bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Detection and Identification of Human Adenovirus Species by Adenoplex, a Multiplex PCR-Enzyme Hybridization Assay
Authors: Pehler-Harrington Karen , Khanna Marilyn , Waters Chris R. , Henrickson Kelly J.
Published: September 2004

Human adenoviruses (AdV) have been implicated in a wide variety of diseases and are ubiquitous in populations worldwide. These agents are of concern particularly in immunocompromised patients, children, and military recruits, resulting in severe disease or death. Clinical diagnosis of AdV is usually achieved through routine viral cell culture, which can take weeks for results. Immunofluorescence and enzyme-linked immunosorbent assay-based techniques are more timely but lack sensitivity. The ability to distinguish between the six different AdV species (A to F) is diagnostically relevant, as infections with specific AdV species are often associated with unique clinical outcomes and epidemiological features. Therefore, we developed a multiplex PCR-enzyme hybridization assay, the Adenoplex, using primers to the fiber gene that can simultaneously detect all six AdV species A through F in a single test. The limit of detection (LOD) based on the viral 50% tissue culture infective dose/ml for AdV A, B, C, D, E, and F was 10
−2
, 10
−1
, 10
−1
, 10
−2
, 10
−1
, and 10
−2
, respectively. Similarly, the LOD for the six DNA controls ranged from 10
2
to 10
3
copies/ml. Twelve common respiratory pathogens were tested with the Adenoplex, and no cross-reactivity was observed. We also validated our assay using clinical specimens spiked with different concentrations of AdV strains of each species type and tested by multiplex PCR and culture. The results demonstrated an overall sensitivity and specificity of Adenoplex of 100%. This assay can be completed in as few as 5 h and provides a rapid, specific, and sensitive method to detect and subtype AdV species A through F.
Isoenzymatic Analysis of 712 Strains of
Leishmania infantum
in the South of France and Relationship of Enzymatic Polymorphism to Clinical and Epidemiological Features
Authors: Pratlong Francine , Rioux Jean-Antoine , Marty Pierre , Faraut-Gambarelli Françoise , Dereure Jacques , Lanotte Geneviève , Dedet Jean-Pierre
Published: September 2004

In the south of France, leishmaniasis due to
Leishmania infantum
occurs in the following five foci of endemicity (from west to east): Pyrénées-Orientales, Cévennes, Provence, Côte d'Azur, and Corsica. Between 1981 and 2002, 712
Leishmania
strains obtained from humans, dogs, cats, and sand flies were studied by isoenzyme analysis. In total, seven zymodemes were identified: MON-1, MON-11, MON-24, MON-29, MON-33, MON-34, and MON-108. The Pyrénées-Orientales focus is characterized by a predominance of human cutaneous leishmaniasis and a high enzymatic polymorphism (five zymodemes). In the other foci, where human visceral leishmaniasis is predominant, only two zymodemes are present.
L. infantum
MON-1 is the parasite most frequently found, in patients both with and without concomitant human immunodeficiency virus infection. MON-1 is the only zymodeme present in dogs, which act as the reservoir host in all of the foci. In Cévennes, where the complete life cycle of zymodeme MON-1 has been identified,
Phlebotomus perniciosus
and
Phlebotomus ariasi
are vectors. The enzymatic polymorphism is compared to that of neighboring countries (Spain and Italy). In Pyrénées-Orientales, small variant zymodemes with electromorphs of heterozygote-like and homozygotic patterns can be explained by different genetic hypotheses.
Development and Validation of a Diagnostic DNA Microarray To Detect Quinolone-Resistant
Escherichia coli
among Clinical Isolates
Authors: Yu Xiaolei , Susa Milorad , Knabbe Cornelius , Schmid Rolf D. , Bachmann Till T.
Published: September 2004

The incidence of resistance against fluoroquinolones among pathogenic bacteria has been increasing in accordance with the worldwide use of this drug.
Escherichia coli
is one of the most relevant species for quinolone resistance. In this study, a diagnostic microarray for single-base-mutation detection was developed, which can readily identify the most prevalent
E. coli
genotypes leading to quinolone resistance. Based on genomic sequence analysis using public databases and our own DNA sequencing results, two amino acid positions (83 and 87) on the A subunit of the DNA gyrase, encoded by the
gyrA
gene, have been identified as mutation hot spots and were selected for DNA microarray detection. Oligonucleotide probes directed against these two positions were designed so that they could cover the most important resistance-causing and silent mutations. The performance of the array was validated with 30 clinical isolates of
E. coli
from four different hospitals in Germany. The microarray results were confirmed by standard DNA sequencing and were in full agreement with phenotypic antimicrobial susceptibility testing.
Real-Time Fluorescence PCR Assays for Detection and Characterization of Heat-Labile I and Heat-Stable I Enterotoxin Genes from Enterotoxigenic
Escherichia coli
Authors: Reischl Udo , Youssef Mohammad T. , Wolf Hans , Hyytia-Trees Eija , Strockbine Nancy A.
Published: September 2004

To facilitate the diagnosis of enterotoxigenic
Escherichia coli
(ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161
E. coli
isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.
Use of Recombinant E Protein Domain III-Based Enzyme-Linked Immunosorbent Assays for Differentiation of Tick-Borne Encephalitis Serocomplex Flaviviruses from Mosquito-Borne Flaviviruses
Authors: Holbrook Michael R. , Shope Robert E. , Barrett Alan D. T.
Published: September 2004

The serological diagnosis of infection by flaviviruses is complicated by the presence of flavivirus cross-reactive antibodies that produce false-positive results for flavivirus infections, especially in regions where more than one virus is endemic. Current diagnostic reagents for tick-borne flavivirus infection have been found to cross-react with yellow fever- or dengue virus-positive sera. This study utilized recombinant flavivirus E protein domain 3 (rE-D3) as a diagnostic reagent to differentiate between infection by mosquito- and tick-borne flaviviruses. This study found that the use of rE-D3 in an enzyme-linked immunosorbent assay (ELISA)-based format allowed the differentiation between serum specific for either mosquito- or tick-borne flaviviruses, but not among the members of the tick-borne encephalitis (TBE) serocomplex of flaviviruses. Sera derived against several TBE serocomplex rE-D3 were found to cross-react with heterologous rE-D3 within the TBE serocomplex, but not with those from mosquito-borne flaviviruses, in both Western blots and ELISAs. Mouse hyperimmune sera generated against TBE serocomplex viruses were also found to react specifically with TBE serocomplex rE-D3, but not with rE-D3 from mosquito-borne viruses and vice versa. When a similar test using virus-derived antigen was performed, a loss of both specificity and sensitivity was observed. These results indicate that flavivirus rE-D3 would be a useful reagent for the detection of infection by TBE serocomplex flaviviruses, several of which are potential biothreat agents, but would not provide the ability to differentiate among infections by separate members of the serocomplex.
Virulence Characteristics and Molecular Epidemiology of Enteroaggregative
Escherichia coli
Isolates from Hospitalized Diarrheal Patients in Kolkata, India
Authors: Kahali Soumen , Sarkar Bhaswati , Rajendran K. , Khanam Jasmina , Yamasaki Shinji , Nandy Ranjan K. , Bhattacharya S. K. , Ramamurthy T.
Published: September 2004

Enteroaggregative
Escherichia coli
(EAEC) is an important diarrheal enteropathogen defined by aggregative adherence to cultured epithelial cells. We have detected EAEC from 121 (6.6%) of 1,826 hospitalized patients admitted with diarrhea to the Infectious Diseases Hospital in Kolkata, India. Watery diarrhea was recorded significantly (
P
= 0.0142) more often in children. The majority of the EAEC isolates were not serotypeable (62%) and showed resistance to five or more antibiotics (76%). We studied different virulence genes and the molecular epidemiology of 121 EAEC isolates recovered from diarrheal patients. A PCR assay for detection of virulence genes, an assay for determination of clump formation in liquid culture, and a HeLa cell adherence assay were carried out to characterize the EAEC isolates. Investigations were also conducted to correlate the virulence gene profiles with diarrheal symptoms and molecular epidemiology by pulsed-field gel electrophoresis (PFGE). Two or more virulence genes were detected in 109 (90.1%) EAEC isolates. In the cluster analysis, some isolates with specific gene profiles and phenotypes formed a group or subcluster. This study highlights the comparative distributions of three fimbrial adhesins and other virulence genes among EAEC isolates. The diverse virulence gene and PFGE profiles, along with the existence of diverse serotypes and antibiograms, suggests that the EAEC isolates are genetically heterogeneous in Kolkata.
Correlation of
wbiI
Genotype, Serotype, and Isolate Source within Species of the
Burkholderia cepacia
Complex
Authors: Vinion-Dubiel Arlene D. , Spilker Theodore , Dean Charles R. , Monteil Henri , LiPuma John J. , Goldberg Joanna B.
Published: September 2004

Gram-negative bacteria of the
Burkholderia cepacia
complex (Bcc) are opportunistic pathogens that can infect the lungs of cystic fibrosis (CF) patients and can be transmitted among these patients, causing epidemics in the CF community. Lipopolysaccharide (LPS) is an important virulence factor of many gram-negative bacteria, with the O antigen component of LPS being responsible for serotype specificity. The goal of this work was to develop a genetic method of determining the serotype of Bcc isolates based on the conserved gene
wbiI
. Homologues of
wbiI
are found in polysaccharide biosynthesis gene clusters in other bacteria. Primers to a conserved region of the Bcc
wbiI
gene were able to amplify by PCR a single product in 67 of 80 Bcc isolates tested. Sequencing and restriction enzyme digestion of this
wbiI
PCR product revealed sufficient DNA polymorphisms to distinguish and group various isolates. In five of nine instances, Bcc isolates of a single serotype had a single
wbiI
restriction fragment length polymorphism (RFLP) pattern, while isolates of the other four serotypes could have multiple
wbiI
RFLP types. Species determination of the Bcc isolates revealed no obvious correlation between
wbiI
RFLP type and species. There was also no apparent correlation between
wbiI
RFLP type and the ability of a single Bcc isolate to infect an individual with CF. However three of five Bcc outbreaks involved isolates with the same
wbiI
RFLP type, indicating that
wbiI
RFLP typing may be a useful tool to help track Bcc outbreaks.
Isolation of Metronidazole-Resistant
Bacteroides fragilis
Carrying the
nimA
Nitroreductase Gene from a Patient in Washington State
Authors: Schapiro Jeffrey M. , Gupta Rachna , Stefansson Eric , Fang Ferric C. , Limaye Ajit P.
Published: September 2004

Members of the
Bacteroides fragilis
group are among the most common anaerobic bacterial isolates in clinical specimens. Metronidazole, a 5-nitroimidazole, is often used as empirical therapy for anaerobic infections. Susceptibility testing is not routinely performed because of nearly universal susceptibility of
Bacteroides
spp. to this agent. We report a case of metronidazole-resistant
Bacteroides fragilis
in the United States and demonstrate the presence of the
nimA
gene, encoding a nitroreductase previously shown to mediate resistance to 5-nitroimidazole antimicrobial agents in
B. fragilis
strains from Europe and Africa. Because clinical failures in
Bacteroides
infections have been associated with the use of inactive antimicrobial agents, clinicians need to be aware of the possibility of metronidazole-resistant
B. fragilis
strains in the United States and the importance of susceptibility testing in selected situations.
Use of the MagNA Pure LC Automated Nucleic Acid Extraction System followed by Real-Time Reverse Transcription-PCR for Ultrasensitive Quantitation of Hepatitis C Virus RNA
Authors: Cook Linda , Ng Ka-Wing , Bagabag Arthur , Corey Lawrence , Jerome Keith R.
Published: September 2004

Hepatitis C virus (HCV) infection is an increasing health problem worldwide. Quantitative assays for HCV viral load are valuable in predicting response to therapy and for following treatment efficacy. Unfortunately, most quantitative tests for HCV RNA are limited by poor sensitivity. We have developed a convenient, highly sensitive real-time reverse transcription-PCR assay for HCV RNA. The assay amplifies a portion of the 5′ untranslated region of HCV, which is then quantitated using the TaqMan 7700 detection system. Extraction of viral RNA for our assay is fully automated with the MagNA Pure LC extraction system (Roche). Our assay has a 100% detection rate for samples containing 50 IU of HCV RNA/ml and is linear up to viral loads of at least 10
9
IU/ml. The assay detects genotypes 1a, 2a, and 3a with equal efficiency. Quantitative results by our assay correlate well with HCV viral load as determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay. In clinical use, our assay is highly reproducible, with high and low control specimens showing a coefficient of variation for the logarithmic result of 2.8 and 7.0%, respectively. The combination of reproducibility, extreme sensitivity, and ease of performance makes this assay an attractive option for routine HCV viral load testing.
Identification of the Emerging Pathogen
Vibrio vulnificus
Biotype 3 by Commercially Available Phenotypic Methods
Authors: Colodner Raul , Raz Raul , Meir Irit , Lazarovich Tsilia , Lerner Larisa , Kopelowitz June , Keness Yoram , Sakran Waheeb , Ken-Dror Shifra , Bisharat Naiel
Published: September 2004

Identification of the emerging pathogen
Vibrio vulnificus
biotype 3 has become a challenge for clinical laboratories in the last few years. In this study, the abilities of five commercial systems to identify this new species have been evaluated for the first time, using a unique collection of strains. Fifty-one well-documented wild strains of
V. vulnificus
biotype 3 were processed using API 20 NE, GNI+ Vitek 1 cards, ID-GNB Vitek 2 cards, Neg Combo 20 Microscan panels, and NMIC/ID-5 BD Phoenix panels. The numbers of strains identified as
V. vulnificus
by ID-GNB, NMIC/ID-5, and GNI+ were 50 (98.0%), 46 (90.2%), and 7 (13.7%), respectively. Neg Combo 20 Microscan panels and API 20 NE were unable to identify any of the strains of this emerging pathogen to the species level and mostly misidentifies them as other species of the
Vibrionaceae
family. Data on the phenotypic pattern of
V. vulnificus
biotype 3 when processed in all five systems as presented here could help clinical laboratories in identifying this new pathogen.
Prevalence of Periodontal Pathogens in Dental Plaque of Children
Authors: Gafan Gavin P. , Lucas Victoria S. , Roberts Graham J. , Petrie Aviva , Wilson Michael , Spratt David A.
Published: September 2004

Porphyromonas gingivalis
,
Actinobacillus actinomycetemcomitans
, and
Tannerella forsythensis
have been implicated as the main etiological agents of periodontal disease. The purpose of this work was to estimate the prevalence of these organisms in plaque from children without gingivitis (group 1;
n
= 65) and from those with gingivitis (group 2;
n
= 53). Extracted DNA from plaque was subjected to two rounds of PCR targeting the 16S rRNA gene using both universal primers and species-specific primers. The results were as follows: group 1,
P. gingivalis
, 49%;
A. actinomycetemcomitans
, 55%; and
T. forsythensis
, 65%; group 2,
P. gingivalis
, 47%;
A. actinomycetemcomitans
, 59%; and
T. forsythensis
, 45%.
T. forsythensis
was detected more frequently in children with no gingivitis than in those with gingivitis (
P
= 0.03). There was no significant difference between the two groups with respect to the presence of
P. gingivalis
or
A. actinomycetemcomitans
in either group (
P
> 0.05). Logistic regression analysis revealed that the odds of a patient having gingivitis were 2.3 times greater in the absence of
T. forsythensis
. In conclusion, the results of this study have shown that the three pathogens can be detected in the dental plaque of healthy children and of those with gingivitis and that
T. forsythensis
is associated with dental plaque at sites with no gingivitis.
Field Evaluation of a Rapid Human Immunodeficiency Virus (HIV) Serial Serologic Testing Algorithm for Diagnosis and Differentiation of HIV Type 1 (HIV-1), HIV-2, and Dual HIV-1-HIV-2 Infections in West African Pregnant Women
Authors: Rouet François , Ekouevi Didier K. , Inwoley André , Chaix Marie-Laure , Burgard Marianne , Bequet Laurence , Viho Ida , Leroy Valériane , Simon François , Dabis François , Rouzioux Christine
Published: September 2004

We evaluated a two-rapid-test serial algorithm using the Determine and Genie II rapid assays, performed on-site in four peripheral laboratories during the French Agence Nationale de Recherches sur le SIDA (ANRS) 1201/1202 Ditrame Plus cohort developed for prevention of mother-to-child transmission of human immunodeficiency virus (HIV) infection in Côte d'Ivoire. A total of 1,039 specimens were retested by two commercial enzyme-linked immunosorbent assays (ELISAs). The following specimens were tested: 315 specimens found on-site to be infected with HIV type 1 (HIV-1), 8 specimens found on-site to be infected with HIV-2, 71 specimens found on-site to be infected with both HIV-1 and HIV-2, 40 specimens found on-site to have indeterminate results for HIV infection, and 605 specimens taken during a quality assurance program. For HIV discrimination, 99 positive serum samples (20 with HIV-1, 8 with HIV-2, and 71 with HIV-1 and HIV-2 on the basis of our rapid test algorithm) were retested by the Peptilav test, Western blot (WB) assays, and homemade monospecific ELISAs. Real-time DNA PCRs for the detection of HIV-1 and HIV-2 were performed with peripheral blood mononuclear cells from 35 women diagnosed on-site with HIV-1 and HIV-2 infections. Compared to the results of the ELISAs, the sensitivities of the Determine and Genie II assays were 100% (95% lower limit [95% LL], 99.1%) and 99.5% (95% confidence interval [95% CI], 98.2 to 99.9%), respectively. The specificities were 98.4% (95% CI, 96.9 to 99.3%) and 100% (95% LL, 99.3%), respectively. All serological assays gave concordant results for infections with single types. By contrast, for samples found to be infected with dual HIV types by the Genie II assay, dual reactivity was detected for only 37 samples (52.1%) by WB assays, 34 samples (47.9%) by the Peptilav assay, and 23 samples (32.4%) by the monospecific ELISAs. For specimens with dual reactivity by the Genie II assay, the rates of concordance between the real-time PCR assays and the serological assays were 25.7% for the Genie II assay, 82.9% for the Peptilav assay, 74.3% for WB assays, and 80% for the homemade ELISAs. Our algorithm provided high degrees of sensitivity and specificity comparable to those of ELISAs. Even if they are rare, women identified by the Genie II assay as being infected with HIV-1 and HIV-2 mostly appeared to be infected only with HIV-2.
Diagnosing Invasive Aspergillosis during Antifungal Therapy by PCR Analysis of Blood Samples
Authors: Lass-Flörl Cornelia , Gunsilius Eberhard , Gastl Günther , Bonatti Hugo , Freund Martin C. , Gschwendtner Andreas , Kropshofer Gabriele , Dierich Manfred P. , Petzer Andreas
Published: September 2004

We evaluated the value of
Aspergillus
PCR as a tool for diagnosing invasive aspergillosis from whole-blood samples during antifungal therapy. In a 3-year study, 36 patients receiving antifungal therapy due to chest radiographic findings highly suggestive of fungal pneumonia were evaluated. The PCR results from whole-blood samples were compared to those obtained from bronchoalveolar lavage fluids and/or tissue specimens. A total of 205 whole-blood samples, 15 fine-needle aspirations or tissue biopsy specimens, and 21 bronchoalveolar lavage fluids and tracheal secretions were analyzed using PCR. Of the 36 patients, 15 had proven, 9 had probable, and 12 had possible invasive
Aspergillus
infection according to European Organization for Research and Treatment of Cancer/Mycosis Study Group definitions. For patients with proven infection the sensitivity values of PCR in lung and blood samples were 100 and 40%, respectively. The negative predictive value of blood monitoring under conditions of antifungal treatment was 44%. Clearance of fungal DNA from blood was associated with resolution of clinical symptoms in six of nine patients with proven infection. Repeated positive PCR results for
Aspergillus
were associated with fatal outcome, as three of six patients died. For patients with probable infection the sensitivity values of PCR in lung fluid and blood were 66 and 44%, respectively. The benefit of PCR diagnosis using whole-blood samples is limited when sampling takes place after treatment has been started. Performance of
Aspergillus
PCR using tissue samples is recommended in addition to microscopic examination and culture technique for sensitive detection of fungal infection.
Continuing Diversification of
Neisseria meningitidis
W135 as a Primary Cause of Meningococcal Disease after Emergence of the Serogroup in 2000
Authors: Taha Muhamed-Kheir , Giorgini Dario , Ducos-Galand Magaly , Alonso Jean-Michel
Published: September 2004

The occurrence of a clonal outbreak of serogroup W135 (of the electrophoretic type 37 [ET-37] clonal complex) meningococcal disease among Hajj pilgrims in 2000 has led to enhanced surveillance of the evolution of this particular serogroup, formerly considered rare, in invasive infections. Since the first case of meningococcal disease due to a serogroup W135 strain was detected in France in 1994, all isolates were characterized phenotypically. We further used phenotypic and genotypic approaches to type the 101 serogroup W135 strains isolated from patients with invasive meningococcal diseases in France in 2001 and 2002. Overall, 55% of these isolates had Hajj strain-related phenotypes (60 and 52% in 2001 and 2002, respectively), although only 45% belonged to the ET-37 clonal complex. Moreover, pulsed-field gel electrophoresis of the ET-37 clonal complex isolates showed that only 32% of the serogroup W135 isolates were indistinguishable from the 2000 Hajj-related strain. Our results suggest the continuous emergence of new genetic lineages of serogroup W135 independently of the 2000 global outbreak.
Complementation in Cells Cotransfected with a Mixture of Wild-Type and Mutant Human Immunodeficiency Virus (HIV) Influences the Replication Capacities and Phenotypes of Mutant Variants in a Single-Cycle HIV Resistance Assay
Authors: Mo Hongmei , Lu Liangjun , Pithawalla Ron , Kempf Dale J. , Molla Akhteruzzaman
Published: September 2004

The impact of cotransfection of mixtures of mutant and wild type (WT) virus on the observed phenotype and replication capacity (RC) in a single-cycle human immunodeficiency virus (HIV) phenotypic assay has been investigated by cotransfecting mutant HIV clones expressing the firefly luciferase expression gene with a WT clone expressing
Renilla
luciferase. Four mutant constructs with different genotypes displayed <1% RC when transfected alone. Cotransfection of as little as 9% of the WT clone resulted in an 18- to 33-fold increase in the RC of the mutant clones. In addition, the 50% inhibitory concentration (IC
50
) of lopinavir against seven mutant clones decreased by up to 97% after incremental cotransfection of 9 to 50% of the WT clone. The enhancement of RC and decrease in IC
50
for mutant variants following cotransfection with the WT variant appear to be due to complementation rather than genetic recombination. These findings suggest that the RC and susceptibility of plasma isolates from patients who are off therapy or not adherent to treatment, in which WT virus may expand to significant levels, should be interpreted with caution.
Automated High-Throughput Mycobacterial Interspersed Repetitive Unit Typing ofMycobacterium tuberculosisStrains by a Combination of PCR and Nondenaturing High-Performance Liquid Chromatography
Authors: Evans Jason T. , Hawkey Peter M. , Smith E. Grace , Boese Kerstin A. , Warren Roderic E. , Hong George
Published: September 2004

Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing ofMycobacterium tuberculosiscomplex isolates is portable, 100% reproducible, and highly discriminatory. Nondenaturing high-performance liquid chromatography (non-dHPLC) with use of a WAVE microbial analysis system is a promising method of PCR amplicon analysis as it is low cost and requires no preanalysis processing. The aims of this study were to validate the application of WAVE microbial analysis system technology to MIRU-VNTR typing. A collection of 70 strains were cultivated in liquid culture and extracted using the QIAamp DNA minikit. Novel primers were designed to target the 12 MIRU-VNTR loci (P. Supply et al., J. Clin. Microbiol.39:3563-3571, 2001). After amplification, each PCR product was analyzed on a WAVE microbial analysis system. The fragment size was calculated from the chromatogram, and the number of tandem repeats at each locus was determined. For the collection of 70 strains 100% concordance was achieved when comparing MIRU-VNTR profiles obtained from agarose gel electrophoresis and PCRs analyzed on a WAVE microbial analysis system. The calculated fragment sizes, obtained from the WAVE microbial analysis system, were sufficiently accurate to ensure 100% confidence when assigning the number of tandem repeats to a MIRU-VNTR locus. This study is the first to report the successful use of non-dHPLC for screening for variations in the number of MIRU-VNTRs in mycobacterial DNA. Non-dHPLC analysis was demonstrated to be a rapid, low-labor input method for the detection and analysis of MIRU-VNTR amplicons. The combination with non-dHPLC further enhances the utility of MIRU-VNTR typing.
Oligonucleotide Chip for Detection of Lamivudine-Resistant Hepatitis B Virus
Authors: Jang Hyunjung , Cho Mong , Heo Jeong , Kim Hyunghoi , Jun Hongki , Shin Woowon , Cho Byungman , Park Heekyung , Kim Cheolmin
Published: September 2004

Hepatitis B virus (HBV) is one of the major causes of liver disease worldwide. It is important to conduct antiviral therapy against chronic hepatitis B to minimize the amount of liver damage. Lamivudine has been known to be an effective antiviral agent for the treatment of HBV infection. However, the emergence of viral mutants resistant to lamivudine is the main concern during the treatment of HBV-infected patients. Therefore, the detection of lamivudine-resistant mutants is of clinical importance. We have developed an oligonucleotide chip for the detection of lamivudine-resistant HBV which is rapid and accurate. The oligonucleotide chip consists of quality control probes, negative control probes, and specific oligonucleotide probes for the detection of lamivudine-resistant HBV. The specific probes consist of five probes for the detection of wild-type rtL180, rtM204, and rtV207 sequences and seven probes for the detection of HBV mutations. We tested 123 serum samples from patients with chronic HBV infection who had received lamivudine therapy. Eighty samples contained mutants with YMDD mutations. Among these, 17 contained rtM204V (YVDD), 24 contained rtM204I3 (YIDD3), 3 contained rtM204I2 (YIDD2), and 36 contained mixed types. We compared the results obtained with our oligonucleotide chip with those obtained by PCR-restriction fragment length polymorphism (RFLP) analysis and sequencing. The rate of concordance between the assay with the oligonucleotide chip and PCR-RFLP analysis for detection of the YMDD motif was 96.7%. The rate of concordance between the results obtained with the oligonucleotide chip for the detection of rtL180 and rtV207 and the results obtained by sequencing was 100%. Thus, the oligonucleotide chip is a reliable and useful tool for the detection of antiviral-resistant HBV.
Identification of Some Charcoal-Black-Pigmented CDC Fermentative Coryneform Group 4 Isolates asRothia dentocariosaand Some asCorynebacterium aurimucosum: Proposal ofRothia dentocariosaemend. Georg and Brown 1967,Corynebacterium aurimucosumemend. Yassin et al. 2002, andCorynebacterium nigricansShukla et al. 2003 pro synon.Corynebacterium aurimucosum
Authors: Daneshvar Maryam I. , Hollis Dannie G. , Weyant Robbin S. , Jordan Jean G. , MacGregor John P. , Morey Roger E. , Whitney Anne M. , Brenner Don J. , Steigerwalt Arnold G. , Helsel Leta O. , Raney Patti M. , Patel Jean B. , Levett Paul N. , Brown June M.
Published: September 2004

Sixty-three clinical isolates of charcoal-black-pigmented, gram-positive coryneform rods were received for identification by the Centers for Disease Control and Prevention (CDC) and were provisionally designated CDC fermentative coryneform group 4 (FCG4). Forty-five of these were characterized by morphological, physiologic, antimicrobial susceptibility, cellular fatty acids, 16S rRNA gene sequencing, and DNA-DNA hybridization analyses. Nitrate reduction, cellular fatty acid analysis, 16S rRNA gene sequencing, and DNA-DNA hybridization studies segregated these strains into two groups: FCG4a (8 strains) and FCG4b (37 strains). The FCG4a strains, only one of which was from a female genitourinary source, produced cellular fatty acid and biochemical profiles similar to those observed with reference strains ofRothia dentocariosaandRothia mucilaginosa, while the FCG4b strains were similar toCorynebacteriumspecies. DNA-DNA hybridization analysis demonstrated species-level relatedness among six FCG4a tested strains and showed that they were a charcoal-black-pigmented variant ofR. dentocariosa. Sixteen isolates of the FCG4b group, mainly from female genitourinary tract specimens, as well as the type strains of two recently named species,Corynebacterium aurimucosumandCorynebacterium nigricans, were shown by DNA-DNA hybridization analysis and the sequencing of the 16S rRNA gene to be related at the species level and unrelated to the type strain ofR. dentocariosa; therefore, theCorynebacterium-like strains were classified as a charcoal-black-pigmented variant ofC. aurimucosum, because this name has nomenclatural priority overC. nigricans. These findings indicate that FCG4 represents a heterogeneous group that contains pigmented variants of bothR. dentocariosaandC. aurimucosum; hence, the descriptions of bothR. dentocariosaandC. aurimucosumhave been amended to include charcoal-black-pigmented variants, andC. nigricansis a pro synonym ofC. aurimucosum.
Correlation of Cerebrospinal Fluid (CSF) Cell Counts and Elevated CSF Protein Levels with Enterovirus Reverse Transcription-PCR Results in Pediatric and Adult Patients
Authors: Mulford William S. , Buller Richard S. , Arens Max Q. , Storch Gregory A.
Published: September 2004

During the 2001, 2002, and 2003 enterovirus seasons, we investigated the correlations between cerebrospinal fluid (CSF) nucleated cell counts and elevated CSF protein levels and the detection of enteroviral RNA by reverse transcription (RT)-PCR. Our objective was to determine if pleocytosis and/or elevated protein levels were predictive of positive RT-PCR results for enterovirus. We were also interested in determining if the presence of West Nile virus during the 2002 enteroviral season contributed to a change in these correlations. We found that in the group of patients aged >2 months, the absence of pleocytosis was highly predictive of a negative RT-PCR result. Elevated CSF protein level was not a good predictor of RT-PCR positivity for enterovirus and did not add to the diagnostic sensitivity or specificity of pleocytosis.
Rapid Detection of Isoniazid and Rifampin Resistance Mutations in
Mycobacterium tuberculosis
Complex from Cultures or Smear-Positive Sputa by Use of Molecular Beacons
Authors: Lin S.-Y. Grace , Probert William , Lo Melanie , Desmond Edward
Published: September 2004

The slow-growing nature of
Mycobacterium tuberculosis
complex hinders the improvement of turnaround time for phenotypic drug susceptibility testing. We designed a set of molecular beacons for the detection of isoniazid and rifampin resistance mutations in
M. tuberculosis
complex organisms from cultures or from
N
-acetyl-
l
-cysteine-NaOH-treated, smear-positive specimens. The performance of the molecular beacons was characterized by studying a total of 196 clinical isolates (127 drug-resistant isolates and 69 drug-susceptible isolates). For detection of isoniazid resistance, the sensitivity and specificity of the assay were 82.7 and 100%, and the positive predictive value (PPV) and negative predictive value (NPV) at a resistance prevalence of 10% were 100 and 98.11%, respectively. For detection of rifampin resistance, the sensitivity and specificity of the assay were 97.5 and 100%, and the PPV and NPV at a resistance prevalence of 2.0% were 100 and 99.95%, respectively.
Laboratory Reporting of Tuberculosis Test Results and Patient Treatment Initiation in California
Authors: Pascopella Lisa , Kellam Steffi , Ridderhof John , Chin Daniel P. , Reingold Arthur , Desmond Edward , Flood Jennifer , Royce Sarah
Published: September 2004

Prompt laboratory reporting of tuberculosis (TB) test results is necessary for TB control. To understand the extent of and factors contributing to laboratory reporting delays and the impact of reporting delays on initiation of treatment of TB patients, we analyzed data from 300 consecutive culture-positive TB cases reported in four California counties in 1998. Laboratory reporting to the specimen submitter was delayed for 26.9% of smear-positive patients and 46.8% of smear-negative patients. Delays were associated with the type of laboratory that performed the testing and with delayed transport of specimens. Referral laboratories (public health and commercial) had longer median reporting time frames than hospital and health maintenance organization laboratories. Among patients whose treatment was not started until specimens were collected, those with delayed laboratory reporting were more likely to have delayed treatment than patients with no laboratory reporting delays (odds ratio [OR] of 3.9 and 95% confidence interval [CI] of 1.6 to 9.7 for smear-positive patients and OR of 13.1 and CI of 5.3 to 32.2 for smear-negative patients). This relation remained after adjustment in a multivariate model for other factors associated with treatment delays (adjusted OR of 25.64 and CI of 7.81 to 83.33 for smear-negative patients). These findings emphasize the need to reduce times of specimen transfer between institutions and to ensure rapid communication among laboratories, health care providers, and health departments serving TB patients.
Accurate Representation of the Hepatitis C Virus Quasispecies in 5.2-Kilobase Amplicons
Authors: Liu Zhi , Netski Dale M. , Mao Qing , Laeyendecker Oliver , Ticehurst John R. , Wang Xiao-Hong , Thomas David L. , Ray Stuart C.
Published: September 2004

Hepatitis C virus (HCV) exists as a swarm of genetically distinct but related variants, or a quasispecies, whose complexity and sequence evolution are critical to studies of viral pathogenesis. Because most studies of the HCV quasispecies have focused on a relatively small genomic segment, the first hypervariable region of the E2 gene, it is possible that viral complexity is occasionally underestimated (due to primer mismatch) and that sequence evolution is misperceived due to unrecognized covariation. This report describes a sensitive and reproducible method to amplify most of the HCV genome as a single 5.2-kb amplicon by using primers directed at relatively conserved genomic segments. Using 52 specimens obtained during acute infection over a range of viral RNA concentrations, the overall rate of successful amplification was 94% and varied in a concentration-dependent manner, with successful amplification in 26 of 26 (100%) specimens at greater than 10
5
IU/ml, 15 of 16 (94%) at 10
4
to 10
5
IU/ml, 6 of 7 (86%) at 10
3
to 10
4
IU/ml, and 2 of 3 (67%) at less than 10
3
IU/ml. Quasispecies complexity, determined by using this novel long-amplicon method followed by heteroduplex mobility assay combined with single-stranded conformational polymorphism (HDA+SSCP) analysis, was very high, even during acute HCV infection, when 10 to 21 (median, 16) different HDA+SSCP patterns were detected among 33 cDNA clones examined. Replicate analyses indicate that this diversity is not dominated by random errors generated during amplification. Therefore, the HCV quasispecies is highly complex even during acute infection and is accurately represented in amplicons representing more than half of the viral genome.
Use of the Minimum Spanning Tree Model for Molecular Epidemiological Investigation of a Nosocomial Outbreak of Hepatitis C Virus Infection
Authors: Spada Enea , Sagliocca Luciano , Sourdis John , Garbuglia Anna Rosa , Poggi Vincenzo , De Fusco Carmela , Mele Alfonso
Published: September 2004

The minimum spanning tree (MST) model was applied to identify the history of transmission of hepatitis C virus (HCV) infection in an outbreak involving five children attending a pediatric oncology-hematology outpatient ward between 1992 and 2000. We collected blood samples from all children attending since 1992, all household contacts, and one health care worker positive for antibody to HCV (anti-HCV). HCV RNA detection was performed with these samples and with smears of routinely collected bone marrow samples. For all isolates, we performed sequence analysis and phylogenetic tree analysis of hypervariable region 1 of the E2 gene. The MST model was applied to clinical-epidemiological and molecular data. No additional cases were detected. All children, but not the health care worker, showed genotype 3a. On six occasions, all but one child had shared the medication room with another patient who later seroconverted. HCV RNA detection in bone marrow smears revealed, in some cases, a delay of several months in anti-HCV responses. Sequence analysis and phylogenetic tree analysis revealed a high identity among the isolates. The MST model applied to molecular data, together with the clinical-epidemiological data, allowed us to identify the source of the outbreak and the most probable patient-to-patient chain of transmission. The management of central venous catheters was suspected to be the probable route of transmission. In conclusion, the MST model, supported by an exhaustive clinical-epidemiological investigation, appears to be a useful tool in tracing the history of transmission in outbreaks of HCV infection.
Persistent Histidine-Rich Protein 2, Parasite Lactate Dehydrogenase, and Panmalarial Antigen Reactivity after Clearance of
Plasmodium falciparum
Monoinfection
Authors: Iqbal Jamshaid , Siddique Ahmed , Jameel Mohammad , Hira Persotum R.
Published: September 2004

We tested 240 patients with
Plasmodium falciparum
monoinfection for persistent parasite antigenemia after successful standardized antimalarial therapy by using the ICT Malaria Pf/Pv and OptiMAL-IT assays that detect the malaria antigens
Plasmodium falciparum
histidine-rich protein 2 (HRP2) and parasite lactate dehydrogenase (pLDH), respectively, as well as a panmalarial antigen (PMA). The patients were screened for antigenemia on days 0, 3, 7, and 14 of follow-up. On day 0, all 240 patients showed positive reactivity with both assays. Of the 229 cases with negative parasitemia on day 3, persistent antigenemia was observed in 207 (90.4%) of the cases: 188 (82.1%) for HRP2 antigen and 75 (32.8%) for PMA. There was a gradual decrease in antigenemia on follow-up to day 14; however, the drop in reactivity to PMA was less than that for HRP2 antigen. In contrast to HRP2 antigenemia, there was a significant decrease in pLDH antigenemia to 38.4% and to 14.8% (PMA) on day 3 (
P
< 0.03). The pLDH antigenemia level dropped further to 14.8% on day 7. There was no significant association of persistent antigenemia with gametocytemia. One case with gametocytemia was negative for both the antigens. In conclusion, the OptiMAL-IT assay is more sensitive than the ICT Malaria Pf/Pv test for monitoring therapeutic responses after antimalarial therapy since the LDH activity ceases when the malarial parasite dies.
Risk Factors for Colonization and Infection in a Hospital Outbreak Caused by a Strain of
Klebsiella pneumoniae
with Reduced Susceptibility to Expanded-Spectrum Cephalosporins
Authors: Cartelle Monica , del Mar Tomas Maria , Pertega Sonia , Beceiro Alejandro , Dominguez M. Angeles , Velasco David , Molina Francisca , Villanueva Rosa , Bou German
Published: September 2004

Between February 2001 and January 2002, an increase in the number of
Klebsiella pneumoniae
isolates with reduced susceptibility to expanded-spectrum cephalosporins (RSKp) was detected in the neonatal unit of the Juan Canalejo Hospital, and 21 patients were either colonized or infected by the bacterial isolates. The current “gold standard” method for typing
K. pneumoniae
isolates is pulsed-field gel electrophoresis. However, this technique is expensive and time-consuming. In a search for faster and accurate alternatives to this method, we investigated PCR-based fingerprinting techniques (enterobacterial repetitive intergenic consensus sequence PCR [ERIC-PCR], repetitive extragenic palindromic sequence-based PCR [REP-PCR], and RAPD [randomly amplified polymorphic DNA]) for their ability to characterize
K. pneumoniae
isolates. The causal agent of the nosocomial outbreak was characterized by these techniques and was found to be a single epidemic strain (RSKp). A multiple regression logistic model was developed to identify potential independent factors associated with colonization and/or infection by RSKp. Logistic regression analysis was applied to all significant variables (
P
< 0.05) in the univariate analysis, and it was revealed that intubation (odds ratio [OR], 27.0; 95% confidence interval [95%CI], 5.39 to 135.14) and prematurity (OR, 4.4; 95%CI, 0.89 to 21.89) were such independent factors. Moreover, oxime cephalosporins did not appear to be statistically significant. Overall, the results showed that PCR-based techniques are expeditious and useful methods for typing
K. pneumoniae
isolates. Of the techniques studied, ERIC-PCR showed the highest discriminatory index (D = 0.828), followed by RAPD (D = 0.826) and REP-PCR (D = 0.773)
Identification and Typing of
Malassezia
Species by Amplified Fragment Length Polymorphism and Sequence Analyses of the Internal Transcribed Spacer and Large-Subunit Regions of Ribosomal DNA
Authors: Gupta Aditya K. , Boekhout Teun , Theelen Bart , Summerbell Richard , Batra Roma
Published: September 2004

Malassezia
yeasts are associated with several dermatological disorders. The conventional identification of
Malassezia
species by phenotypic methods is complicated and time-consuming, and the results based on culture methods are difficult to interpret. A comparative molecular approach based on the use of three molecular techniques, namely, amplified fragment length polymorphism (AFLP) analysis, sequencing of the internal transcribed spacer, and sequencing of the D1 and D2 domains of the large-subunit ribosomal DNA region, was applied for the identification of
Malassezia
species. All species could be correctly identified by means of these methods. The results of AFLP analysis and sequencing were in complete agreement with each other. However, some discrepancies were noted when the molecular methods were compared with the phenotypic method of identification. Specific genotypes were distinguished within a collection of
Malassezia furfur
isolates from Canadian sources. AFLP analysis revealed significant geographical differences between the North American and European
M. furfur
strains.
Simple Subtyping Assay for Human Immunodeficiency Virus Type 1 Subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC
Authors: Wei Min , Guan Qi , Liang Hao , Chen Jianping , Chen Zhao , Hei Faxin , Feng Yi , Hong Kunxue , Huang Hailong , Xing Hui , Shao Yiming
Published: September 2004

After the initial development of a human immunodeficiency virus type 1 (HIV-1) subtype-screening tool by nested multiplex PCR, we further improve it through the redesign of subtype-specific primers based on subtype signature pattern (SSP) analyses and optimization of the PCR conditions. Extracted RNA from plasma samples was used in reverse transcription and the cDNA products were added to the first round PCR, in which universal primers in the
gag
region were used to detect HIV-1 M group isolates. In the second round of PCR, three pairs of subtype-specific primers, detecting subtypes B, C, and CRF01-AE, were added in one tube. Subtype determination was based on the different size of PCR products on the agarose gel electrophoresis. An additional set of primers detecting only the prevalent recombinant strains CRF07-BC and CRF08-BC was used to discriminate CRF07- and CRF08-BC from pure subtype C. Testing for all kinds of HIV subtype reference strains indicated that this assay was applicable. A panel of 252 HIV-positive samples and 30 HIV-negative samples was further used to evaluate and validate this assay. Compared to the assay of sequence-based phylogenetic analysis, the newly developed assay has an adequate designated subtype sensitivity, 93.2% (69 of 74) for subtype B, 95.1% (117 of 123) for subtype C, 94.0% (47 of 50) for CRF01-AE, and 95.0% (115 of 121) for CRF07-BC and CRF08-BC. Most importantly, the intersubtype specificity of the assay was found to be 100%. The assay specificity was also found to be 100% when used to test 30 HIV-negative samples. The average reproducibility was 96.0% for subtype B, 96.7% for subtype C, and 95.0% for CRF01-AE. We have developed a simple, rapid, and low cost assay for screening subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC in China.