bluuebunny/crossref_metadata_2025 huggingface dataset:
BacillusSpecies Are Present in Chewing Tobacco Sold in the United States and Evoke Plasma Exudation from the Oral Mucosa
Authors: Rubinstein Israel , Pedersen Gerald W.
Published: August 2002

FiveBacillusspecies, predominantlyBacillus megateriumandBacillus pumilus, were isolated from two popular brands of commercially available chewing tobacco [(5.0 ± 1) × 106CFU/ml of supernatant; results for four experiments]. Moreover, the supernatant of theBacillusculture evoked plasma exudation from postcapillary venules in the intact hamster cheek pouch, exudation that was mediated by the kallikrein/kinin metabolic pathway. Taken together, these data indicate thatBacillusspecies contaminate chewing tobacco commercially available in the United States and elaborate a potent exogenous virulence factor(s) that injures the oral mucosa.
Affordable CD4
+
-T-Cell Counting by Flow Cytometry: CD45 Gating for Volumetric Analysis
Authors: Janossy George , Jani Ilesh V. , Bradley Nicholas J. , Bikoue Arsene , Pitfield Tim , Glencross Debbie K.
Published: August 2002

The flow cytometers that are currently supported by industry provide accurate CD4
+
-T-cell counts for monitoring human immunodeficiency virus disease but remain unaffordable for routine service work under resource-poor conditions. We therefore combined volumetric flow cytometry (measuring absolute lymphocyte counts in unit volumes of blood) and simpler protocols with generic monoclonal antibodies (MAbs) to increase cost efficiency. Volumetric absolute counts were generated using CD45/CD4 and CD45/CD8 MAb combinations in two parallel tubes. The percentage values for the various subsets were also determined within the leukocyte and lymphocyte populations utilizing a fully automated protocol. The levels of agreement between the newly developed method and the present industry standards, including both volumetric and bead-based systems using a full MAb panel for subset analysis, were tested by Bland-Altman analyses. The limits of agreement for CD4 counts generated by the volumetric methods using either CD45/CD4 (in a single tube) or the full Trio MAb panel (in three tubes) on the CytoronAbsolute flow cytometer were between −29 and +46 cells/mm
3
with very little bias for CD4 counts (in favor of the Trio method: +8 CD4
+
lymphocytes/mm
3
; 0.38% of lymphocytes). The limits of agreement for absolute CD4 counts yielded by the volumetric CD45/CD4 method and the bead-based method were between −118 and +98 cells/mm
3
, again with a negligible bias (−10 CD4
+
lymphocytes/mm
3
). In the volumetric method using CD45/CD8, the strongly CD8
+
cells were gated and the levels of agreement with the full Trio showed a minor bias (in favor of the Trio; +40 CD8
+
cells/mm
3
; 5.2% of lymphocytes) without a significant influence on CD4/CD8 ratios. One trained flow cytometrist was able to process 300 to 400 stained tubes per day. This workload extrapolates to a throughput of >30,000 samples per year if both CD45/CD4 and CD45/CD8 stainings are performed for each patient or a throughput of >60,000 samples if only CD45/CD4 counts are tested in a single tube. Thus, on the basis of the high efficiency and excellent agreement with the present industry standards, volumetric flow cytometers with automated gating protocols and autobiosamplers, complemented by generic CD45, CD4, and CD8 MAbs used in two-color immunofluorescence, represent the most suitable arrangements for large regional laboratories in resource-poor settings.
Double-Blind Study To Evaluate Flow Cytometry Analysis of Anti-Live Trypomastigote Antibodies for Monitoring Treatment Efficacy in Cases of Human Chagas' Disease
Authors: Martins-Filho Olindo Assis , Eloi-Santos Silvana Maria , Carvalho Andréa Teixeira , Oliveira Rodrigo Corrêa , Rassi Anis , Luquetti Alejandro Ostemayer , Rassi Gustavo Gabriel , Brener Zigman
Published: August 2002

The validation of flow cytometry analysis of anti-live trypomastigote antibodies (FC-ALTA) to monitor cure after treatment of Chagas' disease was evaluated with serum samples from treated and nontreated chagasic patients. After optimization of the original technique, toward better sensitivity and applicability to field surveys, we design a double blind study of 94 coded samples classified into the following categories: patients not treated (NT) and patients treated but not cured (TNC), both presenting positive conventional serology and xenodiagnosis; patients treated and cured (TC), showing negative serology and xenodiagnosis; and patients treated under evaluation (TUE), who presented positive or oscillating conventional serology (CSA) but negative xenodiagnosis. Coded samples, diluted 1:256, were assayed by incubation with live cell culture trypomastigotes, which were subsequently stained with fluorescein isothiocyanate-conjugated anti-human immunoglobulin G, with prior fixation and analysis by flow cytometry. The results were expressed as the percentages of positive fluorescent parasites (PPFP) for each individual sample, establishing 20% PPFP as the cutoff between negative and positive results. Our data demonstrated that all NT and TNC presented positive results while all but one TC had a PPFP lower than 20%. Analysis of TUE demonstrated a wide degree of reactivity, with PPFP values that were negative (PPFP ≤ 20%), low positive (20% < PPFP ≤ 50%), and high positive (PPFP > 50%). As TUE with negative PPFP presented negative xenodiagnosis and positive or oscillating CSA, they were classified as dissociated according to the criteria of Krettli and Brener (J. Immunol.128:2009-2012, 1982) and could indeed be considered cured after chemotherapy. This study demonstrates and validates the use of FC-ALTA to easily identify anti-live trypomastigote membrane-bound antibodies, offering another approach for investigating and monitoring the efficacy of specific chemotherapy in cases of human Chagas' disease.
Modulation of Human Immunodeficiency Virus (HIV)-Specific Immune Response by Using Efavirenz, Nelfinavir, and Stavudine in a Rescue Therapy Regimen for HIV-Infected, Drug-Experienced Patients
Authors: Trabattoni Daria , Lo Caputo Sergio , Biasin Mara , Seminari Elena , Di Pietro Massimo , Ravasi Giovanni , Mazzotta Francesco , Maserati Renato , Clerici Mario
Published: August 2002

Analysis of the virologic and immunomodulatory effects of an association of efavirenz (EFV), nelfinavir (NFV), and stavudine (d4T) was performed in 18 human immunodeficiency virus (HIV)-infected and highly active antiretroviral therapy (HAART)-experienced patients who failed multiple therapeutic protocols. Patients (<500 CD4
+
cells/μl; >10,000 HIV copies/ml) were nonnucleoside reverse transcriptase inhibitor (NNRTI)-naive and were treated for 10 months with EFV (600 mg/day) in association with NFV (750 mg three times daily) and d4T (30 or 40 mg twice daily). Measurement of HIV peptide- and mitogen-stimulated production of interleukin-2 (IL-2), gamma interferon (IFN-γ), IL-4, and IL-10 as well as quantitation of mRNA for the same cytokines in unstimulated peripheral blood mononuclear cells were performed at baseline and 2 weeks (t1), 2 months (t2), and 10 months (t3) into therapy. The results showed that HIV-specific (but not mitogen-stimulated) IL-2 and IFN-γ production was augmented and IL-10 production was reduced in patients who received EFV, NFV, and d4T. Therapy was also associated with a reduction in HIV RNA in plasma and an increase in CD4
+
cell count. These changes occurred in the first year of therapy (t2 and t3) and were confirmed by quantitation of cytokine-specific mRNA. Therapy with EFV, NFV, and d4T increases HIV-specific type 1 cytokine production as well as CD4 counts and reduces plasma viremia. This therapeutic regimen may be considered for use in cases of advanced HIV infection.
Immunoblot Analysis of the Humoral Immune Response toLeishmania donovaniPolypeptides in Cases of Human Visceral Leishmaniasis: Its Usefulness in Prognosis
Authors: Kumar Promod , Pai Kalpana , Tripathi Kiran , Pandey H. P. , Sundar Shyam
Published: August 2002

Sera from Indian patients with parasitologically confirmed visceral leishmaniasis were studied by immunoblot analysis in order to identify a specific pattern forLeishmaniainfection. A soluble extract ofLeishmania donovaniwas used as antigen. At diagnosis the sera from patients with visceral leishmaniasis specifically recognized fractions represented by bands of 201 kDa (50% of serum samples), 193 kDa (60%), 147 kDa (50%), 120 kDa (60%), 100 kDa (50%), 80 kDa (80%), 70 kDa (70%), 65 kDa (100%), 50 kDa (50%), 36 kDa (50%), 20 kDa (70%), and 18 kDa (50%). The 65-kDa band, common to all patients infected withLeishmaniaparasites, was found at the time of diagnosis. However, the immunoblot pattern changed after patients were treated and cured with sodium antimony gluconate (SAG;n=10) or miltefosine (n=10), as was evident from blots of sera obtained pretreatment and at 1, 3, and 6 months posttreatment. At 6 months posttreatment, immunoblots of sera from patients on the SAG regimen showed the disappearance of all bands except the 70-kDa band. Similarly, sera from those on the miltefosine regimen showed the disappearance of all bands except the 65- and 70-kDa bands. This study shows that Western blot analysis is a sensitive test for detection of anti-Leishmaniaantibodies. Moreover, the persistence of reactivity with the 65- and 70-kDa bands in the sera of all groups shows its promise as a diagnostic and prognostic tool.
Helicobacter pylori
-Specific Immune Responses of Children: Implications for Future Vaccination Strategy
Authors: Bode Günter , Piechotowski Isolde , Rothenbacher Dietrich , Brenner Hermann
Published: August 2002

We analyzed the specific anti-
Helicobacter pylori
immunoglobulin G (IgG) antibody profile for a sample of 824 asymptomatic schoolchildren in southern Germany (mean age, 10.7 ± 0.65 years) with an H. pylori-specific IgG enzyme-linked immunosorbent assay and Western blot analysis. The prevalence of infection was 19.8% (95% confidence interval, 17.1 to 22.7%). The immunoresponses were characterized predominantly by antibodies against low-molecular-mass antigens of 14 and 29 kDa, with a significant difference between children of German and Turkish nationalities (
P
= 0.0012 and
P
< 0.0001, respectively).
CD4
+
CD25
+
T-Cell Production in Healthy Humans and in Patients with Thymic Hypoplasia
Authors: Sullivan Kathleen E. , McDonald-McGinn Donna , Zackai Elaine H.
Published: August 2002

Regulatory T cells are found primarily in the CD4
+
CD25
+
fraction of T cells and play an important role in the prevention of autoimmunity. We examined CD4
+
CD25
+
T cells in 33 healthy children and adults and compared them to a population with an inherited form of thymic hypoplasia and a predisposition to autoimmune disease. Absolute numbers of CD4
+
CD25
+
T cells were markedly higher in healthy infants than in infants with chromosome 22q11.2 deletion syndrome.
Peripheral Blood Lymphocyte Subsets in Adolescents: a Longitudinal Analysis from the REACH Project
Authors: Rudy Bret J. , Wilson Craig M. , Durako Stephen , Moscicki Anna-Barbara , Muenz Larry , Douglas Steven D.
Published: August 2002

Flow cytometry analysis of lymphocyte subset markers was performed for a group of sexually active, human immunodeficiency virus (HIV)-negative adolescents over a 2-year period to establish normative data. Data were collected in the REACH Project (Reaching for Excellence in Adolescent Care and Health), a multicenter, longitudinal study of HIV-positive and high-risk HIV-negative adolescents. Two- and three-color flow cytometry data were collected every 6 months for these subjects. We determined the effects of gender, race, and age on the following lymphocyte subset markers: total CD4
+
cells, CD4
+
naïve cells, CD4
+
memory cells, all CD8
+
cells, CD8
+
naïve cells, CD8
+
memory cells, CD16
+
natural killer cells, and CD19
+
B cells. Gender was the demographic characteristic most frequently associated with differences in lymphocyte subset measures. Females had higher total CD4
+
cell and CD4
+
memory cells counts and lower CD16
+
cell counts than males. Age was associated with higher CD4
+
memory cell counts as well as higher CD8
+
memory cell counts. For CD19
+
cells, there was an interaction between age and gender, with males having significantly lower CD19
+
cell counts with increasing age, whereas there was no age effect for females. Race and/or ethnicity was associated with differences in total CD8
+
cell counts and CD8
+
memory cell counts, although both of these associations involved an interaction with gender.
Evaluation of an In-House-Developed Radioassay Kit for Antibody Detection in Cases of Pulmonary Tuberculosis and Tuberculous Meningitis
Authors: Kameswaran M. , Shetty K. , Ray M. K. , Jaleel M. A. , Kadival G. V.
Published: August 2002

A radioassay for the detection of antitubercular antibody has been developed. The technique involves the addition of
125
I-labeled
Mycobacterium tuberculosis
antigen as a tracer, diluted clinical sample (serum or cerebrospinal fluid [CSF]), and heat-inactivated
Staphylococcus aureus
to capture the antibody, incubation for 4 h, and quantitation of the amount of antibody present in the sample. A total of 330 serum samples from patients with pulmonary tuberculosis and 138 control serum samples from individuals who were vaccinated with
M. bovis
BCG and from patients with pulmonary disorders of nontubercular origin were analyzed. Also, 26 CSF samples from patients with tuberculous meningitis and 24 CSF samples as controls from patients with central nervous system disorders of nontuberculous origin were analyzed. Sensitivities of 80 and 73% were observed for patients with pulmonary tuberculosis and tuberculous meningitis, respectively, and specificities of 90 and 88% were seen for the two groups of patients, respectively. The sensitivity was lower, however, for human immunodeficiency virus-infected patients coinfected with
M. tuberculosis
. The control population could be differentiated from the patient population. This assay is rapid and user friendly and, with its good sensitivity and specificity, should benefit the population by providing diagnoses early in the course of disease and, hence, permit the early administration of appropriate chemotherapy.
Functional and Phenotypic Changes in Circulating Lymphocytes from Hospitalized Zambian Children with Measles
Authors: Ryon Judith J. , Moss William J. , Monze Mwaka , Griffin Diane E.
Published: August 2002

Measles is associated with immunosuppression and increased susceptibility to secondary infections and is a particular problem in developing countries. Lymphocyte changes accompanying immune activation and regulation of the immune response may contribute to immunosuppression. To evaluate lymphocyte changes during measles, children (n= 274) hospitalized with measles in Lusaka, Zambia, were evaluated at entry, discharge, and 1-month follow-up and compared to healthy Zambian children (n= 98). Lymphopenia was present on hospital admission and reflected decreased CD4 and CD8 T cells but resolved quickly. Lymphopenia was most marked in girls, in those with temperatures of >38.5°C, and in malnourished children. CD4/CD8 ratios were decreased at all time points and were lower in boys than in girls at discharge and follow-up. Spontaneous death occurred in cultured lymphocytes, and the proportions of freshly isolated cells undergoing apoptosis, based on annexin V and propidium iodide staining, were increased. Surface Fas was increased on both CD4 and CD8 T cells compared to controls, and expression was greater on CD4 T cells and was inversely correlated with lymphocyte viability in culture at study entry. Mitogen stimulation of lymphocytes improved viability, but inhibitors of Fas, tumor necrosis factor (TNF)-related apoptosis-inducing ligand, and TNF did not. Plasma levels of β2microglobulin and soluble Fas, Fas ligand, CD8, CD4, and TNF receptor were increased, and soluble CD8 was higher in boys than in girls. The multiple effects of measles on lymphocytes from Zambian children include decreased numbers in circulation, increased activation, and increased susceptibility to cell death, with substantive differences in the magnitude of these changes between boys and girls.
Emergence of
Klebsiella pneumoniae
Isolates Producing Inducible DHA-1 β-Lactamase in a University Hospital in Taiwan
Authors: Yan Jing-Jou , Ko Wen-Chien , Jung Yun-Chih , Chuang Chin-Luan , Wu Jiunn-Jong
Published: August 2002

Ten nonrepetitive clinical isolates of
Klebsiella pneumoniae
exhibiting an unusual inducible β-lactam resistance phenotype were identified between January 1999 and September 2001 in a university hospital in Taiwan. In the presence of 2 μg of clavulanic acid, the isolates showed a one to four twofold concentration increase in the MICs of ceftazidime, cefotaxime, and aztreonam but remained susceptible to cefepime (MICs, ≤0.5 μg/ml) and imipenem (MICs, ≤0.5 μg/ml). PCR, sequence analysis, and isoelectric focusing revealed production by these isolates of TEM-1, SHV-11, and DHA-1, a plasmid-encoded inducible AmpC β-lactamase originally found in a
Salmonella enterica
serovar Enteritidis strain. Transfer of the resistance by conjugation experiments was not successful, but Southern hybridization showed that
bla
DHA-1
was located on 70-kb plasmids, suggesting that the
bla
DHA-1
-containing plasmids in the
K. pneumoniae
isolates were non-self-transmissible. Five isolates were recovered from patients in two surgery wards and two intensive care units. Acquisition of the DHA-1 producers could be traced back to previous hospitalizations 1 to 5 months earlier for the other five patients. Six and seven patterns among the isolates were demonstrated by plasmid analysis and ribotyping, respectively, indicating that the spread of the DHA-1 producers was due to both horizontal transfer of
bla
DHA-1
and dissemination of endemic clones.
Hepatitis C Virus Genotyping: Interrogation of the 5′ Untranslated Region Cannot Accurately Distinguish Genotypes 1a and 1b
Authors: Chen Zhenyu , Weck Karen E.
Published: August 2002

Although the 5′ untranslated region (5′ UTR) is the most conserved region of the hepatitis C virus (HCV) genome, it has been suggested that interrogation of this region is sufficient for determination of the HCV genotype. We compared two methods of determination of the HCV genotype: (i) direct sequencing of the DNA of the NS-5b region and (ii) reverse line probe assay (LiPA; INNO-LiPA HCV II; Innogenetics N.V.) of the 5′ UTR. There was 100% concordance between the two methods for genotype but only 80% concordance for subtype. A significant percentage of genotype 1a isolates were misclassified by LiPA as genotype 1b. Sequence analysis revealed that the only consistent difference in the 5′ UTR for these genotype 1a isolates misclassified as genotype 1b was a single nucleotide (A/G) at position −99 of the HCV genome. All isolates with discordant results analyzed had a G at this position, consistent with LiPA determination of these samples as subtype 1b. However, sequence analysis of 222 nucleotides in the NS-5b region clearly identified all of these isolates as subtype 1a. Population distribution data from the University of Pittsburgh Medical Center of over 200 samples analyzed by sequencing of the NS-5b region and over 1,000 samples analyzed by LiPA also indicated that INNO-LiPA HCV II cannot accurately differentiate HCV genotype 1a isolates from HCV genotype 1b isolates. We provide evidence that the A/G at position −99 represents a sequence polymorphism in the HCV genome that cannot differentiate subtype 1a from subtype 1b isolates. In conclusion, the 5′ UTR is not heterogeneous enough for use in determination of the HCV subtype and cannot be used for differentiation of HCV genotypes 1a and 1b.
Replacement of
Candida albicans
with
C. dubliniensis
in Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis Treated with Fluconazole
Authors: Martinez Marcos , López-Ribot José L. , Kirkpatrick William R. , Coco Brent J. , Bachmann Stefano P. , Patterson Thomas F.
Published: August 2002

Candida dubliniensis
is an opportunistic yeast that has been increasingly implicated in oropharyngeal candidiasis (OPC) in human immunodeficiency virus (HIV)-infected patients but may be underreported due to its similarity with
Candida albicans.
Although most
C. dubliniensis
isolates are susceptible to fluconazole, the inducibility of azole resistance in vitro has been reported. Thus, the use of fluconazole prophylaxis in the treatment of these patients may have contributed to the increasing rates of isolation of
C. dubliniensis.
In this study, yeast strains were collected from the oral cavities of HIV-infected patients enrolled in a longitudinal study of OPC. Patients received fluconazole for the suppression or treatment of OPC, and isolates collected at both study entry and end of study were chosen for analysis. Samples were plated on CHROMagar
Candida
medium for initial isolation and further identified by Southern blot analysis with the species-specific probes Ca3 (for
C. albicans
) and Cd25 (for
C. dubliniensis
). Fluconazole MICs were determined by using NCCLS methods. At study entry, susceptible
C. albicans
isolates were recovered from oral samples in 42 patients who were followed longitudinally (1 to 36 months).
C. albicans
strains from 12 of these patients developed fluconazole resistance (fluconazole MIC, ≥64 μg/ml).
C. dubliniensis
was not detected at end of study in any of these patients. Of the remaining 30 patients, eight (27%) demonstrated a replacement of
C. albicans
by
C. dubliniensis
when a comparison of isolates obtained at baseline and those from the last culture was done. For the 22 of these 30 patients in whom no switch in species was detected, the fluconazole MICs for initial and end-of-study
C. albicans
isolates ranged from 0.125 to 2.0 μg/ml. For the eight patients in whom a switch to
C. dubliniensis
was detected, the fluconazole MICs for
C. dubliniensis
isolates at end of study ranged from 0.25 to 64 μg/ml: the fluconazole MICs for isolates from six patients were 0.25 to 2.0 μg/ml and those for the other two were 32 and 64 μg/ml, respectively. In conclusion, a considerable number of patients initially infected with
C. albicans
strains that failed to develop fluconazole resistance demonstrated a switch to
C. dubliniensis. C. dubliniensis
in this setting may be underestimated due to lack of identification and may occur due to the impact of fluconazole on the ecology of oral yeast species.
Weak Association between SEN Virus Viremia and Liver Disease
Authors: Yoshida Hideo , Kato Naoya , Shiratori Yasushi , Shao Runxuan , Wang Yue , Shiina Shuichiro , Omata Masao
Published: August 2002

Recently, a novel DNA virus designated SEN virus (SEN-V), which is thought to be related to posttransfusion hepatitis, was discovered. The aim of the present study was to clarify the relationship between SEN-V infection and the development of liver disease. We examined SEN-V from the sera of 21 patients with non-B, non-C hepatocellular carcinoma (HCC) and 13 patients with non-B, non-C chronic liver disease (CLD) without HCC who were admitted to our hospital between 1995 and 1997. Thirty-two patients without liver disease served as controls and were also examined for SEN-V. SEN-V DNA was detected by the nested PCR method after extraction of DNA from serum. SEN-V DNA was detected in 74% (25 of 34) of patients with CLD with or without HCC who were negative for both hepatitis B virus surface antigen and anti-hepatitis C virus antibody. SEN-V DNA was detected in 69% (9 of 13) of CLD patients without HCC and in 76% (16 of 21) of HCC patients. The prevalence of SEN-V was no higher in patients with liver disease than in patients without liver disease (24 of 32; 75%). There were no significant differences in age, sex, liver function, history of blood transfusion, or amount of alcohol intake between SEN-V-positive and SEN-V-negative CLD and HCC patients. Genetic analysis suggested that SEN-V is closely related to the TT virus family. SEN-V was detected at almost the same frequency in patients with and without liver disease. SEN-V does not seem to contribute either to the pathogenesis of liver disease or to the development of HCC from chronic liver disease.
Cavitary Pneumonia in an AIDS Patient Caused by an Unusual
Bordetella bronchiseptica
Variant Producing Reduced Amounts of Pertactin and Other Major Antigens
Authors: Lorenzo-Pajuelo Benito , Villanueva José Luis , Rodríguez-Cuesta Juan , Vergara-Irigaray Nuria , Bernabeu-Wittel Máximo , Garcia-Curiel Andrés , Martínez de Tejada Guillermo
Published: August 2002

Although
Bordetella bronchiseptica
can infect and colonize immunocompromised humans, its role as a primary pathogen in pneumonia and other respiratory processes affecting those patients remains controversial. A case of cavitary pneumonia caused by
B. bronchiseptica
in an AIDS patient is presented, and the basis of the seemingly enhanced pathogenic potential of this isolate (designated 814) is investigated.
B. bronchiseptica
was the only microorganism recovered from sputum, bronchoalveolar lavage fluid, and samples taken through the protected brush catheter. Unlike previous work reporting the involvement of
B. bronchiseptica
in cases of pneumonia, antibiotic treatment selected on the basis of in vitro antibacterial activity resulted in clearance of the infection and resolution of the pulmonary infiltrate. Although isolate 814 produced reduced amounts of several major antigens including at least one Bvg-activated factor (pertactin), the molecular basis of this deficiency was found to be BvgAS independent since the defect persisted after the
bvgAS
locus of isolate 814 was replaced with a wild-type
bvgAS
allele. Despite its prominent phenotype, isolate 814 displayed only a modest yet a significant deficiency in its ability to colonize the respiratory tracts of immunocompetent rats at an early time point. Interestingly, the antibody response elicited by isolate 814 in these animals was almost undetectable. We propose that isolate 814 may be more virulent in immunocompromised patients due, at least in part, to its innate ability to produce low amounts of immunogenic factors which may be required at only normal levels for the interaction of this pathogen with its immunocompetent natural hosts.
Detection of Human Papillomavirus DNA in Urine Specimens from Human Immunodeficiency Virus-Positive Women
Authors: Brinkman Joeli A. , Jones W. Elizabeth , Gaffga Ann M. , Sanders Jonathan A. , Chaturvedi Anil K. , Slavinsky Joseph , Clayton John L. , Dumestre Jeanne , Hagensee Michael E.
Published: August 2002

Human immunodeficiency virus (HIV)-positive women may represent one of the fastest-growing populations at risk for acquiring cervical cancer and thus require frequent screening. The purpose of the present studies was to validate a PCR-based urine assay by comparing detection and genotyping of human papillomavirus (HPV) DNA in urine samples and matching cervical swab specimens of HIV-positive women. Despite a difference in amplifiability, the prevalence of any HPV genotype (58% for the cervical swab specimens and 48% for the urine specimens) was not significantly different in this population. The levels of concordance were 70, 71, and 78% for detection of any HPV type, any high-risk HPV type, or any low-risk HPV type in the two specimen types, respectively. While instances of discordant detection were greater for the cervical swab specimens than for the urine specimens, this was not statistically significant. The distributions of HPV genotypes were similar in the cervix and the urine for the majority of types examined. Importantly, detection of HPV DNA in urine was associated with an abnormal Papanicolaou smear to the same extent that detection of HPV DNA in a cervical swab specimen was. These data provide preliminary support for the proposal to use urine testing as a primary or secondary screening tool for cervical cancer in HIV-positive women or as an epidemiological tool. Additional studies with larger sample sizes must be conducted in order to further verify these findings.
Reactive Nitrogen Intermediates Have a Bacteriostatic Effect onMycobacterium tuberculosisIn Vitro
Authors: Firmani Marcia A. , Riley Lee W.
Published: August 2002

Susceptibility of six isolates ofMycobacterium tuberculosis(CB3.3, CDC1551, RJ2E, C.C.13, H37Rv, and H37Ra) and two isolates ofMycobacterium bovis(Ravenel and BCG) to reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) was determined by standard in vitro survival assays. After 21 days of incubation, the survival of most strains exposed to either acidified sodium nitrite (ASN) or hydrogen peroxide (H2O2) was significantly lower than the same strains unexposed to these RNI or ROI products. However, after 50 days of incubation, these differences in susceptibility became less apparent for strains exposed to ASN but not for strains exposed to H2O2. The recovery of these strains after exposure to RNI suggests that the effect of RNI onM. tuberculosisis bacteriostatic. The in vitro concentrations of ROI and RNI used in these assays were higher than those expected in vivo. These observations suggest that, in vivo, RNI expression at physiologically achievable concentrations may keepM. tuberculosisfrom proliferating but that removal of RNI may allow the organisms to proliferate. Furthermore, the ability of someM. tuberculosisstrains to cause rapidly progressive disease may relate to their intrinsic levels of RNI and ROI resistance.
Detection of Simian Immunodeficiency Virus in Diverse Species and of Human Immunodeficiency Virus Type 2 by Using Consensus Primers within the
pol
Region
Authors: Masciotra Silvina , Yang Chunfu , Pieniazek Danuta , Thomas Chanda , Owen Sherry M. , McClure Harold M. , Lal Renu B.
Published: August 2002

Human immunodeficiency virus type 2 (HIV-2) is the result of cross-species transmission of simian immunodeficiency virus (SIV) from sooty mangabey monkeys to humans. Primer pairs (intHIV-2/SIV) based on a region of integrase that has considerable homology across HIV-2 and SIV lineages were designed to develop a broadly cross-reactive molecular assay to detect lentivirus infection in primates. The intHIV-2/SIV primers detect HIV-2 and simian viruses SIVcpz, SIVsmm, SIVsyk, SIVagm, and SIVmnd. The primers are also capable of amplifying some HIV-1 strains. Additionally, sequences from the integrase amplicons were of sufficient genetic diversity to permit not only phylogenetic clustering of all simian viruses to their respective lineages but also HIV type and group classification. Thus, the primers described here provide a method to detect primate lentiviruses from diverse species of nonhuman primates, as well as from persons infected with HIV-1 and HIV-2.
groESL
Sequence Determination, Phylogenetic Analysis, and Species Differentiation for Viridans Group Streptococci
Authors: Teng Lee-Jene , Hsueh Po-Ren , Tsai Jui-Chang , Chen Pin-Wun , Hsu Jia-Chuan , Lai Hsin-Chih , Lee Chun-Nan , Ho Shen-Wu
Published: August 2002

The full-length sequences of the
groESL
genes (also known as
cpn10/60
) of
Streptococcus anginosus
,
Streptococcus constellatus
,
Streptococcus gordonii
, and
Streptococcus sanguis
and the near full-length sequence of the
groESL
genes of
Streptococcus intermedius
,
Streptococcus bovis
,
Streptococcus mitis
,
Streptococcus mutans
,
Streptococcus oralis
, and
Streptococcus salivarius
were determined. The lengths of the
groES
genes from the 10 species listed above ranged from 282 to 288 bp, and the full-length sequences of
groEL
determined for 4 species (
S. anginosus
,
S. constellatus
,
S. gordonii
, and
S. sanguis
) revealed that each was 1,623 bp. The intergenic region (spacer) between the
groES
and
groEL
genes varies in size (15 to 111 bp) and sequence between species. The variation of the
groES
sequences among the species tested was greater (62.1 to 95.1% nucleotide sequence identities) than that of the
groEL
sequences (77.2 to 95.2% nucleotide sequence identities). Phylogenetic analysis of the
groES
and
groEL
genes yielded evolutionary trees similar to the tree constructed by use of the 16S rRNA gene. The intraspecies variation of the spacer was minimal for clinical isolates of some species. The
groESL
sequence data provide an additional parameter for identification of viridans group streptococcal species.
Development and Evaluation of Rapid Urinary Antigen Detection Tests for Diagnosis of Penicilliosis Marneffei
Authors: Desakorn Varunee , Simpson Andrew J. H. , Wuthiekanun Vanaporn , Sahassananda Duangjai , Rajanuwong Adul , Pitisuttithum Punnee , Howe Paul A. , Smith Michael D. , White Nicholas J.
Published: August 2002

Penicilliosis, caused by the dimorphic fungus
Penicillium marneffei
, is an important opportunistic systemic fungal infection affecting immunocompromised individuals living in areas where penicilliosis is endemic. We have demonstrated previously that a urinary enzyme-linked immunosorbent assay (ELISA) with purified rabbit polyclonal antibody against killed whole-fission-form arthroconidia of
P. marneffei
was specific and highly sensitive for the diagnosis of penicilliosis. In this study, a dot blot ELISA and a latex agglutination (LA) test were developed with the same polyclonal antibody and compared with the ELISA for the detection of
P. marneffei
urinary antigen. Urine specimens from 37 patients with culture-proven penicilliosis and 300 controls (52 healthy subjects and 248 hospitalized patients without penicilliosis) were tested. Antigen was detected in urine from all 37 (100%) penicilliosis patients by the LA test, 35 (94.6%) penicilliosis patients by the dot blot ELISA, and 36 (97.3%) penicilliosis patients by the ELISA. False-positive results were found by the three assays for 2 (0.7%), 8 (2.7%), and 6 (2%) of 300 controls, respectively. The overall sensitivities of the diagnostic tests were as follows: dot blot ELISA, 94.6%; ELISA, 97.3%; and LA test, 100% (specificities, 97.3, 98, and 99.3%, respectively). The LA test is simple, robust, rapid, and convenient and should prove to be an important addition to the existing diagnostic tests for penicilliosis.
Molecular Characterization of Multiresistant
d
-Tartrate-Positive
Salmonella enterica
Serovar Paratyphi B Isolates
Authors: Miko Angelika , Guerra Beatriz , Schroeter Andreas , Dorn Christina , Helmuth Reiner
Published: August 2002

Since 1996, the National Salmonella Reference Laboratory of Germany has received an increasing number of
Salmonella enterica
subsp.
enterica
serovar Paratyphi B isolates. Nearly all of these belonged to the dextrorotatory tartrate-positive variant (
S
.
enterica
subsp.
enterica
serovar Paratyphi B dT
+
), formerly called
S
.
enterica
subsp.
enterica
serovar Java. A total of 55 selected contemporary and older
S
.
enterica
subsp.
enterica
serovar Paratyphi B dT
+
isolates were analyzed by plasmid profiling, antimicrobial resistance testing, pulsed-field gel electrophoresis, IS
200
profiling, and PCR-based detection of integrons. The results showed a high genetic heterogeneity among 10 old strains obtained from 1960 to 1993. In the following years, however, new distinct multiresistant
S
.
enterica
subsp.
enterica
serovar Paratyphi B dT
+
clones emerged, and one clonal lineage successfully displaced the older ones. Since 1994, 88% of the isolates investigated were multiple drug resistant. Today, a particular clone predominates in some German poultry production lines, poultry products, and various other sources. It was also detected in contemporary isolates from two neighboring countries as well.
Identification ofAnaplasma phagocytophila(FormerlyEhrlichia phagocytophila) Variants in Blood from Sheep in Norway
Authors: Stuen Snorre , Van De Pol Ingrid , Bergström Karin , Schouls Leo M.
Published: August 2002

A total of 41 blood samples were collected from 40Anaplasma phagocytophila-infected sheep in 11 sheep flocks from four different counties of southern Norway. The presence and nature of theAnaplasmaspecies were identified by microscopic detection of morulae, PCR, reverse line blot hybridization, and 16S rRNA gene sequencing.A. phagocytophilawas identified in all of the samples, and sequencing of the 16S rRNA gene revealed the presence of four variants ofA. phagocytophila. Two of these variants have been described before, but two were newly identified 16S rRNA variants of this species.A. phagocytophilavariant 1 was found in nine flocks,A. phagocytophilavariant 2 was found in four flocks, theA. phagocytophilaprototype was found in two flocks, andA. phagocytophilavariant 5 was found in one flock. In two flocks, some sheep were infected withA. phagocytophilavariant 1, whereas others were infected withA. phagocytophilavariant 2, and in three animals a double infection with two variants was registered. Analyses of the blood samples revealed that blood from sheep infected withA. phagocytophilavariant 2 contained nearly twice as many neutrophils and eight times as manyAnaplasma-infected neutrophils as blood from sheep infected with theA. phagocytophilavariant 1. Furthermore, only 43% of theA. phagocytophilavariant 2-infected sheep displayed antibody responses in an immune fluorescence assay, whereas 93% of the sheep with theA. phagocytophilavariant 1-infected sheep were seropositive.
Comparison of Rapid, Automated Ribotyping and DNA Macrorestriction Analysis ofBurkholderia pseudomallei
Authors: Inglis Timothy J. J. , O'Reilly Lyn , Foster Niki , Clair Adele , Sampson Judy
Published: August 2002

An automated ribotyping device (RiboPrinter) was used to determine the ribotypes of a collection ofBurkholderia pseudomalleiisolates. In a preliminary evaluation with the restriction enzymesBamHI andEcoRI, the protocol withEcoRI was more discriminating. The reproducibilities of the ribotypes obtained withEcoRI (EcoRI ribotypes) were determined by testing three levels of bacterial loads. The performance of the manufacturer's software was assessed by comparing the machine-optimized ribotypes with the type determined from the original gel image analyzed with Bionumerics software. The library ofB. pseudomallei EcoRI ribotypes was then compared with the ribotypes obtained by DNA macrorestriction analysis ofXbaI digests by pulsed-field gel electrophoresis. The typeability ofB. pseudomalleibyEcoRI ribotyping was 100%, and the discrimination index was 0.94. The slightly greater discrimination provided by DNA macrorestriction analysis (0.96) was achieved at the expense of a significantly longer processing time of 6 days, although the method was only half the cost of automated ribotyping. Typeability by macrorestriction analysis was lower (97%) unless a thiourea step was added to neutralize the action of Tris-dependent endonucleases. The digital record ofB. pseudomalleiisolates analyzed thus far provides a useful resource for future epidemiological studies and will help shorten the response time in the event of a further melioidosis outbreak or the deliberate release ofB. pseudomalleias a biohazard.
Testing Conditions for Determination of Minimum Fungicidal Concentrations of New and Established Antifungal Agents for
Aspergillus
spp.: NCCLS Collaborative Study
Authors: Espinel-Ingroff A. , Fothergill A. , Peter J. , Rinaldi M. G. , Walsh T. J.
Published: August 2002

Standard conditions are not available for evaluating the minimum fungicidal concentrations (MFCs) of antifungal agents. This multicenter collaborative study investigated the reproducibility in three laboratories of itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B MFCs for 15 selected isolates of
Aspergillus
spp. After MIC determinations for the 15 isolates in each center by the NCCLS M38-A broth microdilution method with four media, standard RPMI 1640 (RPMI), RPMI with 2% dextrose, antibiotic medium 3 (M3), and M3 with 2% dextrose, MFCs were determined for each isolate-medium-drug combination. MFCs were defined as the lowest drug dilutions that yielded <3 colonies (approximately 99 to 99.5% killing activity). The highest reproducibility (96 to 100%) was for amphotericin B MFCs with the four media. Although reproducibility was more variable and medium dependent for the azoles (91 to 98%), agreement was good to excellent for itraconazole, ravuconazole, and voriconazole MFCs with RPMI and M3 (93 to 98%). For posaconazole, the agreement was higher with M3 media (91 to 96%) than with RPMI media (91%). These data extend the refinement of testing guidelines for susceptibility testing of
Aspergillus
spp. and warrant consideration for introduction into future versions of the M38 document. The role of the MFC under these standardized testing conditions as a predictor of clinical outcome needs to be established in clinical trials.
Polyphyletic Strains of Hepatitis E Virus Are Responsible for Sporadic Cases of Acute Hepatitis in Japan
Authors: Mizuo Hitoshi , Suzuki Kazuyuki , Takikawa Yasuhiro , Sugai Yoshiki , Tokita Hajime , Akahane Yoshihiro , Itoh Keiichi , Gotanda Yuhko , Takahashi Masaharu , Nishizawa Tsutomu , Okamoto Hiroaki
Published: August 2002

Among 87 patients who were previously treated for acute hepatitis of unknown etiology between 1992 and 2001 at five hospitals in Japan, 11 (13%) patients were positive for immunoglobulin M-class antibodies to hepatitis E virus (HEV) by enzyme immunoassay and had detectable HEV RNA by reverse transcription-PCR with two independent sets of primers derived from well-conserved genomic areas in open reading frames 1 and 2. Clinical HEV infection was significantly associated with male sex (9 of 11 versus 29 of 76 patients [
P
< 0.01]) and older age (52 ± 11 [mean ± standard deviation] versus 41 ± 17 years [
P
< 0.05]), and its prevalence differed by geographic region (6 to 25%), with a higher rate in the northern part of Japan. At admission, the 11 patients with HEV-associated hepatitis had elevated alanine aminotransferase levels of 914 to 4,850 IU/liter, and all but 1 had elevated bilirubin levels of 1.5 to 24.0 mg/dl. The 11 HEV isolates were of genotype III or IV and were segregated into three groups with intergroup nucleotide differences of 9.5 to 22.0%. Phylogenetic analysis revealed that four isolates of genotype III were closely related to a Japanese isolate, while the other four isolates of the same genotype were nearest those from the United States. The remaining three isolates were close to known isolates of genotype IV in China and Taiwan but shared less than 88% identity with them. These results indicate that multiple genotypes of HEV cocirculate in Japan and contribute to the development of sporadic acute hepatitis, with the prevalence differing by age, sex, and geographic region.
Comparison of C
18
-Carboxypropylbetaine and Standard
N
-Acetyl-
l
-Cysteine-NaOH Processing of Respiratory Specimens for Increasing Tuberculosis Smear Sensitivity in Brazil
Authors: Scott Cherise P. , dos Anjos Filho Luciano , de Queiroz Mello Fernanda Carvalho , Thornton Charles G. , Bishai William R. , Fonseca Leila S. , Kritski AfrÂnio L. , Chaisson Richard E. , Manabe Yukari C.
Published: August 2002

Techniques to improve the sensitivity of smear microscopy would facilitate early tuberculosis (TB) diagnosis and disease control, especially in low-income countries where the positive predictive value is high. C
18
-carboxypropylbetaine (CB-18) is a zwitterionic detergent that helps to compensate for the innate buoyancy of mycobacteria, potentially enhancing recovery by centrifugation. Previous data suggest that CB-18 may increase the sensitivity of smear, culture, and molecular amplification diagnostic testing. The goal of the present study was to evaluate if the sensitivity of the smear technique using light microscopy could be improved by treating respiratory samples with CB-18. In the first phase, respiratory specimens were collected consecutively from patients with suspected pulmonary tuberculosis in a tertiary-care hospital in Rio de Janeiro, Brazil (236 specimens were analyzed). After protocol modifications, another 120 respiratory specimens were evaluated. The standard technique was
N
-acetyl-
l
-cysteine with sodium hydroxide (NALC-NaOH) treatment, smear concentration with centrifugation, and Ziehl-Neelsen staining. Culture on Löwenstein-Jensen slants was performed on all specimens for use as the “gold standard.” No specimens from patients undergoing active TB treatment were included. The initial protocol for CB-18 processing resulted in a sensitivity of 59.6% and specificity of 96.8% compared to standard processing with a sensitivity of 66.0% and specificity of 96.8%. Using the modified protocol, the sensitivity of CB-18 increased to 71.4% with a specificity of 97.0% versus standard processing with a sensitivity of 61.9% and a specificity of 99.0%. The diagnostic yield of acid-fast bacillus smear with CB-18 in the absence of fluorescence microscopy and PCR compared to standard processing with NALC-NaOH was not significantly different, although the power to detect a difference by the modified assay was low.
PCR-Based Identification of Bacteria Associated with Endodontic Infections
Authors: Fouad Ashraf F. , Barry Jody , Caimano Melissa , Clawson Michael , Zhu Qiang , Carver Rachaele , Hazlett Karsten , Radolf Justin D.
Published: August 2002

PCR primers that target the bacterial 16S rRNA genes (or the
tuf
gene for the genus
Enterococcus
) were used to identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were investigated. Microbial samples from the root canals of 24 teeth with necrotic pulp were included in the study. PCR with universal bacterial primers identified bacterial DNA in 22 specimens; the remaining 2 specimens were from intact teeth that had been traumatized 6 months prior to treatment. PCR with specific primers showed that preoperative symptoms were significantly associated with the presence of
Streptococcus
spp. (
P
< 0.001 by chi-square analysis). There was also a nonsignificant trend for symptoms to be associated with
Fusobacterium nucleatum
and
Porphyromonas gingivalis
(odds ratio, >2) and for diabetes mellitus to be associated with
P. gingivalis
and
Porphyromonas endodontalis
(odds ratio, >2). Cloning and sequencing of the universal PCR product in one specimen revealed the presence of an organism related to the genus
Olsenella
, which has not previously been described in endodontic infections.
Sensitivity of Three Urinary Antigen Tests Associated with Clinical Severity in a Large Outbreak of Legionnaires' Disease in The Netherlands
Authors: Yzerman Ed P. F. , Boer Jeroen W. den , Lettinga Kamilla D. , Schellekens Joop , Dankert Jacob , Peeters Marcel
Published: August 2002

In 1999 an outbreak involving 188 patients with Legionnaires' disease (LD) occurred among visitors to a flower show in the Netherlands. Two enzyme immunoassays (Binax and Biotest) and one immunochromatographic assay (Binax NOW) were tested, using urine samples from LD patients from the 1999 outbreak. Sensitivity was calculated using positive culture and/or seroconversion as the “gold standard” in outbreak-related patients with radiographically confirmed pneumonia who fulfilled the epidemiological critera. The Binax EIA, Biotest EIA, and Binax NOW assay showed overall sensitivities of 69, 71, and 72%, respectively. When the tests were performed with concentrated urine samples, the overall sensitivities increased to 79, 74, and 81%, respectively. Using multiple logistic regression analysis with backward elimination, a statistically significant association was found between clinical severity and test sensitivity for all tests. For patients with mild LD, the test sensitivities ranged from 40 to 53%, whereas for patients with severe LD who needed immediate special medical care, the sensitivities reached 88 to 100%. These findings have major implications for the diagnostic process in patients with mild pneumonia and suggest that patients with mild pneumonia may go underdiagnosed if urine antigen tests alone are used.