Biomedical subjects

L W Riley

Publications and source records attributed to L W Riley.

At least 37 records · Page 2Linked to original sources

PubMed2001

Nested restriction site-specific PCR to detect and type hepatitis C virus (HCV): a rapid method to distinguish HCV subtype 1b from other genotypes.

Genotypic differentiation of hepatitis C virus (HCV) has become an integral part of clinical management and epidemiologic studies of hepatitis C infections. Thus, it is extremely important in areas such as the Czech Republic, where current instrumentation and kits for assessing HCV infection are too costly for widespread use. We describe a new and relatively inexpensive method called nested restriction site-specific PCR (RSS-PCR) that generates a "fingerprint" pattern to represent an HCV genotype without the use of restriction endonucleases and that specifically differentiates HCV genotype 1b from the other HCV genotypes. The RSS-PCR method was applied directly to serum samples from patients with hepatitis C from the Czech Republic and from patients with known HCV genotypes from the United States. The method was validated by comparison of the subtype determined by RSS-PCR to the subtype determined from analysis of the 5' noncoding region (NC) or the nonstructural protein gene (NS5b) nucleotide sequence of HCV in these clinical samples. From 75 Czech samples containing HCV RNA, three distinct RSS-PCR patterns were observed; 54 were predicted to contain subtype 1b, 19 were predicted to contain subtype 1a, and 2 were predicted to contain subtype 3a. Among 54 samples predicted to contain HCV genotype 1b, all were confirmed by their 5' NC or NS5b sequences to be subtype 1b. Thus, both the sensitivity and specificity of the RSS-PCR test for the differentiation of HCV subtype 1b from the others were 100%. While the assay described here was designed to specifically differentiate HCV subtype 1b from the other HCV genotypes, the RSS-PCR method can be modified to differentiate any HCV genotype or subtype of interest. Its simplicity and speed may provide new opportunities to study the epidemiology of HCV infections and the relationship between HCV genotypes and clinical outcome by more laboratories throughout the world.

Genotype↗

PubMed2001

Evaluation of recombinant Leptospira antigen-based enzyme-linked immunosorbent assays for the serodiagnosis of leptospirosis.

There is an urgent need for development of new serodiagnostic strategies for leptospirosis, an emerging zoonosis with worldwide distribution. We have evaluated the diagnostic utility of five recombinant antigens in enzyme-linked immunosorbent assays (ELISAs) for serodiagnosis of leptospirosis. Sera from 50 healthy residents of a high-incidence region were used to determine cutoff values for 96% specificity. In paired sera from 50 cases of leptospirosis confirmed by the microscopic agglutination test, immunoglobulin G (IgG) but not IgM reacted with the recombinant leptospiral proteins. The recombinant LipL32 IgG ELISA had the highest sensitivities in the acute (56%) and convalescent (94%) phases of leptospirosis. ELISAs based on recombinant OmpL1, LipL41, and Hsp58 had sensitivities of 16, 24, and 18% during the acute phase and 72, 44, and 32% during convalescence, respectively. Compared to sera from healthy individuals, patient sera did not react significantly with recombinant LipL36 (P > 0.05). Recombinant LipL32 IgG ELISA demonstrated 95% specificity among 100 healthy individuals, and specificities ranging from 90 to 97% among 30 dengue patients, 30 hepatitis patients, and 16 patients with diseases initially thought to be leptospirosis. Among 39 Venereal Disease Research Laboratory test-positive individuals and 30 Lyme disease patients, 13 and 23% of sera, respectively, reacted positively with the rLipL32 antigen. These findings indicate that rLipL32 may be an useful antigen for the serodiagnosis of leptospirosis.

Antibodies, Bacterial↗

PubMed2001

Referral pattern of leptospirosis cases during a large urban epidemic of dengue.

During heavy seasonal rainfall in 1996, concurrent epidemics of dengue and leptospirosis occurred in an urban center in northeastern Brazil. We interviewed 110 cases of leptospirosis hospitalized a median of seven days after the onset of illness to evaluate the impact of the dengue epidemic on the triage of suspected leptospirosis from ambulatory clinics to the infectious disease reference hospital. Within the first three days of illness, 46 (42%) cases sought their first medical evaluation, and 28 (61% of 46) received a diagnosis of dengue. Dengue diagnoses were associated with a median of five days delay in referral to the infectious disease hospital. Patients who reported initial diagnoses of dengue were more likely than other patients to have required admission to the intensive care unit (odds ratio [OR] = 2.7, 95% confidence interval [CI] = 0.8-9.5) and to have died during hospitalization (OR = 5.1, 95% CI = 0.8-55.0). These findings indicate that diagnostic confusion between the early symptoms of leptospirosis and dengue may have contributed to the high mortality observed during the leptospirosis epidemic.

Adolescent↗

PubMed2001

[DRE-PCR (Double Repetitive Element-Polymerase Chain Reaction)--a new molecular-epidemiologic method in the detection of tuberculosis].

In a pilot study Double Repetitive Element-Polymerase Chain Reaction 20 clinical isolates of Mycobacterium tuberculosis from Guatemala and 49 strains from Prague were typed. This technique is based on direct evidence of repetitive elements IS6110 or PGRS and does not require DNA purification, digestion by endonuclease nor Southern blot hybridization. Preliminary examination of Guatemalian strains revealed a striking identity or similarity of DRE-PCR profiles while the Prague strains were characterized by conspicuous polymorphism. The Prague strains were examined in a total number of 13 series of electrophoreograms and subsequently subjected to automated analysis with GelCompar software. The DRE-PCR method is suitable for screening of a major number of clinical isolates of M. tuberculosis in laboratories equipped with a minimum of technical facilities for the PCR method, furthermore it requires facilities for synthesis of the necessary primers and at least basic knowledge of molecular biology.

Bacterial Typing Techniques↗

PubMed2000

Deletion of the putative antioxidant noxR1 does not alter the virulence of Mycobacterium tuberculosis H37Rv.

SETTING: The cloned M. tuberculosis noxR1 gene has been shown to confer resistance to reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) upon Escherichia coli and Mycobacterium smegmatis. OBJECTIVE: To investigate the role of noxR1 in resistance to RNI and virulence of M. tuberculosis. DESIGN: The noxR1 gene was deleted from M. bovis BCG and M. tuberculosis H37Rv by allelic exchange. The mutants were compared to wild type strains with respect to resistance to chemically generated RNI. The virulence of the M. tuberculosis mutant was investigated in a murine model of infection. RESULTS: The NoxR1 mutants grew normally in Sautons and 7H9 broths. The BCG mutant demonstrated decreased resistance to in vitro generated RNI compared to the wild type. Resistance to RNI could be restored to the mutant by reintroduction of the noxR1 locus on a replicating plasmid. However, deletion of noxR1 from M. tuberculosis H37Rv did not result in decreased resistance to RNI nor a difference in growth and survival of the bacterium during murine infection. CONCLUSION: The noxR1 gene locus in M. bovis BCG bestows ability to resist RNI generated in vitro. In M. tuberculosis H37Rv, however, noxR1 is either not involved in RNI resistance and virulence or is better compensated for by other mechanisms.

Animals↗

PubMed2000

Clonally related penicillin-nonsusceptible Streptococcus pneumoniae serotype 14 from cases of meningitis in Salvador, Brazil.

Active hospital-based surveillance in the city of Salvador, Brazil, from December 1995 through October 1998, identified 221 patients with confirmed pneumococcal meningitis. Of these 221 patients, 29 (13%) had isolates with intermediate-level resistance to penicillin. Infection with these penicillin-nonsusceptible isolates was significantly associated with age of <2 years (P<.0019), previous antibiotic use (P<.0006), and coresistance to trimethoprim-sulfamethoxazole (P<.0000). Serotype 14 was the most prevalent serotype (55.2%) of penicillin-nonsusceptible isolates. Strain typing by repetitive element BOX polymerase chain reaction (PCR) analysis showed that penicillin-nonsusceptible serotype 14 isolates had closely related BOX PCR patterns, whereas penicillin-susceptible serotype 14 isolates each had distinct, unrelated patterns. Penicillin-nonsusceptible serotype 14 isolates from Salvador and other Brazilian cities had similar BOX PCR patterns. These observations indicate that in Brazil a large proportion of cases of penicillin-nonsusceptible pneumococcal meningitis appear to be caused by a closely related group of serotype 14 strains that may have disseminated to widely separate geographic areas.

Adolescent↗

PubMed2000

Restriction-site-specific PCR as a rapid test to detect enterohemorrhagic Escherichia coli O157:H7 strains in environmental samples.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield "fingerprint" patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number of E. coli isolates from environmental samples.

Animal Feed↗

PubMed2000

Rapid subtyping of dengue virus serotypes 1 and 4 by restriction site-specific PCR.

We previously reported a simple subtyping method, restriction site-specific PCR (RSS-PCR), for dengue virus serotypes 2 and 3; here we describe its application for subtyping dengue virus serotypes 1 and 4. Three major RSS-PCR types were observed for dengue virus serotype 1 and two types were observed for dengue virus serotype 4, in agreement with previous strain classifications based on sequence analysis. Because of its simplicity, this method is amenable to rapid subtyping and application to epidemiological studies of dengue in countries where dengue is endemic.

DNA Primers↗

PubMed2000

Transmission of tuberculosis in an endemic urban setting in Brazil.

SETTING: Two out-patient facilities in São Paulo, Brazil. OBJECTIVE: To study the transmission pattern of tuberculosis (TB) among human immunodeficiency virus (HIV) infected and uninfected persons in a setting endemic for TB. DESIGN: A prospective study comparing HIV-seropositive and -seronegative TB patients identified consecutively between 1 March 1995 and 1 April 1997. The patients were stratified according to their Mycobacterium tuberculosis isolate IS6110 RFLP patterns. Risk factors were sought for infection with an RFLP cluster pattern strain, inferred to represent recent transmission. RESULTS: Fifty-eight (38%) of 151 HIV-seropositive patients and 36 (25%) of 142 HIV-seronegative patients were infected with M. tuberculosis isolates that belonged to cluster patterns (OR 1.84, 95% CI 1.08-3.13). Multidrug-resistant (MDR) strains were isolated from 19 patients, all of whom were HIV seropositive; 12 (63%) of these, and 46 (35%) of 132 drug-susceptible isolates had cluster patterns (OR 3.20, 95% CI 1.08-9.77). CONCLUSION: In a TB-endemic urban setting in Brazil, the proportion of cases resulting from recent transmission appears to be greater among HIV-seropositive than among HIV-seronegative patients. A large proportion of MDR-TB (63%) cases was caused by strains that had cluster RFLP patterns, suggesting recent transmission of already resistant organisms. This type of knowledge regarding TB transmission may help to improve locally appropriate TB control programs.

AIDS-Related Opportunistic Infections↗

PubMed1999

Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group.

BACKGROUND: Leptospirosis has, traditionally, been considered a sporadic rural disease. We describe a large urban outbreak of leptospirosis. METHODS: Active surveillance for leptospirosis was established in an infectious-disease referral hospital in Salvador, Brazil, between March 10 and Nov 2, 1996. Patients meeting case criteria for severe manifestations of leptospirosis were recruited into the study. The diagnosis was confirmed in the laboratory with the microagglutination test and identification of leptospires in blood or urine. Risk factors for death were examined by multivariate analyses. FINDINGS: Surveillance identified 326 cases of which 193 (59%) were laboratory-confirmed (133) or probable (60) cases. Leptospira interrogans serovar copenhageni was isolated from 87% of the cases with positive blood cultures. Most of the cases were adult (mean age 35.9 years [SD 15.9]), and 80% were male. Complications included jaundice (91%), oliguria (35%), and severe anaemia (26%). 50 cases died (case-fatality rate 15%) despite aggressive supportive care including dialysis (in 23%). Altered mental status was the strongest independent predictor of death (odds ratio 9.12 [95% CI 4.28-20.3]), age over 37 years, renal insufficiency, and respiratory insufficiency were also significant predictors of death. Before admission to hospital, 42% were misdiagnosed as having dengue fever in the outpatient clinic; an outbreak of dengue fever was taking place concurrently. INTERPRETATION: An epidemic of leptospirosis has become a major urban health problem, associated with high mortality. Diagnostic confusion with dengue fever, another emerging infectious disease with a similar geographic distribution, prevents timely intervention that could minimise mortality.

Adult↗

PubMed1999

Expression of a pilin subunit BfpA of the bundle-forming pilus of enteropathogenic Escherichia coli in an aroA live salmonella vaccine strain.

Enteropathogenic Escherichia coli (EPEC) is a major cause of childhood diarrhea in developing countries and is a leading cause of severe diarrheal illness among Brazilian infants. As one approach to constructing a vaccine candidate against diarrhea caused by EPEC, we evaluated whether the pilin subunit (BfpA) of the bundle-forming pilus (BFP) could be expressed by a live Salmonella vaccine strain. Several copies of the coding region of BfpA (bfpA) were amplified by PCR from a preparation of the EAF plasmid of EPEC strain B171 and cloned into plasmid vectors. An intact copy of bfpA was subcloned into the heat inducible prokaryotic expression vector pCYTEXP1, and the resulting pBfpA was used to transform the aroA S. typhimurium strain SL3261, generating SL3261(pBfpA). The recombinant vaccine strain was able to express, but not to process, rBfpA as evidenced by a prominent 21 kDa protein that crossreacted with anti-BFP antiserum found only in extracts of heat-treated SL3261(pBfpA), but not in strains of untreated SL3261(pBfpA) or SL3261 not carrying the plasmid. Furthermore, rBfpA accumulation was not toxic to the Salmonella host, as evidenced by similar plating efficiencies between induced and uninduced strains of SL3261(pBfpA). Finally, SL3261(pBfpA) orally administered to BALB/c mice was capable of eliciting a sustained and vigorous humoral immune response to BfpA, achievable even with a single oral dose of approximately 10(9) organisms. Therefore, this pilin product may serve as a potential immunogen as part of a live combined vaccine strategy to prevent two of the major public health problems in Brazil--salmonellosis and EPEC childhood diahrrea.

Administration, Oral↗

PubMed1999

Rapid subtyping of dengue viruses by restriction site-specific (RSS)-PCR.

Dengue is a major public health problem worldwide. It is caused by four dengue virus serotypes, each further divided into distinct genetic subtypes. Strain typing is important for understanding the epidemiology and viral factors associated with disease transmission. However, most of the existing subtyping methods are expensive and technically unwieldy for timely, practical applications in developing countries. Here we describe a simple, rapid, PCR-based subtyping method, restriction site-specific (RSS)-PCR, which we used to analyze dengue virus serotypes 2 and 3. For each serotype, four primers targeted to sequences spanning polymorphic endonuclease restriction sites in the envelope gene were used to reverse transcribe and amplify viral RNA. These RT-PCR products generated distinct electrophoretic band patterns for different strains. Analysis of 73 dengue-2 strains and 54 dengue-3 strains representing a broad geographic distribution over several decades revealed that the RSS-PCR fingerprints reproducibly fell into 7 and 3 groups, respectively. These groups correlated well with previous phylogenetic classifications. This one-step assay should be widely accessible and allow more detailed epidemiologic investigations in dengue-endemic countries. This novel PCR approach to subtyping organisms based on restriction site polymorphisms should be applicable to other pathogens.

Dengue Virus↗

PubMed1999

IS1245 genotypic analysis of Mycobacterium avium isolates from patients in Brazil.

OBJECTIVE: Disseminated Mycobacterium avium infection is an emerging opportunistic disease among patients with acquired immunodeficiency syndrome (AIDS) in Brazil. The mode of transmission of M. avium in a developing country setting needs to be better characterized. METHODS: Mycobacterium avium strain collections in São Paulo and Rio de Janeiro were analyzed according to the strains' IS1245 DNA gel electrophoretic migration patterns. Medical records of the patients from whom M. avium isolates were available were reviewed, and their demographic characteristics were stratified according to the isolates' IS1245 DNA fingerprint patterns. RESULTS: Of 105 patients, 33 (31%) with M. avium isolated between 1990 and 1994 had strains having IS1245 patterns identical in patterns seen in isolates from two or more patients (designated as cluster pattern strains). Cluster pattern strains were isolated from 21 (39%) of 54 patients with disseminated infection (defined as infection due to M. avium isolated from a sterile site in an adult patient). Six of the cluster pattern strains were isolated only from sterile sites. In São Paulo, cluster pattern strains were significantly more likely to be isolated from patients with disseminated disease. CONCLUSIONS: These preliminary observations suggest that in large cities of Brazil, a high proportion (at least 39%) of disseminated M. avium infections in patients with AIDS results from a recent transmission. Some strains of M. avium may be more likely to cause disseminated disease than others after an infection.

AIDS-Related Opportunistic Infections↗

PubMed1999

Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis.

Porins form channels in the mycolic acid layer of mycobacteria and thereby control access of hydrophilic molecules to the cell. We purified a 100 kDa protein from Mycobacterium smegmatis and demonstrated its channel-forming activity by reconstitution in planar lipid bilayers. The mspA gene encodes a mature protein of 184 amino acids and an N-terminal signal sequence. MALDI mass spectrometry of the purified porin revealed a mass of 19 406 Da, in agreement with the predicted mass of mature MspA. Dissociation of the porin by boiling in 80% dimethyl sulphoxide yielded the MspA monomer, which did not form channels any more. Escherichia coli cells expressing the mspA gene produced the MspA monomer and a 100 kDa protein, which had the same channel-forming activity as whole-cell extracts of M. smegmatis with organic solvents. These proteins were specifically detected by a polyclonal antiserum that was raised to purified MspA of M. smegmatis. These results demonstrate that the mspA gene encodes a protein of M. smegmatis, which assembles to an extremely stable oligomer with high channel-forming activity. Database searches did not reveal significant similarities to any other known protein. Southern blots showed that the chromosomes of fast-growing mycobacterial species contain homologous sequences to mspA, whereas no hybridization could be detected with DNA from slow growing mycobacteria. These results suggest that MspA is the prototype of a new class of channel-forming proteins.

Amino Acid Sequence↗

PubMed1999

Analysis of virulence of clinical isolates of Salmonella enteritidis in vivo and in vitro.

Salmonella enterica serotype Enteritidis (S. enteritidis) is a major food-borne pathogen, and its incidence among all Salmonella serotypes has increased dramatically in the last two decades. To study the virulence characteristics of clinical isolates of S. enteritidis, we determined the 50% lethal doses (LD(50)) in mice of isolates of two major phage types (4 and 8). Isolates of both phage types showed a wide range of LD(50) after oral inoculation, varying from under 10(2) organisms to over 10(8) organisms. No significant difference in LD(50) was observed between the phage types. These observations indicated that clinical isolates of S. enteritidis are highly heterogeneous in their ability to cause death in mice. We compared the LD(50)s of these isolates to the results observed from in vitro pathogenicity assays. We also analyzed these isolates for recognized Salmonella virulence loci (spv, sodCI, sopE, and sef). The in vitro phenotypes of the isolates showed no obvious correlation with their LD(50) in any given assay, and the virulence genes tested were present in all isolates. However, the isolate with the lowest LD(50) (isolate 97A 2472) was resistant to acidified sodium nitrite (ASN). Moreover, the most acid-susceptible, macrophage-susceptible, and ASN-susceptible isolates were attenuated for virulence in mice. These results, based on extensive analysis of clinical isolates of S. enteritidis, demonstrate the complex nature of Salmonella pathogenesis in mice. Our results also indicate the limitation of in vitro assays in predicting in vivo virulence.

Animals↗

PubMed1999

Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility.

In this study, the currently known typing methods for Mycobacterium tuberculosis isolates were evaluated with regard to reproducibility, discrimination, and specificity. Therefore, 90 M. tuberculosis complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays. In all methods, one or more repetitive DNA elements were targeted. The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method. To examine intralaboratory reproducibility, blinded duplicate samples were included. The specificities of the various methods were tested by inclusion of 10 non-M. tuberculosis complex strains. All five RFLP typing methods were highly reproducible. The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping. In contrast, the double repetitive element PCR (DRE-PCR), IS6110 inverse PCR, IS6110 ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible. The 90 strains were best discriminated by IS6110 RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns). We conclude that for epidemiological investigations, strain differentiation by IS6110 RFLP or mixed-linker PCR are the methods of choice. A strong association was found between the results of different genetic markers, indicating a clonal population structure of M. tuberculosis strains. Several separate genotype families within the M. tuberculosis complex could be recognized on the basis of the genetic markers used.

Bacterial Typing Techniques↗

PubMed1997

A novel antioxidant gene from Mycobacterium tuberculosis.

Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI). Selection of recombinants in acidified nitrite led to the cloning of a novel gene, noxR1, from a pathogenic clinical isolate of Mycobacterium tuberculosis. Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages. These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.

Amino Acid Sequence↗

PubMed1997

Widespread dissemination of a drug-susceptible strain of Mycobacterium tuberculosis.

In New York City, a large proportion of new tuberculosis cases has been caused by 1 drug-susceptible strain (called C strain) of Mycobacterium tuberculosis. Between 1991 and 1994, among >600 tuberculosis patients consecutively identified in four large hospitals in the city, 54 with C strain, 69 with non-C cluster pattern strains, and 42 with noncluster pattern strains were studied. Susceptibility to reactive nitrogen intermediates (RNI) of selected isolates was compared. In a case-control analysis, 51% of patients with C strain, 28% with non-C cluster strains (P < .05), and 14% with noncluster strains (P < .01) were found to be injection drug users. C strain but not 13 other unrelated isolates were resistant to RNI. Injection drug use may provide a selective pressure for an RNI-resistant tubercle bacillus to emerge, which may give the organism a biologic advantage and explain the widespread dissemination of C strain M. tuberculosis within the city.

Adolescent↗