Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand. Chiang Mai HEPS Working Group.

Characterization of persons highly exposed to human immunodeficiency virus (HIV)-1 who remain uninfected may help define protective immunity. Seventeen HIV-1-seronegative Thai female sex workers (CSWs) with epidemiologic evidence of exposure to HIV-1 were studied for humoral immune responses and phenotypic and genotypic analyses of HLA class I and CCR5 allelic profiles. Infected CSWs and low-risk HIV-1-seronegative Thai women were controls. Highly exposed, persistently seronegative (HEPS) CSWs did not differ from HIV-infected CSWs in HIV risks, condom use, or sexually transmitted diseases. Significant differences were seen in humoral immune responses: gp160-specific IgA responses were detected in cervicovaginal lavage fluids in 6 of 13 HEPS CSWs but 0 of 21 seronegative subjects. All women had wild-type CCR5. HEPS CSWs were more likely to have the HLA-B18 phenotype and genotype than were matched controls (corrected P=.018). Epidemiologic exposure to HIV-1 without apparent infection, an unusual distribution of HLA class I alleles, and HIV-1 gp160-specific IgA responses suggest a biologic basis for this phenomenon.

Collection and Processing of Mucosal Secretions from HIV-1 Infected Women.

The major route of transmission of the human immunodeficiency virus type 1 (HIV-1) worldwide, like other agents of sexually transmitted disease, is via mucosal surfaces of the genital tract through sexual exposures (1-4). It has been hypothesized that immune responses at these sites may be important determinants of protection against HIV-1 (1,2,5). Because of the potential relationship of mucosal immune responses to protection against HIV-1 infection, an assessment of immune responses at these sites in vaccine trial participants will be critical. Proper collection and processing of specimens from mucosal sites is of vital importance to the measurement of mucosal immune responses. This chapter details the collection and processing procedures of specimens from three distinct mucosal compartments in female subjects. Collection techniques for additional mucosal compartments (e.g. semen, rectal, etc.) can be found in ref. 6 (see Note 1).

HIV type 1 subtypes in Malaysia, determined with serologic assays: 1992-1996.

We investigated the molecular epidemiology of HIV-1 subtypes in Malaysia among injecting drug users (IDUs) and sexual transmission risk groups, using serologic and genetic techniques. Frozen sera collected at a general hospital, a blood bank, several drug treatment centers, and an STD clinic in Kuala Lumpur, between 1992 and 1996, were investigated retrospectively. V3 peptide serotyping and monomeric gp120 capture serotyping were used to study 89 known HIV-1-infected subjects. The methods differentiate subtypes B, E, and C. V3 peptide and gp120 capture results were comparable. No subtype C-specific reactive sera were found; one specimen was dually reactive for subtypes C and B, using the V3 peptide ELISA; and four were durally reactive for subtypes E and C using this assay. Genotypic analysis of HIV-1 gag RNA in serum was done on a subset of subjects and confirmed serologic findings. HIV-1 subtypes differed significantly by risk category: of 53 IDUs, 29 (55%) were infected with subtype B and 19 (36%) were infected with subtype E, 3 (6%) were dually reactive, and 2 (4%) were not typable. Of 36 persons with heterosexual risks, 29 (81%) were infected with subtype E, 5 (14%) were infected with subtype B, and 2 (5%) were not typable. Persons with IDU risks were significantly more likely to be infected with subtype B than were those with sexual risks (OR 5.89; 95% CI, 1.94-18.54; p < 0.001). Subtypes B and E of HIV-1 appear to predominate in Malaysia; subtype B was more prevalent among IDUs; subtype E was more prevalent among all other groups. These results may have important HIV-1 vaccine implications.

The molecular epidemiology of HIV-1 among male sex workers in northern Thailand.

Male commercial sex workers (CSWs) in northern Thailand are at high risk for HIV-1 infection and may be exposed to multiple subtypes of HIV-1 through sexual contact with men and women from Thailand, other Asian nations, and the West. We studied 103 male Thai nationals working in gay bars and clubs in Chiang Mai city who participated in the 1994 sentinel surveillance of the Thai Ministry of Public Health using HIV serologic testing, DNA polymerase chain reaction methods, molecular cloning, differential colony hybridization, nucleotide sequencing, and a third hypervariable region (V3) peptide enzyme-linked immunoabsorbent assay to differentiate subjects infected with HIV-1 subtypes B, E, both, or neither. The majority (58%) reported heterosexual orientation, and 35 (34%) were married. Seventeen (16.5%) were HIV infected: 16 (94%) of 17 had HIV-1 subtype E; 1 had HIV-1 subtype B. Two subjects had dually reactive results by molecular techniques but colony hybridization sequencing revealed only HIV-1 subtype E. HIV-1 subtype E appears to be the predominant clade among male CSWs in northern Thailand. This may have important implications for the global kinetics of the HIV epidemic.

PIP: Male prostitutes in northern Thailand are at high risk for HIV-1 infection and may be exposed to multiple subtypes of HIV-1 through sexual contact with men and women from Thailand and elsewhere. Blood samples of 103 male Thai nationals working in gay bars and clubs in Chiang Mai collected during the Thai Ministry of Public Health's 1994 sentinel surveillance were studied to determine the prevalences of HIV-1 subtypes B and E in the population. The men reported having an average of 2.5 male clients per week and had been in the business from 1 month to 7 years, with a median duration of 4 months. HIV serologic testing, DNA polymerase chain reaction methods, molecular cloning, differential colony hybridization, nucleotide sequencing, and a third hypervariable region (V3) peptide enzyme-linked immunoabsorbent assay were used to differentiate subjects infected with HIV-1 subtypes B, E, both, or neither. 58% of the men reported a heterosexual orientation outside of work and 34% were married. 72.1% reported inconsistent or no condom use with sex partners. 17 (16.5%) were infected with HIV. Of those infected, 16 (94%) had HIV-1 subtype E and 1 had subtype B.

Comparison of serum and plasma viral RNA measurements in primary and chronic human immunodeficiency virus type 1 infection.

We sought to define the relation between serum and plasma HIV-1 viral RNA load in patients with primary and chronic HIV-1 disease. HIV-1 viral load was determined from 116 serum and plasma samples, including 33 matched pairs, from five patients with primary and three patients with chronic HIV disease using the Roche HIV Monitor assay. The mean +/- standard deviations of the serum and plasma viral RNA levels from the 33 matched pairs were 4.372 +/- 0.885 and 4.478 +/- 0.950 log10 (copies/ml), respectively. This -0.106 log difference between serum and plasma viral RNA levels, which equates to 21% of non-log-transformed values, was not statistically significant by the Wilcoxon sign rank test (p = 0.09). The distributions of serum and plasma viral load slopes, calculated from all available viral RNA load data for each patient, were also not statistically different (p = 0.07). The levels of HIV-1 RNA measured in the serum or plasma of HIV-seropositive patients yield equivalent biologic information.

Mucosal immune responses in four distinct compartments of women infected with human immunodeficiency virus type 1: a comparison by site and correlation with clinical information.

Because mucosal immune responses may be important in protection against human immunodeficiency virus type 1 (HIV-1), HIV-1-specific immune responses at mucosal sites in natural infection were compared. Total antibody concentrations and HIV-1-specific binding antibody responses in four distinct mucosal sites and serum were assessed in 41 HIV-infected and 19 HIV-seronegative women. HIV-1 gp160-specific IgG responses were detected in >99% of mucosal samples in infected subjects, with the highest titers in genital secretions. HIV-1-specific IgA was detected in the majority of endocervical secretions (94%) and nasal washes (95%) but less often in vaginal washes (51%) and parotid saliva (38%). There was no significant correlation between mucosal immune response and most clinical factors. Based on methodologic considerations, frequencies of detection, and HIV-1-specific responses, nasal washes and genital secretions may each provide important measures of HIV-1-specific mucosal immune responses in infected women.

Correlation of genetic and serologic approaches to HIV-1 subtyping in Thailand.

The aim of this study was to compare the performance of differential polymerase chain reaction (PCR) typing and peptide enzyme-linked immunosorbent assay (V3-EIA) for human immunodeficiency virus type 1 (HIV-1) subtyping in Thailand using heteroduplex mobility assay (HMA) as the reference standard. Paired peripheral blood mononuclear cells (PBMC) and sera were collected from 38 HIV-1 seropositive persons in Thailand. HMA was done by standard methods; differential PCR employs primer pairs that differentially amplify either subtype E or B. V3-EIA used peptides specific for subtypes E or B. Thirty-two cases (84%) were found by HMA to be infected with subtype E: and six with (16%) subtype B. The results obtained with differential PCR were 100% concordant with those of HMA; V3 EIA correctly predicted the subtype in 95% (36 of 38). Six samples that molecularly subtyped as E were repeatedly dual reactive by screening V3-EIA, but these resolved to subtype E using an antigen-limiting EIA. Two samples were serologically nontypeable because of overall low levels of V3 antibody. Using HMA as the standard, differential PCR was shown to subtype HIV-1 reliably from patient PBMC samples. V3-EIA correctly predicted HIV-1 subtype in most (95%) of our cases. Because of the less rigorous sampling requirements, specimen processing, and logistical and technical requirements of serotyping compared with molecular techniques, it appears to be practical for screening purposes in a field environment. Samples that cannot be definitively subtyped serologically should undergo differential PCR and antigen-limiting V3 EIA. These approaches to HIV-1 subtyping should be used in complementary fashion in Thailand, where subtypes B and E are currently known to cocirculate.

Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand.

Human immunodeficiency virus type 1 isolates of envelope genotype E are contributing substantially to the global pandemic. These strains appear to be mosaics, with the gag gene from clade A and the envelope from clade E; the parental clade E strain has not been found. Here we report the first full genomic sequence of one such mosaic virus, isolate CM240 from Thailand. Multiple breakpoints between the two parental genotypes have been found in a CM240 virus. The entire gag-pol region and most, if not all, of the accessory genes vif, vpr, tat, rev, and vpu appear to derive from clade A. The genotype switches to E shortly after the signal peptide of the envelope and back to clade A near the middle of gp41; thus, the portion of the envelope that lies on the cytoplasmic side of the membrane appears to be principally derived not from clade E, as previously thought, but from clade A. Another small segment not belonging to any recognized clade and presumably also contributed by the parental E strain has been found in the long terminal repeat. It may be significant that the implied virion structure resembles a pseudotype virus with the matrix and core from one clade and the outer envelope from another. In the long terminal repeat, differences were observed between CM240 and other clades in the number of NF-kappa B binding sites, the sequence of the TATA box, and the putative secondary structure of the transactivation response region stem-loop. The mosaic structure of a CM240 virion is suggestive of phenotypic differences which might have contributed to the emergence of this variant.

PIP: A new variant of human immunodeficiency virus (HIV)-1 with a mosaic genomic structure was identified in Thailand in 1992. This variant, termed genotype E, was characterized by an envelope gene sequence equidistant from genotypes A through D. The gag gene, encoding the matrix and core virus proteins, grouped with genotype A rather than forming a new clade. More than 500,000 Thais are estimated to be infected with the envelope clade E virus and its type 1 isolates, previously assumed to be rare outliers, are contributing substantially to the global acquired immunodeficiency syndrome pandemic. Reported here is the first complete genomic analysis of one such mosaic virus, isolate CM240 from Thailand. The entire gag-pol region and most of the accessory genes vif, vpr, tat, rev, and vpu appear to derive from clade A. The genotype switches to E shortly after the signal peptide of the envelope and back to clade A near the middle of gp41. Thus, the portion of the envelope that lies on the cytoplasmic side of the membrane appears to be derived from clade A. Another small segment presumably contributed by the parental E strain has been found in the long terminal repeat. The multiple crossover points detected in CM240 may reflect a common mechanism of frequent strand switching by reverse transcriptase. Full genomic analyses of other mosaic HIV-1 genomes are recommended to determine whether the breakpoints found in CM240 are recurrent and to identify the functional implications of the virion alterations.

Diversity of the envelope glycoprotein among human immunodeficiency virus type 1 isolates of clade E from Asia and Africa.

Human immunodeficiency virus type 1 isolates of clade E, known to be largely responsible for the fulminating epidemic in Southeast Asia, have been derived exclusively from Asia and Africa. Here we provide full or partial sequences of the envelope glycoprotein gene from 13 additional clade E isolates from Asia representing patients in both early and late stages of disease. More extensive comparison of isolates within clade E by geographic locale, stage of disease, and year of isolation is now possible. The genetic diversity of clade E isolates from Asia, particularly among those derived from early-stage patients, is restricted compared with African isolates (mean interisolate distances in gp120, 5.4 and 20.2%, respectively). However, patients hospitalized with AIDS-related illnesses in Thailand harbored clade E isolates exhibiting broader interisolate diversity and with highly heterogeneous third hypervariable loop sequences. An additional pair of cysteine residues, predicting a novel disulfide bridge and present in 80% of clade E isolates from Asia, was uniformly absent from six African isolates. Clade E isolates in Thailand from early-stage subjects continue to be genetically similar to potential vaccine prototype strains, providing a favorable environment for the evaluation of genotype E candidate vaccines. However, evidence of increasing interisolate diversity is appearing among late-stage patients in Asia. This diversification of the clade E virus, if sustained, may impact preventive vaccine development strategies.

Sequences and phylogenetic analysis of the nef gene from Thai subjects harboring subtype E HIV-1.

PIP: It has been demonstrated that nef-defective SIV can cause attenuated disease in rhesus monkeys and that animals immunized with a similar construct can be protected when challenged with wild-type, pathogenic SIV. This finding has sparked interest in the nef gene of HIV-1. It remains unclear, however, whether nef serves an analogous role in human HIV-1 infection; Deacon et al. have reported an association of deletions in the nef gene with an attenuated disease course in humans, although other work does not support a strict correlation between the structure or function of nef and rates of disease progression. The nef gene product has been shown to influence the infectivity and pathogenicity of HIV-1. nef may also have a role in the immune response to HIV-1. The current database of nef sequences is derived almost exclusively from subtype B viruses. The authors therefore sought to develop a database of nef sequences from subjects infected with HIV-1 subtype E. Whole-blood samples were collected from 103 male commercial sex workers in Chiang Mai, Thailand. 17 tested positive for infection with HIV-1, 16 of whom were infected with subtype E by gp120 characterization. Intersubject variation in subtype E nef genes at the nucleotide level ranged from 2.1-7.8%. Thai subtype E nef sequences are more tightly clustered than subtype B nef sequences, analogous to what is seen in env. The authors note that their subtype E nef sequences do not share the genotypic polymorphism in the area of residues 10-30 noted with subtype B nef. These data confirm the need to develop a set of subtype E-specific reagents.^ieng

Detection of diverse HIV-1 genetic subtypes in the USA.

Of the nine genetic subtypes of HIV-1 that exist world wide, subtype B predominates in North America and Europe. Thus, most knowledge about HIV-1 and most vaccine development efforts are based on subtype B viruses. We document here the detection of HIV-1 subtypes A, D, and E in five US servicemen who acquired these non-subtype-B infections during overseas deployments. The dispersal of diverse HIV-1 subtypes into regions of the world with previously restricted genetic diversity may have important implications for the epidemiology of the epidemic and for the design and implementation of vaccine trials.

Multiple introductions of HIV-1 subtype E into the western hemisphere.

There are nine recognised genetic subtypes of HIV-1, and the epidemic in Southeast Asia is largely due to subtype E. We have investigated HIV-1 viral subtypes in 11 Uruguayan military personnel, six with infection acquired during a United Nations deployment to Cambodia and five with infection acquired in South America. We found subtype E in five of the six infections acquired in Southeast Asia, and subtype B in all five of the domestically acquired cases. These findings document multiple introductions of HIV-1 subtype E into the western hemisphere and mean that the genetic diversity of the global HIV-1 pandemic must be considered in strategies for epidemic control.

PIP: The genetic analysis of viruses from 11 HIV-infected Uruguayan military personnel, 6 of whom are thought to have acquired their infection while deployed as part of the UN Transitional Authority in Cambodia, is reported. They were screened for antibodies to HIV-1 before deployment, on return, and one month after return. 10 (.8%) of 1300 individuals acquired HIV-1 infection during overseas deployment. 6 of these 10 and 5 military personnel with domestically acquired infections volunteered for this study. The five had been diagnosed when tested as part of sentinel screening or at blood donation. Medical histories indicated that for all but 1 of the 11 subjects (who did not deploy to Cambodia), transmission most likely occurred through heterosexual exposures. The virus was successfully isolated by coculture in six individuals (four nondeployed, two deployed), and the genetic analyses were carried out on DNA prepared from cocultured peripheral blood mononuclear cells (PBMC) from these subjects. Genetic analyses of viruses from the other five subjects were done on DNA from primary PBMC. Phylogenetic analysis of the DNA sequences from the gp 120 fragment obtained from the five subjects who did not deploy and had not traveled outside South America revealed that all clustered within the B subtype of HIV-1. Of the six subjects who were infected while deployed to Cambodia, five harbored HIV-1 subtype E, while the sixth isolate (UR5) was subtype B. Cross-sectional surveys in several populations in Uruguay have revealed a low overall seroprevalence of HIV-1, with the highest prevalence (1.26% of 868 patients tested) found in a population from a sexually transmitted diseases clinic in Montevideo. The biological consequences of the introduction of subtype E HIV-1 into the western hemisphere are not known, but data from Thailand suggest that subtype E may be associated with a higher risk of heterosexual transmission than B.

Isolated peripheral tuberculous lymphadenitis in adults: current clinical and diagnostic issues.

Eight cases of isolated peripheral tuberculous lymphadenitis occurred over a 16-month period at our institution, prompting a literature review to establish the epidemiology, clinical manifestations, and current approaches to diagnosis and treatment of this disorder. It occurs most commonly in young adult women who either are immigrants from areas where tuberculous is endemic or have histories of travel that are suggestive of exposure to Mycobacterium tuberculosis. The disease is indolent and usually presents as a unilateral painless neck mass. Constitutional symptoms are rare, except in individuals infected with the human immunodeficiency virus (HIV). The results of skin testing with purified protein derivative are invariably positive. Excisional biopsy for histopathologic and microbiological evaluations provides the highest diagnostic yield, although fine needle aspiration may be useful for HIV-infected individuals and in areas of the world with a high prevalence of disease. A 6-month course of combination antituberculous therapy is adequate for disease due to susceptible organisms. This infection may be readily diagnosed if suggestive epidemiological and clinical findings lead to expeditious tissue sampling.

Dual infection with human immunodeficiency virus type 1 of distinct envelope subtypes in humans.

Multiple genetic subtypes of human immunodeficiency virus type 1 (HIV-1) have been identified among internationally collected isolates. The HIV-1 epidemic in Thailand is largely due to B and E subtypes of virus. Dual infection with distinct HIV-1 subtypes would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. Polymerase chain reaction typing and serologic typing were used to screen a panel of specimens from HIV-1-infected subjects in Thailand. Two persons simultaneously harbored HIV-1 of env subtypes B and E, and this was confirmed by colony hybridization with subtype-specific probes and nucleotide sequence analysis of a 630-bp fragment of gp120 from multiple molecular clones. In addition, both subtypes were identified in cocultured peripheral blood mononuclear cells from 1 individual. These data provide the first evidence of dual HIV-1 infection in humans and reinforce the need for polyvalent vaccines.

Infection due to Mycobacterium haemophilum identified by whole cell lipid analysis and nucleic acid sequencing.

A patient with indolent, non-Hodgkin's lymphoma developed a pretibial soft tissue abscess caused by a fastidious mycobacterium. Because the organism could not be definitively identified by standard microbiologic testing, whole cell fatty acid analysis and 16S rDNA sequencing were performed. These procedures identified the organism as Mycobacterium haemophilum. We review the diagnostic considerations with regard to this pathogen.

Safety and immunogenicity of a polyvalent Escherichia coli vaccine in human volunteers.

Since a limited number of O serogroups account for nearly 70% of bacteremic and meningitic Escherichia coli isolates, a polyvalent vaccine was made by conjugating a Pseudomonas aeruginosa exotoxin A carrier protein to the O polysaccharide of 12 serogroups of E. coli (O1, O2, O4, O6-O8, O12, O15, O16, O18, O25, O75). No serious reactions occurred in 88 vaccinees. Four-fold or greater increases in ELISA antibody levels over baseline were greatest (> 60% of vaccinees) for O1, O2, O6-O8 and O15; intermediate (approximately 50%) for O18 and O75, and poorest (> or = 45%) for O4, O12, O16, and O25. Responses with functionally active opsonophagocytic antibody generally paralleled ELISA antibody responses. With the availability of a safe, immunogenic E. coli vaccine, active and passive immunization strategies merit further development as adjunctive treatment for E. coli bacteremia and neonatal meningitis.

Chronic dacryocystitis caused by Mycobacterium fortuitum.

PURPOSE: To characterize the clinical presentation of Mycobacterium fortuitum dacryocystitis, an unusual microbial cause of this disease. METHODS: The authors present a detailed description of a case of M. fortuitum chronic dacryocystitis and a review of the literature. FINDINGS: Although M. fortuitum is a well-recognized cause of chronic keratitis and corneal ulcer, it has only been reported once previously as a cause of dacryocystitis. Multiple factors that alter the ocular environment of the host may predispose to infection with this organism. The diagnosis requires isolation of the organism in the appropriate clinical setting. The authors' case represents the first patient successfully treated with dacryocystectomy. CONCLUSION: M. fortuitum is a rare cause of chronic dacryocystitis that may respond favorably to surgical therapy alone.