , Flemmig T F , Schmidt H , Mutters R , Karch H
Published: January 2020

The Porphyromonas gingivalis collagenase-specific serum immunoglobulin A (IgA), IgM, and IgG responses from 20 patients with early-onset periodontitis (EOP), 20 patients with adult periodontitis, (AP), and 20 age- and sex-matched healthy controls were examined by immunoblot analysis. A recombinant collagenase antigen used for the immunoblot analysis was produced by using the plasmid pGEX-2T, which allows the fusion between the collagenase and glutathione S-transferase. There was no significant difference in collagenase-specific IgG antibody detection between samples from the EOP, AP, and control groups. In contrast, 85% of AP and EOP sera had collagenase-specific IgA antibodies, whereas only 20% of control sera showed collagenase-specific IgA reactivity. Plaque samples from all groups were assessed by PCR with primers complementary to the collagenase-encoding gene prtC. The results indicated that 90% of AP and EOP plaque samples and 10% of control samples were positive for P. gingivalis. All patients with collagenase-specific IgA antibodies were PCR positive. The results of the study indicate a nearly complete concordance (k = 0.856) between the presence of collagenase-specific IgA antibodies and PCR detection of P. gingivalis. By using PCR as the "gold standard," the sensitivity and specificity of the IgA immunoblot test were 94.7 and 90.9%, respectively. Therefore, the recombinant collagenase is a potential candidate for use in the serodiagnosis of periodontitis.
Encephalopathy associated with enteroinvasive Escherichia coli 0144:NM infection
Authors: Ephros M , Cohen D , Yavzori M , Rotman N , Novic B , Ashkenazi S
Published: January 2020

Central nervous system manifestations typically occur with Shigella gastroenteritis and also in enteric Salmonella and Campylobacter infections. To date no association between enteroinvasive Escherichia coli infection and neurologic symptoms has been described. Two children with diarrhea caused by E. coli 0144:NM had otherwise unexplained encephalopathy manifested by profound stupor in one child and by obtundation and meningismus in the other one. These cases of infection occurred in northern Israel during a period of an unusually high rate of enteric infection caused by this organism. None of the microbiologic properties studied were uniquely attributable to the encephalopathic cases. The two encephalopathic as well as all eight nonencephalopathic isolates studied possessed the 140-MDa invasive plasmid. All 10 isolates examined produced small amounts of cytotoxin by the HeLa cell assay, all were nonmotile, and all had identical antibiograms. Eight of 10 of the isolates had identical plasmid profiles, while 2 isolates (from nonencephalopathic patients) had slightly different plasmid profiles. This is the first report of encephalopathy associated with enteroinvasive E. coli.
Patterns of detection of Strongyloides stercoralis in stool specimens: implications for diagnosis and clinical trials
Authors: Dreyer G , Fernandes-Silva E , Alves S , Rocha A , Albuquerque R , Addiss D
Published: January 2020

Reported efficacies of drugs used to treat Strongyloides stercoralis infection vary widely. Because diagnostic methods are insensitive, therapeutic trials generally require multiple negative posttreatment stool specimens as evidence of drug efficacy. However, only a single positive stool specimen is usually required for study enrollment. To determine the reproducibility of detection of S. stercoralis larvae in the stool, 108 asymptomatic infected men submitted 25 g of fresh stool once a week for eight consecutive weeks for examination by the Baermann technique. During the 8-week study, 239 (27.7%) of 864 stool specimens were positive for S. stercoralis. Rates of detection of larvae in the stool specimens ranged from eight of eight specimens in 3 (2.8%) men to none of eight specimens in 36 (33.3%) men. Of 43 men for whom S. stercoralis was detected in at least two of the first four stool specimens, only 1 (2.3%) man tested negative on all of the next four specimens. In comparison, of 29 men who had detectable larvae in only one of the first four specimens, 22 (75.9%) tested negative on all of the next four samples. Thus, if these 29 men had been enrolled in a therapeutic trial between the first and second sets of four specimens, the efficacy of a drug with no activity against this parasite would have been estimated to be 76%. These data suggest that patterns of S. stercoralis detection vary widely among infected persons and that intermittent larval shedding can lead to inflated estimates of drug efficacy. Before a patient is entered in a clinical trial of drug efficacy, four consecutive stool specimens should be examined for S. stercoralis; only persons with two or more positive specimens should be enrolled.
Survey of enterococcal susceptibility patterns in Belgium
Authors: Vandamme P , Vercauteren E , Lammens C , Pensart N , Ieven M , Pot B , Leclercq R , Goossens H
Published: January 2020

A national surveillance study was performed in order to identify the enterococci causing important infections, to determine their susceptibilities to first-choice agents for treatment, and to characterize the phenotypes and genotypes of the glycopeptide-resistant strains. A total of 472 isolates were collected between 15 January and 15 April 1993. The ability of the API rapid ID 32 STREP gallery to identify enterococci was evaluated. The majority of the Belgian enterococci were identified as E. faecalis (89.4%). E. faecium and other enterococci were present in small percentages only (9.1 and 1.5%, respectively). The API rapid ID 32 STREP system identified 88.6% of the strains with an excellent or very good identification score. For the majority of the strains with uncertain identification scores, the results of a single test only were aberrant. Only 2.3% of the strains remained unidentified. High-level aminoglycoside resistance was widespread in E. faecalis (streptomycin, 50.8%; gentamicin, 8.7%), and the emergence of ciprofloxacin resistance was found to be associated with aminoglycoside resistance. E. faecium is generally more resistant to a wide range of antibiotics, but glycopeptide-resistant strains (1.5%) have not yet become widespread.
Gastric mucormycosis due to Rhizopus oryzae in a renal transplant recipient
Authors: Winkler S , Susani S , Willinger B , Apsner R , Rosenkranz A R , Pötzi R , Berlakovich G A , Pohanka E
Published: January 2020

Gastric mucormycosis is a rare disease with a reported fatal outcome of 98%. Manifestations range from colonization of peptic ulcers to infiltrative disease with vascular invasion and dissemination. In our renal transplant patient a deep gastric ulceration infected with Rhizopus oryzae (class Zygomycetes), which is known to be an agent of mucormycosis, was diagnosed in the early posttransplant period after antirejection therapy. The infection was successfully managed with amphotericin B and omeprazole.
Rapid preparation of bacterial DNA for pulsed-field gel electrophoresis
Authors: Matushek M G , Bonten M J , Hayden M K
Published: January 2020

A disadvantage of genotyping bacterial strains by pulsed-field gel electrophoresis is that the procedure requires up to 6 days to complete. We modified a standard pulsed-field gel electrophoresis method (B.E. Murray, K.V. Singh, J.D. Health, B.R. Sharma, and G.M. Weinstock, J.Clin. Microbiol. 28:2059-2063, 1990) so that it could be completed in less than 3 days. We successfully applied this method to the analysis of a variety of gram-positive and gram-negative bacteria.
Comparison of the MUREX C. albicans, Albicans-Sure, and BactiCard Candida test kits with the germ tube test for presumptive identification of Candida albicans
Authors: Crist A E , Dietz T J , Kampschroer K
Published: January 2020

The MUREX C. albicans (MC)(Murex Diagnostics), Albicans-Sure (AS) (Clinical Standards Laboratories), and BactiCard Candida (BC) (Remel) test kits were compared with the germ tube (GT) test for the rapid, presumptive identification of Candida albicans. All three test kits detect the enzymes L-proline aminopeptidase and beta-galactosaminidase in yeast cells grown on culture media and are based on the principle that C. albicans produces both enzymes whereas other yeasts produce only one or neither of the enzymes. The organisms evaluated were fresh clinical isolates identified by methods routinely used in our laboratory (API 20C system and conventional methods) and included 303 C. albicans isolates, 153 Candida glabrata isolates, 70 Candida tropicalis isolates, 36 Candida parapsilosis isolates, 13 isolates of other Candida spp., 5 Cryptococcus neoformans isolates, and 3 Saccharomyces cerevisiae isolates. The MC, AS, BC, and GT tests detected 299 (98.7%), 300 (99.0%), 301 (99.3%), and 287 (94.7%) C. albicans isolates, respectively. There was one false-positive result with both the MC and BC kits and two false-positive results with the GT test. The enzymatic methods evaluated in this study provide rapid and accurate alternatives to the GT test for the presumptive identification of C. albicans.
Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay
Authors: Chiu C H , Ou J T
Published: January 2020

In order to make a rapid and definite diagnosis of Salmonella enteritis in children, an enrichment broth culture-multiplex PCR combination assay was devised to identify Salmonella serovars directly from fecal samples. Two pairs of oligonucleotide primers were prepared according to the sequences of the chromosomal invA and plasmid spvC genes. PCR with these two primers would produce either one amplicon (from the invA gene) or two amplicons (from the invA and spvC genes), depending on whether or not the Salmonella bacteria contained a virulence plasmid. The fecal sample was diluted 10- to 20-fold into gram-negative enrichment broth and incubated to eliminate inhibitory compounds and also to allow selective enrichment of the bacteria. One or two amplicons were obtained, the expected result if Salmonella bacteria were present. The detection limit of this PCR was about 200 bacteria per reaction mixture. The primers were specific, as no amplification products were obtained with 18 species and 22 isolates of non-Salmonella bacteria tested which could be present in the feces or cause contamination. In contrast, when 23 commonly seen Salmonella serovars (38 isolates) were tested, all were shown to carry the invA gene and seven concomitantly harbored the spvC gene of the virulence plasmid. This assay was applied to the diagnosis of Salmonella enteritis in 57 children who were suffering from mucoid and/or bloody diarrhea. Of the 57 children, 38 were PCR positive and 22 were culture positive. There were two culture-positive samples that were not detected by PCR. Thus, this PCR assay showed an efficiency of 95% (38 of 40), which is much higher than the 60% (24 of 40) by culture alone. Not only is this method more sensitive, rapid, and efficient but it will cause only an incremental increase in the cost of stool processing, since enrichment cultivation of fecal samples from diarrheal patients using gram-negative enrichment broth is a routine practice for identification in many diagnostic microbiology laboratories. This PCR method, therefore, has clinical application.
Salivary antibodies to cytomegalovirus (CMV) glycoprotein B accurately predict CMV infections among preschool children
Authors: Wang J B , Adler S P
Published: January 2020

Among preschool children, we found an association between cytomegalovirus (CMV) infection and salivary immunoglobulin G antibodies to CMV glycoprotein B. All of the 20 infected children had immunoglobulin G to CMV glycoprotein B in their saliva, whereas 38 of 38 uninfected children lacked these antibodies. Testing saliva provides a sensitive and specific alternative to obtaining serum.
Multiplex PCR-based assay for detection of Bordetella pertussis in nasopharyngeal swab specimens
Authors: Wadowsky R M , Michaels R H , Libert T , Kingsley L A , Ehrlich G D
Published: January 2020

A multiplex PCR-based assay was developed for the detection of Bordetella pertussis in nasopharyngeal swab specimens. The assay simultaneously amplified two separate DNA targets (153 and 203 bp) within a B. pertussis repetitive element and a 438-bp target within the beta-actin gene of human DNA (PCR amplification control). PCR products were detected by a sensitive and specific liquid hybridization gel retardation assay. A total of 496 paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture. Although 30 (6%) of the specimens inhibited the amplification of the beta-actin target, in all 29 specimens studied, the inhibition disappeared on repeat testing or was easily overcome with a 1:8 dilution or less of specimen digest. Of the 495 specimen pairs yielding a final evaluable result by the PCR-based assay, 19.0% were positive by the PCR-based assay, whereas 13.9% were positive by culture (P < 0.0001). After resolving the PCR-positive, culture-negative results by testing an additional aliquot from these specimens by the multiplex PCR-based assay, the PCR-based assay had a sensitivity and specificity of 98.9 and 99.7%, respectively, compared with values of 73.4 and 100%, respectively, for culture. In comparison with patients with culture-confirmed pertussis, those with PCR-positive, culture-negative results were older and more likely to have had prolonged cough, immunization with pertussis vaccine, or treatment with erythromycin. This multiplex PCR-based assay is substantially more sensitive than culture and identifies specimens that contain inhibitors of PCR.
Modified technique to recover microsporidian spores in sodium acetate-acetic acid-formalin-fixed fecal samples by light microscopy and correlation with transmission electron microscopy
Authors: Carter P L , MacPherson D W , McKenzie R A
Published: January 2020

Microsporidia are an emerging cause of significant disease, particularly in the immunocompromised host. Until recently, the diagnosis of enteric infections has required invasive sampling, the use of expensive technology, and considerable technological expertise. The purpose of the present study was to examine three modifications to the processing of fecal specimens for light microscopy (LM) examination for microsporidian spores: the use of pretreatment with potassium hydroxide, modified centrifugation conditions, and a modified staining technique. A sodium acetate-acetic acid-formalin-fixed fecal sample containing numerous microsporidian spores confirmed to be positive by transmission electron microscopy (TEM) was used in all studies performed. A simulation of a heavy to lightly infected individual was used. The results of LM were correlated with those of TEM. Duplicate smears were stained with Weber's modified trichrome and Giemsa (GS) stains. The stained slides were randomized and examined blindly by LM at x 625 and x 1,250 magnifications. A portion of the dilutions after centrifugation were fixed for TEM. The Weber modified trichrome stain performance rating was higher than the Giemsa stain rating because of ease of interpretation, and material stained with Weber modified trichrome stain required less examination time at a lower magnification. The number of positive smears and the quantity of spores detected were significantly higher following pretreatment of the sample with KOH. TEM was positive only when numerous spores were present, but the quality of the photomicrographs was superior after pretreatment with KOH. Pretreatment of sodium acetate-acetic acid-formalin-fixed fecal samples with 10% KOH and then a 5-min centrifugation time and staining with Weber modified trichrome stain provide for the excellent recovery of microsporidia in the routine diagnostic parasitology laboratory.
Comparison of rapid testing methods for enzyme production with the germ tube method for presumptive identification of Candida albicans
Authors: Heelan J S , Siliezar D , Coon K
Published: January 2020

The observation of germ tube production as a method for the presumptive identification of Candida albicans has been in use for many years. Methods have recently been developed for detecting the production of the enzymes L-proline aminopeptidase and beta-galactosaminidase by yeast isolates grown in culture. Both enzymes are produced by C. albicans; other yeasts may produce either L-proline aminopeptidase or beta-galactosaminidase but not both enzymes. One hundred thirty-three clinical yeast isolates, including 55 C. albicans, 27 Candida tropicalis, 22 Torulopsis (Candida) glabrata, and 29 other yeast isolates were tested by the germ tube production method and three tests for enzyme production, with the API 20C method used as a "gold standard." All three enzymatic methods evaluated provided more objective and rapid nonmicroscopic alternatives to the germ tube test and may be used to accurately distinguish C. albicans from other yeasts.
Characterization of nonmotile variants of Escherichia coli O157 and other serotypes by using an antiflagellin monoclonal antibody
Authors: Feng P , Fields P I , Swaminathan B , Whittam T S
Published: January 2020

An antiflagellin monoclonal antibody (15D8) was used to detect the presence of flagella in nonmotile variants of several pathogenic Escherichia coli serotypes. Of the 48 isolates examined, 15 reacted with monoclonal antibody 15D8 and were culturally confirmed to be motile. Of the 38 clinical strains designated O157:NM or O157:H-, 7 were antibody reactive and motile and agglutinated with anti-H7 sera.
Use of cleaved amplified polymorphic sequences to distinguish strains of Staphylococcus epidermidis
Authors: Calderwood S B , Baker M A , Carroll P A , Michel J L , Arbeit R D , Ausubel F M
Published: January 2020

We examined the utility of a PCR-based method termed cleaved amplified polymorphic sequences (CAPS) to type 35 well-characterized isolates of Staphylococcus epidermidis. The results were compared with detailed epidemiologic information and typing obtained by using pulsed-field gel electrophoresis (PFGE). To identify CAPS markers for this study, eight pairs of oligonucleotide primers corresponding to five previously sequenced S. epidermidis genes were synthesized and then used to amplify DNA sequences from the S. epidermidis strains by using PCR. Amplified products were reproducibly obtained for seven of eight primer pairs from chromosomal DNA of 33 of the 35 isolates. Seven restriction site polymorphisms were found in five of the amplified products when they were subjected to digestion with a panel of restriction endonucleases. Each fragment-enzyme combination that was polymorphic demonstrated only two alleles in the 33 S. epidermidis isolates analyzed, corresponding to the presence or absence of a single restriction site. Overall, five distinct combinations of alleles were detected and were designated CAPS types A through E. There was a close correlation between the CAPS grouping, the epidemiologic information for the strains, and grouping by PFGE following SmaI digestion of chromosomal DNA. Although PFGE analysis was more discriminatory than typing based on the limited number of CAPS markers used in this study (isolates from the same CAPS group were sometimes distributed into more than one PFGE group), no isolates from the same PFGE group were found in more than one CAPS group. The CAPS procedure was highly reproducible, in contrast to published experience with arbitrarily primed PCR. These preliminary data suggest that CAPS represents a PCR-based technique for strain typing that is highly reproducible, rapid, utilizes widely available technologies, and provides results that are relatively easy to interpret and express.
Differential prevalence of hepatitis C virus subtypes in healthy blood donors, patients on maintenance hemodialysis, and patients with hepatocellular carcinoma in Surabaya, Indonesia
Authors: Soetjipto  , Handajani R , Lusida M I , Darmadi S , Adi P , Soemarto  , Ishido S , Katayama Y , Hotta H
Published: January 2020

Determination of the prevalence of liver disease caused by hepatitis C virus (HCV) of various genotypes helps provide an understanding of the virulences of these genotypes. Differences in the prevalences of these genotypes are known to exist in the various geographical regions of the world. Hence, we performed seroepidemiological and molecular epidemiological analyses of HCV in Surabaya, Indonesia. The prevalences of anti-HCV antibodies were 2.3, 76.3 and 64.7% in healthy blood donors, patients on maintenance hemodialysis, and patients with hepatocellular carcinoma (HCC), respectively. HCV-2a was the most common (52%) among the HCV clones obtained from blood donors; this was followed by HCV-1b (15%), HCV-1a (7%), and HCV-1d (7%), a unique Indonesian subtype. The high prevalence of HCV-2a in blood donors was further supported by serotyping analysis that could discriminate HCV type 2 (HCV-2a and -2b) from HCV type 1 (HCV-1a, -1b, and -1d). HCV-1a, -1b, and -1d were strongly associated with elevated serum alanine aminotransferase (ALT) levels in blood donors, suggesting a possibly more pathogenic feature of those subtypes than HCV-2a. In patients on maintenance hemodialysis, HCV-1a and -1b (each 31%) were among the most common subtypes, and in contrast to the case with blood donors, HCV-1a, -1b, and -1d were found in those with normal ALT as well as those with elevated ALT levels. Impaired immune responses of hemodialyzed patients might be responsible for the apparently decreased hepatocytic injury caused by infection with HCV type 1. In patients with HCC, HCV-1b (57%) was the most common; this was followed by HCV-1d (19%) and HCV-2a (5%). Subtype prevalence was not different between HCC patients with advanced liver cirrhosis and those without advanced cirrhosis.
Rapid method for detection of Coxiella burnetii antibodies using high-density particle agglutination
Authors: Nguyen S V , Otsuka H , Zhang G Q , To H , Yamaguchi T , Fukushi H , Noma A , Hirai K
Published: January 2020

A high-density particle agglutination test, using erythrocyte-sensitizing substance from phase II Coxiella burnetii adsorbed to high-density composite particles, was developed for rapid serodiagnosis of Q fever. The test was compared with the microimmunofluorescence test for sensitivity and specificity by using 3,036 human serum samples collected in Gifu Prefecture, Japan. An excellent agreement was found between the two tests for the acute-phase group and paired serum samples, but some discordant results were observed in the single-sample group. The sensitivity and specificity of the high-density particle agglutination test were both 100% in the former group and 81.6 and 99.9%, respectively, in the latter group. The test is a very promising tool for routine serodiagnosis of Q fever because of its simplicity, sensitivity, and specificity.
Characterization of an SAV organism and proposal of Mycobacterium triplex sp. nov
Authors: Floyd M M , Guthertz L S , Silcox V A , Duffey P S , Jang Y , Desmond E P , Crawford J T , Butler W R
Published: January 2020

Polyphasic taxonomic methods were employed to characterize a new species of slowly growing, nonpigmented mycobacteria. We propose the name Mycobacterium triplex sp. nov. for this new taxon. Conventional identification testing demonstrated a group of similar organisms that were geographically widespread in the United States. Commercially available nucleic-acid probes specific for the Mycobacterium avium complex were unreactive for these strains. High-performance liquid chromatography analysis of the mycolic acids revealed mycolate profiles that closely resembled Mycobacterium simiae. Comparative 16S rRNA sequence data confirmed the phylogenetic relationship of the strains with the slowly growing mycobacteria. Representative-type strains have been deposited in the American Type Culture Collection as strain ATCC 700071 [corrected].
Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction
Authors: Beggs M L , Cave M D , Marlowe C , Cloney L , Duck P , Eisenach K D
Published: January 2020

Cycling probe technology (CPT) is a unique and simple method for the detection of specific target sequences. CPT utilizes a chimeric DNA-RNA-DNA probe providing an RNase H-sensitive scissile linkage when bound to a complementary target sequence. For this study a diagnostic assay based on CPT was developed for the detection of the 36-bp direct repeat (DR) region in Mycobacterium tuberculosis. To determine the feasibility of using the DR for detecting M. tuberculosis by CPT, a wide variety of mycobacteria were tested by Southern blot hybridization with three DR probes to verify their specificity. The entire DR region of Mycobacterium bovis 401 was sequenced, and the data were used to design a PCR assay that would allow us to estimate the number of DRs present in a variety of strains. A CPT assay which uses a probe complementary to the DR region was developed and evaluated with synthetic targets and genomic DNA from mycobacteria. In summary, the 36-bp DR provides an attractive target for detecting M. tuberculosis because the sequence is present in high copy numbers in the genome, is specific for the M. tuberculosis complex, and is found in strains that lack IS6110.
Detection of PCR inhibitors in cervical specimens by using the AMPLICOR Chlamydia trachomatis assay
Authors: Verkooyen R P , Luijendijk A , Huisman W M , Goessens W H , Kluytmans J A , van Rijsoort-Vos J H , Verbrugh H A
Published: January 2020

To determine that susceptibility of AMPLICOR Chlamydia trachomatis PCR to inhibitory factors possibly present in cervical specimens, we obtained cervical specimens from 200 gynecology patients attending our outpatient clinic. The prevalence of C. trachomatis infection was 4.1%, as determined by cell culture. All AMPLICOR specimens were tested in one procedure as described by the manufacturer, and after the specimen was spiked with C. trachomatis, several other pretreatment protocols were used. Complete inhibition of the PCR was observed in 38 (19%) cervical specimens. Heat treatment at 95 degrees C, freeze-thawing, or 10-fold dilution of the samples reduced the initial inhibition to 9, 16, or 9%, respectively. A combination of heat treatment and 10-fold dilution reduced the inhibition to 4% of the samples. A second specimen type (swabs inoculated in 0.2 M sucrose phosphate buffer [2SP]) was also evaluated. A 10-fold dilution of the spiked 2SP specimen resulted in an inhibition rate of 6%, which was comparable to that obtained by centrifugation of the 2SP specimen prior to processing. Furthermore, it was shown that the inhibition was not correlated with blood contamination. Processing the specimens on the day of collection or the day after resulted in a higher inhibition rate than did delayed processing (27.6 versus 15.5%, respectively). An inverse correlation was found between the concentration of C. trachomatis added to the sample and the rate of inhibition observed. The inhibition was partly correlated with the pH of the cervical mucosa. Decreased inhibition was found at pH values of > or = 7.5. The effects of blood, pH, and delay in processing were all evaluated by using the AMPLICOR specimen. We conclude that the susceptibility of AMPLICOR C. trachomatis PCR to inhibiting factors in cervical specimens can be significantly reduced if the pretreatment procedure includes heat treatment or the use of 2SP transport medium. Also, a 10-fold dilution of the clinical specimen followed by heat treatment will largely prevent the inhibition of this PCR.
Long PCR and its application to hepatitis viruses: amplification of hepatitis A, hepatitis B, and hepatitis C virus genomes
Authors: Tellier R , Bukh J , Emerson S U , Miller R H , Purcell R H
Published: January 2020

In this study we amplified virtually the entire genomes of hepatitis A virus (a member of the Picornaviridae family), hepatitis B virus (a member of the Hepadnaviridae family), and hepatitis C virus (a member of the Flaviviridae family) by using the recently described technique of long PCR. In order to do this, we first demonstrated, using the lambda phage, that long PCR can be made highly sensitive and the sensitivity can be further enhanced by nested long PCR. We also showed, using tobacco mosaic virus as a model, that a reverse transcriptase reaction can be linked to a long PCR, enabling the nearly full-length amplification of the genomes of RNA viruses. We then applied these techniques to serial dilutions of titrated stocks of well-characterized strains of hepatitis A, B, and C viruses. We amplified the nearly full-length sequence of each of these viruses from a small number of viral genomes, demonstrating the sensitivity of the process.
O serogroups, biotypes, and eae genes in Escherichia coli strains isolated from diarrheic and healthy rabbits
Authors: Blanco J E , Blanco M , Blanco J , Mora A , Balaguer L , Mouriño M , Juarez A , Jansen W H
Published: January 2020

A total of 305 Escherichia coli strains isolated from diarrheic and healthy rabbits in 10 industrial fattening farms from different areas of Spain were serotyped, biotyped, and tested for the presence of the eae gene and toxin production. The characteristics found in strains isolated from healthy rabbits were generally different from those observed in E. coli strains associated with disease. Thus, strains with the eae gene (74% versus 22%); strains belonging to serogroups O26, O49, O92, O103, and O128 (64% versus 12%); rhamnose-negative strains (51% versus 5%); and rhamnose-negative O103 strains with eae genes present (41% versus 1%) were significantly (P < 0.001 in all cases) more frequently detected in isolates from diarrheic animals than in those from healthy rabbits. Whereas a total of 35 serogroups and 17 biotypes were distinguished, the majority of the strains obtained from diarrheic rabbits belonged to only four serobiotypes, which in order of frequency were O103:B14 (72 strains), O103:B6 (16 strains), O26:B13 (12 strains), and O128:B30 (12 strains). These four serobiotypes accounted for 48% (112 of 231) and 5% (4 of 74) of the E. coli strains isolated from diarrheic and healthy rabbits, respectively. Only six strains were toxigenic (three CNF1+, two CNF2+, and one VT1+). We conclude that enteropathogenic E. coli strains that possess the eae gene are a common cause of diarrhea in Spanish rabbit farms and that the rhamnose-negative highly pathogenic strains of serotype O103:K-:H2 and biotype B14 are especially predominant. Detection of the eae gene is a useful method for the identification of enteropathogenic E. coli strains from rabbits. However, a combination of serogrouping and biotyping may be sufficient to accurately identify the highly pathogenic strains for rabbits.
RNA amplification by nucleic acid sequence-based amplification with an internal standard enables reliable detection of Chlamydia trachomatis in cervical scrapings and urine samples
Authors: Morré S A , Sillekens P , Jacobs M V , van Aarle P , de Blok S , van Gemen B , Walboomers J M , Meijer C J , van den Brule A J
Published: January 2020

In the present study, the suitability of RNA amplification by nucleic acid sequence-based amplification (NASBA) for the detection of Chlamydia trachomatis infection was investigated. When comparing different primer sets for their sensitivities in NASBA, use of both the plasmid and omp1 targets resulted in a detection limit of 1 inclusion-forming unit (IFU), while the 16S rRNA appeared to be the most sensitive RNA target for amplification (10(-3) IFU). In contrast, for DNA amplification by PCR, the plasmid target was optimal (10(-2) IFU), which is 10 times less sensitive than rRNA NASBA. To exclude false negativity in NASBA detection because of inhibition of amplification and/or inefficient sample preparation, an internal standard was developed. The internal control was added prior to sample preparation. This 16S rRNA NASBA with an internal control was compared with a plasmid DNA PCR by using a group of C. trachomatis-negative (n = 41) and -positive (n = 37) cervical scrapings, as determined by enzyme immunoassay (EIA). In addition, urine samples from the EIA-positive women were tested (n = 17). Both NASBA and PCR assays were able to detect C. trachomatis in all EIA-positive cervical scrapings, the corresponding urine samples, and two samples from the EIA-negative group. The internal NASBA standard was found clearly in all EIA-negative samples. In conclusion, these results indicate that detection of C. trachomatis by RNA amplification by NASBA with an internal standard is a suitable and highly sensitive detection method, with potential use in the diagnosis of urogenital C. trachomatis infections with cervical scrapings as well as urine specimens.
Characterization of cytotoxic, hemolytic Aeromonas caviae clinical isolates and their identification by determining presence of a unique hemolysin gene
Authors: Wang G , Tyler K D , Munro C K , Johnson W M
Published: January 2020

Aeromonas caviae has recently been recognized as an important enteropathogen and its hemolysin is purported to be one of the virulence factors, In this study, a total of 80 clinical isolates of Aeromonas spp. were investigated by PCR with synthetic oligonucleotides targeting a cloned hemolysin-encoding sequence from an A. caviae isolate of clinical origin. Of the 35 clinical A. caviae isolates tested, only 6 contained the target sequence.
Detection of the microsporidian parasite Enterocytozoon bieneusi in specimens from patients with AIDS by PCR
Authors: Velásquez J N , Carnevale S , Guarnera E A , Labbé J H , Chertcoff A , Cabrera M G , Rodríguez M I
Published: January 2020

Microsporidia are protozoa parasites responsible for significant gastrointestinal disease in patients infected with human immunodeficiency virus. We evaluated a PCR assay of stool samples, duodenal aspirates, and biopsy specimens from patients with Enterocytozoon bieneusi infection. A 210-bp DNA fragment of the unique rRNA intergenic spacer could be amplified from all samples infected with E. bieneusi, but no amplification was seen by using DNA purified from samples with Septata intestinalis or other parasites and from negative control human cells. These results suggest that the PCR in stool samples may be a useful tool for the diagnosis of intestinal microsporidiosis in patients with AIDS.
Synthetic peptide-based enzyme-linked immunosorbent assay for serodiagnosis of visceral leishmaniasis
Authors: Fargeas C , Hommel M , Maingon R , Dourado C , Monsigny M , Mayer R
Published: January 2020

Synthetic peptides, derived from the amino acid sequence of a Leishmania donovani clone, were used to develop an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against L. donovani. For this purpose, five peptides were conjugated to a protein carrier, human serum albumin (HSA), by using a heterobifunctional reagent, epsilon-maleimidocaproic acid N-hydroxysuccinimide ester, to obtain a well-defined product. The sensitivity and the specificity of the peptide-specific ELISA were determined with a panel of 106 serum samples from individuals living in areas where visceral leishmaniasis is endemic; sera from post-kala azar dermal leishmaniasis-infected patients and from individuals suffering from other infectious diseases were also included. ELISAs were performed with either a single peptide-HSA conjugate or a mixture of two peptide-HSA conjugates. Ninety-seven percent of the serum samples from patients with visceral leishmaniasis had detectable antibodies to one or more of the single synthetic peptides. ELISA with a single peptide-HSA conjugate proved to be less sensitive (less than 71%) but more specific (up to 93%) than ELISA with crude promastigote antigens (80% sensitivity and 79% specificity); when a combination of two different peptide-HSA conjugates was used, the test increased both in sensitivity and in specificity. Chemically defined peptide-protein conjugates improve the reproducibility and reliability of ELISA for the serodiagnosis of L. donovani infection.
Comparison of a modified DNA hybridization assay with standard culture enrichment for detecting group B streptococci in obstetric patients
Authors: Kircher S M , Meyer M P , Jordan J A
Published: January 2020

Infection with group B streptococcus (GBS) results in 12,000 to 15,000 cases of neonatal sepsis annually in the United States. GBS is transmitted vertically in up to 70% of infants born to colonized women. Early-onset GBS disease (septicemia, pneumonia, or meningitis occurring within 7 days of life) has a mortality rate of up to 50%, with permanent neurologic sequelae occurring in 15 to 50% of infants surviving meningeal infection. Because of the fulminant nature of neonatal infection, it would be useful to have a rapid assay for determining the GBS status of laboring women. This study illustrated how a commercially available DNA probe-based test was modified to achieve this goal. Modifications included the use of mixed cultures rather than pure isolates for detecting GBS, along with a shorter culture enrichment time and a sample concentration step. To this end, vaginal and rectal swabs from 402 pregnant women during their third trimester were cocultured and tested for GBS rRNA. The 8-h enrichment protocol resulted in an assay with a sensitivity of 95% and specificity of 98%, while the 3-h enrichment protocol revealed a sensitivity of 73% and specificity of 99%. In summary, GBS was detected in the majority of colonized women in less than 4 h. This study illustrated the usefulness of the approach in identifying the most heavily colonized women, who are at the highest risk of transmitting GBS to their neonates. The modified test would have a significant impact on both the medical management and antibiotic therapy for these women and their newborns.
Equine granulocytic ehrlichiosis in Connecticut caused by an agent resembling the human granulocytotropic ehrlichia
Authors: Madigan J E , Barlough J E , Dumler J S , Schankman N S , DeRock E
Published: January 2020

The first recognized cases of equine granulocytic ehrlichiosis in New England are described. The DNA sequence of the 16S rRNA gene of the causative ehrlichia was found to be identical to that of the human granulocytotropic ehrlichia, the agent of human granulocytic ehrlichiosis.
Direct identification and recognition of yeast species from clinical material by using albicans ID and CHROMagar Candida plates
Authors: Baumgartner C , Freydiere A M , Gille Y
Published: January 2020

Two chromogenic media, Albicans ID and CHROMagar Candida agar plates, were compared with a reference medium, Sabouraud-chloramphenicol agar, and standard methods for the identification of yeast species. This study involved 951 clinical specimens. The detection rates for the two chromogenic media for polymicrobial specimens were 20% higher than that for the Sabouraud-chloramphenicol agar plates. The rates of identification of Candida albicans for Albicans ID and CHROMagar Candida agar plates were, respectively, 37.0 and 6.0% after 24 h of incubation and 93.6 and 92.2% after 72 h of incubation, with specificities of 99.8 and 100%. Furthermore, CHROMagar Candida plates identified 13 of 14 Candida tropicalis and 9 of 12 Candida krusei strains after 48 h of incubation.