bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorescent Whole-Cell Hybridization with 16S rRNA-Targeted Oligonucleotide Probes To Identify
Brucella
spp. by Flow Cytometry
Authors: Fernández-Lago Luis , Vallejo F. Javier , Trujillano Ignacio , Vizcaíno Nieves
Published: December 2019

A whole-cell hybridization assay with fluorescent oligonucleotide probes derived from the 16S rRNA sequence of
Brucella abortus
in combination with flow cytometry has been developed. With the three fluorescent probes selected, a positive signal was observed with all the representative strains of the species and biovars of
Brucella
and with a total of nine different
Brucella
clinical isolates. Using the B9 probe in the hybridization assay, it was possible to discriminate between
Brucella suis
biovars 2, 3, 4, and 5 and almost all the other
Brucella
spp. On the basis of differences in fluorescence intensities, no discrimination was established between
Brucella
spp. and other phylogenetically related microorganisms. No positive fluorescence signals were detected with any of the bacteria showing serological cross-reactions with
Brucella
spp. and with a total of 17 clinical isolates not belonging to the genus
Brucella
. These results suggest that the 16S rRNA whole-cell hybridization technique could be a valuable diagnostic tool for the detection and identification of
Brucella
spp.
Bacteriological and Serological Findings in a Further Case of Transfusion-MediatedYersinia enterocoliticaSepsis
Authors: Strobel E. , Heesemann J. , Mayer G. , Peters J. , Müller-Weihrich S. , Emmerling P.
Published: December 2019

A 13-year-old patient developed severe shock due to administration of aYersinia enterocolitica-contaminated red blood cell concentrate.Y. enterocolitica(serotype O:9, biotype II) was cultivated from the residual blood in the blood bag and from a stool sample of the blood donor. In the donor's plasma immunoglobulin M (IgM), IgA, and IgG antibodies againstYersiniaouter proteins (YopM, -H, -D, and -E) were found. Since the donor remembered a short-lasting, mild diarrhea 14 days prior to blood donation, a transient attack ofYersiniaenteritis may be associated with a longer than expected period of asymptomatic bacteremia that causes contamination of donor blood. Serological screening for IgM antibodies againstYersiniaouter proteins might offer a way to reduce the risk of transfusion-associatedY. enterocoliticasepsis.
Comparison and Application of a Novel Genotyping Method, Semiautomated Primer-Specific and Mispair Extension Analysis, and Four Other Genotyping Assays for Detection of Hepatitis C Virus Mixed-Genotype Infections
Authors: Hu Yu-Wen , Balaskas Evan , Furione Milena , Yen Pei-Hua , Kessler Garry , Scalia Vito , Chui Linda , Sher Graham
Published: December 2019

To date the true prevalence of hepatitis C virus (HCV) mixed-genotype infections has not been established mainly because currently available methods are not suitable for the detection of mixed genotypes in a viral population. A novel semiautomated genotyping method, primer-specific and mispair extension analysis (S-PSMEA), which is more reliable than other genotyping assays was developed for detection of HCV mixed-genotype infections. A genotype present at levels as low as 0.8% in a defined mix of HCV genotypes was detected, showing a 20-fold increase in sensitivity over that of direct DNA sequencing. A total of 434 HCV isolates were genotyped and analyzed for a comparative study of the accuracy between S-PSMEA and four current genotyping methods. The results showed that viruses in approximately 40% of the samples from this group determined to be infected with mixed genotypes by S-PSMEA were undetected by direct DNA sequencing due to its low sensitivity. Type-specific PCR, line probe assay, and restriction fragment length polymorphism analysis performed poorly, being able to identify only 38.5, 16.1, and 15.4% of mixed-genotype infections, respectively, that were detected by direct DNA sequencing. The prevalence of mixed-genotype infections detected by S-PSMEA was 7.9% (12 of 152 donors) among HCV-infected blood donors, 14.3% (15 of 105) among patients with chronic hepatitis C, and 17.1% (6 of 36) among thalassemia patients who had received multiple transfusions. The data lead us to conclude that HCV mixed-genotype infections are more common than previously estimated and that S-PSMEA may be the method of choice when detection of genotypes present at low levels in mixed-genotype infections is required due to its higher level of sensitivity.
Proteases as Markers for Differentiation of Pathogenic and Nonpathogenic Species of
Acanthamoeba
Authors: Khan Naveed A. , Jarroll Edward L. , Panjwani Noorjahan , Cao Zhiyi , Paget Timothy A.
Published: December 2019

Acanthamoeba
keratitis is a vision-threatening infection caused by pathogenic species of the genus
Acanthamoeba
. Although not all
Acanthamoeba
spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenic
Acanthamoeba
. Colorimetric assays, using azo-linked protein as a substrate, showed extracellular protease activity in
Acanthamoeba
-conditioned medium and differentiated pathogenic and nonpathogenic
Acanthamoeba
. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic
Acanthamoeba
isolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed with
Acanthamoeba
-conditioned medium as well as with
Acanthamoeba
- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of
Acanthamoeba
. In pathogenic
Acanthamoeba
isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenic
Acanthamoeba
strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic
Acanthamoeba
strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5.
Relative Abundance of Oligosaccharides in
Candida
Species as Determined by Fluorophore-Assisted Carbohydrate Electrophoresis
Authors: Goins Tresa L. , Cutler Jim E.
Published: December 2019

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straightforward, sensitive method for determining the presence and relative abundance of individual oligomannosyl residues in
Candida
mannoprotein, the major antigenic determinant located on the outer surface of the yeast cell wall. The single terminal aldehydes of oligomannosyl residues released by hydrolysis were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis. ANTS fluorescence labeling was not biased by oligomannoside length; therefore, band fluorescence intensity was directly related to the relative abundance of individual oligomannoside moieties in heterogeneous samples. FACE analysis revealed the major oligomannosides released by acid hydrolysis and β-elimination of Fehling-precipitated mannan from
Candida albicans
, which were the same as those previously reported in studies based on mass and nuclear magnetic spectroscopic analysis. FACE was also amenable to the analysis of samples obtained by direct hydrolysis of whole yeast cells. Whole-cell acid hydrolysis and whole-cell β-elimination of two isolates each of
C. albicans
,
C. glabrata
,
C. krusei
,
C. lusitaniae
,
C. parapsilosis
,
C. rugosa
,
C. stellatoidea
, and
C. tropicalis
resulted in oligomannoside gel banding patterns that were species and strain specific for the 16 isolates surveyed. Whereas some bands were specific for an individual isolate or species, other bands were shared by two or three species in various groupings. Differences in the mannoprotein composition of
C. albicans
A9 and four spontaneous cell surface mutants were also detected. Mannan “fingerprints,” or banding pattern profiles, derived from the electrophoretic mobilities of individual bands relative to the migration of acid-hydrolyzed dextran (relative migration index) yielded profiles characteristic of individual isolates not revealed by standard assimilation and biochemical profiles. FACE represents an accessible, sensitive, and quantitative analytical tool enabling the characterization of yeast mannan complexity.
Nosocomial Transmission of Echovirus 30: Molecular Evidence by Phylogenetic Analysis of the VP1 Encoding Sequence
Authors: Bailly Jean-Luc , Béguet Aline , Chambon Martine , Henquell Cécile , Peigue-Lafeuille Hélène
Published: December 2019

We investigated six cases of enterovirus infection in a neonatal unit. The index patient, a 5-day-old boy, was admitted with aseptic meningitis due to echovirus 30 (E30). Secondary infections with E30 occurred in five babies. Comparison of the complete VP1 sequences showed that the isolates recovered from the index patient and his mother were closely related to those recovered from the five babies with secondary infections, demonstrating a nosocomial transmission of the virus. In the phylogenetic tree reconstructed from the VP1 sequences, the isolates formed a monophyletic cluster related to an E30 strain collected in June 1997 during an outbreak of aseptic meningitis.
Estimation of Minimum Sterol 14α-Demethylation-Inhibitory Concentration of Azoles in
Candida
Yeasts Using Acetate-Mediated Growth Inhibition: Potential Utility in Susceptibility Testing
Authors: Shimokawa Osamu , Nakayama Hiroaki
Published: December 2019

We have recently shown that 14α-demethylation-deficient cells of
Candida albicans
are subject to growth arrest by 0.24 M acetate in a yeast extract-peptone-glucose medium and that the minimum concentration of an azole antifungal agent required for total inhibition of sterol 14α-demethylation (MDIC for minimum demethylation-inhibitory concentration) is practically identical to its MIC determined in the acetate-supplemented medium (O. Shimokawa and H. Nakayama, Antimicrob. Agents Chemother. 43:100–105, 1999). In the present study we estimated the MDICs of three different azoles (fluconazole, ketoconazole, and itraconazole) for strains of various
Candida
species using this method and compared them with the MICs determined in the corresponding acetate-free medium. The results demonstrated that the test strains were divided into two classes. One class of strains was characterized by tolerance to 14α-demethylation deficiency (MIC > MDIC) and consisted of strains of
C. albicans
,
C. guilliermondii
,
C. kefyr
, and
C. tropicalis
. The other class was intolerant to 14α-demethylation deficiency (MIC ≈ MDIC) and comprised strains of
C. glabrata
,
C. krusei
, and
C. parapsilosis
. We also showed that replacement of the yeast extract-peptone-glucose medium with RPMI 1640 medium did not affect the results substantially. Furthermore, the 80% inhibitory concentration (IC
80
) in RPMI 1640 medium, recommended as a substitute for the conventional MIC in susceptibility testing, was found to be close to the MDIC.
Development of a Quantitative Real-Time Detection Assay for Hepatitis B Virus DNA and Comparison with Two Commercial Assays
Authors: Pas Suzan D. , Fries Edwin , De Man Robert A. , Osterhaus Albert D. M. E. , Niesters Hubert G. M.
Published: December 2019

A highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B virus (HBV) DNA and compared with two commercially available assays. The assay was validated with the Viral Quality Control panel, which also includes EUROHEP HBV DNA standards. This real-time PCR detection system had a dynamic range of 373 to 10
10
genome copies per ml and showed an excellent correlation with both the commercial HBV Digene Hybrid Capture II microplate assay (Digene Diagnostics) and the HBV MONITOR assay (Roche Diagnostics). To demonstrate its clinical utility, four chronically HBV-infected patients treated with lamuvidine were monitored using the three different assays. From the results we concluded that this assay is an excellent alternative for monitoring of HBV-infected patients in routine diagnostics and clinical practice, enabling the analysis of a large dynamic range of HBV DNA in a single, undiluted sample.
Characterization of
Bartonella clarridgeiae
Flagellin (FlaA) and Detection of Antiflagellin Antibodies in Patients with Lymphadenopathy
Authors: Sander Anna , Zagrosek Anja , Bredt Wolfgang , Schiltz Emile , Piémont Yves , Lanz Christa , Dehio Christoph
Published: December 2019

Cat scratch disease (CSD) is a frequent clinical outcome of
Bartonella henselae
infection in humans. Recently, two case reports indicated
Bartonella clarridgeiae
as an additional causative agent of CSD. Both pathogens have been isolated from domestic cats, which are considered to be their natural reservoir.
B. clarridgeiae
and
B. henselae
can be distinguished phenotypically by the presence or absence of flagella, respectively. Separation of the protein content of purified flagella of
B. clarridgeiae
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis indicated that the flagellar filament is mainly composed of a polypeptide with a mass of 41 kDa. N-terminal sequencing of 20 amino acids of this protein revealed a perfect match to the N-terminal sequence of flagellin (FlaA) as deduced from the sequence of the
flaA
gene cloned from
B. clarridgeiae
. The flagellin of
B. clarridgeiae
is closely related to flagellins of
Bartonella bacilliformis
and several
Bartonella
-related bacteria. Since flagellar proteins are often immunodominant antigens, we investigated whether antibodies specific for the FlaA protein of
B. clarridgeiae
are found in patients with CSD or lymphadenopathy. Immunoblotting with 724 sera of patients suffering from lymphadenopathy and 100 healthy controls indicated specific FlaA antibodies in 3.9% of the patients' sera but in none of the controls.
B. clarridgeiae
FlaA is thus antigenic and expressed in vivo, providing a valuable tool for serological testing. Our results further indicate that
B. clarridgeiae
might be a possible etiologic agent of CSD or lymphadenopathy. However, it remains to be clarified whether antibodies to the FlaA protein of
B. clarridgeiae
are a useful indicator of acute infection.
Urinary Hydatid Antigen Detection by Coagglutination, a Cost-Effective and Rapid Test for Diagnosis of Cystic Echinococcosis in a Rural or Field Setting
Authors: Ravinder P. T. , Parija S. C. , Rao K. S. V. K. Subba
Published: December 2019

We describe here coagglutination (Co-A), a rapid slide agglutination test for the detection of hydatid antigen in the urine for the diagnosis of cystic echinococcosis (CE). Paired urine and serum samples were collected from 16 patients with surgically confirmed CE, 10 patients with ultrasound-proven CE, 14 patients with clinically diagnosed CE, 24 patients with various parasitic diseases other than CE, and 25 healthy control subjects. Co-A detected excreted hydatid antigen in the concentrated urine of 7 of 16 (43.75%) surgically confirmed cases, 6 of 10 (60%) ultrasound-proven cases, and 8 of 14 (57.14%) clinically diagnosed cases of CE. A false-positive reaction was observed with 12.50% of control urine specimens from patients with parasitic diseases other than CE and 12% of urine samples from healthy controls. The circulating antigen was detected in the serum in 13 of 16 (81.25%) surgically confirmed cases, 6 of 10 (60%) ultrasound-proven cases, and 13 of 14 (92.86%) clinically diagnosed cases of CE. False-positive reactions were observed with three sera (12.5%) from controls with other parasitic diseases. The low sensitivity of Co-A for detection of antigen in the urine of a patient whose serum was positive for the antigen is possibly due to low levels of antigen in the urine. Unlike the collection of blood for serum, which is an invasive procedure and also requires technical expertise and disposable syringes, urine can be collected easily and frequently without causing any inconvenience to the patient. Urine as a clinical specimen alternative to serum would be immensely useful in the diagnosis of CE, particularly in a rural or field setting. In such situations as well as in poorly equipped laboratories, Co-A has the potential to be used as a simple, rapid, and economical slide agglutination test for detection of urinary hydatid antigen in the diagnosis of CE.
Molecular Epidemiology of Metronidazole Resistance in a Population of
Trichomonas vaginalis
Clinical Isolates
Authors: Snipes Lauren J. , Gamard Pascale M. , Narcisi Elizabeth M. , Beard C. Ben , Lehmann Tovi , Secor W. Evan
Published: December 2019

Trichomonas vaginalis
, the causative agent for human trichomoniasis, is a problematic sexually transmitted disease mainly in women, where it may be asymptomatic or cause severe vaginitis and cervicitis. Despite its high prevalence, the genetic variability and drug resistance characteristics of this organism are poorly understood. To address these issues, genetic analyses were performed on 109 clinical isolates using three approaches. First, two internal transcribed spacer (ITS) regions flanking the 5.8S subunit of the ribosomal DNA gene were sequenced. The only variation was a point mutation at nucleotide position 66 of the ITS1 region found in 16 isolates (14.7%). Second, the presence of a 5.5-kb double-stranded RNA
T. vaginalis
virus (TVV) was assessed. TVV was detected in 55 isolates (50%). Finally, a phylogenetic analysis was performed based on random amplified polymorphic DNA data. The resulting phylogeny indicated at least two distinct lineages that correlate with the presence of TVV. A band-sharing index indicating relatedness was created for different groups of isolates. It demonstrated that isolates harboring the virus are significantly more closely related to each other than to the rest of the population, and it indicated a high level of relatedness among isolates with in vitro metronidazole resistance. This finding is consistent with the hypothesis that drug resistance to
T. vaginalis
resulted from a single or very few mutational events. Permutation tests and nonparametric analyses showed associations between metronidazole resistance and phylogeny, the ITS mutation, and TVV presence. These results suggest the existence of genetic markers with clinical implications for
T. vaginalis
infections.
Differentiation of Pathogenic
Bartonella
Species by Infrequent Restriction Site PCR
Authors: Handley Scott A. , Regnery Russell L.
Published: December 2019

Infrequent restriction site PCR (IRS-PCR) is a recently described DNA fingerprinting technique based on selective amplification of restriction endonuclease-cleaved fragments.
Bartonella
isolates associated with human disease and related nonhuman isolates were analyzed by IRS-PCR genomic fingerprinting. Preparation of DNA templates began with double digestion using three different restriction endonuclease combinations. Combinations included the frequently cutting endonuclease
Hha
I in conjunction with an infrequently cutting endonuclease,
Eag
I,
Sma
I, or
Xba
I. Digestion was followed by ligation of oligonucleotide adapters designed with ends complementary to the restriction endonuclease sites. Amplification of fragments flanked with an
Eag
I,
Sma
I, or
Xba
I site in combination with an
Hha
I site produced a series of different-sized amplicons resolvable into patterns by polyacrylamide gel electrophoresis (PAGE). The pattern complexity was varied by the addition of selective nucleotides to the 3′ ends of the
Eag
I-,
Sma
I-, or
Xba
I-specific primers. Amplicons were also generated with fluorescently labeled primers and were subsequently resolved and detected by capillary electrophoresis. Analysis by traditional slab PAGE and capillary electrophoresis provided suitable resolution of patterns produced with the enzyme combinations
Eag
I-
Hha
I and
Sma
I-
Hha
I. However, the combination of
Xba
I-
Hha
I produced too many fragments for sufficient resolution by traditional PAGE, thus requiring the better resolving properties of capillary electrophoresis. Due to the flexibility in modulating the pattern complexity and electrophoresis methods, these techniques allow for a high level of experimental optimization. The results provide evidence of the discriminatory power, ease of use, and flexibility of the IRS-PCR method as it applies to the identification of human-pathogenic
Bartonella
species.
Evaluation of LightCycler PCR for Implementation of Laboratory Diagnosis of Herpes Simplex Virus Infections
Authors: Espy Mark J. , Ross Teri K. , Teo Rosaline , Svien Kathleen A. , Wold Arlo D. , Uhl James R. , Smith Thomas F.
Published: December 2019

Five hundred specimens (288 genital, 192 dermal, and 20 ocular) were extracted by technologists, and the DNA was assayed by LightCycler PCR (DNA polymerase and thymidine kinase [TK] gene targets) and by conventional tube and shell vial cell culture. One hundred fifty-eight confirmed (by cell culture and TK target PCR) positive and LightCycler-positive specimens were detected during the first 30 PCR cycles. LightCycler PCR-positive results for cycles 31 to 45 (39 of 67 [58.2%]) required confirmation by another PCR target (TK). LightCycler PCR is more sensitive (
n
= 197; 23.1%) than cell cultures (
n
= 150) for the routine laboratory detection of herpes simplex virus infections.
Small Subunit Ribosomal DNA Sequence ShowsParacoccidioides brasiliensis Closely Related toBlastomyces dermatitidis
Authors: Bialek Ralf , Ibricevic Aida , Fothergill Annette , Begerow Dominik
Published: December 2019

The similarities of paracoccidioidomycosis and blastomycosis are highly suggestive of a close relation of the two etiological agents. Whereas the agent of the first disease is exclusively endemic in Latin America, the agent of the latter one is endemic in North America and Africa. In symptomatic travelers visiting both areas of endemicity, differentiation of the diseases might be impossible, even though therapy and prognosis for these two diseases differ significantly. In order to identify differences in the 18S rRNA gene (rDNA) for use as molecular diagnostic tools, we sequenced this gene from five isolates of Paracoccidioides brasiliensis and compared them to known sequences of other fungi. Neighbor-joining, maximum parsimony, and maximum likelihood analyses and, finally, the Kishino-Hasegawa test revealed that P. brasiliensis, Blastomyces dermatitidis, and Emmonsia parva are more closely related than Histoplasma capsulatum and B. dermatitidis, whose teleomorphic forms belong to one genus,Ajellomyces. In accordance with the work of other investigators who have used internal transcribed spacer and large subunit rDNA sequences, our small subunit rDNA data show that the dimorphic fungus P. brasiliensis must be grouped within the order Onygenales and is closely related to members of the family Onygenaceae. There are hints in the molecular phylogenetic analysis that the family Onygenaceae might be further divided into two families. The subgroup that includes P. brasiliensis comprises all zoopathogenic species. The differences in the 18S rDNAs appear to be too small to allow species identification of the members of the family Onygenaceae pathogenic for humans by use of target sequences within this gene.
Differentiation of Clinical Mycobacterium tuberculosis Complex Isolates by gyrB DNA Sequence Polymorphism Analysis
Authors: Niemann Stefan , Harmsen Dag , Rüsch-Gerdes Sabine , Richter Elvira
Published: December 2019

The discriminatory power of gyrB DNA sequence polymorphisms for differentiation of the species of theMycobacterium tuberculosis complex (MTBC) was evaluated by sequencing and restriction fragment length polymorphism (RFLP) analysis of a 1,020-bp fragment amplified from clinical isolates of M. tuberculosis, Mycobacterium bovis (pyrazinamide [PZA] resistant as well as PZA susceptible), Mycobacterium africanum subtypes I and II, and Mycobacterium microti types vole and llama. We found sequence polymorphisms in four regions described previously and at one additional position. These differences in the gyrB sequences allow an accurate discrimination of M. bovis, M. microti, andM. africanum subtype I. The PZA-susceptible subtypes ofM. bovis shared the M. bovis-specific substitution at position 756 with the PZA-resistant strains, but can be unambiguously differentiated by a characteristic substitution at position 1311. As a drawback, M. tuberculosis and M. africanum subtype II showed an identical gyrBsequence that facilitates discrimination from the other species, but not from each other. A PCR-RFLP technique applying three restriction enzymes could be shown to be a rapid and easy-to-perform tool for the differentiation of the members of the MTBC. Based on these results, we present a clear diagnostic algorithm for the differentiation of species of the MTBC.
Molecular Epidemiology of Mycobacterium avium subsp.paratuberculosis: IS900 Restriction Fragment Length Polymorphism and IS1311 Polymorphism Analyses of Isolates from Animals and a Human in Australia
Authors: Whittington R. J. , Hope A. F. , Marshall D. J. , Taragel C. A. , Marsh I.
Published: December 2019

The distribution and prevalence of strains of Mycobacterium avium subsp. paratuberculosis were determined among sheep, cattle, and other species with Johne's disease in Australia. A total of 328 isolates were evaluated from numerous farms in New South Wales, Victoria, Tasmania, and South Australia, Australia. Restriction fragment length polymorphism (RFLP) analysis of genomic DNA usingBstEII and an IS900 probe and IS1311 polymorphism analysis using PCR and restriction endonuclease analysis (PCR-REA) was used to classify isolates as cattle (C) or sheep (S) strains. IS1311 PCR-REA provided similar information to IS900 RFLP analysis but was more useful than RFLP analysis where DNA was degraded or scarce. Twelve IS900 RFLP types were found. Johne's disease in sheep was always due to S strains, while cattle were infected only with C strains. RFLP type S1 was the dominant strain in sheep in New South Wales (97% of isolates) and was the only strain found in sheep from Victoria. Seven RFLP types were present in cattle. RFLP types C3 and C1 were most common (collectively, 85% of isolates), but C1 was not found in New South Wales and C3 was present in dairy cattle but not in beef cattle in Victoria. These differences may be explained by restricted livestock trading patterns between different segments of the cattle industry. Up to five RFLP types were present in some geographic regions in Victoria, while up to three RFLP types were found among cattle on some farms. Individual cattle usually were infected with only one RFLP type, but one animal was infected with both C5 and CU4. Two isolates from goats were C type as were three from alpacas, one from a rhinoceros, and two from a human with Crohn's disease. The prevalences of specific RFLP types in Australia differ from those reported in Europe and elsewhere. Given the existence of geographical and farm enterprise differences in IS900 RFLP type, this technique may be applied selectively to trace the spread of Johne's disease, at least in the cattle industries. As these observations reflect past exposure of livestock to M. avium subsp.paratuberculosis, the monitoring of strains present in animals in Australia is continuing.
Preclinical Diagnosis of Scrapie by Immunohistochemistry of Third Eyelid Lymphoid Tissue
Authors: O'Rourke K. I. , Baszler T. V. , Besser T. E. , Miller J. M. , Cutlip R. C. , Wells G. A. H. , Ryder S. J. , Parish S. M. , Hamir A. N. , Cockett N. E. , Jenny A. , Knowles D. P.
Published: December 2019

Ovine scrapie is a member of the transmissible spongiform encephalopathies (TSEs), a heterogeneous family of fatal neurologic disorders characterized by deposition of an abnormal isoform (prion protein [PrP] PrP-Sc) of a cellular sialoglycoprotein in neural tissue. PrP-Sc is detectable in some lymphoid tissues of infected sheep months or years before development of clinical disease. Detection of PrP-Sc in these tissues is the basis for live-animal testing. In this study, we characterize the performance of a preclinical diagnostic test for ovine scrapie based on a monoclonal antibody (MAb)-based immunohistochemistry assay of nictitating membrane (“third eyelid”)-associated lymphoid tissue. The results of third eyelid immunohistochemistry assay agreed with the scrapie status of the sheep for 41 of 42 clinical suspects with confirmed scrapie and 174 of 175 sheep without scrapie. Third eyelid sampling agreed with the scrapie status for 36 of 41 clinically normal sheep positive for PrP-Sc immunostaining of brain tissue, including 27 sheep with positive biopsy specimens that progressed to clinical disease with confirmed scrapie 3 to 20 months after biopsy. The assay used MAb F89/160.1.5, which binds to residues 142 to 145 of ovine PrP. This antibody can be used in combination with MAb F99/97.6.1, which binds to residues 220 to 225. One or both MAbs in this cocktail recognize PrP sequences conserved in most mammalian species in which natural TSEs have been reported. Immunohistochemistry assay of routinely formalin-fixed lymphoid tissues with a cocktail of pan-specific MAbs is a practical, readily standardized live-animal and preclinical test for ovine scrapie.
Comprehensive PCR-Based Assay for Detection and Species Identification of Human Herpesviruses
Authors: Johnson Grant , Nelson Susan , Petric Martin , Tellier Raymond
Published: December 2019

The description and evaluation of a PCR-based assay for the detection and species identification of the eight known human herpesviruses are presented. Two primer pairs targeting well-conserved regions of the genome allowed the amplification of the DNAs of all known human herpesviruses at a high level of sensitivity (10 to 100 genome copies for most viruses). Identification of the virus species was achieved through restriction enzyme digestion withBamHI and BstUI, which yielded fragment sizes that were characteristic for each herpesvirus. Furthermore, it was demonstrated that this restriction enzyme panel allowed the discrimination between human herpesvirus 6 variant A and variant B. This assay format was validated over the course of 1 year in a clinical virology laboratory setting, where it was shown that it readily detected human herpesviruses, including occasional multiple infections, in a variety of clinical samples. The PCR assay was compared to isolation and electron microscopy for the detection of herpes simplex (HSV) and varicella-zoster virus (VZV) in clinical samples. All specimens positive by conventional methods were also positive by PCR. However, in a number of clinical specimens in which HSV or VZV could not be detected by conventional methods, PCR was able to demonstrate the presence of the virus.
Molecular and Immunological Characterization ofMycobacterium tuberculosis CFP-10, an Immunodiagnostic Antigen Missing in Mycobacterium bovis BCG
Authors: Dillon Davin C. , Alderson Mark R. , Day Craig H. , Bement Teresa , Campos-Neto Antonio , Skeiky Yasir A. W. , Vedvick Thomas , Badaro Roberto , Reed Steven G. , Houghton Raymond
Published: December 2019

In order to identify antigens that may be used in the serodiagnosis of active tuberculosis (TB), we screened a Mycobacterium tuberculosis genomic expression library with a pool of sera from patients diagnosed with active pulmonary TB. The sera used lacked reactivity with a recombinant form of the M. tuberculosis38-kDa antigen (r38kDa), and the goal was to identify antigens that might complement r38kDa in a serodiagnostic assay. Utilizing this strategy, we identified a gene, previously designated lhp, which encodes a 100-amino-acid protein referred to as culture filtrate protein 10 (CFP-10). The lhp gene is located directly upstream of esat-6, within a region missing in M. bovis BCG. Immunoblot analysis demonstrated that CFP-10 is present in M. tuberculosis CFP, indicating that it is likely a secreted or shed antigen. Purified recombinant CFP-10 (rCFP-10) was shown to be capable of detecting specific antibody in a percentage of TB patients that lack reactivity with r38kDa, most notably in smear-negative cases, where sensitivity was increased from 21% for r38kDa alone to 40% with the inclusion of rCFP-10. In smear-positive patient sera, sensitivity was increased from 49% for r38kDa alone to 58% with the inclusion of rCFP-10. In addition, rCFP-10 was shown to be a potent T-cell antigen, eliciting proliferative responses and gamma interferon production from peripheral blood mononuclear cells in 70% of purified protein derivative-positive individuals without evident disease. The responses to this antigen argue for the inclusion of rCFP-10 in a polyvalent serodiagnostic test for detection of active TB infection. rCFP-10 could also contribute to the development of a recombinant T-cell diagnostic test capable of detecting exposure to M. tuberculosis.
Coronavirus and Pasteurella Infections in Bovine Shipping Fever Pneumonia and Evans' Criteria for Causation
Authors: Storz Johannes , Lin Xiaoqing , Purdy Charles W. , Chouljenko Vladimir N. , Kousoulas Konstantin G. , Enright Frederick M. , Gilmore William C. , Briggs Robert E. , Loan Raymond W.
Published: December 2019

Respiratory tract infections with viruses andPasteurella spp. were determined sequentially among 26 cattle that died during two severe epizootics of shipping fever pneumonia. Nasal swab and serum samples were collected prior to onset of the epizootics, during disease progression, and after death, when necropsies were performed and lung samples were collected. Eighteen normal control cattle also were sampled at the beginning of the epizootics as well as at weekly intervals for 4 weeks. Respiratory bovine coronaviruses (RBCV) were isolated from nasal secretions of 21 and 25 cattle before and after transport. Two and 17 cattle nasally shed Pasteurella spp. before and after transport, respectively. RBCV were isolated at titers of 1 × 103to 1.2 × 107 PFU per g of lung tissue from 18 cattle that died within 7 days of the epizootics, but not from the lungs of the remaining cattle that died on days 9 to 36. Twenty-five of the 26 lung samples were positive for Pasteurella spp., and their CFU ranged between 4.0 × 105 and 2.3 × 109 per g. Acute and subacute exudative, necrotizing lobar pneumonia characterized the lung lesions of these cattle with a majority of pneumonic lung lobes exhibiting fibronecrotic and exudative changes typical of pneumonic pasteurellosis, but other lung lobules had histological changes consisting of bronchiolitis and alveolitis typical of virus-induced changes. These cattle were immunologically naive to both infectious agents at the onset of the epizootics, but those that died after day 7 had rising antibody titers against RBCV andPasteurella haemolytica. In contrast, the 18 clinically normal and RBCV isolation-negative cattle had high hemagglutinin inhibition antibody titers to RBCV from the beginning, while their antibody responses to P. haemolytica antigens were delayed. Evans' criteria for causation were applied to our findings because of the multifactorial nature of shipping fever pneumonia. This analysis identified RBCV as the primary inciting cause in these two epizootics. These viruses were previously not recognized as a causative agent in this complex respiratory tract disease of cattle.
Quantitative Competitive Reverse Transcription-PCR for Quantification of Dengue Virus RNA
Authors: Wang Wei-Kung , Lee Chun-Nan , Kao Chuan-Liang , Lin Yi-Ling , King Chwan-Chuen
Published: December 2019

A quantitative competitive reverse transcription-PCR assay was developed to quantify dengue virus RNA in this study. The main features include a primer pair targeting a highly conserved region in the capsid and the addition of competing RNA that contains an internal deletion to provide a stringent internal control for quantification. It can be utilized to quantify RNA isolated from the four dengue virus serotypes but not RNA isolated from other flaviviruses, including Japanese encephalitis virus and hepatitis C virus, both prevalent in Asia. It can also be used to quantify dengue virus RNA isolated from the plasma of infected individuals. The sensitivity of the assay was estimated to be 10 to 50 copies of RNA per reaction, and twofold differences in virus titer are distinguishable. This assay is a convenient, sensitive, and accurate method for quantification and can be used to further understanding of the pathogenesis of dengue virus infection.
Detection of Giardia lamblia,Entamoeba histolytica/Entamoeba dispar, andCryptosporidium parvum Antigens in Human Fecal Specimens Using the Triage Parasite Panel Enzyme Immunoassay
Authors: Garcia Lynne S. , Shimizu Robyn Y. , Bernard Caroline N.
Published: December 2019

The Triage parasite panel (BIOSITE Diagnostics, San Diego, Calif.) is a new qualitative enzyme immunoassay (EIA) panel for the detection of Giardia lamblia, Entamoeba histolytica/E. dispar, and Cryptosporidium parvum in fresh or fresh, frozen, unfixed human fecal specimens. By using specific antibodies, antigens specific for these organisms are captured and immobilized on a membrane. Panel performance was evaluated with known positive and negative stool specimens (a total of 444 specimens) that were tested by the standard ova and parasite (O&P) examination as the “gold standard,” including staining with both trichrome and modified acid-fast stains. Specimens with discrepant results between the reference and Triage methods were retested by a different method, either EIA or immunofluorescence. A number of samples with discrepant results with the Triage device were confirmed to be true positives. After resolution of discrepant results, the number of positive specimens and the sensitivity and specificity results were as follows: for G. lamblia, 170, 95.9%, and 97.4%, respectively; for E. histolytica/E. dispar, 99, 96.0%, and 99.1%, respectively; and for C. parvum, 60, 98.3%, and 99.7%, respectively. There was no cross-reactivity with other parasites found in stool specimens, including eight different protozoa (128 challenges) and three different helminths (83 challenges). The ability to perform the complete O&P examination should remain an option for those patients with negative parasite panel results but who are still symptomatic.
Automated 5′ Nuclease PCR Assay for Identification of Salmonella enterica
Authors: Hoorfar J. , Ahrens P. , Rådström P.
Published: December 2019

A simple and ready-to-go test based on a 5′ nuclease (TaqMan) PCR technique was developed for identification of presumptiveSalmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica isolates, including 100 problematic “rough” isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonellastrains by resulting in positive end-point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [ΔRn], >0.6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (ΔRn, ≤0.5). All 100 roughSalmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at −20°C. Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method for identification ofSalmonella, particularly in large reference laboratories.
Hepatitis C Virus Core Mutations Reduce the Sensitivity of a Fluorescence Enzyme Immunoassay
Authors: Tokita Hajime , Kaufmann Gilbert R. , Matsubayashi Mamoru , Okuda Isao , Tanaka Tsukasa , Harada Hideharu , Mukaide Motokazu , Suzuki Kazuo , Cooper David A.
Published: December 2019

Four of 107 samples obtained from hepatitis C virus (HCV) carriers showed lower HCV core antigen levels in a fluorescence enzyme immunoassay (FEIA) than expected from corresponding HCV RNA levels. Nucleotide sequencing revealed a mutation in the HCV core region (Thr49Pro) that appears to have reduced the FEIA sensitivity.
Use of Coagulase Gene (coa) Repeat Region Nucleotide Sequences for Typing of Methicillin-Resistant Staphylococcus aureus Strains
Authors: Shopsin B. , Gomez M. , Waddington M. , Riehman M. , Kreiswirth B. N.
Published: December 2019

Coagulase gene (coa) short sequence repeat region sequencing was used to measure relatedness among a collection of temporally and geographically diverse methicillin-resistantStaphylococcus aureus isolates. The results show thatcoa polymorphism is free of strong selective pressure and has a low index of variation that may be useful for long-term epidemiological investigations. coa typing is a useful addition to spa typing for analysis of S. aureus, including methicillin-resistant strains.
Mixed Infection Caused by Two Species ofFusarium in a Human Immunodeficiency Virus-Positive Patient
Authors: Guarro Josep , Nucci Marcio , Akiti Tiyomi , Gené Josepa
Published: December 2019

We report on a case of mixed infection caused by two species ofFusarium in a human immunodeficiency virus-positive patient with lymphoma who was neutropenic due to chemotherapy. The patient showed the typical signs of a disseminated fusarial infection, withFusarium solani isolated from skin lesions and F. verticillioides isolated from blood. The report discusses how difficult it is to make an accurate diagnosis when an immunosuppressed patient is infected with more than one fungal species, especially when the species are morphologically very similar.
Heterogeneity of Influenza B Virus Strains in One Epidemic Season Differentiated by Monoclonal Antibodies and Nucleotide Sequences
Authors: Nakagawa Naoko , Kubota Ritsuko , Maeda Akiko , Nakagawa Toshimasa , Okuno Yoshinobu
Published: December 2019

Seventy-three B/Victoria group strains isolated in the 1996–1997 influenza season were divided into three groups according to the degree of reactivity to monoclonal antibody 8E6. Analysis of nucleotide sequences of the HA1 region clarified that single amino acid substitutions were responsible for the difference in reactivity to 8E6.
Quantitative, Competitive PCR Analysis of Porcine Circovirus DNA in Serum from Pigs with Postweaning Multisystemic Wasting Syndrome
Authors: Liu Qiang , Wang Li , Willson Phil , Babiuk Lorne A.
Published: December 2019

A competitive PCR (cPCR) assay was developed for monitoring porcine circovirus (PCV) DNA in serum samples from piglets. The cPCR was based on competitive coamplification of a 502- or 506-bp region of the PCV type 1 (PCV1) or PCV2 ORF2, respectively, with a known concentration of competitor DNA, which produced a 761- or 765-bp fragment, respectively. The cPCR was validated by quantification of a known amount of PCV wild-type plasmids. We also used this technique to determine PCV genome copy numbers in infected cells. Furthermore, we measured PCV DNA loads in clinical samples. More than 50% of clinically healthy piglets could harbor both types of PCV. While PCV1 was detected in only 3 of 16 pigs with postweaning multisystemic wasting syndrome (PMWS), all the sick piglets contained PCV2. A comparison of the PCV2 DNA loads of healthy and sick animals revealed a significant difference, indicating that the development of PMWS may require a certain amount of PCV2.
Identification of DT104 and U302 Phage Types amongSalmonella enterica Serotype Typhimurium Isolates by PCR
Authors: Pritchett Lori C. , Konkel Michael E. , Gay John M. , Besser Thomas E.
Published: December 2019

A DNA sequence was identified in isolates of Salmonella enterica serotype Typhimurium definitive type 104 (DT104). The PCR amplification of an internal segment of this sequence identified DT104 and the closely related U302 phage type among 146 isolates ofS. enterica serotype Typhimurium tested, thus providing a tool for rapid identification of DT104 and related isolates.
Characterization of Neisseria gonorrhoeae Strains with Decreased Susceptibility to Fluoroquinolones Isolated in Greece from 1996 to 1999
Authors: Mavroidi Angeliki , Tzouvelekis Leonidas S. , Tassios Panayotis T. , Flemetakis Alexandros , Daniilidou Maria , Tzelepi Eva
Published: December 2019

Of the 331 Neisseria gonorrhoeae strains isolated in Greece from 1996 to 1999, 39 (11.8%) exhibited decreased susceptibility to quinolones due to gyrA andparC mutations. Conventional typing and pulsed-field gel electrophoresis showed that 34 of these isolates were clonally related. Epidemiological data indicated that the epidemic clone was sustained in a group of high-frequency transmitters.
Identification of Mycobacterium neoaurumIsolated from a Neutropenic Patient with Catheter-Related Bacteremia by 16S rRNA Sequencing
Authors: Woo Patrick C. Y. , Tsoi Hoi-Wah , Leung Kit-Wah , Lum Peggy N. L. , Leung Andy S. P. , Ma Choi-Ha , Kam Kai-Man , Yuen Kwok-Yung
Published: December 2019

A rapidly growing pigmented mycobacterial strain with an ambiguous biochemical profile was isolated from the blood culture taken through the Hickman catheter of a 9-year-old girl with acute lymphoblastic leukemia. Whole-cell fatty acid analysis showed that the best match profile was that of Mycobacterium aurum, but the similarity index was only 0.217, meaning that there were no good matches between the isolate and the organisms in the database of the Microbial Identification System. The 16S rRNA gene of the mycobacterial strain was amplified, agarose gel purified, and sequenced. There were 44 base differences between the gene sequence of the isolate and that ofM. aurum but only one base difference between the sequence of the isolate and that of Mycobacterium neoaurum, showing that the isolate was indeed a strain of M. neoaurum by using this “gold standard.” This represents the first case ofM. neoaurum infection documented by 16S rRNA sequencing.
A Cytotoxin-Producing Strain of Vibrio choleraeNon-O1, Non-O139 as a Cause of Cholera and Bacteremia after Consumption of Raw Clams
Authors: Namdari Hassan , Klaips Christine R. , Hughes Joan L.
Published: December 2019

We report a case of a cholera-like gastroenteritis subsequent with bacteremia in a healthy man following consumption of raw clams. Although we failed to recover the organism from the patient's stool culture, his blood culture was positive for a non-cholera toxin-producing yet cytotoxin-producing non-O1 and non-O139Vibrio cholerae.