bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of
gyrA
Mutations in Quinolone-Resistant
Salmonella enterica
by Denaturing High-Performance Liquid Chromatography
Authors: Eaves Deborah J. , Liebana Ernesto , Woodward Martin J. , Piddock Laura J. V.
Published: October 2002

Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a rapid screening and identification method for DNA sequence variation detection in the quinolone resistance-determining region of
gyrA
from
Salmonella
serovars. A total of 203 isolates of
Salmonella
were screened using this method. DHPLC analysis of 14 isolates representing each type of novel or multiple mutations and the wild type were compared with LightCycler-based PCR-
gyrA
hybridization mutation assay (GAMA) and single-strand conformational polymorphism (SSCP) analyses. The 14 isolates gave seven different SSCP patterns, and LightCycler detected four different mutations. DHPLC detected 11 DNA sequence variants at eight different codons, including those detected by LightCycler or SSCP. One of these mutations was silent. Five isolates contained multiple mutations, and four of these could be distinguished from the composite sequence variants by their DHPLC profile. Seven novel mutations were identified at five different loci not previously described in quinolone-resistant salmonella. DHPLC analysis proved advantageous for the detection of novel and multiple mutations. DHPLC also provides a rapid, high-throughput alternative to LightCycler and SSCP for screening frequently occurring mutations.
Single-Tube Nested PCR for Detection ofTritrichomonasfoetusin Feline Feces
Authors: Gookin Jody L. , Birkenheuer Adam J. , Breitschwerdt Edward B. , Levy Michael G.
Published: October 2002

Tritrichomonasfoetus, a venereal pathogen of cattle, was recently identified as an inhabitant of the large intestine in young domestic cats with chronic diarrhea. Recognition of the infection in cats has been mired by unfamiliarity withT.foetusin cats as well as misdiagnosis of the organisms asPentatrichomonashominisorGiardiasp. when visualized by light microscopy. The diagnosis ofT.foetuspresently depends on the demonstration of live organisms by direct microscopic examination of fresh feces or by fecal culturing. AsT.foetusorganisms are fastidious and fragile, routine flotation techniques and delayed examination and refrigeration of feces are anticipated to preclude the diagnosis in numerous cases. The objective of this study was to develop a sensitive and specific PCR test for the diagnosis of felineT.foetusinfection. A single-tube nested PCR was designed and optimized for the detection ofT.foetusin feline feces by using a combination of novel (TFITS-F and TFITS-R) and previously described (TFR3 and TFR4) primers. The PCR is based on the amplification of a conserved portion of theT.foetusinternal transcribed spacer (ITS) region (ITS1 and ITS2) and the 5.8S rRNA gene. The absolute detection limit of the single-tube nested PCR was 1 organism, while the practical detection limit was 10 organisms per 200 mg of feces. Specificity was examined by usingP.hominis,Giardialamblia,and feline genomic DNA. Our results demonstrate that the single-tube nested PCR is ideally suited for (i) diagnostic testing of feline fecal samples that are found negative by direct microscopy and culturing and (ii) definitive identification of microscopically observable or cultivated organisms.
Detection of Antibodies Directed against Human Herpesvirus 6 U94/REP in Sera of Patients Affected by Multiple Sclerosis
Authors: Caselli Elisabetta , Boni Michela , Bracci Arianna , Rotola Antonella , Cermelli Claudio , Castellazzi Massimiliano , Di Luca Dario , Cassai Enzo
Published: October 2002

The association between human herpesvirus 6 (HHV-6) and multiple sclerosis (MS) is controversial. In fact, it is difficult to establish a causative role of HHV-6, due to the high prevalence of latently infected individuals in the healthy population. Therefore, the presence of virus sequences in tissue biopsy does not support a viral role, and serological assays do not show significant differences between MS patients and control populations. The only viral gene expressed during latency is U94/rep. Therefore, we have developed a serological assay for the detection of antibodies specifically directed against U94/REP protein. Different populations were analyzed by enzyme-linked immunosorbent assay, including healthy controls, MS patients, and subjects with diseases unrelated to HHV-6 infection, including other neurological diseases. The results show statistically significant differences (P> 0.01) between MS patients and control groups, both in antibody prevalence (87 and 43.9%, respectively) and in geometric mean titer (1:515 and 1:190, respectively). The detection of antibodies specific for HHV-6 U94/REP shows that the immune system is exposed to this antigen during natural infection. The higher prevalence and higher titers of antibodies to U94/REP suggest that MS patients and control groups might experience different exposures to HHV-6.
Performance Assessment of Two Commercial Amplification Assays for Direct Detection ofMycobacterium tuberculosisComplex from Respiratory and Extrapulmonary Specimens
Authors: Piersimoni Claudio , Scarparo Claudio , Piccoli Paola , Rigon Alessandra , Ruggiero Giuliana , Nista Domenico , Bornigia Stefano
Published: October 2002

The new BDProbeTec ETMycobacterium tuberculosisComplex Direct Detection Assay (DTB) was compared with the enhancedM. tuberculosisAmplified Direct Test (AMTDII). The system is an automated walkaway system characterized by simultaneous DNA amplification (strand displacement amplification) and real-time fluorometric detection. It also contains an internal amplification control (IAC) designed to identify inhibition from the processed samples. The AMTDII assay amplifies rRNA by transcription-mediated amplification; it uses hybridization with a chemoluminescent probe as a detection system and is entirely manual. A total of 515N-acetyl-l-cysteine-sodium hydroxide-decontaminated respiratory (n= 331) and extrapulmonary (n= 184) sediments (from 402 patients) were tested in parallel by both assays. The results were compared with those of acid-fast staining and culture (solid plus liquid media), setting the combination of culture and clinical diagnosis as the “gold standard.” Culture results from the tested specimens were as follows: 121Mycobacterium tuberculosiscomplex (MTB) (98 smear-positive), 46 nontuberculous mycobacteria (38 smear-positive), and 338 culture-negative results. After resolution of the discrepant results, the percent sensitivity, percent specificity, and positive and negative likelihood ratios for AMTDII were 88%, 99.2%, 110, and 0.11 for respiratory specimens and 74.3%, 100%, 740, and 0.26 for extrapulmonary specimens, respectively. The corresponding values for DTB were 94.5%, 99.6%, 235, and 0.05 for respiratory specimens and 92.3%, 100%, 920, and 0.07 for extrapulmonary specimens, respectively. The cumulative difference for all tuberculosis-positive extrapulmonary specimens was significant (P= 0.03). The overall inhibition rate for DTB was 5% (26 specimens). We conclude that both amplification assays proved to be rapid and specific for the detection of MTB in clinical samples and particularly feasible for a routine laboratory work flow. DTB combines a labor-intensive specimen preparation procedure with a completely automated amplification and detection. Finally, differences between AMTDII and DTB sensitivities were associated with the presence of inhibitory samples that the former assay, lacking IAC, could not detect.
Rapid and Specific Detection of
Mycobacterium tuberculosis
from Acid-Fast Bacillus Smear-Positive Respiratory Specimens and BacT/ALERT MP Culture Bottles by Using Fluorogenic Probes and Real-Time PCR
Authors: Miller Nancimae , Cleary Tim , Kraus Günter , Young Andrea K. , Spruill Gina , Hnatyszyn H. James
Published: October 2002

A real-time PCR assay using the LightCycler (LC) instrument for the specific identification of
Mycobacterium tuberculosis complex
(MTB) was employed to detect organisms in 135 acid-fast bacillus (AFB) smear-positive respiratory specimens and in 232 BacT/ALERT MP (MP) culture bottles of respiratory specimens. The LC PCR assay was directed at the amplification of the internal transcribed spacer region of the
Mycobacterium
genome with real-time detection using fluorescence resonance energy transfer probes specific for MTB. The results from the respiratory specimens were compared to those from the Amplicor
M. tuberculosis
PCR test. Specimens from MP culture bottles were analyzed by Accuprobe and conventional identification methods. MTB was cultured from 105 (77.7%) respiratory AFB smear-positive specimens; 103 of these samples were positive by LC PCR and Amplicor PCR. Two samples negative in the LC assay contained rare numbers of organisms; both were positive in the Amplicor assay. Two separate samples negative by Amplicor PCR contained low and moderate numbers of AFB, respectively, and both of these were positive in the LC assay. There were 30 AFB smear-positive respiratory specimens that grew mycobacteria other than tuberculosis (MOTT), and all tested negative in both assays. Of the 231 MP culture bottles, 114 cultures were positive for MTB and all were positive by the LC assay. The remaining 117 culture bottles were negative in the LC assay and grew various MOTT. This real-time MTB assay is sensitive and specific; a result was available within 1 h of having a DNA sample available for testing.
Molecular Characterization of Hepatitis A Virus Isolates from a Transcontinental Shellfish-Borne Outbreak
Authors: Sánchez Glòria , Pintó Rosa M. , Vanaclocha Hermelinda , Bosch Albert
Published: October 2002

One hundred eighty-four serologically confirmed cases of hepatitis A were reported in eastern Spain in 1999. A matched case-control study implicated imported coquina clams complying with European Union shellfish standards as the source of infection; this implication was confirmed by the detection by reverse transcription-PCR of hepatitis A virus (HAV) RNA in shellfish samples. In spite of the recognized low variability of HAV, genetic characterization of the complete capsid region of virus isolates from patient serum samples revealed the existence of both synonymous and nonsynonymous variants. Two antigenic variants were detected, one in a discontinuous epitope defined by monoclonal antibody K3-4C8 and a second in a linear VP1 epitope of the virus. In spite of these antigenic variants, all isolates were assigned to genotype IB, providing further evidence that the outbreak originated from a common source, although multiple strains were likely to be involved.
Increasing Genetic Diversity of
Salmonella enterica
Serovar Typhi Isolates from Papua New Guinea over the Period from 1992 to 1999
Authors: Thong Kwai-Lin , Goh Yee-Ling , Yasin Rohani M. , Lau Ming Guek , Passey Megan , Winston Gibson , Yoannes Mition , Pang Tikki , Reeder John C.
Published: October 2002

Pulsed-field gel electrophoresis (PFGE) of
Xba
I-digested chromosomal DNA was performed on 133 strains of
Salmonella enterica
serovar Typhi obtained from Papua New Guinea, with the objective of assessing the temporal variation of these strains. Fifty-two strains that were isolated in 1992 and 1994 were of one phage type, D2, and only two predominant PFGE profiles, X1 and X2, were present. Another 81 strains isolated between 1997 and 1999 have shown divergence, with four new phage types, UVS I (
n
= 63), UVS (
n
= 5), VNS (
n
= 4), and D1 (
n
= 9), and more genetic variability as evidenced by the multiple and new PFGE
Xba
I profiles (21 profiles; Dice coefficient,
F
= 0.71 to 0.97). The two profiles X1 and X2 have remained the stable, dominant subtypes since 1992. Cluster analysis based on the unweighted pair group method using arithmetic averages algorithm identifies two main clusters (at 87% similarity), indicating that the divergence of the PFGE subtypes was probably derived from some genomic mutations of the X1 and X2 subtypes. The majority of isolates were from patients with mild and moderate typhoid fever and had various
Xba
I profiles. A single isolate from a patient with fatal typhoid fever had a unique X11 profile, while four of six isolates from patients with severe typhoid fever had the X1 pattern. In addition, 12 paired serovar Typhi isolates recovered from the blood and fecal swabs of individual patients exhibited similar PFGE patterns, while in another 11 individuals paired isolates exhibited different PFGE patterns. Three pairs of isolates recovered from three individuals had different phage types and PFGE patterns, indicating infection with multiple strains. The study reiterates the usefulness of PFGE in assessing the genetic diversity of
S. enterica
serovar Typhi for both long-term epidemiology and in vivo stability and instability within an individual patient.
Evaluation of an Enzyme-Linked Immunosorbent Assay (ELISA) with Affinity-Purified Em18 and an ELISA with Recombinant Em18 for Differential Diagnosis of Alveolar Echinococcosis: Results of a Blind Test
Authors: Ito Akira , Xiao Ning , Liance Martine , Sato Marcello O. , Sako Yasuhito , Mamuti Wulamu , Ishikawa Yuji , Nakao Minoru , Yamasaki Hiroshi , Nakaya Kazuhiro , Bardonnet Karine , Bresson-Hadni Solange , Vuitton Dominique A.
Published: October 2002

Alveolar echinococcosis (AE) is the most potentially lethal parasitic zoonosis of the nontropical areas in the northern hemisphere, where cystic echinococcosis (CE) is also endemic. Both AE and CE are highly endemic in China, and both serologic detection of echinococcosis, either AE or CE, and differentiation of AE from CE are crucial problems. Evaluation of Western blot analysis (WB) and enzyme-linked immunosorbent assay (ELISA) for the Em18 antigen, using affinity-purified and recombinant Em18, was carried out “blindly” using 60 human sera from patients diagnosed in France. The results were compared with those obtained using a commercially available
Echinococcus
WB immunoglobulin G (IgG) kit developed in France. The Em18 WB and
Echinococcus
WB IgG showed very similar results for detection of AE. Both affinity-purified Em18 or a recombinant Em18 WB and
Echinococcus
WB IgG seem useful for identification of AE, and the latter seems appropriate for both AE and CE, whereas affinity-purified Em18 ELISA and the newly developed recombinant Em18 ELISA appear to be suitable for detection of AE, especially for epidemiological surveys.
Evaluation of the ICT Malaria P.f/P.v and the OptiMal Rapid Diagnostic Tests for Malaria in Febrile Returned Travellers
Authors: Playford E. Geoffrey , Walker John
Published: October 2002

Rapid diagnostic tests (RDTs) are less reliant on expert microscopy and have the potential to reduce errors in malaria diagnosis but have not been extensively evaluated in nonimmune persons or in countries where infection is not endemic. We evaluated the ICT P.f/P.v (ICT-Amrad, Sydney, Australia) and OptiMal (Flow Inc., Portland, Oreg.) assays prospectively for the diagnosis of malaria in 158 specimens from 144 febrile returned travellers in Australia by using expert microscopy and PCR as reference standards. Malaria was diagnosed in 93 specimens from 87 patients by expert microscopy, with 3 additional specimens from recently treated patients testing positive for
Plasmodium falciparum
by PCR. For the diagnosis of asexual-stage
P. falciparum
malaria, the sensitivity and specificity of the ICT P.f/P.v assay were 97 and 90%, respectively, and those of the OptiMal assay were 85 and 96%, respectively. The ICT P.f/P.v assay missed one infection with a density of 45 parasites/μl, whereas the OptiMal assay missed infections up to 2,500/μl; below 1,000/μl, its sensitivity was only 43%. For the diagnosis of
P. vivax
malaria, the sensitivity and specificity of the ICT P.f/P.v assay were 44 and 100%, respectively, and those of the OptiMal assay were 80 and 97%, respectively. Both assays missed infections with parasite densities over 5,000/μl: up to 10,000/μl with the former and 5,300/μl with the latter. Despite the high sensitivity of the ICT P.f/P.v assay for
P. falciparum
malaria, caution is warranted before RDTs are widely adopted for the diagnosis of malaria in nonimmune patients or in countries where malaria is not endemic.
Single and Combination Antibiotic Susceptibilities of Planktonic, Adherent, and Biofilm-Grown
Pseudomonas aeruginosa
Isolates Cultured from Sputa of Adults with Cystic Fibrosis
Authors: Aaron Shawn D. , Ferris Wendy , Ramotar Karam , Vandemheen Katherine , Chan Francis , Saginur Raphael
Published: October 2002

Evidence suggests that
Pseudomonas aeruginosa
bacteria form biofilms within the airways of adults with cystic fibrosis (CF). The objective of this study was to determine whether clinical isolates of
P. aeruginosa
recovered from adults with CF have similar susceptibilities to individual antibiotics and to antibiotic combinations when grown as adherent monolayers or as biofilms compared to when they are grown using planktonic methods. Twelve multiresistant
P. aeruginosa
isolates, one mucoid and one nonmucoid from each of six CF patients, were grown conventionally under planktonic conditions, as adherent bacterial monolayers, and as biofilms. Each bacterial isolate remained genotypically identical despite being cultured under planktonic, adherent, or biofilm growth conditions. Isolates grown as adherent monolayers and as biofilms were less susceptible to bactericidal killing by individual antibiotics compared to those grown planktonically. More importantly, biofilm-grown bacteria, but not adherent monolayer-grown bacteria, were significantly less susceptible to two- and three-drug combinations of antibiotics than were planktonically grown bacteria (
P
= 0.005). We conclude that biofilm-grown bacteria derived from patients with CF show decreased susceptibility to the bactericidal effects of antibiotic combinations than do adherent and planktonically grown bacteria.
Epidemiological Features of Invasive
Kingella kingae
Infections and Respiratory Carriage of the Organism
Authors: Yagupsky Pablo , Peled Nechama , Katz Orna
Published: October 2002

The age, sex, and seasonal distributions of invasive
Kingella kingae
infections in southern Israel were examined and compared to the epidemiology of respiratory carriage of the organism. Medical records of all patients diagnosed between 1988 and 2002 were reviewed, and 2,044 oropharyngeal specimens were cultured on selective media during two periods (February to May and October to December) in 2001. Invasive infections significantly affected children (73 of 74 patients [98.6%] were younger than 4 years), 50 patients (67.8%) were males (
P
= 0.045), and 55 episodes (74.3%) occurred between July and December (
P
= 0.004). Carriage was higher in the 0- to 3-year-old group and decreased with increasing age (
P
for trend = 0.0008). Carriage rates were similar in both sexes and did not significantly differ between the February-to-May and October-to-December periods. The highest rate of carriage of
K. kingae
coincided with the age (less than 4 years) at which invasive infections were especially frequent. The peculiar sex and seasonal distributions of invasive disease, however, cannot be readily explained by the epidemiology of respiratory carriage. Viral infections and other yet-to-be-defined cofactors may play a role in the causation of invasive
K. kingae
infections.
Rate of Occurrence and Pathogenic Effect of Enteroaggregative
Escherichia coli
Virulence Factors in International Travelers
Authors: Jiang Zhi-Dong , Greenberg David , Nataro James P. , Steffen Robert , DuPont Herbert L.
Published: October 2002

One or more putative enteroaggregative
Escherichia coli
(EAEC) virulence factors (
aggA, aggR, aspU,
or
aafA
) were identified in 60 (70%) of 86 EAEC isolates from travelers with diarrhea compared with a rate of 7 (8%) of 90 in patients with diarrhea who were infected with nonadherent
E. coli
(odds ratio, 27.36; 95% confidence interval, 11.30 to 65.91). The presence of
aggR
or one or more virulence factors in EAEC from patients with diarrhea was associated with a statistically increased concentration of interleukin-8 (IL-8) in feces compared with that in EAEC negative for these factors: for
aggR
positive (9 of 12 [75%]; median, 800 pg/ml) versus
aggR
negative (5 of 18 [28%]; median, 0),
P
< 0.05; and for isolates positive for ≥1 virulence factor (13 of 21 [62%]; median, 360 pg/ml) versus those negative for ≥1 virulence factor (1 of 9 [11%]; median, 0),
P
< 0.05. Other fecal cytokines (IL-1β and IL-1ra) were found in increased concentrations (
P
< 0.05 when at least one EAEC virulence factor was present compared with the concentrations when EAEC negative for multiple virulence factors was found in patients with diarrhea. Putative virulence factors were commonly found in EAEC from patients with diarrhea, and the pathogenicity of many strains was suggested by showing an association between the presence of plasmid-borne virulence factors and the presence of fecal cytokines. The different patterns of virulence factors of EAEC revealed several clusters demonstrating diversity among the isolates from the various regions.
Novel Allelic Variants of Mycobacteria Isolated in Brazil as Determined by PCR-Restriction Enzyme Analysis of
hsp65
Authors: da Silva Rocha A. , Werneck Barreto A. M. , Dias Campos C. E. , Villas-Bôas da Silva M. , Fonseca L. , Saad M. H. , Degrave W. M. , Suffys P. N.
Published: October 2002

Human isolates of
Mycobacterium
collected in 16 different states of Brazil were submitted to PCR-restriction analysis (PRA) of a 439-bp fragment of the
hsp65
gene with
Hae
III and
Bst
EII. Fourteen allelic variants not described in clinical isolates so far were observed among 36 (10%) of 356 Brazilian strains, including a new pattern for
Mycobacterium scrofulaceum
,
M. intracellulare
, and
M. flavescens
, two new patterns for
M. fortuitum
, three new patterns each for
M. gordonae
and
M. terrae
, and one new pattern for
M
.
avium
complex-like strains. Two unidentified strains each also presented a new pattern, strongly suggesting that
Mycobacterium
genotypes are distributed biogeographically. The PRA procedure was also performed with 43 reference isolates belonging to 34 species, adding a further six new patterns to the identification algorithm. A database containing the normalized restriction patterns of both enzymes was constructed. Patterns available on the Internet can be introduced into this database, which will make possible the comparison of genotypes from isolates from different parts of the world.
Heterogeneity and Seroprevalence of a Newly Identified Avian Hepatitis E Virus from Chickens in the United States
Authors: Huang F. F. , Haqshenas G. , Shivaprasad H. L. , Guenette D. K. , Woolcock P. R. , Larsen C. T. , Pierson F. W. , Elvinger F. , Toth T. E. , Meng X. J.
Published: October 2002

We recently identified and characterized a novel virus, designated avian hepatitis E virus (avian HEV), from chickens with hepatitis-splenomegaly syndrome (HS syndrome) in the United States. Avian HEV is genetically related to but distinct from human and swine HEVs. To determine the extent of genetic variation and the seroprevalence of avian HEV infection in chicken flocks, we genetically identified and characterized 11 additional avian HEV isolates from chickens with HS syndrome and assessed the prevalence of avian HEV antibodies from a total of 1,276 chickens of different ages and breeds from 76 different flocks in five states (California, Colorado, Connecticut, Virginia, and Wisconsin). An enzyme-linked immunosorbent assay using a truncated recombinant avian HEV ORF2 antigen was developed and used to determine avian HEV seroprevalence. About 71% of chicken flocks and 30% of chickens tested in the study were positive for antibodies to avian HEV. About 17% of chickens younger than 18 weeks were seropositive, whereas about 36% of adult chickens were seropositive. By using a reverse transcription-PCR (RT-PCR) assay, we tested 21 bile samples from chickens with HS syndrome in California, Connecticut, New York, and Wisconsin for the presence of avian HEV RNA. Of the 21 bile samples, 12 were positive for 30- to 35-nm HEV-like virus particles by electron microscopy (EM). A total of 11 of the 12 EM-positive bile samples and 6 of the 9 EM-negative bile samples were positive for avian HEV RNA by RT-PCR. The sequences of a 372-bp region within the helicase gene of 11 avian HEV isolates were determined. Sequence analyses revealed that the 11 field isolates of avian HEV had 78 to 100% nucleotide sequence identities to each other, 79 to 88% identities to the prototype avian HEV, 76 to 80% identities to chicken big liver and spleen disease virus, and 56 to 61% identities to other known strains of human and swine HEV. The data from this study indicated that, like swine and human HEVs, avian HEV isolates are genetically heterogenic and that avian HEV infection is enzoonotic in chicken flocks in the United States.
Molecular Methods for Cytomegalovirus Surveillance in Bone Marrow Transplant Recipients
Authors: Weinberg Adriana , Schissel Debra , Giller Roger
Published: October 2002

Two different methods for detection of cytomegalovirus (CMV), PCR and hybrid capture (HC), were compared by using plasma, peripheral blood leukocytes (PBLs), and whole blood (WB) from allogeneic bone marrow transplant recipients. One hundred specimens were obtained from nine children over an 18-month surveillance period. PCR of plasma for CMV was used for clinical management. The proportions of samples positive for CMV DNA by PCR with plasma, HC with WB, and PCR with PBLs were 21, 28, and 37%, respectively. Among 44 samples that were tested by all three methods, 68% had concordant results. By using a robust definition of true-positive samples (positivity by two or more methods or positivity of sequential samples by one method), the sensitivities of PCR with plasma, HC with WB, and PCR with PBLs were 50, 67, and 83%, respectively, and the specificities were 100, 96, and 96%, respectively. Two patients developed CMV-associated end-organ disease (one developed respiratory disease, and one developed gastrointestinal disease). CMV DNA was not detected in the plasma 1 week prior to the development of symptoms in either patient, whereas HC with WB was positive for both patients and PCR with PBLs was for one patient. These data suggest that WB or PBLs might be the preferred sample for use for surveillance for CMV in immunocompromised patients.
Performance Characteristics and Utilization of Rapid Antigen Test, DNA Probe, and Culture for Detection of Group A Streptococci in an Acute Care Clinic
Authors: Chapin Kimberle C. , Blake Patricia , Wilson Claire D.
Published: October 2002

Group A streptococcus (GAS) antigen testing has become a routine point-of-care (POC) test in acute care settings. Concern about performance parameters (PP) of these tests as well as inappropriate antibiotic use has resulted in various recommendations regarding diagnosis of GAS. There were two objectives in this study. The first was to evaluate the rapid GAS antigen test presently in use (Thermo BioStar, Boulder, Colo.) and the GAS Direct probe test (Gen-Probe, San Diego, Calif.) compared to culture. The second was to define the optimal use of these technologies in a large acute care pediatric clinic. A total of 520 consecutive pediatric patients presenting with symptoms of pharyngitis at any of three Lahey Clinic acute care facilities were evaluated. Pharyngeal specimens were collected using a double-swab collection device (Copan, Corona, Calif.). One swab was used for the antigen test, the second was used for the probe test, and the pledget was placed in the collection device for culture on 5% sheep blood agar, incubated for 48 h anaerobically, and subsequently placed in Todd-Hewitt broth. After discrepant analysis, sensitivity, specificity, and positive and negative predictive values were as follows: 94.8, 100, 100, and 96.9% for the probe test and 86.1, 97.1, 93.7, and 93.4% for the antigen test, respectively. Sensitivity using an enhanced culture technique was 99.4% (163 of 164). False-positive (FP) antigen results were often seen from patients previously diagnosed and/or treated for GAS. No FP results were seen with the probe test. Colony counts for the false-negative (FN) antigen tests were higher than those for the FN probe tests. Compared to culture and DNA probe, the rapid antigen test (RAT) offered a result at the time of the patient's visit, with acceptable PP when prevalence of disease is high. Follow-up testing with the RAT of GAS patients who previously tested as positive should be avoided due to increased FP results. The probe test was comparable to culture in performance. Results indicate the probe test can be used as the primary test or as a backup to negative antigen tests. The probe test offers the advantage over culture of same-day reporting of a final result but, in contrast to a POC test, necessitates follow-up communication to the patient. Preliminary data show the specificity of the probe test to be greater than that of the RAT for patients previously diagnosed with GAS.
Optimal DNA Isolation Method for Detection of Bacteria in Clinical Specimens by Broad-Range PCR
Authors: Rantakokko-Jalava Kaisu , Jalava Jari
Published: October 2002

Broad-range amplification of bacterial DNA from clinical specimens has proved useful for the diagnosis of various bacterial infections, especially during antimicrobial treatment of the patient. Optimal sample processing protocols for diagnostic broad-range bacterial PCR should release DNA from an array of target organisms with equal efficiencies and wash out inhibitory factors from various sample types without introducing bacterial DNA contamination to the amplification reaction. In the present study, two physical cell wall disintegration methods, bead beating and sonication, for enhanced detection of organisms with difficult-to-lyse cell walls were studied. The analytical sensitivities of several commercially available DNA purification kits, which were used with and without additional cell disintegration steps, were compared by using dilution series of model bacteria. Selected purification methods were used to process routine clinical specimens in parallel with the standard phenol-ether DNA extraction, and the results obtained by bacterial PCR and sequencing with the two template preparations were compared. The method with the DNA isolation kit with the lowest detection limits from the bacterial suspensions (Masterpure) did not prove to be superior to the standard method when the two methods were applied to 69 clinical specimens. For another set of 68 clinical specimens, DNA purified with a glass fiber filter column (High Pure) with an additional sonication step yielded results well in accord with those obtained by the standard method. Furthermore, bacterial DNA was detected in four samples that remained PCR negative by the standard method, and three of these contained DNA from gram-positive pathogens. Three samples were positive by the standard method only, indicating the limitations of applying any single method to all samples.
Human Rhinovirus 87 and Enterovirus 68 Represent a Unique Serotype with Rhinovirus and Enterovirus Features
Authors: Blomqvist Soile , Savolainen Carita , Råman Laura , Roivainen Merja , Hovi Tapani
Published: October 2002

It has recently been reported that all but one of the 102 known serotypes of the genus
Rhinovirus
segregate into two genetic clusters (C. Savolainen, S. Blomqvist, M. N. Mulders, and T. Hovi, J. Gen. Virol.
83:
333-340, 2002). The only exception is human rhinovirus 87 (HRV87). Here we demonstrate that HRV87 is genetically and antigenically highly similar to enterovirus 68 (EV68) and is related to EV70, the other member of human enterovirus group D. The partial nucleotide sequences of the 5′ untranslated region, capsid regions VP4/VP2 and VP1, and the 3D RNA polymerase gene of the HRV87 prototype strain F02-3607 Corn showed 97.3, 97.8, 95.2, and 95.9% identity to the corresponding regions of EV68 prototype strain Fermon. The amino acid identities were 100 and 98.1% for the products of the two capsid regions and 97.9% for 3D RNA polymerase. Antigenic cross-reaction between HRV87 and EV68 was indicated by microneutralization with monotypic antisera. Phylogenetic analysis showed definite clustering of HRV87 and EV68 with EV70 for all sequences examined. Both HRV87 and EV68 were shown to be acid sensitive by two different assays, while EV70 was acid resistant, which is typical of enteroviruses. The cytopathic effect induced by HRV87 or EV68 was inhibited by monoclonal antibodies to the decay-accelerating factor known to be the receptor of EV70. We conclude that HRV87 and EV68 are strains of the same picornavirus serotype presenting features of both rhinoviruses and enteroviruses.
Comparison of Serum Hepatitis C Virus RNA and Core Antigen Concentrations and Determination of Whether Levels Are Associated with Liver Histology or Affected by Specimen Storage Time
Authors: Lagging L. Martin , Garcia Clementina E. , Westin Johan , Wejstål Rune , Norkrans Gunnar , Dhillon Amar P. , Lindh Magnus
Published: October 2002

An enzyme immunoassay has recently been developed for the hepatitis C virus (HCV) core antigen. To evaluate the possible association between core antigen and HCV RNA levels with regards to the change in liver histology over time as well as study the effect of duration of storage on viral load results, sequential sera were analyzed from 45 patients with chronic HCV infection who had undergone two or more liver biopsies. A relatively strong association was found between the core antigen and HCV RNA concentrations (

r
s

= 0.8), with a core antigen level of 1 pg/ml corresponding to approximately 1,000 IU/ml. All 42 sera with detectable HCV RNA at the time of the second biopsy had core antigen concentrations above 1 pg/ml, and the three sera without detectable HCV RNA had concentrations below 1 pg/ml. No association was found between HCV RNA or core antigen levels and the stage of fibrosis in biopsy samples, progression of fibrosis, necro-inflammatory grade, steatosis, genotype, alanine aminotransferase level, or alcohol consumption. A significant association was demonstrated between the storage time of the samples and both the HCV RNA and core antigen concentrations. The median log HCV RNA concentrations (international units/milliliter) were 3.92 for the sera obtained at the time of the first biopsy (median storage time, 13.0 years) and 4.41 for the sera obtained at the time of the second biopsy (median storage time, 6.6 years) compared to 5.96, the median for 102 different routine clinical patient samples.
Identification of the Causative Organism of Tuberculous Lymphadenitis in Ethiopia by PCR
Authors: Kidane Dawit , Olobo Joseph O. , Habte Abebe , Negesse Yohannes , Aseffa Abraham , Abate Getahun , Yassin Mohammed A. , Bereda Kiflu , Harboe Morten
Published: October 2002

Tuberculous lymphadenitis (TBLN) is a common form of extrapulmonary tuberculosis with multiple differential diagnoses. Demonstration of the etiologic agent by smear microscopy or culture of fine needle aspirate (FNA) specimens is often unsuccessful. FNA specimens from 40 patients presenting at a rural health center in South Ethiopia and diagnosed as positive for TBLN on the basis of clinical and cytological criteria were analyzed for mycobacterial DNA by PCR. Thirty (75%) had cervical lymphadenitis and 11 (27.5%) were seropositive for human immunodeficiency virus (HIV). Three primer sets were initially used to identify the causative agent at the genus (antigen 85 complex), complex (IS
6110
insertion sequence), and species (
pncA
gene and allelic variation) levels. Among the forty TBLN cases, 35 (87.5%) were positive by PCR at the genus and complex levels. Based on PCR for detection of allelic variation at position 169, 24 (68.6%) of the 35 were positive for
Mycobacterium tuberculosis
and 6 (17.1%) were positive for
M. bovis
. These six were positive in additional PCR assays using the JB21-JB22 primer set, which is highly specific for
M. bovis.
Five (14.1%) showed amplification for both
M. tuberculosis
and
M. bovis
with the allele-specific primer set
.
Cooccurrence of pyrazinamide (PZA)-sensitive and -resistant
M. tuberculosis
in those five cases was indicated, since all were negative in assays with the JB21-JB22 primer set. This feature was seen in 3 of 11 HIV-positive and 2 of 29 HIV-negative individuals (
P
< 0.001). Conclusion: among 35 PCR-positive cases of TBLN from southern Ethiopia, 29 (82.9%) were caused by
M. tuberculosis
and six (17.1%) were caused by
M. bovis.
Serological Diagnosis of Ovine Enzootic Abortion by Enzyme-Linked Immunosorbent Assay with a Recombinant Protein Fragment of the Polymorphic Outer Membrane Protein POMP90 ofChlamydophila abortus
Authors: Longbottom David , Fairley Susan , Chapman Stephanie , Psarrou Evgenia , Vretou Evangelia , Livingstone Morag
Published: October 2002

Ovine enzootic abortion (OEA) resulting from infection of sheep and goats withChlamydophila abortusis of major economic importance worldwide. Over the last 50 years the serological diagnosis of infection has been based mainly on the complement fixation test (CFT), which lacks both sensitivity and specificity because of cross-reactive antibodies to other gram-negative bacteria, including another common chlamydial pathogen of sheep,Chlamydophila pecorum. In the present study, a series of overlapping recombinant antigens representing the polymorphic outer membrane protein POMP90 ofC. abortuswas assessed by enzyme-linked immunosorbent assay (ELISA) with a panel of 143 serum samples from sheep experimentally infected withC. abortus, from sheep clinically free of OEA, and from specific-pathogen-free lambs experimentally infected with different subtypes ofC. pecorum. The results were compared to those obtained by CFT and another recently described test, an indirect ELISA (iELISA) with the recombinant OMP91B (rOMP91B) fragment (rOMP91B iELISA) (D. Longbottom, E. Psarrou, M. Livingstone, and E. Vretou, FEMS Microbiol. Lett. 195:157-161, 2001). The rOMP90-3 and rOMP90-4 ELISAs were identified as being more sensitive and specific than CFT. Assays with both fragments were evaluated further with a panel of 294 field serum samples from flocks with documented histories of abortion, from flocks with no clinical histories of abortion but which had a high proportion of samples seropositive by CFT, and from animals with no histories of abortion but from which variousC. pecorumsubtypes had been isolated. ELISAs with both POMP90 fragments outperformed CFT with serum samples fromC. pecorum-infected animals, producing no false-positive results. However, the ELISA with the rOMP90-4 fragment appeared to be more sensitive than the one with rOMP90-3, as it identified more of the OEA-positive samples. The ELISA with the rOMP90-4 fragment was also able to identify apparently healthy animals that were infected with an enteric strain ofC. abortusin flocks that were probably infected with both entericC. abortusandC. pecorumstrains. The identification of animals infected with entericC. abortusis extremely important in controlling the spread of OEA. Overall, the new rOMP90-4 ELISA was found to be a more sensitive and specific test than CFT for differentiating animals infected withC. abortusfrom those infected withC. pecorum.
Development of a Peptide-Mediated Capture PCR for Detection of
Mycobacterium avium
subsp.
paratuberculosis
in Milk
Authors: Stratmann Janin , Strommenger Birgit , Stevenson Karen , Gerlach Gerald-F.
Published: October 2002

Based on phage display technology, a peptide-mediated magnetic separation technique was developed to facilitate selective isolation of
Mycobacterium avium
subsp.
paratuberculosis
(
M. paratuberculosis
) from bulk milk of naturally infected dairy herds. Nine recombinant bacteriophages binding to
M. paratuberculosis
were isolated from a commercial phage-peptide library encoding random 12-mer peptides. Nucleotide sequencing revealed the deduced sequence of the binding peptides. One peptide with the sequence NYVIHDVPRHPA, designated aMP3, was chemically synthesized with an amino-terminal biotin residue attached via an amino-hexacarbonic acid spacer molecule. Paramagnetic beads coated with the phage or with peptide aMP3 enabled the capture of
M. paratuberculosis
from milk. Combining this peptide-mediated magnetic separation with an IS
Mav2
-based PCR allowed the detection of
M. paratuberculosis
in artificially spiked milk down to a concentration of 10
1
ml
−1
. Experiments using milk from naturally infected cows and bulk milk samples from infected herds demonstrated that the peptide-mediated capture PCR is sufficiently sensitive to detect single strong shedders in pooled milk samples. The method, for the first time, applies phage display technology to microbial diagnostics and has potential value as a completely standardizable tool for the routine
M. paratuberculosis
screening of bulk milk samples at acceptable costs.
Improved Monitoring of Cytomegalovirus Infection after Allogeneic Hematopoietic Stem Cell Transplantation by an Ultrasensitive Plasma DNA PCR Assay
Authors: Kaiser Laurent , Perrin Luc , Chapuis Bernard , Hadaya Karine , Kolarova Lenka , Deffernez Christelle , Huguet Saadia , Helg Claudine , Wunderli Werner
Published: October 2002

Cytomegalovirus (CMV) DNA amplification assays in plasma have shown limited sensitivity compared to the detection of pp65 antigen in leukocytes. Our goal was to increase the sensitivity of a commercial CMV DNA PCR quantitative assay. After modification, the new assay was able to reproducibly detect 20 CMV DNA copies/ml of plasma. We compared this new ultrasensitive PCR assay with the standard PCR and the pp65 test for CMV detection and quantification in 22 consecutive allogeneic hematopoietic stem cell recipients. CMV infection or reactivation was detected in 84 of 319 (26%) samples by the ultrasensitive PCR assay compared to 38 of 319 (12%) samples by the pp65 assay (P< 0.01). All samples positive by the pp65 assay were positive by the ultrasensitive PCR, and CMV episodes were detected on average 4 days earlier and 7 days later than the first and the last pp65-positive test, respectively. In addition, during CMV episodes, the ultrasensitive assay identified positive samples that were inconsistently detected by the pp65 assay. The ultrasensitive assay was also much more sensitive than the standard PCR, with 26 versus 12% of CMV DNA-positive samples (P< 0.01). This assay improved the monitoring of CMV infection or reactivation in hematopoietic allogeneic stem cell recipients.
OspE-Related, OspF-Related, and Elp Lipoproteins Are Immunogenic in Baboons Experimentally Infected withBorrelia burgdorferiand in Human Lyme Disease Patients
Authors: Hefty P. Scott , Brooks Chad S. , Jett Amy M. , White Gary L. , Wikel Stephen K. , Kennedy Ronald C. , Akins Darrin R.
Published: October 2002

Presently, the rhesus macaque is the only nonhuman primate animal model utilized for the study of Lyme disease. While this animal model closely mimics human disease, rhesus macaques can harbor the herpes B virus, which is often lethal to humans; macaques also do not express the full complement of immunoglobulin G (IgG) subclasses found in humans. Conversely, baboons contain the full complement of IgG subclasses and do not harbor the herpes B virus. For these reasons, baboons have been increasingly utilized as the basis for models of infectious diseases and studies assessing the safety and immunogenicity of new vaccines. Here we analyzed the capability of baboons to become infected withBorrelia burgdorferi, the agent of Lyme disease. Combined culture and PCR analyses of tick- and syringe-infected animals indicated that baboons are a sufficient host forB. burgdorferi. Analysis of the antibody responses in infected baboons over a 48-week period revealed that antibodies are generated early during infection against many borrelial antigens, including the various OspE, OspF, and Elp paralogs that are encoded on the ubiquitous 32-kb circular plasmids (cp32s). By using the baboon sera generated by experimental infection it was determined that a combination of two cp32-encoded lipoproteins, OspE and ElpB1, resulted in highly specific and sensitive detection ofB. burgdorferiinfection. An expanded analysis, which included 39 different human Lyme disease patients, revealed that a combination of the OspE and ElpB1 lipoproteins could be the basis for a new serodiagnostic assay for Lyme disease. Importantly, this novel serodiagnostic test would be useful independent of prior OspA vaccination status.
Virus Diversity in a Winter Epidemic of Acute Diarrhea in France
Authors: Chikhi-Brachet Roxane , Bon Fabienne , Toubiana Laurent , Pothier Pierre , Nicolas Jean-Claude , Flahault Antoine , Kohli Evelyne
Published: October 2002

In France, an epidemic peak of acute diarrhea is observed each winter. Previous results suggested a viral etiology for these winter epidemics. We investigated the role of enteric viruses in acute diarrhea and their molecular diversity. One hundred sixty-one patients with acute diarrhea and 45 healthy patients (controls) from the general population were given a standardized questionnaire between December 1998 and May 1999. Stool specimens were screened for group A and C rotaviruses, human caliciviruses, astroviruses, and adenovirus types 40 and 41 by reverse transcription-PCR and/or enzyme immunoassay. Virologic analysis was positive for 63 cases (39%). Caliciviruses and group A rotaviruses were the most frequent (19 and 17% of cases, respectively). Two control stool specimens were found positive for group A rotavirus, and one was found positive for astrovirus. Molecular characterization of the strains disclosed a cocirculation of P[8],G1, P[8],G4, and P[4],G2 rotaviruses; type 1, 2, 3, 4, and 8 astroviruses; and Sapporo-like and Norwalk-like human caliciviruses. These four types of viruses accounted for an attributable risk of acute diarrhea of 34.7% for the general population, under the assumption of a causal role of these viruses.
Disseminated Mycotic Infection Caused by
Colletotrichum acutatum
in a Kemp's Ridley Sea Turtle (
Lepidochelys kempi
)
Authors: Manire Charles A. , Rhinehart Howard L. , Sutton Deanna A. , Thompson Elizabeth H. , Rinaldi Michael G. , Buck John D. , Jacobson Elliott
Published: October 2002

Colletotrichum acutatum
is a cosmopolitan plant pathogen with a wide host range. While the organism's phytopathogenic potential has been well documented, it has never been reported as an etiologic agent of disease in either animals or humans. In this case, a juvenile Kemp's ridley sea turtle,
Lepidochelys kempi
, probably with immune compromise following cold stunning (extended hypothermia), developed a disseminated mycotic infection in the lungs and kidneys. Prophylactic treatment with oral itraconazole did not prevent or cure the infection. This report of a
Colletotrichum acutatum
infection in an animal extends the range of disease caused by this organism beyond that of a phytopathogen.
Presumptive Identification ofCandida kefyron Levine Formulation of Eosin Methylene Blue Agar
Authors: Munson Erik L. , Troy Dennis R. , Weber Joanne K. , Messer Shawn A. , Pfaller Michael A.
Published: October 2002

Three hundred thirty-one yeast and yeast-like isolates were cultivated on eosin methylene blue agar. While the sensitivity rate forCandida kefyrisolates producing a metallic green sheen was 81.8%, the high positive predictive value (100%) for yeast isolates with this phenotype belonging toC. kefyrsuggests that these isolates can be presumptively identified asC. kefyr.