bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a Firefly Luciferase-Based Assay for Determining Antimicrobial Susceptibility ofMycobacterium aviumsubsp.paratuberculosis
Authors: Williams Stephanie L. , Harris N. Beth , Barletta Raúl G.
Published: December 2019

Paratuberculosis (Johne’s disease) is a fatal disease of ruminants for which no effective treatment is available. Presently, no drugs againstMycobacterium aviumsubsp.paratuberculosis(M. paratuberculosis), the causative agent of Johne’s disease, are approved for use in livestock. Additionally,M. paratuberculosishas been linked to a human chronic granulomatous ileitis (Crohn’s disease). To assist in the evaluation of antimicrobial agents with potential activity againstM. paratuberculosis, we have developed a firefly luciferase-based assay for the determination of drug susceptibilities. The microorganism used wasM. paratuberculosisK-10(pYUB180), a clinical isolate carrying a plasmid with the firefly luciferase gene. The MICs determined by the broth macrodilution method were as follows: amikacin, 2 μg/ml; Bay y 3118, 0.015 μg/ml; clarithromycin, 1.25 μg/ml;d-cycloserine, 25 μg/ml; ethambutol, 20 μg/ml; and rifabutin, 0.5 μg/ml. The strain was resistant to isoniazid and kanamycin. The results obtained by the luciferase assay were identical or fell within 1 doubling dilution. These results suggest that a combination of amikacin, clarithromycin, and rifabutin may be the most efficacious therapy for the treatment ofM. paratuberculosisinfections and that the use of fluoroquinolone class of antibiotics deserves further consideration. We demonstrate that the luciferase drug susceptibility assay is reliable forM. paratuberculosisand gives results within 7 days, whereas the broth macrodilution method requires 14 days.
High-Throughput Real-Time Reverse Transcription-PCR Quantitation of Hepatitis C Virus RNA
Authors: Martell María , Gómez Jordi , Esteban Juan I. , Sauleda Silvia , Quer Josep , Cabot Beatriz , Esteban Rafael , Guardia Jaime
Published: December 2019

We describe a rapid and reproducible method for assessment of the hepatitis C virus (HCV) load in serum samples. The method combines Taqman technology (Roche) and the ABI Prism 7700 (Perkin Elmer) real-time sequence detection system. We have optimized a single-tube reverse transcription-PCR (RT-PCR) that contains a dual-labeled fluorogenic probe to quantify the 5′ noncoding region (5′ NCR) of HCV. The probe contains a fluorescent reporter at the 5′ end and a fluorescent quencher at the 3′ end. The use of such a probe combined with the 5′-3′ nuclease activity of
Taq
polymerase allows direct quantitation of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. For accurate quantitation of the number of copies of HCV in samples containing unknown quantities, we have used serial dilutions of a synthetic 5′ NCR RNA standard of HCV that was previously quantified with an isotopic tracer. The method has a 5-log dynamic range (10
3
to 10
7
). The coefficient of regression of the standard curve was, on average, 0.98. The intra-assay and the interassay coefficients of variation of the threshold cycle were 1% and 6.2%, respectively. Seventy-nine RNA samples from the sera of infected patients were quantified by this method. Comparison of the results with those obtained by other quantitation methods (the Quantiplex 2.0 branched-DNA assay and the Superquant assay from the National Genetics Institute) revealed a significant correlation with all of the results. The mean values were also statistically comparable. In conclusion, the high sensitivity, simplicity, and reproducibility of the real-time HCV RNA quantitation which allows the screening of large numbers of samples, combined with its wide dynamic range, make this method especially suitable for monitoring of the viral load during therapy and tailoring of treatment schedules.
Clinical Evaluation of an In-House Reverse Transcription-Competitive PCR for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Zazzi Maurizio , Romano Laura , Catucci Marinunzia , Venturi Giulietta , De Milito Angelo , Valensin Pier E.
Published: December 2019

An in-house reverse transcription (RT)-competitive PCR (RT-cPCR) for the quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma samples was developed and validated. The procedure involves (i) extraction of RNA with spin columns, (ii) ready-to-use bead-mediated RT, (iii) competitive PCR in a microtiter plate, (iv) agarose gel electrophoresis of the reaction products, and (v) densitometric analysis of the digitized image of the gel. Quadruplicate tests and dilution studies showed that the sensitivity and intertest coefficient of variability of the RT-cPCR are comparable to those of the reference AMPLICOR HIV-1 MONITOR test. The results obtained by the two assays with a panel of 45 clinical samples were in good agreement (mean difference, 0.36 ± 0.25 log units). Analysis of 1,982 clinical samples by the in-house RT-cPCR yielded the typical range of plasma HIV-1 RNA levels with the expected inverse correlation between CD4 counts and HIV-1 RNA titers. In addition, testing of plasma from 36 subjects at weeks 0 and 4 with respect to the time of initiation of protease inhibitor therapy detected a significant decrease in HIV-1 viremia. The mean reduction in the HIV-1 RNA level was 0.914 log unit for those receiving saquinavir (
P
= 0.0210), 1.584 log units for those receiving indinavir (
P
= 0.0047), and 1.904 log units for those receiving ritonavir (
P
< 0.0001). The in-house RT-cPCR assay is simple to develop and perform and allows quantitation of HIV-1 RNA in 100 to 200 samples per operator per week. Since the cost is 1/8 to 1/10 of those of reference commercial assays, this procedure could be conveniently used in medium-scale laboratories.
Evaluation of a Colorimetric PCR-Based Assay To Diagnose
Plasmodium falciparum
Malaria in Travelers
Authors: Zhong Kathleen J. Y. , Kain Kevin C.
Published: December 2019

New diagnostic tests are needed to facilitate the diagnosis of
Plasmodium falciparum
malaria in the returned traveler. We performed a blinded evaluation of a nonisotopic colorimetric PCR-based assay (Digene SHARP Signal System) and compared the results with those obtained by microscopy and nested PCR for the detection of
P. falciparum
malaria in 150 febrile travelers. By using nested PCR as the reference standard, the colorimetric assay had a sensitivity of 100% and a specificity of 95.4% for the detection of
P. falciparum
. This PCR-based nonisotopic assay is a rapid, sensitive, and specific method for the detection of falciparum malaria in returned travelers.
Rapid Detection of a
Schistosoma mansoni
Circulating Antigen Excreted in Urine of Infected Individuals by Using a Monoclonal Antibody
Authors: Attallah Abdelfattah M. , Ismail Hisham , El Masry Samir A. , Rizk Hassan , Handousa Aia , El Bendary Mahmoud , Tabll Ashraf , Ezzat Farouk
Published: December 2019

Schistosoma
circulating antigens were used to indicate the infection intensity and to assess cure. An immunoglobulin G2a (IgG2a) mouse monoclonal antibody was used in a fast dot-enzyme-linked immunosorbent assay (ELISA; FDA) for rapid and simple diagnosis of schistosomiasis in the field. Seven hundred Egyptians were parasitologically examined for
Schistosoma mansoni
and other parasitic infections. A rectal biopsy was done as a “gold standard” for individuals showing no
S. mansoni
eggs in their feces. Egg counts were obtained by the Kato smear method for only 100 of 152 individuals with eggs in their feces. Specific anti-schistosome IgG antibodies were evaluated in sera by ELISA. Urine samples from the 700 individuals were tested by FDA for detection of the circulating antigen. The assay showed a sensitivity of 93% among 433 infected individuals and a specificity of 89% among 267 noninfected individuals. FDA showed the highest efficiency of antigen detection (91%) compared with the efficiency of antibody detection by ELISA (75%) and stool analysis (60%). In addition, FDA detected infected patients with 20 eggs/g of feces. Also, the sensitivity of FDA ranged from 90 to 94% among samples from patients with different clinical stages of schistosomiasis. All the assay steps can be completed within 30 min at room temperature for 96 urine samples. The monoclonal antibody identified a 74-kDa antigen in different antigenic extracts of
S. mansoni
and
Schistosoma haematobium
and in the urine of infected individuals. In addition, a 30-kDa degradation product was identified only in the urine samples. On the basis of these results, FDA should be used as a rapid tool for the sensitive and specific diagnosis of
Schistosoma
infection.
Pulsed-Field Gel Electrophoresis Is More Efficient than Ribotyping and Random Amplified Polymorphic DNA Analysis in Discrimination ofPasteurella haemolyticaStrains
Authors: Kodjo Angeli , Villard Laurence , Bizet Chantal , Martel Jean-Louis , Sanchis Richard , Borges Evelyne , Gauthier Dominique , Maurin Françoise , Richard Yves
Published: December 2019

One hundred thirty-three strains ofPasteurella haemolyticaof both biotypes (90 and 43 strains of biotypes A and T, respectively) and almost all the serotypes were subjected to ribotyping, random amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE) analysis for epidemiological purposes. A total of 15 patterns recorded as ribotypes HA to HO were found for theP. haemolyticabiotype A strains, with ribotypes HA, HC, and HD being encountered most often (66 strains [74%]); and 20 ribotypes, designated HA′ to HT′, that were clearly distinct from those observed for biotype A strains were observed for strains of biotype T. RAPD analysis generated a total of 44 (designated Rp1 to Rp44) and 15 (designated Rp1′ to Rp 15′) unique RAPD patterns for biogroup A and biogroup T, respectively. Analysis of the data indicated that a given combined ribotype-RAPD pattern could be observed for biotype A strains of different serotypes, whatever the zoological or geographic origin, whereas this was not the case for biotype T strains. PFGE appeared to be more efficient in strain discrimination since selected strains from various zoological or geographical origins harboring the same ribotype-RAPD group were further separated into unique entities.
Stability of
Mycobacterium tuberculosis
IS
6110
Restriction Fragment Length Polymorphism Patterns and Spoligotypes Determined by Analyzing Serial Isolates from Patients with Drug-Resistant Tuberculosis
Authors: Niemann Stefan , Richter Elvira , Rüsch-Gerdes Sabine
Published: December 2019

The stability of
Mycobacterium tuberculosis
IS
6110
fingerprint patterns and spoligotypes has been assessed by analyzing serial isolates from patients with drug-resistant tuberculosis. Altogether, 165
M. tuberculosis
isolates obtained from 56 patients have been analyzed. The time spans between the first and the last or a changed isolate from one patient ranged from 1 to 772 days. Among the 56 patients, 5 (9%) were infected with isolates with changes in their IS
6110
fingerprint patterns. According to the total number of strains analyzed, 5% of the subsequent isolates showed variations in their IS
6110
restriction fragment length polymorphism patterns compared to the pattern of the first isolates. Up to 10 isolates from one patient sampled at time intervals of up to 772 days with no changes in their IS
6110
patterns have been analyzed. A statistically significant correlation could be found between changes in insertion sequence (IS) patterns and the increased time intervals over which the isolates were obtained, whereas changes in IS patterns are not correlated to changes in the drug resistance of the isolates. In contrast to the observed variations in IS
6110
fingerprint patterns, no changes in the spoligotypes of the isolates analyzed could be found. In conclusion, our results confirm that the IS
6110
fingerprint patterns of
M. tuberculosis
isolates have high degrees of stability. Compared to IS
6110
, the direct repeat (DR) region, which is the basis for spoligotyping, has a lower rate of change. Partial deletions, e.g., deletions induced by homologous recombination between the repetitive DR elements, could not be detected in this study.
Molecular and Phenotypic Characterization of Genotypic
Candida albicans
Subgroups and Comparison with
Candida dubliniensis
and
Candida stellatoidea
Authors: McCullough Michael J. , Clemons Karl V. , Stevens David A.
Published: December 2019

There have been increased reports of the isolation of unusual genotypic groups of
Candida albicans
(groups C and D) based on a well-defined genotypic method; this method uses cellular DNA digested with the
Eco
RI enzyme and the restriction fragment length polymorphisms (RFLPs) generated by agarose gel electrophoresis. The aim of the present study was to use additional molecular tools to characterize these unusual strains and to compare them with authentic strains of
C. dubliniensis
, a recently delineated species, and type I
C. stellatoidea
. The RFLPs of PCR products generated from the intergenic transcribed spacer (ITS) region did not differentiate among
C. albicans
genotypes A, B, and C and type I
C. stellatoidea
. However, this method did differentiate the
C. albicans
genotype D strains, which were identical to
C. dubliniensis
. The RFLPs generated by
Hae
III digestion of the PCR products of the V3 region of the 25S rRNA gene (rDNA) could differentiate the same groups as RFLP analysis of the PCR amplicon of the ITS region.
C. albicans
genotype B isolates have been shown to have a transposable intron in the 25S rDNA, whereas genotype A isolates do not;
C. dubliniensis
strains also have an intron that is larger than that in genotype B
C. albicans
strains but that is in the same location. PCR designed to span this region resulted in a single product for
C. albicans
genotype A (450 bp), B (840 bp), type 1
C. stellatoidea
(840 bp), and
C. dubliniensis
(1,080 bp), whereas the
C. albicans
genotype C isolates had two major products (450 and 840 bp). All
C. albicans
genotype D isolates gave a PCR product identical to that given by
C. dubliniensis
. These results indicate that those strains previously designated
C. albicans
genotype D are in fact
C. dubliniensis
, that no differences were found between type 1
C. stellatoidea
and
C. albicans
genotype B strains, and that the
C. albicans
genotype C strains appear to have the transposable intron incompletely inserted throughout the ribosomal repeats in their genomes. The results of the antifungal susceptibility testing of 105 of these strains showed that, for fluconazole, strains of
C. dubliniensis
were significantly more susceptible than strains of each of the
C. albicans
genotypes (genotypes A, B, and C). The flucytosine susceptibility results indicated that strains of
C. albicans
genotype A were significantly less susceptible than either
C. albicans
genotype B or
C. albicans
genotype C strains. These results indicate that there is a correlation between the
Candida
groups and antifungal susceptibility.
Comparison of NucliSens and Roche Monitor Assays for Quantitation of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Dyer John R. , Pilcher Christopher D. , Shepard Robin , Schock Jody , Eron Joseph J. , Fiscus Susan A.
Published: December 2019

We compared the performance of Organon Teknika’s NucliSens and Roche Diagnostic Systems’ Monitor quantitative human immunodeficiency type 1 RNA assays. Both had similar linearity and sensitivity over most of the dynamic range of the assays, although the Monitor assay was superior at the low range of RNA values while the NucliSens assay was more consistent at higher RNA values. NucliSens generally showed less interassay variability.
Comparison of Randomly Amplified Polymorphic DNA Analysis and Pulsed-Field Gel Electrophoresis for Typing of
Moraxella catarrhalis
Strains
Authors: Vu-Thien Hoang , Dulot Carole , Moissenet Didier , Fauroux Brigitte , Garbarg-Chenon Antoine
Published: December 2019

Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) for the analysis of 13
Moraxella catarrhalis
isolates, 11 successive strains isolated from sputa of five children and 2 isolates obtained the same day from twins, were compared. RAPD and PFGE both yielded nine types from the 13 isolates, showing a chronic colonization with one strain in three patients and a successive colonization with different strains in two patients. The promising results obtained with RAPD should be confirmed with a larger number of strains, but RAPD seems as suitable as PFGE for the typing of
M. catarrhalis
.
Detection of
Cryptosporidium parvum
in Horses: Thresholds of Acid-Fast Stain, Immunofluorescence Assay, and Flow Cytometry
Authors: Cole D. J. , Snowden K. , Cohen N. D. , Smith R.
Published: December 2019

Feces collected from three asymptomatic horses and seeded with
Cryptosporidium parvum
oocysts (10
1
to 10
6
/g of feces) were evaluated by acid-fast staining (AF), an immunofluorescent antibody (IFA) technique, and flow cytometry. The thresholds of detection were 5 × 10
5
oocysts/g of feces for the IFA and AF techniques and 5 × 10
4
oocysts/g for flow cytometry.
Safe Determination of Susceptibility of
Mycobacterium tuberculosis
to Antimycobacterial Agents by Flow Cytometry
Authors: Moore Andrea V. , Kirk Scott M. , Callister Steven M. , Mazurek Gerald H. , Schell Ronald F.
Published: December 2019

We showed previously that susceptibility testing for
Mycobacterium tuberculosis
labeled with fluorescein diacetate could be accomplished rapidly by using flow cytometry. However, safety was a major concern because mycobacteria were not killed prior to flow cytometric analysis. In this study, we developed a biologically safe flow cytometric susceptibility test that depends on detection and enumeration of actively growing
M. tuberculosis
organisms in drug-free and antimycobacterial agent-containing medium. The susceptibilities of 17 clinical isolates of
M. tuberculosis
to ethambutol, isoniazid, and rifampin were tested by the agar proportion and flow cytometric methods. Subsequently, all flow cytometric susceptibility test samples were inactivated by exposure to paraformaldehyde before analysis with a flow cytometer. Agreement between the results from the two methods was 98%. In addition, the flow cytometric results were available 72 h after the initiation of testing. The flow cytometric susceptibility assay is safe, simple to perform, and more rapid than conventional test methods, such as the BACTEC system and the proportion method.
Prospective Comparison of Whole-Blood- and Plasma-Based Hepatitis C Virus RNA Detection Systems: Improved Detection Using Whole Blood as the Source of Viral RNA
Authors: Stapleton Jack T. , Klinzman Donna , Schmidt Warren N. , Pfaller Michael A. , Wu Ping , LaBrecque Douglas R. , Han Jian-qiu , Phillips Mary Jeanne Perino , Woolson Robert , Alden Beth
Published: December 2019

We previously demonstrated that whole blood contains significantly more hepatitis C virus (HCV) RNA than plasma. To validate the whole-blood-based HCV RNA detection method, a prospective comparison of HCV RNA detection in whole blood and plasma from 50 patients with chronic liver disease was undertaken. Whole-blood and plasma aliquots were independently tested for HCV RNA by reverse transcriptase (RT) PCR assay, and plasma was tested by the Roche Amplicor assay. HCV RNA was detected in 35 of 50 (70%) whole-blood samples by RT-PCR but in only 26 of 50 (52%) plasma samples tested by the Amplicor assay (
P
< 0.01). HCV RNA was detected in 85% of HCV antibody-positive patients by the whole-blood method compared with 74% of plasma samples by the Amplicor method. The five HCV antibody-positive subjects who were negative by whole-blood-based RT-PCR assay were all receiving interferon therapy and had normal transaminases at the time of testing. HCV RNA was detected in 38% of HCV antibody-negative subjects by the whole-blood-based RT-PCR assay compared with 6.25% of these patients by the Amplicor assay (
P
< 0.05). There were nine samples in which HCV RNA was detected in whole blood but the Amplicor test was negative. Eight of the nine RNAs prepared from these whole-blood samples tested positive in the Amplicor assay, thus confirming the specificity of our results. This study demonstrates that whole-blood-based HCV RNA detection is more sensitive than currently available commercial tests and that whole-blood RNA is suitable for use in commercial assays.
Dissemination of Two Methicillin-Resistant
Staphylococcus aureus
Clones Exhibiting Negative Staphylase Reactions in Intensive Care Units
Authors: Hsueh Po-Ren , Teng Lee-Jene , Yang Pan-Chyr , Pan Hui-Ju , Chen Yu-Chi , Wang Li-Hua , Ho Shen-Wu , Luh Kwen-Tay
Published: December 2019

From December 1997 to March 1998, 25 methicillin-resistant
Staphylococcus aureus
(MRSA) isolates exhibiting negative Staphylase (Oxoid Ltd., Basingstoke, England) reactions were identified from various clinical specimens from 13 patients in six intensive care units (ICUs) or in wards following a stay in an ICU at the National Taiwan University Hospital. The characteristics of these isolates have not been previously noted in other MRSA isolates from this hospital. Colonies of all these isolates were grown on Trypticase soy agar supplemented with 5% sheep blood and were nonhemolytic and unpigmented. Seven isolates, initially reported as
Staphylococcus haemolyticus
(5 isolates) and
Staphylococcus epidermidis
(2 isolates) by the routine identification scheme and with the Vitek GPI system (bioMerieux Vitek, Inc., Hazelwood, Mo.), were subsequently identified as
S. aureus
by positive tube coagulase tests, standard biochemical reactions, and characteristic cellular fatty acid chromatograms. The antibiotypes obtained by the E test, coagulase types, restriction fragment length polymorphism profiles of the staphylococcal coagulase gene, and random amplified polymorphic DNA patterns generated by arbitrarily primed PCR of the isolates disclosed that two major clones disseminated in the ICUs. Clone 1 (16 isolates) was resistant to clindamycin and was susceptible to trimethoprim-sulfamethoxazole (TMP-SMZ) and was coagulase type II. Clone 2 (eight isolates) was resistant to clindamycin and TMP-SMZ and was coagulase type IV. These two epidemic clones from ICUs are unique and underline the need for caution in identifying MRSA strains with colonial morphologies not of the typical type and with negative Staphylase reactions.
Typing of Clinical
Mycobacterium avium
Complex Strains Cultured during a 2-Year Period in Denmark by Using IS
1245
Authors: Bauer Jeanett , Andersen Åse B. , Askgaard Dorthe , Giese Sten B. , Larsen Birger
Published: December 2019

In the present study restriction fragment length polymorphism analyses with the recently described insertion sequence IS
1245
as a probe was performed with clinical
Mycobacterium avium
complex strains cultured in Denmark during a 2-year period. The overall aim of the study was to disclose potential routes of transmission of these microorganisms. As a first step, the genetic diversity among isolates from AIDS patients and non-human immunodeficiency virus (HIV)-infected patients was described. In addition, a number of isolates from nonhuman sources cultured during the same period were analyzed and compared to the human isolates. A total of 203 isolates from AIDS patients (
n
= 90), non-HIV-infected patients (
n
= 91), and nonhuman sources (
n
= 22) were analyzed. The presence of IS
1245
was restricted to
Mycobacterium avium
subsp.
avium
isolates. The majority of human isolates had large numbers of IS
1245
copies, while nonhuman isolates could be divided into a high-copy-number group and a low-copy-number group. Groups of identical strains were found to be geographically widespread, comprising strains from AIDS patients as well as strains from non-HIV-infected patients. Samples of peat (to be used as potting soil) and veterinary samples were found to contain viable
M. avium
isolates belonging to genotypes also found in humans.
TechLab and Alexon
Giardia
Enzyme-Linked Immunosorbent Assay Kits Detect Cyst Wall Protein 1
Authors: Boone James H. , Wilkins Tracy D. , Nash Theodore E. , Brandon Jill E. , Macias Elizabeth A. , Jerris Robert C. , Lyerly David M.
Published: December 2019

A
Giardia lamblia
antigen detected by the TechLab
Giardia Test
(TechLab, Inc., Blacksburg, Va.) and the Alexon ProSpecT
Giardia
microplate assay (Alexon, Inc., Sunnyvale, Calif.) was purified by immunoaffinity chromatography from supernatant fluids of encystment cultures. Two major proteins (
M
r
22,000 and 26,000) were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining that did not resemble the GSA65 antigen reportedly detected by the Alexon test. These proteins reacted intensely with the monoclonal antibodies used in both commercial enzyme-linked immunosorbent assays (ELISAs). Both proteins had identical N-terminal amino acid sequences and were identified as cyst wall protein 1 (CWP1). The 26-kDa form appeared early during encystment followed by the appearance of the 22-kDa form. Recombinant CWP1 (
M
r
26,000) was strongly positive in both commercial tests. CWP1 was stable in human stool specimens, resistant to degradation by proteases and
N
- and
O
-glycanases, and unaffected by oxidation with sodium periodate. Two minor proteins with
M
r
s of 32,000 and 39,000 were detected in CWP1 preparations by using a sensitive fluorescent protein stain. Both were identified as CWP2, and neither reacted with the monoclonal antibodies from the commercial tests. We analyzed 535 stool specimens for CWP1 by using both commercial ELISAs and resolved discrepant results by using routine ova and parasite examination (O&P) and on immunofluorescence antibody assay. The presence of CWP1 correlated well between both ELISAs (98.7% correlation). Our results demonstrate that both commercial ELISAs detect CWP1, which is a useful diagnostic marker because it is highly stable, is secreted in large amounts by encysting trophozoites, and correlates well with O&P.
Application of
pbp1A
PCR in Identification of Penicillin-Resistant
Streptococcus pneumoniae
Authors: du Plessis Mignon , Smith Anthony M. , Klugman Keith P.
Published: December 2019

A seminested PCR assay, based on the amplification of the pneumococcal
pbp1A
gene, was developed for the detection of penicillin resistance in clinical isolates of
Streptococcus pneumoniae
. The assay was able to differentiate between intermediate (MICs = 0.25 to 0.5 μg/ml) and higher-level (MICs = ≥1 μg/ml) resistance. Two species-specific primers, 1A-1 and 1A-2, which amplified a 1,043-bp region of the
pbp1A
penicillin-binding region, were used for pneumococcal detection. Two resistance primers, 1A-R1 and 1A-R2, were designed to bind to altered areas of the
pbp1A
gene which, together with the downstream primer 1A-2, amplify DNA from isolates with penicillin MICs of ≥0.25 and ≥1 μg/ml, respectively. A total of 183 clinical isolates were tested with the
pbp1A
assay. For 98.3% (180 of 183) of these isolates, the PCR results obtained were in agreement with the MIC data. The positive and negative predictive values of the assay were 100 and 91%, respectively, for detecting strains for which the MICs were ≥0.25 μg/ml and were both 100% for strains for which the MICs were ≥1 μg/ml.
Rapid Detection of Epidemic Strains of Methicillin-ResistantStaphylococcus aureus
Authors: Wichelhaus Thomas A. , Kern Sylvia , Schäfer Volker , Brade Volker
Published: December 2019

Fifty methicillin-resistantStaphylococcus aureus(MRSA) initial isolates obtained from patients hospitalized in the orthopedic clinic of the Frankfurt University Hospital and 150 methicillin-sensitiveStaphylococcus aureus(MSSA) isolates were investigated in this study to determine whether the Slidex Staph-Kit is capable of differentiating between MRSA and MSSA owing to its unique performance characteristics. The Slidex Staph-Kit is a combined latex hemagglutination test designed to detect clumping factor, protein A, and a specific surface immunogen forS. aureus. Clumping factor-positive strains cause erythrocytes sensitized with fibrinogen to hemagglutinate, thereby resulting in visible red clumps.S. aureusstrains deficient in clumping factor agglutinate latex particles sensitized with specific antibodies against surface proteins ofS. aureus, thereby resulting in visible white clumps. Our results demonstrate that white clumping has a 99% specificity as well as a 98% positive predictive value for MRSA. Clumping factor-negative MRSA, which have been reported to occur in several countries, are epidemic in the Frankfurt area and account for 80% of all MRSA initial isolates in the orthopedic clinic of the Frankfurt University Hospital. Genotyping of all MRSA isolates by macrorestriction analysis of chromosomal DNA revealed that 83% of clumping factor-negative MRSA are closely related to the “southern-German” epidemic strain. This is the first study demonstrating the Slidex Staph-Kit’s capability for identifying epidemic clumping factor-negativeS. aureusstrains as methicillin resistant even prior to antimicrobial susceptibility testing.
Lactobacillus
Species Identification, H
2
O
2
Production, and Antibiotic Resistance and Correlation with Human Clinical Status
Authors: Felten Annie , Barreau Claude , Bizet Chantal , Lagrange Philippe Henri , Philippon Alain
Published: December 2019

Lactobacilli recovered from the blood, cerebrospinal fluid, respiratory tract, and gut of 20 hospitalized immunocompromised septic patients were analyzed. Biochemical carbohydrate fermentation and total soluble cell protein profiles were used to identify the species. Hydrogen peroxide production was measured. Susceptibility to 19 antibiotics was tested by a diffusion method, and the MICs of benzylpenicillin, amoxicillin, imipenem, erythromycin, vancomycin, gentamicin, and levofloxacin were determined. A small number of species produced H
2
O
2
, and antibiotic susceptibilities were species related. Eighteen (90%) of the isolates were
L. rhamnosus
, one was
L. paracasei
subsp.
paracasei
, and one was
L. crispatus
.
L. rhamnosus
,
L. paracasei
subsp.
paracasei
isolates, and the type strains were neither H
2
O
2
producers nor vancomycin susceptible (MICs, ≥256 μg/ml).
L. crispatus
, as well as most of the type strains of lactobacilli which belong to the
L. acidophilus
group, was an H
2
O
2
producer and vancomycin susceptible (MICs, <4 μg/ml).
Isolation of
Abiotrophia adiacens
from a Brain Abscess Which Developed in a Patient after Neurosurgery
Authors: Biermann C. , Fries G. , Jehnichen P. , Bhakdi S. , Husmann M.
Published: December 2019

We report the case of a patient who developed a large brain abscess after neurosurgery. Cerebrospinal fluid from the abscess drainage yielded
Abiotrophia adiacens
-specific PCR products and microorganisms that were identified by conventional microbiological methods and by 16S ribosomal DNA analysis as
Abiotrophia adiacens
, which was formerly classified as a member of nutritionally variant streptococci.
Clonal Diversity of Chilean Isolates of Enterohemorrhagic
Escherichia coli
from Patients with Hemolytic-Uremic Syndrome, Asymptomatic Subjects, Animal Reservoirs, and Food Products
Authors: Rios Maritza , Prado Valeria , Trucksis Michele , Arellano Carolina , Borie Consuelo , Alexandre Marcela , Fica Alberto , Levine Myron M.
Published: December 2019

To determine clonal relationship among Chilean enterohemorrhagic
Escherichia coli
(EHEC) strains from different sources (clinical infections, animal reservoirs, and food), 54 EHEC isolates (44 of
E. coli
O157, 5 of
E. coli
O111, and 5 of
E. coli
O26) were characterized for virulence genes by colony blot hybridization and by pulsed-field gel electrophoresis (PFGE). By colony blotting, 12 different genotypes were identified among the 44
E. coli
O157 isolates analyzed, of which the genetic profile
stx
1
+
stx
2
+

hly
+
eae

+
was the most prevalent. All human O157 strains that were associated with sporadic cases of hemolytic-uremic syndrome (HUS) carried both the
stx
1
and
stx
2
toxin-encoding genes and were
eaeA
positive. Only 9 of 13 isolates from human controls were
stx
1
+
stx
2
+
, and 8 carried the
eaeA
gene. Comparison of profiles obtained by PFGE of
Xba
I-digested genomic DNA showed a great diversity among the
E. coli
O157 isolates, with 37 different profiles among 39 isolates analyzed. Cluster analysis of PFGE profiles showed a wide distribution of clinical isolates obtained from HUS cases and asymptomatic individuals and a clonal relationship among O157 isolates obtained from HUS cases and pigs. Analysis of virulence genes showed that a correlation exists among strains with the genotype
stx
1
+
stx
2
+
eae
+
and pathogenic potential. A larger difference in the PFGE restriction patterns was observed among the EHEC strains of serogroups O26 and O111. These results indicate that several different EHEC clones circulate in Chile and suggest that pigs are an important animal reservoir for human infections by EHEC. Guidelines have been proposed for better practices in the slaughter of animals in Chile.
Variation in Structure and Location of VanA Glycopeptide Resistance Elements among Enterococci from a Single Patient
Authors: Tremlett Catherine H. , Brown Derek F. J. , Woodford Neil
Published: December 2019

Forty-six VanA glycopeptide-resistant enterococci (GRE) from a single patient were investigated for variation in structure and location of VanA resistance elements. Together with identification to species level and pulsed-field gel electrophoresis, these data divided the GRE into 10 groups and subgroups. Combining data in this manner appears helpful when investigating the epidemiology of GRE.
Identification of Contaminating Fungal DNA Sequences in Zymolyase
Authors: Rimek Dagmar , Garg Amar P. , Haas Walter H. , Kappe Reinhard
Published: December 2019

When different preparations of Zymolyase were included in the pretreatment protocol of a panfungal PCR assay using a primer system for the 18S rRNA gene, an amplification product occurred in negative controls. The amplified fragment showed 100.0% sequence identity to the
Saccharomyces
sensu stricto complex and
Kluyveromyces lodderae
. Lyticase, lysing enzymes, and proteinase K appeared to be free from fungal DNA.
Quality Control in Nucleic Acid Amplification Methods: Use of Elementary Probability Theory
Authors: Shapiro Daniel S.
Published: December 2019

Since it is not possible to state with certainty that contamination has occurred during a nucleic acid amplification assay in the absence of a positive result for a negative control, methods of elementary probability theory are used to illustrate how to identify those runs in which the possibility of contamination should be considered. The use of binomial and Poisson distributions and an analysis of clusters are presented with illustrative examples to demonstrate their use.
Identification of Seven
Treponema
Species in Health- and Disease-Associated Dental Plaque by Nested PCR
Authors: Willis S. G. , Smith K. S. , Dunn V. L. , Gapter L. A. , Riviere K. H. , Riviere G. R.
Published: December 2019

Species-specific nested PCR was used to detect
Treponema amylovorum
,
Treponema denticola
,
Treponema maltophilum
,
Treponema medium
,
Treponema pectinovorum
,
Treponema socranskii
, and
Treponema vincentii
in dental plaque. Subjects with periodontitis harbored all species, but
T. pectinovorum
and
T. vincentii
were not found in plaque from disease-free subjects.
Comparison of Large Restriction Fragments of
Mycobacterium avium
Isolates Recovered from AIDS and Non-AIDS Patients with Those of Isolates from Potable Water
Authors: Aronson T. , Holtzman A. , Glover N. , Boian M. , Froman S. , Berlin O. G. W. , Hill H. , Stelma G.
Published: December 2019

We examined potable water in Los Angeles, California, as a possible source of infection in AIDS and non-AIDS patients. Nontuberculous mycobacteria were recovered from 12 (92%) of 13 reservoirs, 45 (82%) of 55 homes, 31 (100%) of 31 commercial buildings, and 15 (100%) of 15 hospitals. Large-restriction-fragment (LRF) pattern analyses were done with
Ase
I. The LRF patterns of
Mycobacterium avium
isolates recovered from potable water in three homes, two commercial buildings, one reservoir, and eight hospitals had varying degrees of relatedness to 19 clinical isolates recovered from 17 patients. The high number of
M. avium
isolates recovered from hospital water and their close relationship with clinical isolates suggests the potential threat of nosocomial spread. This study supports the possibility that potable water is a source for the acquisition of
M. avium
infections.
Human Papillomavirus (HPV) DNA Copy Number Is Dependent on Grade of Cervical Disease and HPV Type
Authors: Swan David C. , Tucker Ruth Ann , Tortolero-Luna Guillermo , Mitchell Michele Follen , Wideroff Louise , Unger Elizabeth R. , Nisenbaum Rosane A. , Reeves William C. , Icenogle Joseph P.
Published: December 2019

The association between human papillomavirus (HPV) DNA copy number and cervical disease was investigated. Viral DNA copy number for the most common high-risk HPV types in cervical cancer (types 16, 18, 31, and 45) was determined in cervical cytobrush specimens from 149 women with high-grade cervical intraepithelial neoplasias (CIN II-CIN III), 176 with low-grade CIN (CIN I), and 270 with normal cytology. Quantitative, PCR-based fluorescent assays for each of the HPV genotypes and for the β-globin gene were used. The amount of cellular DNA increased significantly with increasing disease; thus, HPV was expressed as copies per microgram of cellular DNA. The assay had a dynamic range of >10
7
, allowing documentation for the first time of the wide range of HPV copy numbers seen in clinical specimens. Median HPV DNA copy number varied by more than 10
4
among the viral types. HPV16 was present in the highest copy number; over 55% of HPV16-positive samples contained more than 10
8
copies/μg. Median copy number for HPV16 showed dramatic increases with increasing epithelial abnormality, an effect not seen with the other HPV types. HPV16 increased from a median of 2.2 × 10
7
in patients with normal cytology, to 4.1 × 10
7
in CIN I patients, to 1.3 × 10
9
copies/μg in CIN II-III patients. Even when stratified by cervical disease and viral type, the range of viral DNA copies per microgram of cellular DNA was quite large, precluding setting a clinically significant cutoff value for “high” copy numbers predictive of disease. This study suggests that the clinical usefulness of HPV quantitation requires reassessment and is assay dependent.
Use of PCR and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Techniques for Differentiation of
Prevotella intermedia
Sensu Stricto and
Prevotella nigrescens
Authors: Premaraj Thyagaseely , Kato Naoki , Fukui Katsuhito , Kato Haru , Watanabe Kunitomo
Published: December 2019

Primers were designed from 16S rRNA sequences of
Prevotella intermedia
sensu stricto and
Prevotella nigrescens
and were used to discriminate these two species by PCR. The results were compared with those from the PCR technique using primers designed from arbitrarily primed PCR products by Guillot and Mouton (E. Guillot and C. Mouton, J. Clin. Microbiol. 35:1876–1882, 1997). The specificities of both assays were studied by using
P. intermedia
ATCC 25611,
P. nigrescens
ATCC 33563, 174 clinical isolates of
P. intermedia
sensu lato, and 59 reference strains and 58 clinical isolates of other
Prevotella
species and/or common oral flora. In addition, the usefulness and reliability of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the differentiation of the two species were examined by comparing the results with those from PCR assays. The controversial lipase test for distinguishing these species was also carried out. Unambiguous differentiation was made by both PCR assays, and the results matched each other. The SDS-PAGE assay was found to misidentify a few strains tested, compared with the results of PCR assays. The lipase test was positive for both species, including the reference strains of
P. intermedia
and
P. nigrescens
. We conclude that both PCR assays are simple, rapid, reliable, and specific methods which could be used in clinical studies and that the lipase test is not valuable in the differentiation. The reliable discrimination of the two species by SDS-PAGE is questionable.
Use of Molecular Subtyping To Document Long-Term Persistence of
Corynebacterium diphtheriae
in South Dakota
Authors: Popovic Tanja , Kim Chung , Reiss Jonathan , Reeves Mike , Nakao Hiroshi , Golaz Anne
Published: December 2019

Enhanced surveillance of patients with upper respiratory symptoms in a Northern Plains community revealed that approximately 4% of them were infected by toxigenic
Corynebacterium diphtheriae
of both mitis and gravis biotypes, showing that the organism is still circulating in the United States. Toxigenic
C. diphtheriae
was isolated from five members of four households. Four molecular subtyping methods—ribotyping, multilocus enzyme electrophoresis (MEE), random amplified polymorphic DNA (RAPD), and single-strand conformation polymorphism—were used to molecularly characterize these strains and compare them to 17 archival South Dakota strains dating back to 1973 through 1983 and to 5 isolates collected from residents of diverse regions of the United States. Ribotyping and RAPD clearly demonstrated the household transmission of isolates and provided precise information on the circulation of several distinct strains within three households. By MEE, most recent and archival South Dakota strains were identified as closely related and clustered within the newly identified ET (electrophoretic type) 215 complex. Furthermore, three recent South Dakota isolates and eight archival South Dakota isolates were indistinguishable by both ribotyping and RAPD. All of these molecular methods showed that recent South Dakota isolates and archival South Dakota isolates were more closely related to each other than to the
C. diphtheriae
strains isolated in other parts of the United States or worldwide. The data also supported the improbability of importation of
C. diphtheriae
into this area and rather strongly suggest the long-term persistence of the organism in this region.
Strain Variation in Adenovirus Serotypes 4 and 7a Causing Acute Respiratory Disease
Authors: Crawford-Miksza Leta K. , Nang Roberto N. , Schnurr David P.
Published: December 2019

In order to determine the suitability of vaccine strains established in the 1960s for a new vaccine, a comprehensive study of strain variation of adenovirus serotype 4 (AV 4) and AV 7 was undertaken. A 1,500-bp region of the hexon gene containing the AV neutralization epitopes from prototype, vaccine, and community-acquired strains and from wild-type strains from military personnel that cause acute respiratory disease (ARD) was sequenced and analyzed. The whole hexon gene from prototype strains, vaccine strains, and selected isolates was sequenced. AV 7 and AV 7a were found to have distinct genotypes, and all vaccine and wild-type strains recovered from 1963 to 1997 had the AV 7a genotype. There was no significant strain variation in the neutralization epitopes of the AV 7a genotype over a 42-year period. The evolution of AV 4 was more complex, with continuous genetic drift punctuated by replacement with a new strain. The current strain of AV 4, which has been in circulation since 1995, is significantly different from the AV 4 prototype and the vaccine strains. Genetic differences were confirmed to be antigenic differences by neutralization tests, which define the new strain as an AV 4 variant. A type-specific PCR for AV 4, AV 7/7a, and AV 21 was developed, and this PCR facilitated the rapid identification of isolates from outbreaks of ARD.
Genetic Diversity of the 28-Kilodalton Outer Membrane Protein Gene in Human Isolates ofEhrlichia chaffeensis
Authors: Yu Xue-Jie , McBride Jere W. , Walker David H.
Published: December 2019

TheEhrlichia chaffeensis28-kDa outer membrane protein (p28) gene was sequenced completely by genomic walking with adapter PCR. The DNA sequence of the p28 gene was nearly identical to the previously reported sequence (N. Ohashi, N. Zhi, Y. Zhang, and Y. Rikihisa, Infect. Immun. 66:132–139, 1998), but analysis of a further 75 bp on the 5′ end of the gene revealed DNA that encoded a 25-amino-acid signal sequence. The leader sequence was removed from the N terminus of a 30-kDa precursor to generate the mature p28 protein. A monoclonal antibody (MAb), 1A9, recognizing four outer membrane proteins ofE. chaffeensis(Arkansas strain) including the 25-, 26-, 27-, and 29-kDa proteins (X.-J. Yu, P. Brouqui, J. S. Dumler, and D. Raoult, J. Clin. Microbiol. 31:3284–3288, 1993) reacted with the recombinant p28 protein. This result indicated that the four proteins recognized by MAb 1A9 were encoded by the multiple genes of the 28-kDa protein family. DNA sequence alignment analysis revealed divergence of p28 among all five human isolates ofE. chaffeensis. TheE. chaffeensisstrains could be divided into three genetic groups on the basis of the p28 gene. The first group consisted of the Sapulpa and St. Vincent strains. They had predicted amino acid sequences identical to each other. The second group contained strain 91HE17 and strain Jax, which only showed 0.4% divergence from each other. The third group contained the Arkansas strain only. The amino acid sequences of p28 differed by 11% between the first two groups, by 13.3% between the first and third groups, and by 13.1% between the second and third groups. The presence of antigenic variants of p28 among the strains ofE. chaffeensisand the presence of multiple copies of heterogeneous genes suggest a possible mechanism by whichE. chaffeensismight evade the host immune defenses. Whether or not immunization with the p28 of one strain ofE. chaffeensiswould confer cross-protection against other strains needs to be investigated.
Rapid Film-Based Determination of Antibiotic Susceptibilities of
Mycobacterium tuberculosis
Strains by Using a Luciferase Reporter Phage and the Bronx Box
Authors: Riska Paul F. , Su Ya , Bardarov Svetoslav , Freundlich Lawrence , Sarkis Gary , Hatfull Graham , Carrière Christian , Kumar Vanaja , Chan John , Jacobs William R.
Published: December 2019

Detecting antibiotic resistance in
Mycobacterium tuberculosis
is becoming increasingly important with the global recognition of drug-resistant strains and their adverse impact on clinical outcomes. Current methods of susceptibility testing are either time-consuming or costly; rapid, reliable, simple, and inexpensive methods would be highly desirable, especially in the developing world where most tuberculosis is found. The luciferase reporter phage is a unique reagent well-suited for this purpose: upon infection with viable mycobacteria, it produces quantifiable light which is not observed in mycobacterial cells treated with active antimicrobials. In this report, we describe a modification of our original assay, which allows detection of the emitted light with a Polaroid film box designated the Bronx Box. The technique has been applied to 25
M. tuberculosis
reference and clinical strains, and criteria are presented which allow rapid and simple discrimination among strains susceptible or resistant to isoniazid and rifampin, the major antituberculosis agents.