bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of viral DNA to evaluate outcome of antiviral treatment of patients with recurrent genital herpes
Authors: Diaz-Mitoma F , Ruben M , Sacks S , MacPherson P , Caissie G
Published: January 2020

Culture of infectious virus, PCR amplification of viral DNA, and the appearance of genital skin lesions were used as markers to study the course of a recurrence of genital herpes in 40 patients treated with famciclovir or placebo. The highest frequency of patients with skin lesions occurred within the first 36 h following the onset of a recurrence, which also corresponded to the peak in the production of virus. While the timing of the peak in skin lesions was independent of the type of treatment, the frequency of lesions and the release of virus at the lesion site were both reduced by famciclovir treatment. Furthermore, patients receiving this antiviral agent showed a more rapid recovery time and a shorter period during which viral DNA could be detected at the lesion. PCR and then Southern blot hybridization greatly enhanced our ability to detect herpes simplex virus at the lesion site. This procedure proved to be of greater diagnostic value in assessing genital herpes than the standard culture method currently used. In addition, PCR was more sensitive in evaluating treatment effectiveness.
Two distinct human parainfluenza virus type 1 genotypes detected during the 1991 Milwaukee epidemic
Authors: Henrickson K J , Savatski L L
Published: January 2020

The extent of genetic and antigenic variation found in a population of human parainfluenza virus type 1 (HPIV-1) during a single local epidemic was investigated. Fifteen HPIV-1 strains isolated from children in 1991 were analyzed. Nucleotide sequence variation in the hemagglutinin-neuraminidase protein (HN) gene demonstrated two distinct genotypes (genotypes C and D). Unique patterns were identified involving 62 nucleotide and 10 amino acid positions. These patterns represented 40% of all mutations within the HN gene. The remaining mutations were randomly distributed, and 74% involved only one (55%) or two isolates. Genotypes were statistically different from each other at both the nucleotide (P = 0.001) and amino acid (P = 0.001) levels and demonstrated unique potential N-linked glycosylation patterns. Thirty-eight monoclonal antibodies (MAbs) made to four different viral proteins (22 HN, 2 fusion [F], 1 phosphoprotein, and 13 nucleoprotein) (originating from two different genotypes [genotypes A and D]) were compared for their ability to bind to the clinical isolates in enzyme-linked immunosorbent assays (ELISAs) and hemagglutinin-inhibition (HI) assays. Twenty-one MAbs bound well to all clinical isolates in ELISAs and HI assays. The remaining 17 MAbs showed variation in all four structural proteins. Three HN MAbs demonstrated genotype C- and D-specific antigenic and neutralization differences. Evolutionary analysis using parsimony methods confirmed the differences between the two genotypes. No differences in either clinical presentation or disease severity between the two genotypes were found. Geographically localized HPIV-1 epidemics can be caused by at least two distinct genotypes with minor but specific antigenic changes. The clinical and immunologic roles of HPIV-1 genotypes have not been determined.
Immunodetection of Pneumocystis carinii in bronchoalveolar lavage specimens compared with methenamine silver stain
Authors: Lautenschlager I , Lyytikainen O , Jokipii L , Jokipii A , Maiche A , Ruutu T , Tukiainen P , Ruutu P
Published: January 2020

Immunodetection of Pneumocystis carinii, based on immunofluorescence and use of a monoclonal antibody specific for an antigen located within the cyst wall and detectable after trypsin digestion only, was compared with a methenamine silver stain in 553 bronchoalveolar lavage specimens from immunosuppressed patients. P. carinii was found by immunofluorescence in 72 (86%) and by silver stain in 68 (81%) of the total of 84 positive samples detected by either or both of these methods.
Evaluation of mycobacteria growth indicator tube for recovery and drug susceptibility testing of Mycobacterium tuberculosis isolates from respiratory specimens
Authors: Palaci M , Ueki S Y , Sato D N , Da Silva Telles M A , Curcio M , Silva E A
Published: January 2020

The new BBL mycobacteria growth indicator tube (MGIT) was evaluated for its ability to detect mycobacteria directly from patient specimens and to determine the drug susceptibility of Mycobacterium tuberculosis isolates. A total of 85 respiratory specimens were tested. Specimens were digested, concentrated, examined microscopically for acid-fast bacilli, and inoculated into MGITs and onto Lowenstein-Jensen slants by standard procedures. The tubes were incubated at 37 degrees C and were examined daily for fluorescence to 365-nm UV light. All 25 specimens smear positive for acid-fast bacilli were tested for drug susceptibility in MGITs containing 1.0 mu g of rifampin per ml, 0.1 mu g of isoniazid per ml, 2.0 mu g of streptomycin per ml, and 2.0 mu g of ofloxacin per ml. These results were compared with those obtained by testing the same M. tuberculosis isolates by the indirect proportion method at drug concentrations of 4.0 mu g of rifampin per ml, 0.2 mu g of isoniazid per ml, 2.0 mu g of ethambutol per ml. 4.0 mu g of streptomycin per ml, and 2.0 mu g of ofloxacin per ml. No significant difference in the sensitivity of detection of M. tuberculosis isolates was found between the two methods. However, the time to detection was significantly shorter in MGITs. Drug susceptibility test results for M. tuberculosis isolates by the two methods demonstrated an excellent correlation. The mean time to reporting of drug susceptibility results was 5 days for MGITs versus 16 days for Lowenstein-Jensen slants. The results of this preliminary study indicate that the MGIT system appears to have potential for routine use in mycobacteriology for both the detection and the drug susceptibility testing of M. tuberculosis isolates. However, it is important to emphasize that simple nonautomated equipment should be developed to improve the accuracy of fluorescence detection.
Atmospheric growth requirements for Alloiococcus species and related gram-positive cocci
Authors: Miller P H , Facklam R R , Miller J M
Published: January 2020

The growth of Alloiococcus otitis under different atmospheres and nutritional conditions was studied. The growth rates of 25 strains of gram-positive cocci representing five genera on heart infusion agar plates containing 5% rabbit blood and on brucella agar plates with and without sheep blood under aerobic, increased CO2, and anaerobic atmospheres were compared. Eight strains of alloiococci plated on heart infusion agar with rabbit blood and on brucella sheep blood agar grew under aerobic and candle jar atmospheres. Two of these strains showed poor anaerobic growth after 7 days. Strains of Aerococcus viridans, Aerococcus urinae, Helcococci kunzi, Dolosigranulum pigrum, Gemella haemolysans, and Gemella morbillorum grew well under all three atmospheres and on the three types of media and in thioglycolate broth. These results confirm all the aerobic atmospheric requirements for Alloiococcus strains and show that aerobic growth characteristics help distinguish the alloiococci from the other gram-positive cocci that are facultatively anaerobic.
Recombinant antigen-based avidin-biotin microtiter enzyme-linked immunosorbent assay for serodiagnosis of invasive amebiasis
Authors: Shenai B R , Komalam B L , Arvind A S , Krishnaswamy P R , Rao P V
Published: January 2020

An immunoscreening approach was used to isolate a strongly positive cDNA clone from an Entamoeba histolytica HK-9 cDNA expression library in the phage vector lambda ZAP-II. The 1.85-kb cDNA insert was found to be truncated and encoded the cysteine-rich, immunodominant domain of the antigenic 170-kDa subunit of the amebal galactose-N-acetylgalactosamine binding lectin. This domain was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Inclusion bodies of the recombinant protein were solubilized with Sarkosyl, and the protein was enriched from the crude bacterial extract by thiol-affinity chromatography. The recombinant protein was used to develop a rapid, sensitive, and specific avidin-biotin microtiter enzyme-linked immunosorbent assay (ELISA) for invasive amebiasis. Sera from 38 individuals suffering from invasive amebiasis, 12 individuals with noninvasive amebiasis, 44 individuals with other infections, and 27 healthy subjects were screened by the recombinant antigen-based ELISA. The sensitivity and specificity of the assay were 90.4 and 94.3%, respectively, which correlated well with those of an ELISA developed with crude amebal antigen (r = 0.94; P < 0.0001), as well as with those of a commercially available serodiagnostic ELISA (r = 0.92; P < 0.0001). Thus, the bacterially expressed recombinant lectin can replace the crude amebal extract as an antigen in the serodiagnosis of invasive amebiasis by using avidin-biotin microtiter ELISA.
Molecular subtype distribution of Cryptococcus neoformans in four areas of the United States. Cryptococcal Disease Active Surveillance Group
Authors: Brandt M E , Hutwagner L C , Klug L A , Baughman W S , Rimland D , Graviss E A , Hamill R J , Thomas C , Pappas P G , Reingold A L , Pinner R W
Published: January 2020

To improve understanding of the epidemiology of cryptococcal disease, we analyzed the multilocus genotype distribution of 358 Cryptococcus neoformans isolates obtained from 251 patients through active surveillance in four U.S. geographic areas from 1992 through 1994. Isolates of the predominant enzyme electrophoretic type (ET), ET-1, were recovered in significantly greater proportion from Atlanta, Ga., Houston, Tex., and all major metropolitan areas of Alabama than from San Francisco, Calif. ET-2 and ET-7 complex (serotype AD) isolates were recovered predominantly from San Francisco. ET-3 was recovered less frequently from San Francisco than from the three other locations. These findings may reflect geographic differences in exposure to environmental strains or the identification of previously unrecognized C. neoformans clusters. Analysis by random amplified polymorphic DNA-PCR subtyping further divided 67 ET-1 isolates into 19 additional subtypes, none of which could be associated with a particular geographic region. Multiple isolates from the same patient always revealed the same multilocus enzyme electrophoresis and random amplified polymorphic DNA subtypes. No differences in subtype distribution were found when isolates from AIDS patients were compared with those from persons without or with another underlying disease, although one C. noeformans var. gattii isolate was obtained from an AIDS patient. When body site distribution was analyzed, ET-4 was disproportionately recovered from skin or surface body sites. Evidence for linkage disequilibrium in this fungal population suggests that virulent C. neoformans possesses a clonal population structure. Continued application of molecular subtyping methods will be useful in tracking the source, transmission, and relative virulence of different C. neoformans strains.
To culture or not to culture: fecal lactoferrin screening for inflammatory bacterial diarrhea
Authors: Choi S W , Park C H , Silva T M , Zaenker E I , Guerrant R L
Published: January 2020

Because of its low yield in unselected specimens, stool culture is often cost ineffective. We tested 55 fecal samples from Fairfax Hospital (46 patients with diarrhea and 9 from controls without diarrhea) for lactoferrin by latex agglutination (LFLA) with the Leukotest (Techlab, Blacksburg, Va.) as a marker for inflammatory diarrhea. Of the 28 samples with Salmonella, Shigella, or Campylobacter infection, 93% had detectable fecal lactoferrin at > or = 1:50 (61% had LFLA titers of > or = 1:400), while 83% of 18 samples with rotavirus or no detectable pathogen were LFLA negative at a titer of 1:50 (100% were negative at 1:400). All 9 controls without diarrhea were LFLA negative at 1:50. The use of fecal lactoferrin to screen for inflammatory diarrhea selects specimens for which stool culture is fivefold more likely to yield an invasive bacterial pathogen (reducing the cost per positive result by over $800) and thus may greatly enhance a cost-effective approach to evaluating diarrheal illness.
Molecular epidemiology of Escherichia coli O157:H7 by pulsed-field gel electrophoresis and comparison with that by bacteriophage typing
Authors: Krause U , Thomson-Carter F M , Pennington T H
Published: January 2020

One hundred twenty-four Escherichia coli O157:H7 isolates were characterized by pulse-field gel electrophoresis, bacteriophage typing, and PCR of verotoxin genes. Diversity indices obtained--0.786 for phage types and 0.987 for pulsed-field gel electrophoresis types--demonstrated that phage typing falls below the critical value (0.9) required for confident interpretation of results.
Etiological agents of mycobacterioses in pet birds between 1986 and 1995
Authors: Hoop R K , Böttger E C , Pfyffer G E
Published: January 2020

Between May 1986 and June 1995, mycobacteriosis was diagnosed by histology and microscopy in 204 (3.8%) of 5,345 necropsied pet birds. The predominant macroscopic changes were enlargement of the liver and spleen and thickening of intestinal walls. Attempts to cultivate mycobacteria were made in 110 cases. Acid-fact bacilli grew in 66 specimens (60%) only. In 18 cases we failed to obtain subcultures. Therefore, species identification could be performed for only 48 isolates. Identification was carried out by conventional biochemical tests as well as by PCR-mediated sequencing of the 16S rRNA gene. The majority of the isolates were Mycobacterium genavense (34 isolates), followed by M. avium complex (8), M. fortuitum (2), M. tuberculosis (2), M. gordonae (1), and M. nonchromogenicum (1). The significance of M. genavense as a zoonotic agent remains to be determined.
Performance characteristics of recombinant enzyme immunoassay to detect antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 and to measure early antibody responses in seroconverting patients
Authors: Galli R A , Castriciano S , Fearon M , Major C , Choi K W , Mahony J , Chernesky M
Published: January 2020

We investigated the performance of a double-antigen sandwich recombinant enzyme immunoassay (EIA; Abbott Laboratories, North Chicago, Ill.) and compared it with that of a synthetic-peptide-based EIA (Biochem Immunosystems, Montreal, Quebec, Canada) for the detection of human immunodeficiency type 1 (HIV-1) and HIV-2 antibodies in 2,321 clinical serum samples. The results of both EIA methods and Western blot (immunoblot) were in agreement for 1,046 HIV-1 and 10 HIV-2 specimens from a panel of known positives. From a prospective panel of 1,085 specimens, 38 proved to be positive by both EIAs and Western blot, 3 were positive by the recombinant EIA only, and 9 were positive by the peptide EIA only, for calculated specificities of 99.71 and 99.04%, respectively. Of 180 specimens from a seroconversion panel collected from 77 patients, the results for 170 were in agreement by all antibody testing methods and 10 were found to be repeat reactive for HIV antibodies by the recombinant EIA only. All 10 were initial specimens of seroconverting patients; 7 were also reactive for HIV p24 antigen. An examination of four of these sera by radioimmunoprecipitation assay showed gp120 and gp160 bands in each. Analysis of the anti-Env antibody class in three of these samples showed that one consisted of immunoglobulin M (IgM) only and two contained both IgG and IgM antibodies. Although both EIA procedures were sensitive and specific in the detection of antibodies to HIV-1 and HIV-2 and both were capable of detecting early antibodies, the recombinant assay was more sensitive for antibody detection during early seroconversion.
Evaluation of tuberculosis transmission in a community by 1 year of systematic typing of Mycobacterium tuberculosis clinical isolates
Authors: Torrea G , Offredo C , Simonet M , Gicquel B , Berche P , Pierre-Audigier C
Published: January 2020

Interhuman transmission of Mycobacterium tuberculosis was investigated by using molecular typing, including restriction fragment length polymorphism with probes IS6110, DR (direct repeat) and PGRS (polymorphic GC-rich sequence) and a PCR method using the inverted repeat sequences of IS6110 as primers. From 105 patients hospitalized for tuberculosis during a 1-year survey in three hospitals in Paris, France, 111 isolates were collected and analyzed. Eighty-eight patients were infected with genetically different isolates, demonstrating the clonal heterogeneity of M. tuberculosis in these patients originating from various geographical areas. Fourteen patients were infected by strains clustered with identical fingerprints. An epidemiological relatedness was demonstrated for isolates from only seven of these patients. Thus, the typing of isolates from all tuberculous patients in hospitals during 1 year allows the detection of transmission in the general community. This would improve the case findings, thereby further improving the detection of outbreaks.
Serological and genomic characterization of porcine rotaviruses in Thailand: detection of a G10 porcine rotavirus
Authors: Pongsuwanna Y , Taniguchi K , Chiwakul M , Urasawa T , Wakasugi F , Jayavasu C , Urasawa S
Published: January 2020

A total of 557 fecal specimens collected from piglets with diarrhea in Thailand were examined for rotavirus RNA by polyacrylamide gel electrophoresis. Twenty-three, one, and two samples were positive for group A, group B, and group C rotaviruses, respectively. Two samples exhibited two segments found in picobirnavirus RNA. RNA electropherotyping of 23 group A rotaviruses showed that they were classified into five patterns. By serotyping by enzyme-linked immunosorbent assay and PCR, viruses in 3 and 14 specimens were found to be serotype G3 and serotype G10, respectively. For one specimen, containing a serotype G10 virus (strain P343), virus was isolated in MA-104 cells, and the nucleotide sequences of the VP7 and VP4 genes were determined. Comparative sequence analysis and cross-neutralization tests showed that strain P343 has B223-like G10 and UK-like P7 serotype (or VP4 genotype 5) specificities. Rotaviruses having such antigenic specificities have not been detected in piglets. Thus, the interspecies transmission of rotaviruses between cows and pigs was suggested.
Ability of RapID Yeast Plus System to identify 304 clinically significant yeasts within 5 hours
Authors: Kitch T T , Jacobs M R , McGinnis M R , Appelbaum P C
Published: January 2020

The RapID Yeast Plus System (Innovative Diagnostic Systems, Norcross, Ga.) is a qualitative micromethod that uses conventional and chromogenic substrates for the identification of medically important yeasts. The ability of the RapID Yeast Plus system to accurately identify 304 clinical yeast isolates within 5 h was evaluated. The RapID Yeast Plus method correctly identified 286 (94.1%) of strains to the species level without the need for additional tests. A further 12 strains (3.9%) were classified as correct to the genus level or to a low-probability identification with two or more possibilities. In these latter cases, additional tests were required to delineate the correct identification. Organisms in the latter group comprised Candida parapsilosis (n = 1), Candida tropicalis (n = 1), Candida ciferrii (n = 1), Candida guilliermondii (n = 2), Candida humicola (n = 1), Candida kefyr (n = 1), Cryptococcus neoformans (n = 1), and Rhodotorula rubra (n = 4). Six strains (2.0%) were misidentified or did not yield codes in the manufacturer's database. These included one Candida utilis (identified as Candida famata/Candida guilliermondii), one Trichosporon beigelii (identified as Cryptococcus neoformans), one Candida diddensiae (identified as Candida albicans), one Candida membranaefaciens (identified as Candida parapsilosis), one Candida norvegensis (identified as Candida zeylanoides), and one Candida catenulata (no code) isolate; the last four strains are not included in the firm's current database. The RapID Yeast Plus system yielded excellent results and may be recommended for use in the routine laboratory for accurate same-day identification of clinically significant yeasts.
Ribotype diversity of Listeria monocytogenes strains associated with outbreaks of listeriosis in ruminants
Authors: Wiedmann M , Bruce J L , Knorr R , Bodis M , Cole E M , McDowell C I , McDonough P L , Batt C A
Published: January 2020

Ribotyping is a molecular method for the characterization, identification, and typing of bacterial isolates that has value in epidemiological studies. To demonstrate the utility of this technique for typing of Listeria monocytogenes, four outbreaks of epizootic listeriosis in ruminants were investigated through coordinated detection and characterization methods utilizing classical microbiology and nucleic acid-based techniques. L. monocytogenes strains isolated from clinical samples and the silage consumed by the affected animals were ribotyped to establish the causal relationship between feed and the disease outbreak. For all but one outbreak, we were able to isolate L. monocytogenes strains represented by the same ribotype from both clinical and silage samples. Additional L. monocytogenes strains with ribotypes different from those of the respective clinical samples were isolated from all silage samples. This indicates that a diverse population of L. monocytogenes strains exists in farm environments, of which some may be more likely than others to cause disease.
Performance of Meridian ImmunoCard Mycoplasma test in a multicenter clinical trial
Authors: Alexander T S , Gray L D , Kraft J A , Leland D S , Nikaido M T , Willis D H
Published: January 2020

Serology is the principal laboratory method used to diagnose Mycoplasma pneumoniae infection. Meridian Diagnostics has developed the ImmunoCard Mycoplasma kit, a 10-min card-based enzyme-linked immunosorbent assay (ELISA) designed to detect immunoglobulin M (IgM) antibodies to M. pneumoniae. We compared the ImmunoCard with two M. pneumoniae IgM-specific assays (immunofluorescence assay [IFA] and ELISA) and a standard complement fixation (CF) procedure using 896 specimens submitted to clinical laboratories for M. pneumoniae serology. Equivocal results obtained by CF, IFA, or ELISA were resolved by testing with an additional method or by reviewing patient chart information. The ImmunoCard had sensitivities ranging from 74% compared with the ELISA to 96% compared with CF results with IFA. ImmunoCard specificities ranged from 85% compared with the IgM-specific ELISA to 98% compared with IgM-specific IFA results resolved with clinical chart review. We also compared the ImmunoCard results with consensus results of 694 specimens tested on at least two non-ImmunoCard methods because of the lack of a "gold standard" for M. pneumoniae serology. Overall, the ImmunoCard Mycoplasma IgM assay had 90% sensitivity, 93% specificity, and 92% agreement with the consensus results. The ImmunoCard is technically less complex and requires less equipment that the three other assays. Our results indicate that the ImmunoCard Mycoplasma IgM assay is a valid and simple procedure which can reduce technologist time (and, thus, labor cost) and turnaround time for laboratories analyzing small numbers of specimens (< 10 per batch) submitted for IgM anti-M. pneumoniae testing.
Molecular characterization of Vibrio cholerae O1 strains isolated during cholera outbreaks in Guinea-Bissau
Authors: Dalsgaard A , Mortensen H F , Mølbak K , Dias F , Serichantalergs O , Echeverria P
Published: January 2020

In the present study, 19 strains of Vibrio cholerae O1 biotype El Tor isolated during outbreaks of cholera in Guinea-Bissau in 1987, 1994, and 1995 were characterized to investigate a possible epidemiological relationship among the isolates. On the basis of ribotyping with the restriction enzyme BglI, 5 strains isolated in 1987 showed two closely related ribotypes, while 14 strains isolated in 1994 and 1995 showed the same ribotype that was distinct from the ribotypes of strains isolated in 1987. Southern blot hybridization of BglI-digested genomic DNA with a cholera toxin probe demonstrated that the strains isolated in 1987 showed an identical cholera toxin genotype, whereas O1 strains isolated in 1994 and 1995 showed the same genotype that was distinct from the genotype of strains isolated in 1987. These results were supported by the results of antibiotic susceptibility testing, in which strains isolated in 1987 showed resistance to polymyxin B only, while each of the strains from 1994 and 1995 showed resistance to polymyxin B, trimethoprim-sulfamethoxazole, and the vibriostatic agent O/129. Although our results are based on a limited number of V. cholerae O1 strains, they suggest that the epidemic in Guinea-Bissau in 1994 and 1995 was due to the introduction of a new strain to the country.
Sequence capture-PCR improves detection of mycobacterial DNA in clinical specimens
Authors: Mangiapan G , Vokurka M , Schouls L , Cadranel J , Lecossier D , van Embden J , Hance A J
Published: January 2020

The rapid identification of mycobacterial DNA in clinical samples by PCR can be useful in the diagnosis of tuberculous infections, but several large studies have found that the sensitivity of this approach is not better than that of culture. In order to improve the sensitivity of detection of mycobacterial DNA in clinical specimens from patients with paucibacillary forms of tuberculosis, we have developed a procedure permitting the specific capture of mycobacterial DNA in crude samples prior to amplification, thereby concentrating the target sequences and removing irrelevant DNA and other potential inhibitors of the amplification reaction (sequence capture-PCR). By using this approach to capture and amplify two different sequences specific for organisms of the Mycobacterium tuberculosis complex (IS6110 and the direct repeat region), it was possible to detect as little as one genome of mycobacterial DNA in samples containing up to 750 micrograms of total DNA, representing a 10- to 100-fold increase in sensitivity compared with that obtained by purifying total DNA prior to amplification. Detection of the IS6110 sequence in pleural fluid samples from patients with tuberculous pleurisy by sequence capture-PCR gave positive results in 13 of 17 cases, including 3 of 3 culture-positive samples and 10 of 14 culture-negative samples. In contrast, when total DNA was purified from these samples by adsorption to a silica matrix prior to amplification, only the three culture-positive samples were positive by PCR. The sensitivity of detection of the direct repeat sequence in these samples by sequence capture-PCR was similar to that of IS6110 and, in addition, permitted immediate typing of the strains from some patients. We conclude that sequence capture-PCR improves the sensitivity of detection of mycobacterial DNA in paucibacillary samples. This approach should be useful in detecting rare target sequences from organisms implicated in other pathologic processes.
Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie
Authors: van Keulen L J , Schreuder B E , Meloen R H , Mooij-Harkes G , Vromans M E , Langeveld J P
Published: January 2020

The scrapie-associated form of the prion protein (PrPSc) accumulates in the brain and lymphoid tissues of sheep with scrapie. In order to assess whether detecting PrPSc in lymphoid tissue could be used as a diagnostic test for scrapie, we studied the localization and distribution of PrPSc in various lymphoid tissues collected at necropsy from 55 sheep with clinical scrapie. Samples collected from the spleen, palatine tonsil, ileum, and five different lymph nodes were immunohistochemically stained for PrPSc. PrPSc was found to be deposited in a reticular pattern in the center of both primary and secondary lymphoid follicles. In addition, granules of PrPSc were seen in the cytoplasm in macrophages associated with the lymphoid follicles. In 54 (98%) of the 55 scrapie-affected sheep, PrPSc was detected in the spleen, retropharyngeal lymph node, mesenteric lymph node, and the palatine tonsil. However, only in the palatine tonsils was PrPSc present in a consistently high percentage of the lymphoid follicles. PrP was not detected in any of the lymphoid tissues of 12 sheep that had no neurohistopathological signs of a scrapie infection. We conclude that the tonsils are the best-suited lymphoid tissue to be biopsied for the detection of PrPSc in the diagnosis of clinical scrapie in living sheep.
Comparative susceptibilities of human embryonic fibroblasts and HeLa cells for isolation of human rhinoviruses
Authors: Arruda E , Crump C E , Rollins B S , Ohlin A , Hayden F G
Published: January 2020

The recovery of human rhinovirus (HRV) from nasal washings and nasal and pharyngeal swabs from volunteers with naturally acquired colds was compared in different cell types. Human embryonic lung fibroblast (HELF) strain WI-38 (sensitivity, 61 to 84%) and HeLa-I, an HRV-susceptible HeLa clone (sensitivity, 86 to 94%), were the most sensitive cell types used. HELF-WI-38 cells showed a cytopathic effect earlier than the other cells used, and the different strains of HRV-susceptible HeLa cells varied in their sensitivities for HRV isolation. HRV was detected in a single cell type in 20 to 35% of the positive samples, suggesting that use of a combination of different HRV-susceptible cell lines is the best approach for the recovery of HRV. Although nasal washings tended to yield more HRV isolates than nasal and pharyngeal swabs, the two sampling methods were not found to be significantly different.
Multicenter evaluation of broth microdilution method for susceptibility testing of Cryptococcus neoformans against fluconazole
Authors: Sanati H , Messer S A , Pfaller M , Witt M , Larsen R , Espinel-Ingroff A , Ghannoum M
Published: January 2020

We have developed a microdilution method for measuring the susceptibility of Cryptococcus neoformans to fluconazole. The present study evaluated the interlaboratory agreement of the results for the microdilution method obtained at three different sites and compared this method with the National Committee for Clinical Laboratory Standards M27-P reference method. Excellent interlaboratory agreement among the results obtained at the three sites was achieved with this method (83 and 96% agreement within 1 and 2 log2 dilutions, respectively). An overall agreement of 90% between the microdilution method and the M27-P method was observed, demonstrating the comparability of the two methods. However, there are inherent problems with the M27-P method in relation to measuring C. neoformans susceptibility, including suboptimal growth of the organism in RPMI 1640, a longer incubation period, and a narrow range of MICs. On the basis of these data, the microdilution method tested in this study is recommended for inclusion in the National Committee for Laboratory Standards method for testing the antifungal susceptibility of C. neoformans.
Polymorphism in Brucella strains detected by studying distribution of two short repetitive DNA elements
Authors: Mercier E , Jumas-Bilak E , Allardet-Servent A , O'Callaghan D , Ramuz M
Published: January 2020

Thirty-four Brucella reference or field strains representing all the species and biovars were studied by repetitive element sequence-based PCR, a PCR using primers complementary to two enterobacterial short repetitive sequences: repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequences. All the stains showed a positive amplification, suggesting that the Brucella genome contains such sequences. Repetitive extragenic palindromic PCR was less discriminating than enterobacterial repetitive intergenic consensus PCR in terms of distinguishing strains, but a combination of the two methods was able to distinguish all the isolates, except for some strains belonging to biovars 3 and 9 of Brucella abortus. Repetitive element sequence-based PCR appears to be a simple and useful method for the study of brucellosis epidemiology.
Fatal, disseminated Acremonium strictum infection in a neutropenic host
Authors: Schell W A , Perfect J R
Published: January 2020

Disseminated Acremonium strictum infection in a neutropenic patient is reported. Positive fecal cultures preceded positive cutaneous and blood cultures by 18 and 21 days, respectively, which suggests gastrointestinal colonization and invasion as initiating events. Microscopic examination of cutaneous biopsy and pulmonary specimens revealed hyphae, phialides, and phialoconidia in vivo. These adventitious forms also can occur in infections due to other phialidic fungi such as Fusarium and Paecilomyces species and can be misdiagnosed as Candida species. Budding cells also can occur in vivo for species of Fusarium, Paecilomyces, and apparently Acremonium, further adding to the potential for misdiagnosis. The occurrence of adventitious forms in infections caused by species of Acremonium, Fusarium, Paecilomyces, Scedosporium, and Blastoschizomyces is suggested as a mechanism for dissemination of infection and as an explanation of the relatively higher frequency of positive blood cultures in these cases.
Genomic fingerprinting for epidemiological differentiation of Staphylococcus aureus clinical isolates
Authors: Smeltzer M S , Pratt F L , Gillaspy A F , Young L A
Published: January 2020

We used genomic fingerprinting to investigate an outbreak of methicillin-resistant Staphylococcus aureus in the neonatal intensive care units (NICUs) of two hospitals. The hospitals are located in the same city and are part of the same medical care system. Fingerprinting was done by Southern blot hybridization with DNA probes for the genes encoding the S. aureus collagen adhesin (cna), fibronectin-binding proteins (fnbA and fnbB), and beta-toxin (hlb). Genomic DNA was digested with HaeIII (cna and fnbA-fnbB probes) or HindIII (hlb probe). Hybridization patterns could be distinguished on the basis of (i) the presence or absence of cna, (ii) the size of the restriction fragment containing the cna gene, (iii) restriction fragment length polymorphisms within fnbA and fnbB, (iv) the presence of a lysogenic phage within hlb, and (v) the sizes of the restriction fragments containing the phage-bacterial DNA junction fragments. Over a period of 4 months we examined a total of 46 isolates obtained from various wards within each hospital. Among these 46 isolates, we observed a total of 4 cna patterns, 11 fnbA-fnbB patterns, and 11 hlb patterns. Southern blots with HaeIII-digested genomic DNA and a combination of all three gene probes revealed a total of 16 clearly distinguishable patterns. A total of 22 of the 46 isolates were identical with respect to every genomic marker examined. A total of 21 of these 22 isolates were obtained from patients within an NICU. Nineteen of 21 isolates also exhibited identical antibiotic resistance profiles (antibiogram). Although 5 of the remaining 24 strains exhibited an antibiogram identical to those of the NICU isolates, all 24 strains could be distinguished from the NICU isolates by at least one genomic marker. These results suggest that the NICU isolates had a common origin and that genomic fingerprinting with the cna, fnbA, fnbB, and hlb gene probes can provide an important epidemiological tool for the identification of clinical isolates of S. aureus.
Molecular epidemiology of impetiginous group A streptococcal infections in aboriginal communities of northern Australia
Authors: Gardiner D L , Sriprakash K S
Published: January 2020

Group A streptococcal infections among the Aboriginal communities of the Northern Territory of Australia are endemic, with a concurrently high rate of the postinfection sequelae of rheumatic fever and acute post-streptococcal glomerulonephritis. The majority of the group A streptococcal isolates from the Northern Territory are not typeable by M typing. We recently developed a novel genotyping method, Vir typing. A preliminary study using this method discriminated all the M-nontypeable (MNT) isolates. Vir typing is based on restriction fragment length polymorphisms of the 4- to 7-kb Vir regulon of group A streptococci, which contains a number of genes, including emm (the gene for M protein). A total of 407 isolates of group A streptococci obtained from four Aboriginal communities over a 4-year period were typed by this genotyping method. Forty-two distinct genotypes were found among the isolates, including 22 among the MNT isolates. The correlation between Vir type and M type was good. This genotyping method allows the characterization of all group A streptococcal isolates from Aboriginal communities in the Northern Territory. We also propose that Vir typing be used in conjunction with M typing for epidemiological surveillance in geographical regions where the majority of isolates are MNT.
Evaluation of pulsed-field gel electrophoresis as a typing system for Candida rugosa: comparison of karyotype and restriction fragment length polymorphisms
Authors: Dib J C , Dube M , Kelly C , Rinaldi M G , Patterson J E
Published: January 2020

Nosocomial infections with Candida species have emerged as an increasingly important cause of morbidity and mortality in intensive care units. Ten Candida rugosa isolates from a previously documented cluster of C. rugosa infections in one hospital (nine burn unit isolates and one isolate from another hospital ward) and eight C. rugosa isolates recovered in a referral fungus testing laboratory (comparison isolates) from distinct geographic areas were investigated by molecular techniques. Isolates were from multiple anatomic sites. Pulsed-field gel electrophoresis (PFGE) of whole-cell DNA was performed with the 18 C. rugosa isolates as a marker of strain identity. The PFGE karyotypes of the C. rugosa isolates were demonstrated from four to seven chromosome bands. Karyotyping revealed the same PFGE pattern for the nine outbreak isolates from the burn unit, confirming clonal strain transmission. The isolate from the other hospital ward had a distinct karyotype. Distinct PFGE karyotype patterns were demonstrated for the eight comparison isolates. Restriction fragment length polymorphisms (RFLP) generated from whole-cell DNA digested with SfiI demonstrated the same RFLP pattern among outbreak isolates. Among comparison isolates, karyotyping distinguished some isolates that were indistinguishable by RFLP patterns. Karyotyping by PFGE appears to be the most useful molecular typing tool for discrimination among strains of C. rugosa and will be a useful marker for evaluating the epidemiology of future C. rugosa infections.
Genetic variation in Australian spotted fever group rickettsiae
Authors: Baird R W , Stenos J , Stewart R , Hudson B , Lloyd M , Aiuto S , Dwyer B
Published: January 2020

Rickettsiae were isolated by cell culture of buffy coat blood from six patients with spotted fever from southeastern Australia and Flinders Island in Bass Strait. The isolates were genetically compared with two previous Rickettsia australis patient isolates. The genus-specific 17-kDA genes from the isolates were compared after DNA amplification and restriction fragment analysis of the amplified DNA. This comparison revealed that mainland rickettsial isolates from southeastern Australia were identical to two previous isolates of R. australis from northeastern Australia. Rickettsial isolates from Flinders Island were distinct from the mainland isolates. The 16S rRNA gene sequences from the isolates were determined and compared. The Flinders Island rickettsial agent was most closely related (0.3% structural divergence) to Rickettsia rickettsii, Rickettsia conorii, and Rickettsia slovaca. The Flinders Island rickettsial agent was 1.3 and 2.1% structurally divergent from R. australis and Rickettsia akari, respectively. The 16S rRNA gene sequence from the Flinders Island agent shows that this rickettsia is more closely related to the rickettsial spotted fever group than is R. australis. We conclude that there are two populations of spotted fever group rickettsiae in Australia and propose that the genetically distinct causative organism of Flinders Island spotted fever be designated Rickettsia honei. The extent of distribution and animal host reservoirs remain to be elucidated.
Comparison of Binax Legionella Urinary Antigen EIA kit with Binax RIA Urinary Antigen kit for detection of Legionella pneumophila serogroup 1 antigen
Authors: Hackman B A , Plouffe J F , Benson R F , Fields B S , Breiman R F
Published: January 2020

The Legionella Urinary Antigen EIA kit (Binax, Portland, Maine) was compared with the EQUATE RIA Legionella Urinary Antigen kit (Binax) for its ability to detect the presence of urinary antigens to Legionella pneumophila serogroup 1. Urine specimens from patients without Legionnaires' disease (n = 33) were negative by both methods (specificity, 100%). Twenty (77%) of 26 urine specimens from patients with Legionnaires' disease positive by the radioimmunoassay kit were also positive by the enzyme immunoassay (EIA) kit. If the cutoff for a positive EIA result were lowered to a ration of > or = 2.5, 23 of 26 (88%) urine specimens would have been positive by EIA and the specificity would remain 100%. Use of the EIA kit is an acceptable method for detecting L. pneumophila serogroup 1 urinary antigens by laboratories that do not want to handle radioactive materials.
Detection of Pneumocystis carinii DNA in blood by PCR is not of value for diagnosis of P. carinii pneumonia
Authors: Tamburrini E , Mencarini P , Visconti E , Zolfo M , De Luca A , Siracusano A , Ortona E , Wakefield A E
Published: January 2020

A nested PCR which amplified a portion of the mitochondrial large-subunit rRNA gene of Pneumocystis carinii was used to detect P. carinii DNA in blood from patients with P. carinii pneumonia. P. carinii DNA was not detected in serum and was detected at low levels of blood cells.
High incidence of hepatitis C virus infection in hemodialysis patients in units with high prevalence
Authors: Pujol F H , Ponce J G , Lema M G , Capriles F , Devesa M , Sirit F , Salazar M , Vásquez G , Monsalve F , Blitz-Dorfman L
Published: January 2020

The prevalence of hepatitis C virus (HCV) infection was evaluated in 227 hemodialysis patients from four units in Caracas, Venezuela, by using different second- and third-generation enzyme immunoassays (EIAs) and immunoblot assays. HCV antibodies were detected in 162 patients (71%) by the recombinant-based second-generation assays (Abbott and Ortho) and in 161 patients by the synthetic peptide-based EIA (UBI). Of the 162 HCV antibody-positive serum samples, 161 were confirmed to be positive by RIBA 3. HCV RNA was detected in 49 of 68 (72%) of the seropositive patients and in 5 of 21 (24%) of the seronegative ones. HCV RNA was not always correlated with an increase in alanine aminotransferase (ALT) levels. Among 20 patients positive for HCV RNA and for HCV antibodies (without any hepatitis B virus [HBV] marker), only 10 had elevated ALT levels. The possible interference of HBV for HCV replication was evaluated. No significant difference was found between the presence of HCV RNA and the presence of any HBV serological markers. The possible routes of transmission of HCV in hemodialysis patients are multiple, and some of them are still controversial. Of the HCV-positive patients, 30% received a blood transfusion, significantly more than the 15% found for the HCV-negative group. However, blood transfusions alone could not account for the high incidence observed in this group of patients (38% from 1994 to 1995). In conclusion, about one-quarter of the apparently non-HCV-infected patients were probably seroconverting, ALT may not be a useful indicator of HCV infection in hemodialysis patients, and nosocomial transmission of HCV may play a role in the spread of HCV in this group.
Detection of a transforming fragment of herpes simplex virus type 2 in clinical specimens by PCR. The Canadian Women's HIV Study Group
Authors: Guibinga G H , Coutlée F , Kessous A , Hankins C , Lapointe N , Richer G , Tousignant J
Published: January 2020

A PCR assay for the sensitive detection of a transforming fragment of herpes simplex virus type 2 (HSV-2) was developed. Oligonucleotide primers were selected in Xho-2, a transforming region of the BglII N fragment of HSV-2. The assay reached a sensitivity endpoint of 10 copies of the Xho-2 subfragment and did not show cross-reactivity with other herpesviruses, including HSV-1. All 42 HSV-2 isolates scored positive in the assay. The Xho-2 PCR assay was evaluated with 216 clinical specimens and the results were compared with those of cell culture. The best protocol for processing specimens contained in viral transport medium included a centrifugation step followed by cell lysis. Of the 107 specimens positive for HSV-2 by culture, 105 were PCR positive (sensitivity, 98.1%). For one of the two falsely negative samples, beta-globin as well as sequences from the HSV-2 DNA polymerase gene could not be amplified. The other sample scored positive in both of these reactions but was indeterminate in duplicate tests by Xho-2 PCR. Two of 109 HSV-2 culture-negative specimens tested positive in the PCR assay (specificity, 98.2%). The latter two samples tested positive in a PCR test for the HSV-2 DNA polymerase gene. This novel tool was shown to be sensitive and specific for HSV-2 sequences and should allow for the investigation of the role of HSV-2 in genital cancers.
Heterogeneity of diphtheria toxin gene, tox, and its regulatory element, dtxR, in Corynebacterium diphtheriae strains causing epidemic diphtheria in Russia and Ukraine
Authors: Nakao H , Pruckler J M , Mazurova I K , Narvskaia O V , Glushkevich T , Marijevski V F , Kravetz A N , Fields B S , Wachsmuth I K , Popovic T
Published: January 2020

Diphtheria toxin (tox) and its regulatory element (dtxR) from 72 Corynebacterium diphtheriae strains isolated in Russia and Ukraine before and during the current diphtheria epidemic were studied by PCR-single-strand conformation polymorphism analysis (PCR-SSCP). Twelve sets of primers were constructed (eight for tox and four for dtxR), and three regions within tox and all four regions of dtxR showed significant variations in the number and/or sizes of the amplicons. Two to four different SSCP patterns were identified in each of the variable regions; subsequently, tox and dtxR could be classified into 6 and 12 different types, respectively. The great majority of epidemic strains from both Russia and Ukraine had tox types 3 and 4, and only in a single preepidemic strain isolated in Russia were all eight tox regions identical to those of C. diphtheriae Park-Williams No. 8 (tox type 1). Epidemic strains from Ukraine can easily be identified by dtxR type 5, while the majority of the Russian epidemic strains have dtxR of types 2 and 8. No differences in the tox regions between mitis and gravis biotype strains were observed. However, dtxR types 2, 5, and 8 were identified only in the gravis biotype, and dtxR type 1 was characteristic for the mitis biotype strains. PCR-SSCP is a simple and rapid method for the identification of variable tox and dtxR regions that allows for the clear association of tox and dtxR types with strains of distinct temporal and/or geographic origins.