Chlamydia trachomatis OmpA genotyping as a tool for studying the natural history of genital chlamydial infection.

OBJECTIVE: To investigate the relationship of Chlamydia trachomatis (CT) outer membrane protein A (OmpA) type to the clearance of CT infection before treatment. METHODS: CT OmpA genotyping, with amplification and sequencing of ompA, was utilised to study the natural history of CT infection (spontaneous resolution vs persistence) in 102 individuals with chlamydia-positive screening tests returning for treatment. RESULTS: CT OmpA distribution was associated with spontaneous resolution of CT, most notably with CT OmpA genotype J/Ja detected more often from the initial screening CT test than other genotypes in those who then had spontaneous resolution of CT noted at the time of treatment. Five individuals with presumed persisting CT infection had discordant CT OmpA genotypes at the screening and treatment visits, suggesting possible new interval CT infection. CONCLUSIONS: Clearance of chlamydia by the host before treatment may be influenced by the CT OmpA genotype infecting the host. CT OmpA genotyping may be a valuable tool in understanding the natural history of chlamydial infections.

Typing of Chlamydia trachomatis strains from urine samples by amplification and sequencing the major outer membrane protein gene (omp1).

OBJECTIVES: To develop a novel protocol for the extraction, amplification, and sequencing of Chlamydia trachomatis MOMP gene (omp1) from urine, a non-invasive source, and apply it to an epidemiological study on the distribution of C trachomatis strains in a population of pregnant women in Thailand. METHODS: The C trachomatis DNA was extracted from culture stocks and urine using a slightly modified commercially available kit, the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, IN, USA). The PCR and sequencing primers used for the amplification and sequencing of the omp1 were designed based on the nucleotide sequence of multiple C trachomatis strains found in GenBank. The protocol for the extraction, amplification, and sequencing was tested on laboratory culture stocks of reference strains of all C trachomatis serovars and on urine samples collected in a cross sectional study designed to assess the prevalence of C trachomatis infections in the cities of Bangkok and Chiang Rai, Thailand. RESULTS: The omp1 gene was successfully amplified and sequenced from 18 laboratory C trachomatis reference strains and from 45 C trachomatis positive urine clinical samples collected from asymptomatic pregnant women. Among clinical samples, we found nine different C trachomatis genotypes: F (11, 25%), D (10, 22.6%), H (5, 11.7%), K (5, 11.7%), E (4, 9.3%), Ia (3, 7%), B (3, 7%), Ja (2, 4.5%), and G (1, 2.3%). One specimen generated an omp1 DNA sequence pattern indicating the presence of a mixed infection with at least two different serovars. CONCLUSIONS: Urine is a convenient and reliable source for genotyping C trachomatis strains. A clear advantage of urine over traditional samples, such as cervical swabs, is that urine is a non-invasive source which makes collection easier and thus facilitates the enrolment of patients in clinical and epidemiological studies. In addition to typing, urine is increasingly used for diagnosis of C trachomatis infection by several commercially available nucleic acid amplification assays which represents a distinct advantage for collecting, transport, storage, and laboratory handling of samples.

Predominance of human immunodeficiency virus type 2 subtype B in Abidjan, Ivory Coast.

We analyzed the genetic variability and phylogenetic relationships among 28 HIV-2 strains collected from patients enrolled in an HIV epidemiologic study in Abidjan, Ivory Coast, during 1995-1996. Although both subtype A (n = 8; 29%) and subtype B (n = 20; 71%) were present in this sampling, the majority of infections were caused by subtype B viruses. These findings contrasted with the reported predominance of HIV-2 subtype A in other African countries. The broad genetic diversity identified among protease gene sequences for HIV-2 subtype A (6%; range 3-15%) and subtype B (7%; range, 2-12%), and their presence in Abidjan during the 1980s, document a long coexistence of two viral subtypes in Ivory Coast. Our data indicate that viruses of subtypes A and B have contributed to the HIV-2 epidemic in Ivory Coast.

Dual and recombinant infections: an integral part of the HIV-1 epidemic in Brazil.

We systematically evaluated multiple and recombinant infections in an HIV-infected population selected for vaccine trials. Seventy-nine HIV-1 infected persons in a clinical cohort study in Rio de Janeiro, Brazil, were evaluated for 1 year. A combination of molecular screening assays and DNA sequencing showed 3 dual infections (3.8%), 6 recombinant infections (7.6%), and 70 (88.6%) infections involving single viral subtypes. In the three dual infections, we identified HIV-1 subtypes F and B, F and D, and B and D; in contrast, the single and recombinant infections involved only HIV-1 subtypes B and F. The recombinants had five distinct B/F mosaic patterns: Bgag-p17/Bgag-p24/Fpol/Benv, Fgag-p17/Bgag-p24/Fpol/Fenv, Bgag-p17/B-Fgag-p24/Fpol/Fenv, Bgag-p17/B-Fgag-p24/Fpol/Benv, and Fgag-p17/B-Fgag-p24/Fpol/Fenv. No association was found between dual or recombinant infections and demographic or clinical variables. These findings indicate that dual and recombinant infections are emerging as an integral part of the HIV/AIDS epidemic in Brazil and emphasize the heterogenous character of epidemics emerging in countries where multiple viral subtypes coexist.

Neurofilament heavy chain-like messenger RNA and protein are present in benign prostate and down-regulated in prostatic carcinoma.

Differences in gene expression between benign and malignant human prostate specimens were investigated using the differential display technique. RNA samples from paired benign and malignant areas microdissected from opposite sides of the same prostate gland were used for reverse transcription PCR. A 477-bp band was identified that was consistently present in benign prostate but absent or diminished in intensity in malignant tissue. This band was cloned, and the sequence demonstrated 99% identity with a region in the fourth exon of the human neurofilament heavy chain gene (NF-H). Northern blotting with a cDNA probe derived from this band confirmed the presence of a similarly sized message of approximately 3.9 kb in both prostate and brain, and reverse transcription PCR using primers specific to an upstream region of exon 4 confirmed NF-H-like mRNA expression in benign prostatic tissue. Immunostaining with a monoclonal antibody to NF-H showed a positive reaction in benign prostatic epithelial cells but complete absence of staining in prostatic cancer cells. These data demonstrate the presence of a NF-H-like gene product in normal prostatic epithelial cells that is down-regulated or absent in prostatic carcinomas.

Increase in sensitivity to soluble CD4 by primary HIV type 1 isolates after passage through C8166 cells: association with sequence differences in the first constant (C1) region of glycoprotein 120.

Primary isolates of human immunodeficiency virus type 1 (HIV-1) were obtained by coculture of peripheral blood lymphocytes (PBLs) from HIV-1-infected people with PBLs from uninfected donors. These viral stocks tend to be resistant to neutralization/inactivation by soluble CD4 (sCD4). When these stocks were passed through the T cell line C8166, virus stocks emerged that were sensitive to sCD4. Pre- and post-C8166 stocks maintained their sCD4-resistant and -sensitive phenotypes, respectively, with further passage in PBLs. Pre- and post-C8166 stocks were biologically cloned by two cycles of limiting dilution. The majority (14 of 17) of pre-C8166 clones were sCD4 resistant, and, conversely, the majority of post-C8166 clones (11 of 12) were sensitive to sCD4. Nucleotide and predicted amino acid sequence analysis in the env (gp120) region revealed a limited number of differences between the clones. The only differences that sorted with biological phenotype were in the first constant (C1) region of gp120. Adaptation to growth in C8166 cells and conversion from the sCD4-resistant to the sCD4-sensitive phenotype represent the emergence to prominence of viral species in the pre-C8166 stock that have a replication advantage in C8166 coincident with increased sensitivity to sCD4.

Cloning, expression, and primary structure of Metapenaeus ensis tropomyosin, the major heat-stable shrimp allergen.

Shrimp is a common cause of seafood hypersensitivity. To study the mechanism of seafood hypersensitivity at the molecular level, we have determined the primary structure of the major heat-stable allergen of shrimp by cloning, expression, nucleotide sequencing, and amino acid sequence determination of an IgE-reactive cDNA clone, Met e I, isolated from a Metapenaeus ensis expression library in lambda gt 11. We first constructed a cDNA library from the shrimp M. ensis in lambda gt 11. We then screened the library with sera from patients with hypersensitivity reactions to shrimp and identified a positive IgE-reactive clone, designated as Met e I. This cDNA was purified to homogeneity and subsequently expressed in the plasmid pGEX. Serum antibodies from patients with shrimp allergy demonstrated positive IgE reactivity by immunoblotting to a protein encoded by the clone Met e I; sera from nonallergic control subjects were not reactive. The nucleotide sequence of this cDNA clone revealed an open reading frame of 281 amino acid residues, coding for a protein of 34 kd. Comparison of the Met e I amino acid sequence with the Genbank database showed that Met e I is highly homologous to multiple isoforms of tropomyosin.

Transcription function of each base pair in the control region of the adenovirus VARNA1 gene.

Eighty-seven mutants with single-base substitution in the control region (from -44 to +70) of the adenovirus VARNA1 gene were generated, including nearly every base pair, to examine the role of DNA sequences within this control region for regulating transcription. The effect of these mutations on transcription of the gene was determined in vitro using cytoplasmic S100 extracts from human KB cells. Mutations at -37T, -35A, -29T, -28A, -25C, -18A, -17A, -16A, -13A, -9C, -8C, and -1C in the 5'-flanking region reduced transcription of the gene. Thus two positive regulatory elements, from -44 to -25 and from -18 to +2, interspersed with a putative negative regulatory element were defined. Furthermore, a distinct A-rich purine stretch in the -18 to +2 element was identified. Point mutations in the pyrimidine-rich sequence immediately upstream of the A block promoter element reduced transcription of the gene. Mutations in the GTGG direct repeats of the A block promoter element drastically decreased transcription. Furthermore, mutations that altered the AT-rich sequence immediately downstream of the A block element to become less AT-rich decreased transcription. Mutations of the base pairs at +43C, +45T, and +51A in the inter-block element moderately reduced transcription efficiency of the gene. Mutations at the central four base pairs, GTTC, of the B block palindrome severely affected transcription. These unique sequence motifs and their exact base pairs were proven to be important for regulating transcription of the VARNA1 gene.

Characterization of human immunodeficiency virus type 1 isolates from children in Romania: identification of a new envelope subtype.

Human immunodeficiency virus type 1 (HIV-1) isolates recovered from infected children in Romania were characterized for their biologic, serologic, and molecular properties. The isolates were from subjects in different clinical states, and all showed cytopathic properties in peripheral blood mononuclear cells and varying kinetics of replication. The isolates grew to varying titers in macrophages and established T cell lines. Serologic evaluation with Romanian sera indicated stronger antibody response to the gp120 of Romanian isolates than to the envelope protein of HIV-1 isolates from other countries. Although there was cross-neutralization among the Romanian isolates, no substantial activity was noted against HIV-1 prototype strains from the United States, Africa, and Thailand. Genetic analysis of the envelope C2-V3 region strongly suggests that the Romanian isolates are a subtype distinct from those assigned to other HIV-1 strains analyzed to date. This finding raises questions about the origin of HIV-1 in Romania.

Genetic diversity of human immunodeficiency virus type 1 strains in Kinshasa, Zaire.

The envelope (env) gene of human immunodeficiency virus type 1 (HIV-1) was amplified by polymerase chain reaction (PCR) from the peripheral blood mononuclear cells (PBMCs) of 14 HIV-1-infected women from Kinshasa, Zaire. Amplified DNA was directly sequenced with a primer specific for the HIV-1 env C2 region. The predicted amino acid sequences for the C2-V3 region for the 14 specimens are presented. The tetrapeptide sequence, GPGQ, located at the crown of the V3 loop, is conserved in all specimens. The same tetrapeptide sequence is present in the Zairian isolate MAL, but not in other published Zairian isolates (Z6, ELI, Z321, JY1, and NDK). Sequence comparison of the env C2-V3 region among the 14 specimens from Kinshasa revealed a 9-25% range of nucleotide divergence, with an average of 16%. Divergence between the 14 specimens and the Zairian isolates MAL, Z6, ELI, Z321, JY1, and NDK ranged from 13 to 31%. A range of 18-28% nucleotide sequence divergence was demonstrated between the 14 Kinshasa specimens and the North American isolate MN. These results demonstrate the importance of examining HIV-1 samples from diverse geographic origins in the development of effective HIV-1 vaccines.

Mutations in the p53 gene in human astrocytomas: detection by single-strand conformation polymorphism analysis and direct DNA sequencing.

The p53 gene was examined in a series of formalin-fixed paraffin-embedded astrocytic neoplasms of various types by polymerase chain reaction (PCR), single-strand conformation polymorphism analysis (SSCP), and direct sequencing of amplified DNA. PCR primers were designed to amplify three DNA fragments encompassing exons 5, 7, and 8 with splice sites, including all four mutational "hot spots" within this gene. SSCP was performed in a polyacrylamide gel containing 10% glycerol. Two mutations were found among the 20 high and intermediate grade adult astrocytomas studied by this sensitive screening technique and confirmed by sequencing of the PCR product. (1) An anaplastic astrocytoma disclosed a T-A transversion in Codon 246 giving rise to a methionine to lysine amino acid substitution. (2) A giant cell glioblastoma disclosed a G to A transition in Codon 285 resulting in a glutamic acid to lysine substitution. Both mutations were associated with loss of the normal allele. Twenty-three DNA fragments that disclosed no mutation by SSCP analysis were confirmed to be negative by direct sequencing of amplified DNA. No mutations were detected in a series of eight juvenile cerebellar astrocytomas, a biologically distinct form of low-grade astrocytoma. Mutations of the p53 gene may play an important pathogenetic role in a subset of human astrocytomas.

Surveillance of HIV infection and zidovudine use among health care workers after occupational exposure to HIV-infected blood. The CDC Cooperative Needlestick Surveillance Group.

OBJECTIVE: To study the risk for human immunodeficiency virus (HIV) infection and the patterns of use and associated toxicity of zidovudine among health care workers after an occupational exposure to HIV. DESIGN: An ongoing, prospective surveillance project conducted by the Centers for Disease Control and Prevention. PARTICIPANTS: Exposed workers voluntarily reported by 312 U.S. health care facilities from August 1983 to June 1992. RESULTS: Four of 1103 enrolled workers with percutaneous exposure to HIV-infected blood seroconverted (HIV seroconversion rate, 0.36%; upper limit of the 95% Cl, 0.83%); no enrolled workers with mucous membrane (n = 75) or skin (n = 67) contact seroconverted. During October 1988 to June 1992, 31% of 848 enrolled workers used zidovudine after exposure; this proportion increased from 5% during October through December 1988 to 43% during January through June 1992. Despite using zidovudine after exposure, one worker became infected with a strain of HIV that was apparently sensitive to zidovudine. Adverse symptoms, most commonly nausea, malaise or fatigue, and headache, were reported by 75% of workers using zidovudine; 31% of workers did not complete planned courses of zidovudine because of adverse events. CONCLUSIONS: The risk for HIV seroconversion after percutaneous exposure to HIV-infected blood is 0.36%, which is similar to previous estimates. Zidovudine is used after exposure by a sizable proportion of health care workers enrolled in the project despite frequent, minor, associated symptoms. Documented failures of postexposure zidovudine prophylaxis, including in one worker enrolled in this study, indicate that if zidovudine is protective, any protection afforded is not absolute. Postexposure zidovudine, if used, requires careful consideration of possible risks and benefits.

Absence of HIV transmission from an infected dentist to his patients. An epidemiologic and DNA sequence analysis.

OBJECTIVE: To determine if a general dentist with human immunodeficiency virus (HIV) infection transmitted HIV to any of his patients. DESIGN: A cohort study in which all patients treated by a dentist who developed the acquired immunodeficiency syndrome (AIDS) were identified and attempts were made to contact all patients for HIV antibody testing. SETTING: A general dentistry clinic operated by the Department of Veterans Affairs in southeastern Florida. PARTICIPANTS: All patients treated by a dentist during the 5 3/4 years before he developed AIDS were identified in a computerized registry of dental care. MAIN OUTCOME MEASURES: Attempts were made to contact all living patients for counseling and HIV antibody testing. Living patients with newly identified HIV infection were interviewed, and DNA sequence analysis was performed to compare genetic relatedness of their HIV to that of the dentist. Death certificates were obtained for decreased patients, and the medical records of those with diagnoses suggestive of HIV disease or drug abuse and those dying under the age of 50 years were reviewed in detail. RESULTS: There were 1192 patients who had undergone 9267 procedures, of whom 124 were deceased. A review of the death certificates of the deceased patients identified five who had died with HIV infection, all of whom were either homosexuals or users of illicit intravenous drugs. We were able to locate 962 (92%) of the remaining 1048 patients, and 900 agreed to be tested. Infection with HIV was documented in five of the 900 patients, including four who had clear evidence of risk factors for acquiring HIV infection. One patient who had only a single evaluation by the dentist denied high-risk behavior. Comparative DNA sequence analysis demonstrated that the viruses from the dentist and these five patients were not closely related. CONCLUSION: This study indicates that the risk for transmission of HIV from a general dentist to his patients is minimal in a setting in which universal precautions are strictly observed. Programs to ensure compliance with universal precautions would appear preferable to programs for widespread testing of dentists.

Adenovirus VARNA1 gene B block promoter element sequences required for transcription and for interaction with transcription factors.

We constructed mutants with a deletion of either half of the 18 base-pair B block palindrome in the VARNA1 gene, mutants with different intra-palindromic spacings, a complete set of mutants with single base substitutions, and mutants with double and triple base substitutions in the palindrome. The transcription efficiencies of these mutants were determined in human KB cell-free cytoplasmic S100 extracts. The relative competing strength of each mutant, as determined by a sequential competition experiment, was used to assess each mutant's ability to sequester factors into formation of a stable preinitiation complex. The ability of each mutant to assemble transcriptionally active preinitiation complexes was also determined by direct transcription of the isolated complexes. Finally, the ability of each mutant to interact with the transcription factor(s) TFIIIC and form a distinct gel-resolved complex was also determined. From the results of the above assays, we concluded that the two seemingly identical halves of the palindrome did not contribute equally to transcription, or to assembly of the functional preinitiation complex, nor to interaction with TFIIIC. The anterior half (B1) of the B block palindrome, which is proximal to the A block promoter element, played a stronger role in transcription and in assembly of the functional preinitiation complex than the posterior half (B2) of the palindrome. Consistent with this observation, the point mutations in four base-pairs, GTTC, from +60 to +63 in the anterior half of the B block palindrome, has the most severe effect on transcription. In contrast, we showed that the central sequence and the posterior half (B2) played a stronger role than the anterior half (B1) of the B block palindrome in the interaction of the promoter with TFIIIC. This was corroborated by the observation that base substitutions in the central four base-pair sequence of the palindrome, TCGA, from +62 to +65, had the most severe effect on interaction with TFIIIC, and that mutations in most of the sequences in the posterior half of the B block palindrome had more drastic effects than mutations in the anterior half of the palindrome in this interaction. Furthermore, the spacing between the two halves of the B block palindrome had a drastic effect on the overall transcription efficiency and the interaction of the promoter with TFIIIC, suggesting that the interaction between the two halves of the B block palindrome is not only essential, but also synergistic for the interaction with TFIIIC as well as the assembly of a transcriptionally active preinitiation complex and efficient transcription.

Wide distribution of two subtypes of HIV-1 in Thailand.

PIP: Scientists wanted to identify the genetic characteristics of 2 HIV-1 subtypes in Thailand. Staff from regional laboratories of the Ministry of Public Health took blood samples from people in various high risk groups and from all regions of the country. Staff at the National Institutes of Health in Bangkok then did lymphocyte separation, DNA extraction, and virus culture. They took the extracted DNA specimens and sent them to the US Centers for Disease Control where scientists did serologic testing, polymerase chain reaction, and sequence determination. They used Kimura's method to study sequence variations. They sequenced 300 nucleotides, including the C2-V3 domains of HIV-1 envelope gene and/or hybridization. Every risk group had HIV-1 subtype A, but subtype B was mostly found in drug users. Subtype A had spread mainly among heterosexuals. The mean intraperson variation for subtypes A and B stood at 2% and 2.7%, respectively, while the interperson variation within subtype A and B stood at 3.8% and 3.7%, respectively. The mean interperson variation between subtypes A and B from different persons was 18.1%. Phylogenetic tree analysis showed that subtype B identified with about 85% of the sequence as that of the North American isolates, making it more closely related to them than to African isolates (about 75% sequence identity). On the other hand, subtype A had a GPGQ motif at the V3 crown which was common among African HIV-1 isolates. Antibodies which usually recognize HIV-1 MN strains (which have the GPGR motif) may not react wholly with the V3 loop from the Thailand subtype A viruses, thus the GPGQ motif at the V3 crown may pose a problem. Now for the first time, scientists can follow the natural history of 2 HIV-1 subtypes and determine their relative pathogenicity and transmission efficiency between adults or from mother to infant. The relative homogeneity of the HIV-1 strains in Thailand presents a theoretical advantage in designing vaccines for potential large-scale clinical trials.^ieng

Molecular epidemiology of HIV transmission in a dental practice.

Human immunodeficiency virus type 1 (HIV-1) transmission from infected patients to health-care workers has been well documented, but transmission from an infected health-care worker to a patient has not been reported. After identification of an acquired immunodeficiency syndrome (AIDS) patient who had no known risk factors for HIV infection but who had undergone an invasive procedure performed by a dentist with AIDS, six other patients of this dentist were found to be HIV-infected. Molecular biologic studies were conducted to complement the epidemiologic investigation. Portions of the HIV proviral envelope gene from each of the seven patients, the dentist, and 35 HIV-infected persons from the local geographic area were amplified by polymerase chain reaction and sequenced. Three separate comparative genetic analyses--genetic distance measurements, phylogenetic tree analysis, and amino acid signature pattern analysis--showed that the viruses from the dentist and five dental patients were closely related. These data, together with the epidemiologic investigation, indicated that these patients became infected with HIV while receiving care from a dentist with AIDS.