Corneo-conjunctival carcinoma in Uganda.

AIMS: Since 1990, the incidence of conjunctival neoplasia has more than tripled in Uganda. It is known to be associated with exposure to solar ultraviolet radiation and to infection with the human immunodeficiency virus-1 (HIV). However, little is known about the most effective treatments. In this study, we report surgical outcomes among people with corneo-conjunctival squamous neoplasia in Uganda and investigate the role of HIV infection and other factors in the aetiology of the tumour. METHODS: Country-wide enrolment of participants; removal and histology of suspect lesions; HIV counselling and testing; home visiting of participants to determine outcomes. RESULTS: In 67 months between 1995 and 2001, 476 participants were enrolled (262 female, 214 male, median age 32 years). A total of 463 (97%) had eye-conserving removal of the lesion and 13 had other surgery. For 414, the histology was squamous neoplasia (184 invasive carcinoma, 230 intraepithelial). The prevalence of HIV infection in cases was 64%. In all, 96% were followed up for a median period of 32 months (range 0-81) after eye-conserving surgery during which time 13 (3.2%) had a recurrence. CONCLUSIONS: Surgery resulted in a low recurrence rate during the follow-up period and had minimal complications. The prevalence of HIV among cases was higher than expected on the basis of data from the general population, although about a third of cases were HIV-negative and had normal CD4 counts. No new cofactors were identified.

Predominance of HIV-1 subtype A and D infections in Uganda.

To better characterize the virus isolates associated with the HIV-1 epidemic in Uganda, 100 specimens from HIV-1-infected persons were randomly selected from each of two periods from late 1994 to late 1997. The 200 specimens were classified into HIV-1 subtypes by sequence- based phylogenetic analysis of the envelope (env) gp41 region; 98 (49%) were classified as env subtype A, 96 (48%) as D, 5 (2.5%) as C, and 1 was not classified as a known env subtype. Demographic characteristics of persons infected with the two principal HIV-1 subtypes, A and D, were very similar, and the proportion of either subtype did not differ significantly between early and later periods. Our systematic characterization of the HIV-1 epidemic in Uganda over an almost 3-year period documented that the distribution and degree of genetic diversity of the HIV subtypes A and D are very similar and did not change appreciably over that time.

Optimizing the delivery of HIV counseling and testing services: the Uganda experience using rapid HIV antibody test algorithms.

The AIDS Information Center (AIC) was established in Kampala, Uganda in 1990 in response to increasing interest by members of the general public who wished to know their HIV serostatus. By 1996, >300,000 clients had been seen. HIV serologic testing was performed at a central laboratory and results reported back to AIC after 2 weeks. Approximately 25% of clients failed to learn their HIV serostatus as a result of failure to return or late arrival of results. To address these issues, AIC carried out an evaluation of 3 rapid HIV assays, Sero-Strip, SeroCard, and Capillus, against a standard criterion to identify a testing algorithm that could be used as an on-site confirmatory testing strategy. The study was carried out over a period of 5 working days and 325 clients were seen. An algorithm was identified, which gave no indeterminate results with unambiguously positive or negative specimens, which was 100% sensitive and specific, and which could be integrated with minimal disruption into existing counseling procedures. All clients left AIC knowing their HIV serostatus and having spent <2 hours at the Center. The results of this evaluation demonstrate that "same-day" results can be provided in counseling and testing settings without compromising the quality of counseling or the accuracy of HIV testing.

PIP: An evaluation conducted at the AIDS Information Center in Kampala, Uganda, demonstrated that same-day HIV results can be provided in counseling and testing centers without compromising service quality. The Center was established in 1990 in response to widespread public interest in HIV serodiagnosis. By 1996, more than 300,000 clients had been tested. However, 25% of these clients never received their result because of failure to report back to the Center after 2 weeks (the time required for results to be returned from an off-site laboratory) or late arrival of results. To address this problem, the Center evaluated three rapid HIV assays (Sero-Strip, SeroCard, and Capillus) against a standard criterion to identify a testing algorithm that could be used as an on-site confirmatory testing strategy. 325 clients were enrolled in the 5-day evaluation. Individually, all three rapid tests performed well when compared with the standard criterion result. The resulting algorithm (a combination of Capillus as the screening assay and SeroCard as a supplementary assay for initially positive specimens and Multispot as a tie-breaker assay) gave no indeterminate results, was 100% sensitive and specific, and could be integrated easily into existing counseling protocols. The entire process (registration, test decision counseling, phlebotomy, laboratory testing, prevention counseling, and post-test counseling) took an average of 2 hours to complete.

The development and evaluation of a probe hybridization method for subtyping HIV type 1 infection in Uganda.

We developed a method for large-scale screening of HIV-1 genotypic variation based on DNA probe hybridization. Nested PCR amplifications were performed to generate fragments in the env C2-V3 region and also in the gp41 region, which encompasses the immunodominant domain. The proviral DNA sequences were derived from 68 samples and phylogenetically analyzed. For comparison, the C2-V3 fragment was used in DNA probe hybridization to rapidly determine the infecting HIV subtype. The hybridizing probes were designed on the basis of the two most prevalent subtypes in Uganda, A and D. The results were compared to evaluate the feasibility of using this hybridization method for large-scale genotypic screening. Sequence analysis of the 68 amplified PCR fragments showed that 39 were subtype A and 29 were subtype D. The results of DNA hybridization to the amplified products with A and D subtype-specific probes were more than 90% concordant with the subtypes determined by sequence analysis. Our findings suggest that probe hybridization with subtype-specific probes is effective for large-scale screening of HIV-infected populations. Application of this method will significantly reduce the time needed for large, population-based investigations.

A molecular epidemiologic survey of HIV in Uganda. HIV Variant Working Group.

OBJECTIVE: Previous data, based on a small sampling of convenience, reported subtypes A, B, C, D, and G in Uganda, but neither the extent nor the proportion of these subtypes could be evaluated. To establish correctly the prevalence and distribution of HIV-1 subtypes, we analysed viral clades in 739 HIV-1-seropositive specimens from different areas of Uganda. METHODS: Blood specimens from 1100 patients were collected in five districts of Uganda. Within this collection, 929 HIV-1-seroreactive samples underwent analysis of viral DNA, and 739 were selected for further subtyping in env or pol regions. RESULTS: Using a combination of subtype A- and D-specific probes to C2-V3 region and DNA sequencing, HIV-1 env subtypes were determined in 594 specimens: 341 were of subtype A (57.4%), 250 of subtype D (42.1%), and three of subtype C (0.5%). Sixty-two samples showed reactivity with both probes, suggesting potential mixed infections, cross-reactivity to probes, or possibly other subtypes. Subsequent sequence analysis of 19 randomly selected specimens revealed subtypes A (n = 4), D (n = 12), and C (n = 3). Sequence analysis of the 27 samples chosen from the remaining 83 samples, which could be amplified only with viral gp41 or protease gene primers, classified them as subtypes A (n = 13) and D (n = 14). No significant clinical, demographic, or geographic differences were found between HIV-1 infections with viruses of subtypes A and D, despite considerable genetic diversity within these clades. CONCLUSIONS: This is the first major population-based study of the prevalent HIV-1 strains in an African country selected for vaccine trials. The subtyping methods we describe should be of use to investigators seeking to conduct large-scale screening for HIV variants in other populations.

Binding of lithium and boron to human plasma proteins.

The binding of lithium and boron, at normal physiological levels, to plasma proteins has been investigated by the techniques of precipitation with ethyl alcohol and gel chromatography. Assays of lithium and boron were made by thermal neutron activation and mass spectrometric assay of 3He and 4He. Results of alcohol precipitation experiments for plasma from two apparently healthy donors showed that 13+/-4% and 16+/-3% of the lithium in plasma is protein bound, but essentially no boron is bound under the conditions used. We believe that because of denaturation of proteins which occurs during alcohol precipitation, these percentages represent lithium and boron tightly bound to protein molecules. The results of the gel-chromatography experiment, on the other hand, showed that lithium and boron are bound to a wide range of plasma proteins, from low (approximately 60,000 amu) to high (approximately 1,000,000 amu) molecular weights, and to very low- (approximately 6000 amu) molecular-weight ligands. Although a clear identification of the specific proteins which bind lithium and boron cannot be made at present, some possibilities can be suggested.

A round-robin determination of boron in botanical and biological samples.

The accurate determination of boron (B) at trace and ultratrace concentrations is an important step toward establishing the role of B in biological functions. However, low-level B concentrations are difficult to determine accurately, especially for many botanical and biological matrices. A round-robin study was conducted to assess analytical agreement for low-level B determinations. Ten experienced research groups from analytical laboratories extending across Europe, Asia, and the US participated in this study. These groups represent a cross-section of academic, commercial, and government facilities. The researchers employed both ion-coupled plasma and neutron techniques in the study. Results from this round-robin study indicate good agreement between participating laboratories at the mg/kg level, but at the lowest levels, microg/kg, only three laboratories participated, and agreement was poor. By encouraging discussion among scientists over these data, the secondary goal of this round-robin study is to stimulate continued improvement in analytical procedures and techniques for accurate low-level B determinations. Furthermore, it is intended to encourage the development of a variety of low-level (low mg/kg and microg/kg) B certified reference samples in biological and botanical matrices. The results from the round-robin analyses were compiled and are summarized in this article.

Considerations in the determination of boron at low concentrations.

Experimental evidence now supports the nutritional essentiality of boron (B) in some biological systems, and accordingly, the need for reliable analytical B data is increasing. However, the accurate determination of B in biological materials is a formidable challenge at low concentrations (<1 mg B/kg). Recent studies still show significant analytical discrepancies in the analysis of animal tissues and fluids, despite the development of instrumental techniques such as TIMS, ICP-MS, ICP-ES, ICAP, SIMS, NA-MS, PGAA, NRA, and so forth, which have demonstrated detection limits approaching or exceeding (microg B/kg concentrations. Since boric acid is both volatile and ubiquitous in nature, the chemical and physical pathways for B contamination and its loss are manifold, especially during sample preparation. An added obstacle is the inadequacy of biological reference materials certified for B below mg B/kg. With an emphasis toward sample preparation and ICP-MS analysis, examples are provided in this article to help the analyst avoid common problems associated with the analysis of B from biological sources. Topics that are discussed include contamination from Teflon vessels during microwave digestion, losses owing to freeze-drying, B isotopic variations, standards preparation, reagent backgrounds, and instrumental interferences.

New crown motif of an HIV-1 V3 loop sequence from a Ugandan AIDS patient.

PIP: HIV-1 V3 loop sequences from Ugandan patients include motifs from subtypes A, B, and D. To characterize further HIV isolates, V3 loop sequences were amplified from HIV-1 isolated in 1987 from peripheral blood mononuclear cells (PBL) of three patients with full-blown AIDS from Kampala, Uganda. The PBL were separated by Ficoll Paque gradients and cocultivated with noninfected donor lymphocytes for two weeks. The HIV was then transferred to HUT-78 cells. From extracted DNA of the permanently-infected HUT-78 cells, nested polymerase chain reaction (PCR) was conducted, with V3 loop sequencing performed directly upon PCR fragments derived from two independent DNA preparations and on cloned fragments. Isolates MVP-9801, -9802, and -9803 show 35.6%, 32.4%, and 29.7% nucleotide sequence divergence from the ELI subtype D sequence; 31.5%, 25.7%, and 18.9% divergence from the Z2Z6 subtype D sequence; and 21.9%, 12.2%, and 12.2% divergence from the subtype D consensus sequence. All three deduced amino acid sequences fit into the subtype D consensus sequence rather than into other V3 loop sequences described for Ugandan subtype A isolates. MVP-9802 and MVP-9803 contain the GSGQA pentapeptide motif at the tip of the V3 loop, while MVP-9801 contains GGRA. This may be explained by a deletion of proline codon between the codons for the two glycine residues. The authors believe that this deletion has not been previously reported. They also note that the deletion does not appear to be associated with a growth difference in vitro or with a difference in pathogenicity in vivo. The immunogenic implications of this altered V3 loop crest remain unclear. The Western blot profiles for the gp160, gp120, and gp41 proteins of the three Ugandan isolates manifest normal molecular weights.^ieng

Ugandan HIV-1 strains with unusual antigenicity

Recent reports have documented that certain HIV-1 antibody tests may not reliably detect divergent variants of HIV-1; provisionally classified as HIV-1 group O. Although most known cases of group O infections are found in Cameroon; the prevalence and worldwide distribution of these unusual variants is unclear. In Uganda; where serveral HIV-1 subtypes have been identified; approximately 15of confirmed HIV-1-positive specimens tested negative using an in-house peptide enzyme immunoassay (PLEA) based on the immuno-dominant region of gp41 (QQLLGIWGCSGKLICTT) of the prototypic HIV-1 strain LAI. This result prompted us to further investigate these specimens using PEIA's based on the representative V3-loops of each known serotype; including group O (ANT70 and MVP5180) sequences; and other immuno-dominant petides representing the genetic groups M (LAI and ELI) and O (MVP518 and ANT70). No group O infections were found among these samples. This was confirmed by genetic analysis of the C2V3 region of the envelope protein (gp 20) and/or the immuno-dominant region pf the gp41. Our analysis showed that a single amino-acid substitution (leuu to His) in position607 of gp41; was associated with the diminished sero-reactivity with the immuno-dominant gp41 peptide baserd on LAI. Since synthetic peptides with similar sequences are being used in many commercial third-generation assays for HIV-1; our findings indicate the need to evaluate the performance of these assays before they are used in Uganda. The results of these evaluations may be useful in developing improved diagnostic assays for HIV-1 in Uganda and other African countries

An evaluation of the probe hydridization method for large scale population studies of HIV-1 strain variation in Uganda

A strategy was developed for a long-term; large scale study of HIV-1 genetic variation in Uganda. Our approach was based on the premisethat DNA sequencing is costly and time consiming with respect to screening largenumbers of specimens. We adopted a dot-blod hybridization method using oligonucleotide probes specific for HIV-1 subtypes A and D in the envC2V3 region to screen for HIV-1 subtypes in Uganda. Specicimens which could not be subtyped in this way were sequenced directly. Genomic DNA from 72 HIV-1 seropositive subjects were amplified by PCR in the envC2V3 region. The results of dot-blot hybridization indicated that 40 were subtype A and 29 were subtype D. The remaining three specimens could not be typed due to either non-binding to probes of both subtypes or non specific binding. Hybdridization results were compared with results obtained by sequence phylogenetic analysis. Sequences were generated from 50 DNA specimens and analysis in the C2V3 region showed that 29 were subtype A and 21 were subtype D. Sequences were also generated from the immuno-dominant env gp41 region (384bp fragment) from specimens for which C2V3 sequences were not available. Phylogenetic analysis of these sequences indicated that 8 were subtype A and 8 were subtype D. Regardless of the region of the HIV genome under study sequence data fully supports the subtype assignment derived from hybridization data. Our findings suggest that probe hybridization using A and D subtype specific probes will be effective for large scale screening og the HIV-infected populationin Uganda. Application of this method should lead to significant savings in cost and time for large; population-based investigations

Computer-assisted quality assurance in an HIV serology laboratory.

A computer-based quality assurance programme for an HIV-1 serology laboratory is described. The programme was designed to minimise transcription errors and to provide rapid feedback on laboratory performance. Similar systems could readily be introduced to any laboratory with access to simple computing facilities.

Reactions of Ugandan antisera with peptides encoded by V3 loop epitopes of human immunodeficiency virus type 1.

The specificities of antibodies reacting with peptides encoded by V3 loop apical epitopes were determined for sera from 230 seropositive Ugandans, including asymptomatic persons and AIDS patients, sampled between 1986 and 1992. Most (71%) of the sera reacted with the peptide encoded by HIV-MN, 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the HIV-1 global subtype A (referred to as the Uganda A consensus), 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the global subtype D (the Uganda D consensus); 19% of the sera also reacted with peptides encoded by the divergent Ugandan variant U31. There was no obvious correlation between the specificities of antibody binding and the V3 loop sequence of the corresponding virus isolate or provirus. Competitive inhibition and antibody adsorption experiments indicated that the MN peptide, the Uganda A consensus peptide, the Uganda D consensus peptide, and the U31 peptide were recognized by different sets of antibodies. Eighteen percent of the sera from AIDS patients and 26% of the sera from asymptomatic persons were monospecific for one of the MN, Uganda A, or Uganda D peptides. Whereas all except one of the singly reactive AIDS sera were specific for MN, 39% of the singly reactive asymptomatic sera were specific for MN, 39% for the Uganda A peptide, and 21% for the Uganda D peptides. We conclude that analysis of the specificities of antibodies against the V3 loop epitopes in sera from asymptomatic persons could provide useful epidemiological data about the prevalence of viral subtypes within a population.

PIP: The specificities of antibodies reacting with peptides encoded by V3 loop apical epitopes were determined for sera from 230 HIV seropositive Ugandans, including 123 asymptomatic persons and 107 AIDS patients, mostly mothers attending prenatal clinics, sampled between 1986 and 1992. 71% of the sera reacted with the peptide encoded by HIV-MN, 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the HIV-1 global subtype A (referred to as the Uganda A consensus); 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the global subtype D (the Uganda D consensus); and 19% of the sera also reacted with peptides encoded by the divergent Ugandan variant U31. Although 70% of the 1986 sera reacted with the Uganda A consensus peptide, only 49% of the 1991/92 sera reacted with this peptide (p 0.005). 20% of the 1991/92 sera, compared with only 7% of the 1986 sera, did not react with any of the peptides (p 0.05). There was no obvious correlation between the specificities of antibody binding and the V3 loop sequence of the corresponding virus isolate or provirus. Competitive inhibition and antibody adsorption experiments indicated that the MN peptide, the Uganda A consensus peptide, the Uganda D consensus peptide, and the U31 peptide were recognized by different sets of antibodies. 18% of the sera from AIDS patients and 26% of the sera from asymptomatic persons were monospecific for one of the MN, Uganda A, or Uganda D peptides. Whereas all except one of the singly reactive AIDS sera were specific for MN, 39% of the singly reactive asymptomatic sera were specific for MN, 39% for the Uganda A peptide, and 21% for the Uganda D peptides. The analysis of the specificities of antibodies against the V3 loop epitopes in sera from asymptomatic persons could provide useful epidemiological data about the prevalence of viral subtypes within a population.

Algorithms for detecting antibodies to HIV-1: results from a rural Ugandan cohort.

OBJECTIVE: To evaluate an algorithm using two enzyme immunoassays (EIA) for anti-HIV-1 antibodies in a rural African population and to assess alternative simplified algorithms. METHODS: Sera obtained from 7895 individuals in a rural population survey were tested using an algorithm based on two different EIA systems: Recombigen HIV-1 EIA and Wellcozyme HIV-1 Recombinant. Alternative algorithms were assessed using negative or confirmed positive sera. RESULTS: None of the 227 sera classified as unequivocably negative by the two assays were positive by Western blot. Of 192 sera unequivocably positive by both assays, four were seronegative by Western blot. The possibility of technical error cannot be ruled out in three of these. One of the alternative algorithms assessed classified all borderline or discordant assay results as negative had a specificity of 100% and a sensitivity of 98.4%. The cost of this algorithm is one-third that of the conventional algorithm. CONCLUSIONS: Our evaluation suggests that high specificity and sensitivity can be obtained without using Western blot and at a considerable reduction in cost.

PIP: Although the Western blot test is widely used to confirm HIV-1 serostatus, concerns over its additional cost have prompted review of the need for supplementary testing and the evaluation of alternative test algorithms. Serostatus tends to be confirmed with this additional test especially when tested individuals will be informed of their serostatus or when results will be used for research purposes. The confirmation procedure has been adopted as a means of securing suitably high levels of specificity and sensitivity. With the goal of exploring potential alternatives to Western blot confirmation, the authors describe the use of parallel testing with a competitive and an indirect enzyme immunoassay with and without supplementary Western blots. Sera were obtained from 7895 people in the rural population survey and tested with an algorithm based on the Recombigen HIV-1 EIA and Wellcozyme HIV-1 Recombinant; alternative algorithms were assessed on negative or confirmed positive sera. None of the 227 sera classified as negative by the 2 assays were positive by Western blot. Of the 192 identified ass positive by both assays, 4 were found to be seronegative with Western blot. The possibility of technical error does, however, exist for 3 of these latter cases. One of the alternative algorithms assessed classified all borderline or discordant assay results as negative with 100% specificity and 98.4% sensitivity. This particular algorithm costs only one-third the price of the conventional algorithm. These results therefore suggest that high specificity and sensitivity may be obtained without using Western blot and at a considerable reduction in cost.

Regulation of lithium and boron levels in normal human blood: environmental and genetic considerations.

Blood lithium levels may be both genetically and environmentally regulated. The genetic component is evidenced mainly from studies in twins who were either normal or had a manic-depressive disorder. An environmental contribution is adduced from the relationship between the blood lithium level and the amount of the element ingested. No such information is available for boron, another element present in ultra trace amounts in human blood. Unusually high levels of lithium and boron in the waters of northern Chile offer an opportunity to study the genetic and environmental regulation of these elements in the blood of healthy subjects. Samples of blood (n = 40) and water (n = 47) were collected at seven locations in the province of Tarapaca. Most of the healthy subjects were Aymara who had been resident in the respective communities for at least 3 years. The samples were transported to Canada and then freeze-dried. Neutron irradiation was performed in a highly thermalized flux to induce the reactions 6Li (n, alpha) t and 10B (n,7Li) alpha. Assays of 6Li and 10B were conducted in a static mass spectrometer by measurement, respectively, of 3He, produced from decay of tritium, and 4He from alpha-particles. Lithium concentrations in water and blood exhibited a linear relationship, as did the boron concentrations in these fluids. Because some of the individual subjects (n = 15) were first-degree relatives, a genetic component to the regulation of blood levels was explored. The variance in blood levels of lithium and boron was significantly greater between than within families (p < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Neutron Depth Profiling: Overview and Description of NIST Facilities.

The Cold Neutron Depth Profiling (CNDP) instrument at the NIST Cold Neutron Research Facility (CNRF) is now operational. The neutron beam originates from a 16 L D2O ice cold source and passes through a filter of 135 mm of single crystal sapphire. The neutron energy spectrum may be described by a 65 K Maxwellian distribution. The sample chamber configuration allows for remote controlled scanning of 150 × 150 mm sample areas including the varying of both sample and detector angle. The improved sensitivity over the current thermal depth profiling instrument has permitted the first nondestructive measurements of 17O profiles. This paper describes the CNDP instrument, illustrates the neutron depth profiling (NDP) technique with examples, and gives a separate bibliography of NDP publications.

Sequence analysis of the V3 loop regions of the env genes of Ugandan human immunodeficiency proviruses.

Ugandan strains of human immunodeficiency virus type 1 (HIV-1) were isolated by cocultivation of peripheral blood lymphocytes from infected individuals with cord blood lymphocytes. Sequences from the V3 region of the env gene were amplified by the polymerase chain reaction (PCR) from chromosomal DNA obtained from low passage virus cultures. The PCR products from 13 Ugandan isolates were cloned into a phagemid vector and sequenced. Many isolates contained divergent V3 loop sequences and adjacent regions: diversity was associated with codon deletions or duplications and with nucleotide substitutions, especially G----A transitions. Proviruses from some of the cultures showed extensive diversity within the V3 loop sequences but others were more homogeneous. The V3 loop apices were conserved in 6 of the Ugandan proviruses and these were very similar to the equivalent regions of several Zairean proviruses. The V3 loop apices of African isolates of HIV-1 are divergent from those of North American isolates. The possible biological consequences of this divergence are discussed.

Nucleotide sequence of a Ugandan HIV-1 provirus reveals genetic diversity from other HIV-1 isolates.

A Ugandan isolate of human immunodeficiency virus type 1 (HIV-1), designated U455, was adapted to growth in U937 cells, the provirus cloned into the lambda L47.1 vector, and its DNA sequence determined. The sequences of some of the U455 genes showed a marked divergence from those of North American and other African isolates. The sequenced clone was defective with single in-phase stop codons in the vpr and env genes and frame shift, resulting in a stop codon, within the vpu gene.