bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of NCCLS and 3-(4,5-Dimethyl-2-Thiazyl)-2,5-Diphenyl-2H-Tetrazolium Bromide (MTT) Methods of In Vitro Susceptibility Testing of Filamentous Fungi and Development of a New Simplified Method
Authors: Meletiadis Joseph , Meis Jacques F. G. M. , Mouton Johan W. , Donnelly J. Peter , Verweij Paul E.
Published: December 2019

The susceptibility of 30 clinical isolates belonging to six different species of filamentous fungi (
Aspergillus fumigatus
,
Aspergillus flavus
,
Scedosporium prolificans
,
Scedosporium apiospermum
,
Fusarium solani
, and
Fusarium oxysporum
) was tested against six antifungal drugs (miconazole, voriconazole, itraconazole, UR9825, terbinafine, and amphotericin B) with the microdilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (M38-P). The MICs were compared with the MICs obtained by a colorimetric method measuring the reduction of the dye 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan by viable fungi. The levels of agreement between the two methods were 96 and 92% for MIC-0 (clear wells) and MIC-1 (75% growth reduction), respectively. The levels of agreement were always higher for
Aspergillus
spp. (97% ± 2.5%), followed by
Scedosporium
spp. (87% ± 10.3%) and
Fusarium
spp. (78% ± 7.8%). The NCCLS method was more reproducible than the MTT method: 98 versus 95% for MIC-0 and 97 versus 90% for MIC-1. However, the percentage of hyphal growth as determined visually by the NCCLS method showed several discrepancies when they were compared with the percentages of MTT reduction. A new simplified assay that incorporates the dye MTT with the initial inoculum and in which the fungi are incubated with the dye for 48 h or more was developed, showing comparable levels of agreement and reproducibility with the other two methods. Furthermore, the new assay was easier to perform and more sensitive than the MTT method.
Detection of Adenoviruses (AdV) in Culture-Negative Environmental Samples by PCR during an AdV-Associated Respiratory Disease Outbreak
Authors: Echavarria Marcela , Kolavic Shellie A. , Cersovsky Steven , Mitchell Felicia , Sanchez Jose L. , Polyak Christina , Innis Bruce L. , Binn Leonard N.
Published: December 2019

Since 1954, adenoviruses (AdV) have been recognized as an important cause of acute respiratory disease (ARD) among U.S. military recruits. Until recently, routine oral vaccination for AdV serotypes 4 and 7 eliminated epidemic AdV-associated ARD in this population. Now that the manufacturer has ceased production, vaccination has ended and AdV epidemics have reappeared. As part of a prospective epidemiological study during the high-risk ARD season, serial samples were obtained from ventilation system filters and tested for AdV by culture and PCR. An outbreak occurred during this surveillance. Of 59 air filters, 26 (44%) were AdV positive only by PCR. Sequence analysis confirmed the presence of AdV serotype 4, the implicated outbreak serotype. The number of AdV-related hospitalizations was directly correlated with the proportion of filters containing AdV; correlation coefficients were 0.86 (Pearson) and 0.90 (Spearman's rho). This is the first report describing a PCR method to detect airborne AdV during an ARD outbreak. It suggests that this technique can detect and quantify AdV-associated ARD exposure and may enable further definition of environmental effects on AdV-associated ARD spread.
Outbreak of Methicillin-Resistant
Staphylococcus aureus
with Reduced Susceptibility to Glycopeptides in a Parisian Hospital
Authors: Guerin François , Buu-Hoï Annie , Mainardi Jean-Luc , Kac Guillaume , Colardelle Nathalie , Vaupré Sabine , Gutmann Laurent , Podglajen Isabelle
Published: December 2019

Epidemiological relationships were investigated between 40 methicillin-resistant
Staphylococcus aureus
(MRSA) strains with decreased glycopeptide susceptibility isolated from November 1998 to March 1999 from 39 patients (17 infected and 22 colonized patients) in nine wards of the Broussais Hospital, Paris, France. Reduced glycopeptide susceptibility was readily detected on brain heart infusion (BHI) agar containing 6 μg of teicoplanin per ml and on gradient plates, but not by the standard disk diffusion method. The MICs of vancomycin and teicoplanin, determined on BHI agar, were 4 and 8 to 32 μg/ml, respectively (standard antibiotic dilution), and 4 to 8 and 8 to 32 μg/ml, respectively (E-test). All strains were resistant to macrolides, aminoglycosides, tetracycline, rifampin, sulfonamides, and pefloxacin, showed reduced susceptibility to fusidic acid and fosfomycin, and were susceptible to trimethoprim and chloramphenicol. Pulsed-field gel electrophoresis and lysotyping revealed that a multidrug-resistant MRSA clone with decreased susceptibility to glycopeptides has been discretely endemic since at least 1996 in our institution, where it was responsible for an outbreak in November and December 1998.
Comparison of BACTEC MYCO/F LYTIC and WAMPOLE ISOLATOR 10 (Lysis-Centrifugation) Systems for Detection of Bacteremia, Mycobacteremia, and Fungemia in a Developing Country
Authors: Archibald Lennox K. , McDonald L. Clifford , Addison Rachel M. , McKnight Celeste , Byrne Terry , Dobbie Hamish , Nwanyanwu Okey , Kazembe Peter , Reller L. Barth , Jarvis William R.
Published: December 2019

In less-developed countries, studies of bloodstream infections (BSI) have been hindered because of the difficulty and costs of culturing blood for bacteria, mycobacteria, and fungi. During two study periods (study period I [1997] and study period II [1998]), we cultured blood from patients in Malawi by using the BACTEC MYCO/F LYTIC (MFL), ISOLATOR 10 (Isolator), Septi-Chek AFB (SC-AFB), and Septi-Chek bacterial (SC-B) systems. During study period I, blood was inoculated at 5 ml into an MFL bottle, 10 ml into an Isolator tube for lysis and centrifugation, and 10 ml into an SC-B bottle. Next, 0.5-ml aliquots of Isolator concentrate were inoculated into an SC-AFB bottle and onto Middlebrook 7H11 agar slants, chocolate agar slants, and Inhibitory Mold Agar (IMA) slants. During study period II, the SC-B and chocolate agar cultures were discontinued. MFL growth was detected by fluorescence caused by shining UV light (λ = 365 nm) onto the indicator on the bottom of the bottle. During study period I, 251 blood cultures yielded 44 bacterial isolates. For bacteremia, the MFL was similar to the Isolator concentrate on chocolate agar (34 of 44 versus 27 of 44;
P
, not significant [NS]), but more sensitive than the SC-B bottle (34 of 44 versus 24 of 44;
P
= 0.05). For both study periods combined, 486 blood cultures yielded 37 mycobacterial and 13 fungal isolates. For mycobacteremia, the sensitivities of the MFL and Isolator concentrate in the SC-AFB bottle were similar (30 of 37 versus 29 of 37;
P
, NS); the MFL bottle was more sensitive than the concentrate on Middlebrook agar (30 of 37 versus 15 of 37;
P
= 0.002). For fungemia, the MFL bottle was as sensitive as the SC-B bottle or Isolator concentrate on chocolate agar or IMA slants. We conclude that the MFL bottle, inoculated with just 5 ml of blood and examined under UV light, provides a sensitive and uncomplicated method for comprehensive detection of BSI in less-developed countries.
Simple and Rapid Detection of
Candida albicans
DNA in Serum by PCR for Diagnosis of Invasive Candidiasis
Authors: Wahyuningsih Retno , Freisleben Hans-Joachim , Sonntag Hans-Günther , Schnitzler Paul
Published: December 2019

A rapid and sensitive PCR assay for the detection of
Candida albicans
DNA in serum was established. DNA from human serum samples was purified using the QIAamp blood kit, which proved to be a fast and simple method for isolating minute amounts of
Candida
DNA from clinical specimens for diagnosis of invasive candidiasis. Universal primer sequences used in the PCR assay are derived from the internal transcribed spacer rRNA gene of fungi, whereas the biotinylated hybridization probe used in a DNA enzyme immunoassay (DEIA) binds specifically to
C. albicans
DNA. The sensitivity of this PCR-DEIA method is very high; the detection limit for genomic
Candida
DNA is one
C. albicans
genome per assay. Blood from uninfected and infected persons, ranging from healthy volunteers, patients with mucocutaneous infections, and patients at risk to develop a systemic
Candida
infection to patients with an established systemic candidiasis, was analyzed for the presence of
C. albicans
to diagnose fungal infection.
Candida
DNA could not be detected in sera of 16 culture-negative controls and from 11 nonsystemic candidal infections by PCR or DEIA. Blood cultures from patients at risk were all negative for
Candida
, whereas all blood cultures from systemic candidiasis patients were positive. However,
Candida
DNA could be detected by PCR and DEIA in the serum from three out of nine patients who were at risk for a systemic infection and in the serum of all seven patients who had already developed an invasive
Candida
infection. PCR is more sensitive than blood culture, since some of the patients at risk for invasive yeast infection, whose blood cultures were all negative for
Candida
, tested positive in the PCR amplification. These results indicate the potential value of PCR for detecting
C. albicans
in serum samples and for identifying patients at risk for invasive candidiasis.
Comparative Evaluation of Three Human Immunodeficiency Virus Genotyping Systems: the HIV-GenotypR Method, the HIV PRT GeneChip Assay, and the HIV-1 RT Line Probe Assay
Authors: Wilson John W. , Bean Pamela , Robins Terry , Graziano Frank , Persing David H.
Published: December 2019

Evaluation of drug resistance by human immunodeficiency virus (HIV) genotyping has proven to be useful for the selection of drug combinations with maximum antiretroviral activity. We compared three genotyping methods for identification of mutations known to confer drug resistance in the reverse transcriptase (RT) and protease genes of HIV type 1 (HIV-1). The HIV-GenotypR method (GenotypR; Specialty Laboratories, Inc., Santa Monica, Calif.) with the ABI 377 DNA sequencer (Applied Biosystems Inc.), the HIV PRT GeneChip assay (GeneChip; Affymetrix, Santa Clara, Calif.), and the HIV-1 RT Line Probe Assay (LiPA; Innogenetics, Alpharetta, Ga.) were used to genotype plasma samples from HIV-infected patients attending the University of Wisconsin Hospitals and Clinics and the Mayo Clinic. At the time of analysis, patients were failing combination therapy (
n
= 18) or were treatment naive (
n
= 6). Forty codons of the RT and protease genes were analyzed by GenotypR and GeneChip for resistance-associated mutations. LiPA analyzed seven RT codons for mutations. Each sample was genotyped by all three assays, and each assay was subjected to pairwise comparisons. At least 92% of the codons tested (by the three assays) in paired comparisons were concordant. GenotypR and GeneChip demonstrated 96.6% concordance over the 40 codons tested. GenotypR identified slightly more mutations than GeneChip and LiPA; GeneChip identified all primary mutations that corresponded to failing treatment regimens. Each assay identified at least 84% of the mutations identified by the other assays. Mutations that were discordant between the assays mainly comprised secondary mutations and natural polymorphisms. The assays had better concordance for mutations that corresponded to current failing regimens, present in the more predominant viral quasispecies. In the treatment-naive patients, GenotypR, GeneChip, and LiPA mainly identified wild-type virus. Only the LiPA identified K70R, a possible transmitted zidovudine resistance mutation, in the RT gene of a treatment-naive patient. We conclude that although discrepancies in results exist between assays, each assay showed a similar capacity to identify potentially clinically relevant mutations related to patient treatment regimens.
Duplex PCR for Differential Identification ofMycobacterium bovis,M. avium, andM. aviumsubsp.paratuberculosisin Formalin- Fixed Paraffin-Embedded Tissues from Cattle
Authors: Coetsier Christophe , Vannuffel Pascal , Blondeel Nathalie , Denef Jean-Francois , Cocito Carlo , Gala Jean-Luc
Published: December 2019

We previously isolated and sequenced two genomic segments ofMycobacterium aviumsubsp.paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) fromM. aviumsubsp.paratuberculosisandM. tuberculosisshowed a 79-base deletion inM. tuberculosis. Sequence analysis of the p34 genes in another two species,M. bovis(strain BCG) andM. avium(strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis,M. bovis, andM. africanum), for both nontuberculous mycobacteriaM. aviumandM. intracellulare, or forM. aviumsubsp.paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of theM. aviumcomplex.
High-Resolution Genotyping of
Chlamydia trachomatis
from Recurrent Urogenital Infections
Authors: Pedersen Lisbeth Nørum , Kjær Holger Olaf , Møller Jens Kjølseth , Ørntoft Torben Falck , Ostergaard Lars
Published: December 2019

A method for
Chlamydia trachomatis
restriction fragment length polymorphism (RFLP) analysis and complete sequencing of
omp1
was developed for use on samples collected at home, and results were compared. Genotyping by sequencing was superior to RFLP analysis. The
omp1
gene in 31 clinical strains harbored few mutations compared to the same gene in ATCC reference strains. Follow-up samples obtained during a 24-week period from 31 patients showed recurrence with the same genotype in five cases and a new genotype in one case.
Validation of the Abbreviated
Brucella
AMOS PCR as a Rapid Screening Method for Differentiation of
Brucella abortus
Field Strain Isolates and the Vaccine Strains, 19 and RB51
Authors: Ewalt Darla R. , Bricker Betsy J.
Published: December 2019

The
Brucella
AMOS PCR assay was previously developed to identify and differentiate specific
Brucella
species. In this study, an abbreviated
Brucella
AMOS PCR test was evaluated to determine its accuracy in differentiating
Brucella abortus
into three categories: field strains, vaccine strain 19 (S19), and vaccine strain RB51/parent strain 2308 (S2308). Two hundred thirty-one isolates were identified and tested by the conventional biochemical tests and
Brucella
AMOS PCR. This included 120 isolates identified as
B. abortus
S19, 9 identified as
B. abortus
strain RB51, 57 identified as
B. abortus
biovar 1, 15 identified as
B. abortus
bv. 2, 1 identified as
B. abortus
bv. 2 (M antigen dominant), 7 identified as
B. abortus
bv. 4, and 22 identified as
B. abortus
S2308 and isolated from experimentally infected cattle. The
Brucella
AMOS PCR correctly identified each isolate as RB51/S2308, S19, or a field strain of
Brucella
.
Lactoferrin and Eosinophilic Cationic Protein in Nasal Secretions of Patients with Experimental Rhinovirus Colds, Natural Colds, and Presumed Acute Community-Acquired Bacterial Sinusitis
Authors: Niehaus Mark D. , Gwaltney Jack M. , Hendley J. Owen , Newman Mercy J. , Heymann Peter W. , Rakes Gary P. , Platts-Mills Thomas A. E. , Guerrant Richard L.
Published: December 2019

To distinguish sinusitis from uncomplicated “colds,” we examined lactoferrin and eosinophilic cationic protein (ECP) in nasal secretions. Lactoferrin titers were ≥1:400 in 4% of persons with uncomplicated colds and controls but in 79% of persons with sinusitis or purulent sputa. ECP levels were >200 ng/ml in 61% of persons with colds and >3,000 ng/ml in 62% of persons with sinusitis. Nasal lactoferrin helps distinguish sinusitis from colds.
Recovery and Identification of West Nile Virus from a Hawk in Winter
Authors: Garmendia Antonio E. , Van Kruiningen Herbert J. , French Richard A. , Anderson John F. , Andreadis Theodore G. , Kumar Asok , West A. Brian
Published: December 2019

West Nile virus was recovered from the brain of a red-tailed hawk that died in Westchester County, N.Y., in February 2000. Multiple foci of glial cells, lymphocytes, and a few pyknotic nuclei were observed in the brain. Three to 4 days after inoculation of Vero cells with brain homogenates, cytopathic changes were detected. The presence of West Nile virus antigen in fixed cells or cell lysates was revealed by fluorescent antibody testing or enzyme-linked immunosorbent assay, respectively. Furthermore, Reverse transcriptase-PCR with primers specific for the NS3 gene of West Nile virus resulted in an amplicon of the expected size (470 bp). Electron microscopy of thin sections of infected Vero cells revealed the presence of viral particles approximately 40 nm in diameter, within cytoplasmic vesicles. The demonstration of infection with the West Nile virus in the dead of the winter, long after mosquitoes ceased to be active, is significant in that it testifies to the survival of the virus in the region beyond mosquito season and suggests another route of transmission: in this case, prey to predator.
Utility of Commercial Systems for Identification of
Burkholderia cepacia
Complex from Cystic Fibrosis Sputum Culture
Authors: Shelly Deborah Blecker , Spilker Theodore , Gracely Edward J. , Coenye Tom , Vandamme Peter , LiPuma John J.
Published: December 2019

Performances of several commercial test systems were reviewed to determine their relative levels of accuracy in identifying
Burkholderia cepacia
complex isolates recovered from cystic fibrosis sputum culture. Positive predictive values ranged from 71 to 98%; negative predictive values ranged from 50 to 82%. All systems misidentified
B. cepacia
complex. The species most frequently misidentified as
B. cepacia
was
Burkholderia gladioli
. These data support the results of previous studies that recommend confirmatory testing, including the use of DNA-based methods, for sputum culture isolates presumptively identified as
B. cepacia
.
Two Successive Hepatitis C Virus Infections in an Intravenous Drug User
Authors: Proust Barbara , Dubois Frederic , Bacq Yannick , Le Pogam Sophie , Rogez Sylvie , Levillain Romuald , Goudeau Alain
Published: December 2019

We report the case of an occasional intravenous drug user who developed two successive hepatitis C virus (HCV) infections. The first infection led to seroconversion (anti-HCV antibodies detected) and the detection of HCV RNA in serum. After a spontaneous recovery (normalization of alanine aminotransferase levels and HCV RNA clearance), a second HCV infection was observed, with the recurrence of HCV viremia. Antibody directed against HCV serotype 1 was detected throughout the follow-up monitoring, but two different HCV strains were identified during the two infectious episodes: genotype 1a for the first and genotype 3a for the second. This observation shows that primary HCV infection does not confer protective immunity against subsequent infection with viruses of other genotypes. This may hamper the development of a vaccine. Conflicting results were obtained in genotyping and serotyping assays, suggesting that the serotyping method cannot be used to identify the HCV type in patients, such as intravenous drug users, who are exposed to successive HCV infections.
Clinical Relevance of Direct Quantification of pp65 Antigenemia Using Flow Cytometry in Solid Organ and Stem Cell Transplant Recipients
Authors: Poirier-Toulemonde Anne-Sophie , Milpied Noel , Cantarovich Diego , Morcet Jean-François , Billaudel Sylviane , Imbert-Marcille Berthe-Marie
Published: December 2019

A total of 1,305 blood samples from 85 solid organ transplant (SOT) recipients and 25 stem cell transplant (SCT) recipients at risk for cytomegalovirus (CMV) infection were prospectively collected and tested using the shell vial assay (SVA) and a leukocytic qualitative PCR (q-PCR). Of these, 462 specimens were further tested by direct quantification of CMV antigenemia by flow cytometry (FC-Ag), 125 were tested with a quantitative competitive PCR, and 200 were tested for pp65 antigenemia using the slide method (S-Ag). Laboratory data were statistically analyzed according to the presence of CMV-related symptoms. In SOT and SCT recipients, active CMV infection occurred in 63.5 and 36%, respectively, and CMV disease occurred in 53 and 24%, respectively. FC-Ag results correlated better with q-PCR and S-Ag than with SVA. The first test found to be positive during follow-up was FC-Ag in 73% of cases. In SOT recipients, FC-Ag showed the highest sensitivity and negative predictive value for the diagnosis of any grade of CMV disease. For FC-Ag, the threshold beyond which CMV disease was highly probable seemed to lie at 0.20% positive polymorphonuclear leukocytes. FC-Ag appears to be a useful test for the early detection of CMV infection and the prediction of CMV disease.
Improved Identification and Differentiation of Varicella-Zoster Virus (VZV) Wild-Type Strains and an Attenuated Varicella Vaccine Strain Using a VZV Open Reading Frame 62-Based PCR
Authors: Loparev Vladimir N. , Argaw Takele , Krause Philip R. , Takayama Michiko , Schmid D. Scott
Published: December 2019

A new method was developed to identify and differentiate varicella-zoster virus (VZV) wild-type strains from the attenuated varicella Oka vaccine strain. The PCR technique was used to amplify a VZV open reading frame (ORF) 62 region. A single specific amplicon of 268 bp was obtained from 71 VZV clinical isolates and several laboratory strains. Subsequent digestion of the VZV ORF 62 amplicons with SmaI enabled accurate strain differentiation (threeSmaI sites were present in amplicons of vaccine strain VZV, compared with two enzyme cleavage sites for all other VZV strains tested). This method accurately differentiated the Oka vaccine strain from wild-type VZV strains circulating in countries representing all six populated continents. Moreover, the assay more reliably distinguished wild-type Japanese strains from the vaccine strain than did previously described methods.
Relationship between Mutations in Dihydropteroate Synthase of Pneumocystis carinii f. sp.hominis Isolates in Japan and Resistance to Sulfonamide Therapy
Authors: Takahashi Takashi , Hosoya Noriaki , Endo Tokiomi , Nakamura Tetsuya , Sakashita Hiroyuki , Kimura Kyoko , Ohnishi Kenji , Nakamura Yoshikazu , Iwamoto Aikichi
Published: December 2019

We examined mutations in the dihydropteroate synthase (DHPS) genes of Pneumocystis carinii f. sp. hominis(P. carinii) strains isolated from 24 patients withP. carinii pneumonia (PCP) in Japan. DHPS mutations were identified at amino acid positions 55 and/or 57 in isolates from 6 (25.0%) of 24 patients. The underlying diseases for these six patients were human immunodeficiency virus type 1 infection (n= 4) or malignant lymphoma (n = 2). This frequency was almost the same as those reported in Denmark and the United States. None of the six patients whose isolates had DHPS mutations were recently exposed to sulfa drugs before they developed the current episode of PCP, suggesting that DHPS mutations not only are selected by the pressure of sulfa agents but may be incidentally acquired. Co-trimoxazole treatment failed more frequently in patients whose isolates had DHPS mutations than in those whose isolates had wild-type DHPS (n = 4 [100%] versus n = 2 [11.1%]; P = 0.002). Our results thus suggest that DHPS mutations may contribute to failures of co-trimoxazole treatment for PCP.
Time-Resolved Fluorometry PCR Assay for Rapid Detection of Herpes Simplex Virus in Cerebrospinal Fluid
Authors: Hukkanen Veijo , Rehn Tiina , Kajander Ritva , Sjöroos Minna , Waris Matti
Published: December 2019

We have introduced a time-resolved fluorometry (TRF)-based microwell hybridization assay for PCR products in detection of herpes simplex virus (HSV) in cerebrospinal fluid (CSF) specimens. TRF is a sensitive nonradioactive detection technique which involves the use of lanthanide chelates as fluorescent labels. We used PCR primers from the glycoprotein D genes of HSV type 1 (HSV-1) and HSV-2. The biotinylated PCR products were collected on streptavidin-coated microtitration wells and hybridized with short oligonucleotide probes, europium labeled for HSV-1 and samarium labeled for HSV-2. The TRF results were obtained as counts per second and as signal-to-noise (S/N) ratios. The sensitivity of the assay was 0.1 infectious units (PFU) of HSV in CSF specimens, and the S/N values increased with the virus amount, up to 68.5 for 103 PFU of HSV-1 and to 58.5 for 103 PFU of HSV-2, allowing semiquantitation of HSV in CSF. The primers and probes recognized all the studied 48 HSV wild-type samples, with S/N ratios of 12.4 to 190 (HSV-1) and 5.1 to 248 (HSV-2). We tested CSF specimens, 100 for each HSV type, which were HSV PCR negative by Southern blot and 22 CSF specimens which were HSV-1 or -2 PCR blot positive. In the TRF test, the mean S/N ratio for the HSV-1-negative CSF was 1.37 (standard deviation [SD] = 0.513) and for the HSV-2-negative CSF it was 1.03 (SD = 0.098). The HSV-1 blot-positive CSF yielded S/N ratios of 3.6 to 85.9, and the HSV-2 blot-positive CSF yielded ratios from 1.9 to 13. Using the mean S/N ratio for negative CSF specimens + 3 SD as the cutoff yielded all the previously HSV-positive specimens as TRF positive. The TRF PCR assay for HSV in CSF specimens is a rapid and sensitive method, improves interpretation of PCR results, and is well suited for automation.
Diagnosis of Amebic Liver Abscess and Intestinal Infection with the TechLab Entamoeba histolytica II Antigen Detection and Antibody Tests
Authors: Haque Rashidul , Mollah Nasir Uddin , Ali Ibne Karim M. , Alam Khorshed , Eubanks Aleida , Lyerly David , Petri William A.
Published: December 2019

A noninvasive diagnostic test for amebic liver abscess is needed, because amebic and bacterial abscesses appear identical on ultrasound or computer tomography and because it is rarely possible to identifyEntamoeba histolytica in stool specimens from patients with amebic liver abscess. Here we report a method of detection in serum of circulating E. histolytica Gal/GalNAc lectin to diagnose amebic liver abscess, which was used in patients from Dhaka, Bangladesh. The TechLab E. histolytica II test (which differentiates the true pathogen E. histolytica fromEntamoeba dispar) detected Gal/GalNAc lectin in the sera of 22 of 23 (96%) amebic liver abscess patients tested prior to treatment with the antiamebic drug metronidazole and 0 of 70 (0%) controls. After 1 week of treatment with metronidazole, 9 of 11 (82%) patients became serum lectin antigen negative. The sensitivity of the E. histolytica II antigen detection test for intestinal infection was also evaluated. Antigen detection identified E. histolytica infection in 50 samples from 1,164 asymptomatic preschool children aged 2 to 5 years, including 16 of 16 (100%) culture-positive specimens. PCR analysis of stool specimens was used to confirm that most antigen-positive but culture-negative specimens were true-positive: PCR identified parasite DNA in 27 of 34 (79%) of the antigen-positive, culture-negative stool specimens. Antigen detection was a more sensitive test for infection than antilectin antibodies, which were detected in only 76 of 98 (78%) amebic liver abscess patients and in 26 of 50 (52%) patients with intestinal infection. We conclude that the TechLab E. histolytica II kit is a sensitive means to diagnose hepatic and intestinal amebiasis prior to the institution of metronidazole treatment.
False-Positive Results of Plasma PCR for Cytomegalovirus DNA due to Delayed Sample Preparation
Authors: Schäfer Peter , Tenschert Werner , Schröter Matthias , Gutensohn Kai , Laufs Rainer
Published: December 2019

Positive results by cytomegalovirus (CMV) PCR of plasma are considered predictive of active CMV infection in kidney allograft recipients. To assess whether contamination with leukocyte-derived CMV DNA can distort the results, aliquots of whole-blood samples from 60 CMV immunoglobulin G-positive patients with leukocyte CMV DNAemia were stored for up to 24 h at room temperature (RT) and at 4°C before plasma preparation. Native and ultrafiltered plasma samples were tested by CMV and β-globin PCRs. Among 30 latently infected patients (negative for CMV pp65 antigens), low baseline rates (10%) and levels (median number of copies, 10 [per 10 μl]) of CMV plasma DNAemia in native plasma samples increased significantly over time (after 4 h at RT, 37% [P < 0.001]; median number of copies, 45 [P < 0.001]). Similar effects were found during storage at 4°C. Ultrafiltration reduced the levels of CMV plasma DNAemia, but by 6 h of storage the levels were significantly elevated as well. CMV and β-globin DNA kinetics in plasma were parallel. In contrast, 30 actively infected patients (pp65 positive) had high baseline rates (87% in native samples) and levels (median number of copies, 75) of CMV plasma DNAemia. No significant effects of storage or ultrafiltration and no concordance with β-globin DNA kinetics were seen. In conclusion, delayed preparation of plasma samples bears a significant risk of false-positive CMV PCR results, probably due to leukocyte lysis. This has important implications in the clinical setting and for PCR standardization.
Development of a Rapid PCR Assay Specific forStaphylococcus saprophyticus and Application to Direct Detection from Urine Samples
Authors: Martineau Francis , Picard François J. , Ménard Christian , Roy Paul H. , Ouellette Marc , Bergeron Michel G.
Published: December 2019

Staphylococcus saprophyticus is one of the most frequently encountered microorganisms associated with acute urinary tract infections (UTIs) in young, sexually active female outpatients. Conventional identification methods based on biochemical characteristics can efficiently identify S. saprophyticus, but the rapidities of these methods need to be improved. Rapid and direct identification of this bacterium from urine samples would be useful to improve time required for the diagnosis of S. saprophyticus infections in the clinical microbiology laboratory. We have developed a PCR-based assay for the specific detection ofS. saprophyticus. An arbitrarily primed PCR amplification product of 380 bp specific for S. saprophyticus was sequenced and used to design a set of S. saprophyticus-specific PCR amplification primers. The PCR assay was specific for S. saprophyticus when tested with DNA from 49 gram-positive and 31 gram-negative bacterial species. This assay was also able to amplify efficiently DNA from all 60 strains of S. saprophyticus from various origins tested. This assay was adapted for direct detection from urine samples. The sensitivity levels achieved with urine samples was 19 CFU with 30 cycles of amplification and 0.5 CFU with 40 cycles of amplification. This PCR assay for the specific detection of S. saprophyticus is simple and rapid (approximately 90 min, including the time for urine specimen preparation).
Characterization of a Nosocomial Outbreak Caused by a Multiresistant Acinetobacter baumannii Strain with a Carbapenem-Hydrolyzing Enzyme: High-Level Carbapenem Resistance inA. baumannii Is Not Due Solely to the Presence of β-Lactamases
Authors: Bou Germán , Cerveró Gonzalo , Domínguez M. Angeles , Quereda Carmen , Martínez-Beltrán Jesús
Published: December 2019

From February to November 1997, 29 inpatients at Ramón y Cajal Hospital, Madrid, Spain, were determined to be either colonized or infected with imipenem- and meropenem-resistant Acinetobacter baumannii (IMRAB) strains (MICs, 128 to 256 μg/ml). A wide antibiotic multiresistance profile was observed with IMRAB strains. For typing IMRAB isolates, pulsed-field gel electrophoresis was used. For comparative purposes, 30 imipenem- and meropenem-susceptible A. baumannii (IMSAB) strains isolated before, during, and after the outbreak were included in this study. The molecular-typing results showed that the outbreak was caused by a single IMRAB strain (genotype A). By cloning experiments we identified a class D β-lactamase (OXA-24) encoded in the chromosomal DNA of this IMRAB strain which showed carbapenem hydrolysis. Moreover, the outer membrane profile of the IMRAB strain showed a reduction in the expression of two porins at 22 and 33 kDa when compared with genetically related IMSAB isolates. In addition no efflux mechanisms were identified in the IMRAB strains. In summary, we report here the molecular characterization of a nosocomial outbreak caused by one multiresistant A. baumannii epidemic strain that harbors a carbapenem-hydrolyzing enzyme. Although alterations in the penicillin-binding proteins cannot be ruled out, the reduction in the expression of two porins and the presence of this OXA-derived β-lactamase are involved in the carbapenem resistance of the epidemic nosocomial IMRAB strain.
Use of the National Committee for Clinical Laboratory Standards Guidelines for Disk Diffusion Susceptibility Testing in New York State Laboratories
Authors: Kiehlbauch Julia A. , Hannett George E. , Salfinger Max , Archinal Wendy , Monserrat Catherine , Carlyn Cynthia
Published: December 2019

Accurate antimicrobial susceptibility testing is vital for patient care and surveillance of emerging antimicrobial resistance. The National Committee for Clinical Laboratory Standards (NCCLS) outlines generally agreed upon guidelines for reliable and reproducible results. In January 1997 we surveyed 320 laboratories participating in the New York State Clinical Evaluation Program for General Bacteriology proficiency testing. Our survey addressed compliance with NCCLS susceptibility testing guidelines for bacterial species designated a problem (Staphylococcus aureus and Enterococcusspecies) or fastidious (Streptococcus pneumoniae,Haemophilus influenzae, and Neisseria gonorrhoeae) organism. Specifically, we assessed compliance with guidelines for inoculum preparation, medium choice, number of disks per plate, and incubation conditions for disk diffusion tests. We also included length of incubation for S. aureus andEnterococcus species. We found overall compliance with the five characteristics listed above in 80 of 153 responding laboratories (50.6%) for S. aureus and 72 of 151 (47.7%) laboratories for Enterococcus species. The most common problem was an incubation time shortened to less than 24 h. Overall compliance with the first four characteristics was reported by 92 of 221 (41.6%) laboratories for S. pneumoniae, 49 of 163 (30.1%) laboratories for H. influenzae, and 11 of 77 (14.3%) laboratories for N. gonorrhoeae. Laboratories varied from NCCLS guidelines by placing an excess number of disks per plate. Laboratories also reported using alternative media forEnterococcus species, N. gonorrhoeae, andH. influenzae. This study demonstrates a need for education among clinical laboratories to increase compliance with NCCLS guidelines.
In Vitro Susceptibility Testing of Filamentous Fungi: Comparison of Etest and Reference Microdilution Methods for Determining Itraconazole MICs
Authors: Pfaller M. A. , Messer S. A. , Mills K. , Bolmström A.
Published: December 2019

The performance of the Etest for itraconazole susceptibility testing of 50 isolates of filamentous fungi was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS) proposed standard microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose and with Casitone agar and were read after incubation for 24 h (Aspergillus spp. and Rhizopus spp.) and 48 h (all species except Rhizopus spp.) at 35°C. The isolates included Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Fusarium spp., Pseudallescheria boydii, Rhizopus spp., Paecilomyces variotii, and an Acremonium sp. Overall agreement between Etest and microdilution MICs was 96% with RPMI agar and 80% with Casitone agar. The agreement was 100% for all species exceptRhizopus spp. (83%) and Paecilomyces varioti(0%) with RPMI agar. When Casitone agar was used, the agreement ranged from 50% with Rhizopus spp. to 100% withFusarium spp., P. boydii, P. varioti, and an Acremonium sp. Notably, forAspergillus spp., the agreement between itraconazole Etest MICs read at 24 h and reference microdilution MICs read at 48 h was 100% with both RPMI and Casitone agar. Both media supported the growth of all filamentous fungi tested. Where a discrepancy was observed between Etest and the reference method, the Etest MIC was generally higher. The Etest method using RPMI agar appears to be a useful method for determining itraconazole susceptibilities ofAspergillus spp. and other filamentous fungi.
Comparison of Escherichia coli O157:H7 Antigen Detection in Stool and Broth Cultures to That in Sorbitol-MacConkey Agar Stool Cultures
Authors: Stapp Jennifer R. , Jelacic Srdjan , Yea Yoo-Lee , Klein Eileen J. , Fischer Marc , Clausen Carla R. , Qin Xuan , Swerdlow David L. , Tarr Phillip I.
Published: December 2019

We evaluated the Meridian IC-STAT direct fecal and broth culture antigen detection methods with samples from children infected withEscherichia coli O157:H7 and correlated the antigen detection results with the culture results. Stools of 16 children who had recently had stool cultures positive for this pathogen (population A) and 102 children with diarrhea of unknown cause (population B) were tested with the IC-STAT device (direct testing). Fecal broth cultures were also tested with this device (broth testing). The results were correlated to a standard of the combined yield from direct culture of stools on sorbitol-MacConkey (SMAC) agar and culture of broth on SMAC agar. Eleven (69%) of the population A stool specimens yieldedE. coli O157:H7 when plated directly on SMAC agar. Two more specimens yielded this pathogen when the broth culture was similarly plated. Of these 13 stool specimens, 8 and 13 were positive by direct and broth testing (respective sensitivities, 62 and 100%). Compared to the sensitivity of a simultaneously performed SMAC agar culture, the sensitivity of direct testing was 73%. Three (3%) of the population B stool specimens contained E. coli O157:H7 on SMAC agar culture; one and three of these stool specimens were positive by direct and broth testing, respectively. The direct and broth IC-STAT tests were 100% specific with samples from children from population B. Direct IC-STAT testing of stools is rapid, easily performed, and specific but is insufficiently sensitive to exclude the possibility of infection with E. coli O157:H7. Performing the IC-STAT test with a broth culture increases its sensitivity. However, attempts to recover E. coli O157:H7 by culture should not be abandoned but, rather, should be increased when the IC-STAT test result is positive.
PCR for Diagnosis of Paracoccidioidomycosis
Authors: Gomes Glauce M. , Cisalpino Patrícia S. , Taborda Carlos P. , de Camargo Zoilo P.
Published: December 2019

A PCR assay based on oligonucleotide primers derived from the sequence of the gene coding for the 43,000-Da (gp43) antigen was developed to detect Paracoccidioides brasiliensis DNA in sputa. In the standardized conditions, it could detect 10 cells/ml of sputum, providing sufficient accuracy to be useful for diagnosis of paracoccidioidomycosis.
Molecular Cloning, Expression, and Antigenicity of Seto Virus Belonging to Genogroup I Norwalk-Like Viruses
Authors: Kobayashi Shinichi , Sakae Kenji , Suzuki Yasumoto , Shinozaki Kuniko , Okada Mineyuki , Ishiko Hiroaki , Kamata Kunio , Suzuki Kenji , Natori Katsuro , Miyamura Tatsuo , Takeda Naokazu
Published: December 2019

The viral capsid protein of the Seto virus (SeV), a Japanese strain of genogroup I Norwalk-like viruses (NLVs), was expressed as virus-like particles using a baculovirus expression system. An antigen detection enzyme-linked immunosorbent assay based on hyperimmune antisera to recombinant SeV was highly specific to homologous SeV-like strains but not heterologous strains in stools, allowing us type-specific detection of NLVs.
The Papillomavirus Minor Capsid Protein, L2, Induces Localization of the Major Capsid Protein, L1, and the Viral Transcription/Replication Protein, E2, to PML Oncogenic Domains
Authors: Day Patricia M. , Roden Richard B. S. , Lowy Douglas R. , Schiller John T.
Published: December 2019

We have used immunofluorescent staining and confocal microscopy to examine the subcellular localization of structural and nonstructural bovine papillomavirus (BPV) proteins in cultured cells that produce infectious virions. When expressed separately, L1, the major capsid protein, showed a diffuse nuclear distribution while L2, the minor capsid protein, was found to localize to punctate nuclear regions identified as promonocytic leukemia protein (PML) oncogenic domains (PODs). Coexpression of L1 and L2 induced a relocation of L1 into the PODs, leading to the colocalization of L1 and L2. The effect of L2 expression on the distribution of the nonstructural viral proteins E1 and E2, which are required for maintenance of the genome and viral DNA synthesis, was also examined. The localization of the E1 protein was unaffected by L2 expression. However, the pattern of anti-E2 staining was dramatically altered in L2-expressing cells. Similar to L1, E2 was shifted from a dispersed nuclear locality into the PODs and colocalized with L2. The recruitment of full-length E2 by L2 occurred in the absence of other viral components. L2 was shown previously to be essential for the generation of infectious BPV. Our present results provide evidence for a role for L2 in the organization of virion components by recruiting them to a distinct nuclear domain. This L2-dependent colocalization probably serves as a mechanism to promote the assembly of papillomaviruses either by increasing the local concentration of virion constituents or by providing the physical architecture necessary for efficient packaging and assembly. The data also suggest a role for a nonstructural viral protein, E2, in virion assembly, specifically the recruitment of the viral genome to the sites of assembly, through its high-affinity interaction with specific sequences in the viral DNA.
The 131-Amino-Acid Repeat Region of the Essential 39-Kilodalton Core Protein of Fowlpox Virus FP9, Equivalent to Vaccinia Virus A4L Protein, Is Nonessential and Highly Immunogenic
Authors: Boulanger Denise , Green Philip , Smith Tim , Czerny Claus-Peter , Skinner Michael A.
Published: December 2019

The immunodominant, 39,000-molecular weight core protein (39K protein) of fowlpox virus (FP9 strain), equivalent to the vaccinia virus A4L gene product, contains highly charged domains at each end of the protein and multiple copies of a 12-amino-acid serine-rich repeat sequence in the middle of the protein. Similar repeats were also detected in other fowlpox virus strains, suggesting that they might confer a selective advantage to the virus. The molloscum contagiosum virus homolog (MC107L) also contains repeats, unlike the vaccinia virus protein. The number of repeats in the fowlpox virus protein does not seem to be crucial, since some strains have a different number of repeats, as shown by the difference in the size of the protein in these strains. The repeat region could be deleted, indicating that it is not essential for replication in vitro. It was not possible to delete the entire 39K protein, indicating that it was essential (transcriptional control signals for the flanking genes were left intact). The repeat region is partly responsible for the immunodominance of the protein, but the C-terminal part of the protein also contains highly antigenic linear epitopes. A role for the 39K protein in immune system modulation is discussed.
The N Terminus of Rotavirus VP2 Is Necessary for Encapsidation of VP1 and VP3
Authors: Zeng Carl Q.-Y. , Estes Mary K. , Charpilienne Annie , Cohen Jean
Published: December 2019

The innermost core of rotavirus is composed of VP2, which forms a protein layer that surrounds the two minor proteins VP1 and VP3, and the genome of 11 segments of double-stranded RNA. This inner core layer surrounded by VP6, the major capsid protein, constitutes double-layered particles that are transcriptionally active. Each gene encoding a structural protein of double-layered particles has been cloned into baculovirus recombinants and expressed in insect cells. Previously, we showed that coexpression of different combinations of the structural proteins of rotavirus double-layered particles results in the formation of virus-like particles (VLPs), and each VLP containing VP1, the presumed RNA-dependent RNA polymerase, possesses replicase activity as assayed in an in vitro template-dependent assay system (C. Q.-Y. Zeng, M. J. Wentz, J. Cohen, M. E. Estes, and R. F. Ramig, J. Virol. 70:2736–2742, 1996). This work reports construction and characterization of VLPs containing a truncated VP2 (VPΔ2, containing amino acids [aa] Met-93 to 880). Expression of VPΔ2 alone resulted in the formation of single-layered Δ2-VLPs. Coexpression of VPΔ2 with VP6 produced double-layered Δ2/6-VLPs. VLPs formed by coexpression of VPΔ2 and VP1 or VP3, or both VP1 and VP3, resulted in the formation of VLPs lacking both VP1 and VP3. The presence of VP6 with VPΔ2 did not result in encapsidation of VP1 and VP3. To determine the domain of VP2 required for binding VP1, far-Western blot analyses using a series of truncated VP2 constructs were performed to test their ability to bind VP1. These analyses showed that (i) full-length VP2 (aa 1 to 880) binds to VP1, (ii) any N-terminal truncation lacking aa 1 to 25 fails to bind VP1, and (iii) a C-terminal 296-aa truncated VP2 construct (aa 1 to 583) maintains the ability to bind VP1. These analyses indicate that the N terminus of rotavirus VP2 is necessary for the encapsidation of VP1 and VP3.
Cells with High Cyclophilin A Content Support Replication of Human Immunodeficiency Virus Type 1 Gag Mutants with Decreased Ability To Incorporate Cyclophilin A
Authors: Ackerson Bradley , Rey Osvaldo , Canon Jude , Krogstad Paul
Published: December 2019

Gag polyprotein-mediated incorporation of cellular cyclophilin A (CyPA) into virions is essential for the formation of infectious human immunodeficiency virus type 1 (HIV-1) virions. Either a point mutation in Gag (P222A) or drugs which bind CyPA decrease virion incorporation of CyPA and interfere with HIV-1 replication. We have found that lymphoid cells varied greatly in their CyPA content and that cells with high CyPA content supported the replication of P222A HIV-1 Gag mutants. These experiments demonstrated that a higher cellular CyPA content of some cells was able to compensate for the decreased binding affinity of P222A mutant Gag for CyPA, allowing virus replication to occur.