Characteristics of HIV-1-associated Kaposi's sarcoma among women and men in South Africa.

Despite the increase of HIV-1-associated Kaposi's sarcoma (KS), little is known about HIV-associated KS in the African setting, particularly among women. A descriptive study of the demographic, clinical, immunological and virological features of AIDS-associated KS from KwaZulu-Natal, South Africa was undertaken. Consecutively, recruited patients were clinically staged; CD4/CD8 cell counts, HIV-1 viral loads and clinical parameters were evaluated. Of the 152 patients (77 male and 75 female) 99% were black. Females were significantly younger (P = 0.02) and had poorer disease prognosis (odds ratio [OR] = 2.7, 95% confidence interval [CI] = 1.4-5.4, P = 0.003) and were more likely to have extensive cutaneous KS when compared with males (OR = 3.1, 95% CI = 1.4-6.7, P = 0.003). One-third of patients had coexisting HIV-related disease, most commonly tuberculosis, and these were more frequent in females (56.7 vs. 43.3%). In conclusion, HIV-associated KS in South Africans has an equal female-to-male ratio. Females are younger and have more severe disease than males.

Histopathology and genotyping in infectious spondylitis of HIV- and HIV+ patients.

Approximately 2 million South Africans are HIV/TB coinfected, and many develop skeletal disease. The resurgence of spinal tuberculosis, including atypical forms, is due largely to HIV-associated immune suppression. We investigated the impact of HIV coinfection on the histological features of the disease and the occurrence of atypical opportunistic organisms in infectious spondylitis in an HIV/TB endemic region. We analyzed blood and tissue biopsies from 60 patients with tuberculous spondylitis. Investigations included full blood counts, CD4/CD8 counts, HIV-1 serology and RNA quantification (tissue and plasma), acid-fast bacilli localization and routine TB culture, histopathologic evaluation of biopsies, and bacterial genotyping using the 16S rDNA gene. Twenty-two patients (37%) were HIV positive with a mean age of 29 years (range, 2-65 years). Forty-one (68%) tissue specimens were culture negative for Mycobacterium tuberculosis (Mtb), although nontuberculous mycobacteria (NTM) were identified in three HIV-negative patients. Histopathologic features were characteristic of TB infection in 91.4% of all specimens tested and 100% of the HIV-infected group. Genotyping of 10 culture-positive isolates identified Mtb (3/10), NTMs (2/10), and environmental bacilli (3/10). Our observations suggest HIV-induced immune suppression impacts the histological and clinical features of infectious spondylitis but has no impact on the incidence of NTMs in this setting.

Atypical mycobacterial spondylitis in HIV-negative patients identified by genotyping.

Non-tuberculous mycobacterial infections pose a significant diagnostic and therapeutic challenge. We report two cases of such infection of the spine in HIV-negative patients who presented with deformity and neurological deficit. The histopathological features in both specimens were diagnostic of tuberculosis. The isolates were identified as Mycobacterium intracellulare and M. fortuitum by genotyping (MicroSeq 16S rDNA Full Gene assay) and as M. tuberculosis and a mycobacterium other than tuberculosis, respectively, by culture. There is a growing need for molecular diagnostic tools that can differentiate accurately between M. tuberculosis and atypical mycobacteria, especially in regions of the developing world which are experiencing an increase in non-tuberculous mycobacterial infections.

The HIV-1 exposed neonate: outcome of intensive care management in the first week of life.

A prospective study was carried out to determine if the outcome in HIV-exposed neonates requiring intensive care (n=30) is different from that in HIV-unexposed neonates (n=40) requiring intensive care in the first week of postnatal life. It was noted that the outcome in terms of incidence of death and intensive care stay do not differ significantly in these two groups although some hematological parameters may be significantly different. Considering the fact that the outcome is not worse in HIV-exposed babies and that most of these babies ultimately turn out to be HIV-uninfected, these babies should not be deprived of intensive care, whenever necessary.

Molecular characteristics of human immunodeficiency virus type 1 subtype C viruses from KwaZulu-Natal, South Africa: implications for vaccine and antiretroviral control strategies.

The KwaZulu-Natal region of South Africa is experiencing an explosive outbreak of human immunodeficiency virus type 1 (HIV-1) subtype C infections. Understanding the genetic diversity of C viruses and the biological consequences of this diversity is important for the design of effective control strategies. We analyzed the protease gene, the first 935 nucleotides of reverse transcriptase, and the C2V5 envelope region of a representative set of 72 treatment-naïve patients from KwaZulu-Natal and correlated the results with amino acid signature and resistance patterns. Phylogenetic analysis revealed multiple clusters or "lineages" of HIV-1 subtype C that segregated with other C viruses from southern Africa. The same pattern was observed for both black and Indian subgroups and for retrospective specimens collected prior to 1990, indicating that multiple sublineages of HIV-1 C have been present in KwaZulu-Natal since the early stages of the epidemic. With the exception of three nonnucleoside reverse transcriptase inhibitor mutations, no primary resistance mutations were identified. Numerous accessory polymorphisms were present in the protease, but none were located at drug-binding or active sites of the enzyme. One frequent polymorphism, I93L, was located near the protease/reverse transcriptase cleavage site. In the envelope, disruption of the glycosylation motif at the beginning of V3 was associated with the presence of an extra protein kinase C phosphorylation site at codon 11. Many polymorphisms were embedded within cytotoxic T lymphocyte or overlapping cytotoxic T-lymphocyte/T-helper epitopes, as defined for subtype B. This work forms a baseline for future studies aimed at understanding the impact of genetic diversity on vaccine efficacy and on natural susceptibility to antiretroviral drugs.

Diagnosis of paediatric HIV infection in a primary health care setting with a clinical algorithm.

OBJECTIVE: To determine the validity of an algorithm used by primary care health workers to identify children with symptomatic human immunodeficiency virus (HIV) infection. This HIV algorithm is being implemented in South Africa as part of the Integrated Management of Childhood Illness (IMCI), a strategy that aims to improve childhood morbidity and mortality by improving care at the primary care level. As AIDS is a leading cause of death in children in southern Africa, diagnosis and management of symptomatic HIV infection was added to the existing IMCI algorithm. METHODS: In total, 690 children who attended the outpatients department in a district hospital in South Africa were assessed with the HIV algorithm and by a paediatrician. All children were then tested for HIV viral load. The validity of the algorithm in detecting symptomatic HIV was compared with clinical diagnosis by a paediatrician and the result of an HIV test. Detailed clinical data were used to improve the algorithm. FINDINGS: Overall, 198 (28.7%) enrolled children were infected with HIV. The paediatrician correctly identified 142 (71.7%) children infected with HIV, whereas the IMCI/HIV algorithm identified 111 (56.1%). Odds ratios were calculated to identify predictors of HIV infection and used to develop an improved HIV algorithm that is 67.2% sensitive and 81.5% specific in clinically detecting HIV infection. CONCLUSIONS: Children with symptomatic HIV infection can be identified effectively by primary level health workers through the use of an algorithm. The improved HIV algorithm developed in this study could be used by countries with high prevalences of HIV to enable IMCI practitioners to identify and care for HIV-infected children.

An integrated genetic data environment (GDE)-based LINUX interface for analysis of HIV-1 and other microbial sequences.

MOTIVATION: Sequence databases encode a wealth of information needed to develop improved vaccination and treatment strategies for the control of HIV and other important pathogens. To facilitate effective utilization of these datasets, we developed a user-friendly GDE-based LINUX interface that reduces input/output file formatting. DESIGN AND RESULTS: GDE was adapted to the Linux operating system, bioinformatics tools were integrated with microbe-specific databases, and up-to-date GDE menus were developed for several clinically important viral, bacterial and parasitic genomes. Each microbial interface was designed for local access and contains Genbank, BLAST-formatted and phylogenetic databases. AVAILABILITY: GDE-Linux is available for research purposes by direct application to the corresponding author. Application-specific menus and support files can be downloaded from (http://www.bioafrica.net).

Prevalence, incidence, and mother-to-child transmission of HIV-1 in rural South Africa.

Simple, robust approaches are needed to monitor prevalence, incidence, and mother-to-child transmission of HIV-1 in rural Africa. We have designed a method that uses antibody and viral RNA testing of dried blood spots obtained from mother-infant pairs attending routine immunisation clinics. In our study, prevalence and incidence of HIV-1 was highest in young women in their late teens and early twenties. In children born to infected mothers, prevalence increased from 14% in infants younger than 6 weeks of age to 24% at 3-6 months. The blood-spot approach is an effective method for surveillance of HIV-1 in women and children, and for early identification of incidence of this infection in women of child-bearing age.

Viral levels in newborn African infants undergoing primary HIV-1 infection.

We examined weekly changes in viral levels in seven untreated infants infected with HIV at birth. Viral levels spiked immediately but reverted quickly to plateau levels typical of infant HIV infection within 2 weeks of first detected viraemia. We speculated that the depletion of naive, susceptible cells is responsible for the rapid decrease in spike levels and that the rapid replacement of lymphocytes in infants causes the high plateau viral levels (10(5) copies/ml) to be sustained.

Oral mucosal Langerhans' cells as target, effector and vector in HIV infection.

The mechanism underlying a transition of the oral cavity mucosal epithelium towards susceptibility to opportunistic infections in HIV-seropositive patients was investigated. Phenotypic markers CD1a, HLA-DR, and CD86 of oral mucosal Langerhans' cells (LCs), p17 core protein of human immunodeficiency virus (HIV), and CD45RO of memory T cells were labeled on oral hairy leukoplakia lesional biopsies and clinically normal autologous tissue of HIV-infected patients. HIV p17 protein was detected in association with mucosal LCs, mainly within the lesional epithelium. There were significant correlations between the detection of HIV p17 and the depletion of LCs, and between the depletion of LCs and the presence of hairy leukoplakia lesions. Conjugates of activated LCs and memory T cells were also evident in the submucosal area of lesional biopsies. The findings from this study support the hypothesis that oral mucosal LCs are also the target of HIV infection. Cytopathic changes of LCs caused by productive HIV infection may contribute to selective depletion of LCs, which may impair the mucosal immunologic protection against colonization by microorganisms causing HIV-associated oral mucosal lesions.

Decreased HIV-associated T cell apoptosis by HIV protease inhibitors.

Antiretroviral treatment of patients infected with HIV results in improvements in CD4+ T cell number. Emerging evidence suggests that some of the improvements in CD4+ T cell number that occur in response to protease inhibitor (PI) therapy may not be accounted for solely by enhanced viral suppression, implying that PI may directly affect T cell survival. Since HIV T cell depletion is associated with enhanced apoptosis, we analyzed the effect of PIs on T cell apoptosis. In vitro treatment of peripheral blood lymphocytes (PBLs) from HIV-infected but untreated patients with reverse transcriptase inhibitors (RTIs) does not alter apoptosis, whereas PI treatment rapidly reduces CD4+ and CD8+ T cell apoptosis. In contrast, PI treatment does not alter apoptosis in PBL blasts from HIV-negative patients, or in Jurkat T cells. Consistent with this observation, 8 days of PI therapy in HIV-infected patients does not significantly alter plasma viremia, yet results in significant inhibition of CD4+ and CD8+ T cell apoptosis. The inhibitory effects of PI on apoptosis have implications concerning the treatment of HIV and its pathogenesis.

Association of HIV-1 load and CD4 lymphocyte count with mortality among untreated African children over one year of age.

OBJECTIVE: To examine the association of viral load and CD4 lymphocyte count with mortality among HIV-infected children over one year of age. DESIGN: A prospective study. HIV-infected children were enrolled during the first year of life and followed for more than 2 years at the Queen Elizabeth Central Hospital in Blantyre, Malawi (southeast Africa). METHODS: Morbidity and mortality information was collected every 3 months, and physical examination and blood testing (for viral level and CD4 cell percentage) were performed every 6 months. Kaplan-Meier analyses and proportional hazards models were used to estimate survival and to examine the association of primary predictors with mortality. RESULTS: Of 155 HIV-infected children originally enrolled, 115 (74%) had viral load testing and 82 (53%) had both viral load and CD4 cell percentage testing after their first year. Among children over one year of age, significant associations were found between mortality and the log10 viral load and CD4 cell percentage in both univariate and multivariate models. Independent of the CD4 cell value, a one unit log10 increase in HIV RNA level increased the hazard of child mortality by more than twofold. Children with low CD4 cell counts (< 15%) and high viral loads (> or = 250,000 copies/ml median value) had the worst survival; children with high CD4 cell counts (> or = 15%) and low viral loads (< 250,000 copies/ml) had the best survival. CONCLUSION: As in developed countries, viral load and CD4 cell count are the main predictors of mortality among African children. Making these tests available adds to the challenges to be considered if antiviral therapies were to be adopted in these countries.

Virus levels in untreated African infants infected with human immunodeficiency virus type 1.

In developed areas, human immunodeficiency virus (HIV)-infected infants have high virus levels and rapidly progress to death. HIV levels were assessed in 1994-1997 in untreated infants in Malawi by analysis of dried blood spots tested by nucleic acid silica-bound amplification. Of 24 umbilical cord blood (CB)-positive samples, 83% had >10,000 copies/mL. The median virus level was 78,000 copies/mL. First positive sample median levels were 355,000 copies/mL among 52 perinatally infected infants and 130,000 copies/mL among 43 infants infected by breast-feeding. Virus levels were stable, and initial levels predicted levels 1 year after infection (P=.005), at which time levels did not significantly differ among in utero, perinatally, or postnatally infected infants. Thus, neither age at infection nor route of infection significantly influenced HIV levels measured 1 year after infection. Most (87%) CB-positive infants were infected before labor onset, since virus levels greatly exceeded those expected in their mothers.

Cerebrospinal fluid HIV RNA and drug levels with combination ritonavir and saquinavir.

UNLABELLED: Combination antiretroviral therapy with ritonavir and saquinavir has established potent and durable activity on plasma viremia. CNS HIV infection may be sequestered from drug therapy that does not penetrate the blood-brain barrier. Penetration of these protease inhibitors into the cerebrospinal fluid (CSF) and CSF HIV RNA levels on such therapy has not been well described. DESIGN/METHODS: In a cross-sectional study, 28 HIV1-infected study subjects were evaluated either before initiation of or before maximal response to ritonavir-saquinavir therapy, during maximal plasma virologic response, and after virologic failure. Simultaneous samples of plasma and cerebrospinal fluid were obtained from 24 study subjects to measure HIV RNA and protease inhibitor levels. RESULTS: Across the treatment groups, a strong correlation was found between plasma and CSF HIV RNA levels (r = 0.870; p < .001). In each study subject with plasma HIV RNA levels below assay limit (80 copies/ml), the CSF HIV RNA level was also below the limit of quantitation. Low levels of saquinavir (<2 ng/ml) and ritonavir (<25 ng/ml) in the CSF were observed, with a CSF:plasma drug concentration ratio of < or = 0.005 (0.5%) in all study subjects evaluated (n = 11). The plasma:CSF HIV RNA ratio was high before or early in treatment (median, 38; interquartile range [IQR], 13,97), but low (median, 0.29; IQR, 0.17, 7.5) in those failing therapy (group C, p < .001). CONCLUSIONS: CSF ritonavir and saquinavir levels are consistent with the estimated known fraction of unbound drug in plasma (<2%). Across these treatment response groups, suppression of plasma viremia can predict low CSF HIV RNA levels. This correlation may represent HIV RNA transport and equilibrium between CSF and plasma, or it may represent CNS anti-HIV activity of protease inhibitors. The low drug levels and inverted ratio of HIV RNA in the CSF compared with plasma early in plasma virologic breakthrough suggests CSF virologic failure may contribute to failure of plasma virologic response.

Persistently negative HIV-1 antibody enzyme immunoassay screening results for patients with HIV-1 infection and AIDS: serologic, clinical, and virologic results. Seronegative AIDS Clinical Study Group.

OBJECTIVE: To describe persons with HIV infection and AIDS but with persistently negative HIV antibody enzyme immunoassay (EIA) results. DESIGN: Surveillance for persons meeting a case definition for HIV-1-seronegative AIDS. SETTING: United States and Canada. PATIENTS: A total of eight patients with seronegative AIDS identified from July 1995 through September 1997. MAIN OUTCOME MEASURES: Clinical history of HIV disease, history of HIV test results, and CD4 cell counts from medical record review; results of testing with a panel of EIA for antibodies to HIV-1, and HIV-1 p24 antigen; and viral subtype. RESULTS: Negative HIV EIA results occurred at CD4 cell counts of 0-230 x 10(6)/l, and at HIV RNA concentrations of 105,000-7,943,000 copies/ml. Using a panel of HIV EIA on sera from three patients, none of the HIV EIA detected infection with HIV-1, and signal-to-cut-off ratios were < or = 0.8 or all test kits evaluated. Sera from five patients showed weak reactivity in some HIV EIA, but were non-reactive in other HIV EIA. All patients were infected with HIV-1 subtype B. CONCLUSIONS: Rarely, results of EIA tests for antibodies to HIV-1 may be persistently negative in some HIV-1 subtype B-infected persons with AIDS. Physicians treating patients with illnesses or CD4 cell counts suggestive of HIV infection, but for whom results of HIV EIA are negative, should consider p24 antigen, nucleic acid amplification, or viral culture testing to document the presence of HIV.

Diagnosis and Direct Automated Sequencing of HIV-1 From Dried Blood Spots (DBS) Collected on Filter Paper.

Since its discovery in 1981, human immunodeficiency virus type 1 (HIV-1) has rapidly emerged as one of the most devastating infectious pathogens of this century (1-3). The World Health Organization (WHO) estimates that, as of 1995, there were at least 15 million HIV- infected men, women, and children worldwide, with the vast majority of infections occurring in developing countries and isolated rural regions where specimen collection, preparation and shipment are difficult (4). Simple and improved sampling methods that can be widely applied under difficult field conditions are needed to effectively monitor the changing dynamics of the HIV-1/AIDS pandemic, track the spread of HIV-1 variants among different population groups, and ensure that research and interventive activities are directed against biologically important variants of the virus. To date, at least eight major HIV-1 subtypes, designated A through H, have been identified (5,6). More recently, a ninth subtype, I, has been detected (7), as well as several highly divergent, or "outlying" variants of HIV-1 that have been tentatively classified as group O (8,9). This subtyping is based on a relatively small number of specimens collected from a few geographic areas and the full range and distribution of HIV-1 variants remains to be established. The collection of whole blood on filter paper provides an innovative and powerful approach for the systematic and unbiased collection of large numbers of field specimens for diagnostic and surveillance purposes (10-19).

Quantification of HIV-1 RNA in Dried Plasma Spots (DPS) : A Field Approach to Therapeutic Monitoring.

The ability to accurately measure viral RNA in the plasma (1-3) and intracellular (4-7) compartments of HIV-1-infected persons has led to a dramatic improvement in the understanding of the natural history of HIV-1 and AIDS. A number of recent studies have convincingly demonstrated that high levels of viral replication occur at all stages of disease (8-10), and that changes in viral RNA load are predictive of disease outcome (11,12), and response to therapy (13,14). These findings, combined with the introduction of potent new antivirals (15,16), have stimulated a growing interest in viral load monitoring, both as a function of disease status, and as a predictor of disease progression and therapeutic efficacy.