Connected diagnostics: linking digital rapid diagnostic tests and mobile health wallets to diagnose and treat brucellosis in Samburu, Kenya.

BACKGROUND: Despite WHO guidelines for testing all suspected cases of malaria before initiating treatment, presumptive malaria treatment remains common practice among some clinicians and in certain low-resource settings the capacity for microscopic testing is limited. This can lead to misdiagnosis, resulting in increased morbidity due to lack of treatment for undetected conditions, increased healthcare costs, and potential for drug resistance. This is particularly an issue as multiple conditions share the similar etiologies to malaria, including brucellosis, a rare, under-detected zoonosis. Linking rapid diagnostic tests (RDTs) and digital test readers for the detection of febrile illnesses can mitigate this risk and improve case management of febrile illness. METHODS: This technical advance study examines Connected Diagnostics, an approach that combines the use of point-of-care RDTs for malaria and brucellosis, digitally interpreted by a rapid diagnostic test reader (Deki Reader) and connected to mobile payment mechanisms to facilitate the diagnosis and treatment of febrile illness in nomadic populations in Samburu County, Kenya. Consenting febrile patients were tested with RDTs and patient diagnosis and risk information were uploaded to a cloud database via the Deki Reader. Patients with positive diagnoses were provided digital vouchers for transportation to the clinic and treatment via their health wallet on their mobile phones. RESULTS: In total, 288 patients were tested during outreach visits, with 9% testing positive for brucellosis and 0.6% testing positive for malaria. All patients, regardless of diagnosis were provided with a mobile health wallet on their cellular phones to facilitate their transport to the clinic, and for patients testing positive for brucellosis or malaria, the wallet funded their treatment. The use of the Deki Reader in addition to quality diagnostics at point of care also facilitated geographic mapping of patient diagnoses in relation to key risk areas for brucellosis transmission. CONCLUSIONS: This study demonstrates that the Connected Dx approach can be effective even when addressing a remote, nomadic population and a rare disease, indicating that this approach to diagnosing, treatment, and payment for healthcare costs is feasible and can be scaled to address more prevalent diseases and conditions in more populous contexts.

Long-term virological outcomes, failure and acquired resistance in a large cohort of Ugandan children.

OBJECTIVES: To evaluate long-term virological failure (VF) and drug resistance among HIV-infected Ugandan children on first-line ART. METHODS: In a multicentre prospective cohort study, viral load (VL) and drug resistance mutations (DRMs) were investigated at baseline and 6 monthly intervals in children (age ≤ 12 years). VF (two consecutive VLs >1000 copies/mL or death after 6 months of ART) was defined as early VF (0-24 months of ART) or late VF (25-48 months of ART). An active regimen was defined as partially active if the genotypic susceptibility score (GSS) was <3. RESULTS: Between 2010 and 2011, 316 children were enrolled. Viral suppression was achieved in 75.8%, 71.5%, 72.6% and 69.2% at 12, 24, 36 and 48 months. VF occurred in 111/286 (38.8%), of which 67.6% was early and 32.4% late VF. Early VF was associated with a partially active regimen at baseline (OR 6.0, 95% CI 1.9-18.5), poor adherence (OR 3.1, 95% CI 1.3-7.4) and immunodeficiency (OR 3.3, 95% CI 1.1-10.2). Late VF was associated with age >3 years (OR 2.5, 95% CI 1.0-6.6) and WHO stage 3/4 (OR 4.2, 95% CI 1.4-13.4). Acquired DRMs were detected in 27.0% before 24 months, versus 14.4% after 24 months (P < 0.001). A total of 92.2% of the children with early VF, versus 56.2% with late VF, had a partially active regimen (P < 0.001). CONCLUSIONS: VF rates were high, occurred predominantly in the first 24 months and appeared to increase again in year four. Risk factors and patterns of early VF/DRMs were different from those of late VF/DRMs. Virological control may improve by close monitoring and prompt switching to second-line therapy in the first 24 months. Late VF may be prevented by early start of ART.

Alarming increase in pretreatment HIV drug resistance in children living in sub-Saharan Africa: a systematic review and meta-analysis.

BACKGROUND: Children have an augmented risk of pretreatment HIV drug resistance (PDR) due to exposure to antiretroviral drugs for the prevention of mother-to-child transmission (PMTCT). Paediatric data are essential to evaluate the effectiveness of the restricted number of paediatric regimens currently available, but these data are scarce. METHODS: We conducted a systematic review of the literature on PDR in children (median age ≤12 years) in sub-Saharan Africa. We separately extracted the proportion of children with PDR for children with and without prior PMTCT exposure, used random-effects meta-analysis to pool proportions and used meta-regression to assess subgroup differences. RESULTS: We included 19 studies representing 2617 children from 13 countries. The pooled PDR prevalence was 42.7% (95% CI 26.2%-59.1%) among PMTCT-exposed children and 12.7% (95% CI 6.7%-18.7%) among PMTCT-unexposed children (P = 0.004). The PDR prevalence in PMTCT-unexposed children increased from 0% in 2004 to 26.8% in 2013 (P = 0.009). NNRTI mutations were detected in 32.4% (95% CI 18.7%-46.1%) of PMTCT-exposed children and in 9.7% (95% CI 4.6%-14.8%) of PMTCT-unexposed children; PI mutations were uncommon (<2.5%). PDR was more common in children aged <3 years compared with children aged ≥3 years [40.9% (95% CI 27.6%-54.3%) versus 17.6% (95% CI 8.9%-26.3%), respectively (P = 0.025)]. CONCLUSIONS: The PDR prevalence in African children is high and rapidly increasing. Even in PMTCT-unexposed children, the most recent reports indicate that PDR is present in up to a third of children starting first-line therapy. Our data underscore the importance of initiating PI-based first-line ART in young children (<3 years of age) and suggest that older children may also benefit from this approach.

Evaluation of an affordable HIV-1 virological failure assay and antiretroviral drug resistance genotyping protocol.

HIV-1 RNA viral load is the preferred tool to monitor virological failure during antiretroviral therapy (ART) exposure. Timely detection of virological failure can reduce the prevalence and complexity of HIV-1 drug resistance. This field evaluation further characterizes a two-step approach to identify virological failure, as a measure of ART adherence, and detect HIVDR mutations in the reverse transcriptase (RT) gene of HIV-1. Two hundred and forty-eight (248) samples were tested; 225 from South African HIV-1 participants enrolled in the PharmAccess African Studies to Evaluate Resistance (PASER) cohort, forty of which had paired dried blood spot (DBS) samples and 23 HIV-1 negative samples. A newly developed virological failure assay (ARTA-VFA) was used on all samples, and those with a viral load >5000 RNA copies/ml were genotyped with a shortened RT protocol to detect HIVDR (ARTA-HIVDR(ultralight)). The ARTA-VFA showed good precision and linearity as compared to a commercial reference assay (NucliSENS EasyQ v1.2, Roche) with an R(2) of 0.99. Accuracy studies illustrated standard deviations of <1 log RNA copies/ml for plasma and DBS ARTA-VFA results compared to the reference method. The ARTA-VFA's intended use was to deliver qualitative results either < or >5000 RNA copies/ml. No significant differences in the proportion of results < or > either the 5000 RNA copies/ml or 1000 RNA copies/ml cut-off were noted for plasma indicating either cut-off to be useful. Significant differences were noted in these proportions when DBS were used (P=0.0002), where a 5000 RNA copies/ml cut-off was deemed more appropriate. The sensitivity and specificity of the ARTA-VFA with plasma were 95% and 93% and 91% and 95% for DBS using a 5000 RNA copies/ml cut-off. The ARTA HIVDR(ultralight) assay was reliable for plasma and DBS samples with a viral load >5000 RNA copies/ml, with amplification and sequencing success rates of 91% and 92% respectively for plasma, and 95% and 80% respectively for DBS. HIVDR profiles for plasma and DBS were 100% concordant with the reference assay. This study evaluated a previously described combination of two assays potentially useful in assessing HIV-1 virological failure and resistance, showing good concordance with reference assays. These assays are simple to perform and are affordable, viable options to detect virological failures in certain resource limited settings. The assays' compatibility with DBS sampling extends the access of HIV-1 virological monitoring to more remote settings.

The Accelerating Access Initiative: experience with a multinational workplace programme in Africa.

PROBLEM: A multinational company with operations in several African countries was committed to offer antiretroviral treatment to its employees and their dependants. APPROACH: The Accelerating Access Initiative (AAI), an initiative of six pharmaceutical companies and five United Nations' agencies, offered the possibility of obtaining brand antiretroviral drugs (ARVs) at 10% of the commercial price. PharmAccess, a foundation aimed at removing barriers to AIDS treatment in Africa, helped to establish an HIV policy and treatment guidelines, and a workplace programme was rolled out from September 2001. LOCAL SETTING: Private sector employers in Africa are keen to take more responsibility in HIV prevention and AIDS care. An important hurdle for African employers remains the price and availability of ARVs. RELEVANT CHANGES: The programme encountered various hurdles, among them the need for multiple contracts with multiple companies, complex importation procedures, taxes levied on ARVs, lack of support from pharmaceutical companies in importation and transportation, slow delivery of the drugs, lack of institutional memory in pharmaceutical companies and government policies excluding the company from access to ARVs under the AAI. LESSONS LEARNED: The launch of the AAI enabled this multinational company to offer access to ARVs to its employees and dependants. The private sector should have access to these discounted drugs under the AAI. A network of local AAI offices should be created to assist in logistics of drugs ordering, purchase and clearance. No taxes should be levied on ARVs.

[AIDS treatment in Africa: the risk of antiretroviral resistance]. / Aidsbehandeling in Afrika: het risico van antiretrovirale resistentie.

--In recent years, implementation of antiretroviral therapy in developing countries with a high prevalence of HIV-1 has been recognised as a public health priority. Consequently, the availability ofantiretroviral combination therapy for people with HIV is increasing rapidly in sub-Saharan Africa. --HIV treatment programmes are implemented according to the standardised, simplified public health guidelines developed by the World Health Organization (WHO). --However, the implementation of treatment programmes in Africa is hindered by several factors, including the lack of adequate immunological and virological laboratory monitoring, insufficient support for adherence to therapy, vulnerable health care systems and the use of suboptimal drug combinations. --These suboptimal treatment conditions increase the risk that resistant virus strains will emerge that are less susceptible to standard first-line combination therapy, thus threatening the long-term success of the treatment programmes. --The WHO has initiated HIVResNet, an international expert advisory board that has developed a global strategy for surveillance and prevention of antiretroviral drug resistance. --The Dutch initiative known as 'PharmAccess African studies to evaluate resistance' (PASER) is contributing to this strategy by creating a surveillance network in sub-Saharan Africa.

[Health care insurance for Africa]. / Een ziektekostenverzekering voor Afrika.

Long-term substantial development aid has not prevented many African countries from being caught in a vicious circle in health care: the demand for care is high, but the overburdened public supply of low quality care is not aligned with this demand. The majority of Africans therefore pay for health care in cash, an expensive and least solidarity-based option. This article describes an innovative approach whereby supply and demand of health care can be better aligned, health care can be seen as a value chain and health insurance serves as the overarching mechanism. Providing premium subsidies for patients who seek health care through private, collective African health insurance schemes stimulates the demand side. The supply of care improves by investing in medical knowledge, administrative systems and health care infrastructure. This initiative comes from the Health Insurance Fund, a unique collaboration of public and private sectors. In 2006 the Fund received Euro 100 million from the Dutch Ministry of Foreign Affairs to implement insurance programmes in Africa. PharmAccess Foundation is the Fund's implementing partner and presents its first experiences in Africa.

Database-supported teleconferencing: an additional clinical mentoring tool to assist a multinational company HIV/AIDS treatment program in Africa.

BACKGROUND: The lack of human resources for health is presently recognized as a major factor limiting scale-up of antiretroviral treatment (ART) programs in resourcelimited settings. The mobilization of public and private partners, the decentralization of care, and the training of non-HIV specialist nurses and general practitioners could help increase the number of HIV-infected patients receiving ART. In addition to other forms of training, scheduled teleconferences (TCs) have been organized to support a comprehensive HIV treatment program delivered by a private company's health team. OBJECTIVE: To describe the role of the TC as an additional tool in mentoring a company's health care workers (HCWs). METHOD: For this study, all TC reports were retrospectively reviewed and the questions classified by topic. Participating Heineken physicians evaluated the technical quality and scientific relevance of the TCs through an anonymous survey. RESULTS: From October 2001 to December 2003, 10 HCWs working in 14 operating companies in 5 African countries raised 268 problems during 45 TCs. A total of 79 questions (29%) were asked about antiretroviral (ARV) therapy, 53 (20%) about the diagnosis and treatment of opportunistic infection, 43 (16%) about ARV toxicity, 40 (15%) about care organization and policy, 32 (12%) about laboratory or drug supply, and 21 (8%) about biological parameters. The mean TC attendance rate was 70%. The level of satisfaction among local company physicians was 65% for logistics, 89% for scientific relevance, 84% for applicability of advice, and 85% overall. The most common complaints concerned the poor quality of the telephone connection and language problems for francophone participants. CONCLUSION: Database-supported teleconferencing could be an additional tool to mentor company HCWs in their routine care of HIV-infected workers and family members. The role and costeffectiveness of telemedicine in improving health outcomes should be further studied.

Determinants of CD4 counts among HIV-negative Ethiopians: role of body mass index, gender, cigarette smoking, khat (Catha Edulis) chewing, and possibly altitude?

To study the determinants of CD4% and CD4 counts among HIV-negative Ethiopians, and to identify factors susceptible to explain the low CD4 counts observed among Ethiopian subjects. Cohort studies among factory workers in Akaki and Wonji, Ethiopia. Clinical and laboratory examinations, including determination of HIV serological status and T-cell subsets, were performed during follow-up visits every six months. In addition, micronutrients (retinol, carotenoids, tocopherol, transferrin receptor, and selenium) plasma concentrations were determined in a subset of 38 HIV-positive and 121 HIV-negative participants. HIV-negative participants with at least one CD4 count measurement were 157 females in Akaki, 203 males in Akaki, and 712 males in Wonji. CD4 counts were independently and positively associated with body mass index (through an increase in lymphocyte counts), female gender (through an increase in CD4%), cigarette smoking (through an increase in CD4%), khat chewing (through an increase in both lymphocyte counts and CD4%), and Akaki study site (through a large increase in lymphocyte counts compensating a decrease in CD4%). Intestinal parasitic infections were not associated with CD4% or CD4 counts. Retinol, carotenoids, and alpha-tocopherol plasma concentrations decreased with HIV infection and advancing immunosuppression, but were not associated with CD4 counts among HIV-negative subjects. Low body mass index among Ethiopians may have contributed to their overall low CD4 counts. Other factors remain to be elucidated.

Role of incidental and/or cured intestinal parasitic infections on profile of CD4+ and CD8+ T cell subsets and activation status in HIV-1 infected and uninfected adult Ethiopians.

Intestinal parasitic infections have been suggested to cause persistent immune activation leading to an unbalanced immune state. Such a state has been proposed to be a major factor in the pathogenesis of AIDS in an African context. The present study investigated the effect of incidental parasitic infection and treatment on the profile of T cell differentiation and activation markers on CD4+ and CD8+ T cells from HIV-1 infected and uninfected adult Ethiopians. Cryopreserved PBMCs from 64 subjects (41 HIV-negative and 23 HIV-positive) with follow-up visits at 6-monthly intervals were used to compare the effect of incidental intestinal parasites and their treatment upon T cell subset profiles and activation status. The samples were stained with antibodies to various T cell differentiation and activation markers allowing naive, memory, effector, memory/effector, activated and resting CD4+ and CD8+ T cell subsets to be quantified by triple-colour FACScan. Incidental intestinal parasitic infections resulted in a significant increase in memory CD4+ T cell numbers both in HIV-negative and HIV-positive subjects (P < 0.05). There was also a significant increase in the percentage of CD8+ HLA-DR+ T cells (P < 0.05) in HIV-positive subjects co-infected with parasites. In HIV-negative subjects, a significant decline in activated cells and a significant increase in resting CD8+ T cells (P < 0.05) was observed after treatment for parasites. These data suggest that intestinal parasitic infections could result in the alteration of T cell subset counts and also in the up-regulation of T cell activation markers in peripheral blood. Treatment of parasitic infections showed a tendency to reduce the activation suggesting that, together with other community based intervention strategies, such treatment could be used to down-regulate immune activation and hence protect the host from being easily attacked by HIV.

Distribution of lymphocyte subsets in healthy human immunodeficiency virus-negative adult Ethiopians from two geographic locales.

Immunological values for 562 factory workers from Wonji, Ethiopia, a sugar estate 114 km southeast of the capital city, Addis Ababa, Ethiopia, were compared to values for 218 subjects from Akaki, Ethiopia, a suburb of Addis Ababa, for whom partial data were previously published. The following markers were measured: lymphocytes, T cells, B cells, NK cells, CD4(+) T cells, and CD8(+) T cells. A more in depth comparison was also made between Akaki and Wonji subjects. For this purpose, various differentiation and activation marker (CD45RA, CD27, HLA-DR, and CD38) expressions on CD4(+) and CD8(+) T cells were studied in 60 male, human immunodeficiency virus-negative subjects (30 from each site). Data were also compared with Dutch blood donor control values. The results confirmed that Ethiopians have significantly decreased CD4(+) T-cell counts and highly activated immune status, independent of the geographic locale studied. They also showed that male subjects from Akaki have significantly higher CD8(+) T-cell counts, resulting in a proportional increase in each of the CD8(+) T-cell compartments studied: naïve (CD45RA(+)CD27(+)), memory (CD45RA(-)CD27(+)), cytotoxic effector (CD45RA(+)CD27(-)), memory/effector (CD45RA(-)CD27(-)), activated (HLA-DR(+)CD38(+)), and resting (HLA-DR(-)CD38(-)). No expansion of a specific functional subset was observed. Endemic infection or higher immune activation is thus not a likely cause of the higher CD8 counts in the Akaki subjects. The data confirm and extend earlier observations and suggest that, although most lymphocyte subsets are comparable between the two geographical locales, there are also differences. Thus, care should be taken in extrapolating immunological reference values from one population group to another.

Timing of the HIV-1 subtype C epidemic in Ethiopia based on early virus strains and subsequent virus diversification.

OBJECTIVE: To trace the introduction of HIV-1 subtype C into Ethiopia based on virus diversification during the epidemic. DESIGN: A set of 474 serum samples obtained in Ethiopia in 1982-1985 was tested for HIV-1. HIV-1 env gp120 V3 and gag or pol regions were sequenced and analysed together with sequences from later stages of the epidemic. RESULTS: None of 98 samples from 1982-1983, one of 193 samples from 1984, and one of 183 samples from 1985 were HIV-1 positive. Phylogenetic analysis of virus sequences from positive samples revealed that they belong to the Ethiopian C, and not the C', cluster. Analysis of 81 Ethiopian C V3 sequences from 1984-1997 revealed that the consensus sequence of the Ethiopian epidemic has been stable over time. Both the 1984 and 1985 V3 sequences, in contrast with three out of 27 (11%) of the 1988 and none out of 51 of the 1992-1997 sequences, had no synonymous substitutions compared to the reconstructed common ancestor of the Ethiopian C viruses. A highly significant correlation between sampling years of the V3 sequences and their synonymous distances to the common ancestor was demonstrated. CONCLUSIONS: The increasing genetic heterogeneity together with stable consensus sequence of the Ethiopian HIV-1 C population demonstrates that evolution of the virus population is characterized by an unbiased expansion around a stationary consensus. Based on the rate of synonymous diversification of HIV-1 strains within the Ethiopian population, we were able to estimate 1983 (95% confidence interval, 1980-1984) as the year of HIV-1 C introduction into Ethiopia.

[Results from the 'Ethiopia-Netherlands AIDS Research Project'; 1995-2000]. / Resultaten van het 'Ethiopia-Netherlands aids research project'; 1995-2000.

Since 1995 the 'Ethiopia-Netherlands aids research project' (ENARP) has been up and running in Addis Ababa, Ethiopia. Several surveys point towards an HIV seroprevalence of approximately 15% amongst adult Ethiopians in the capital city. Prospective cohort studies initiated since early 1997 indicate that healthy, HIV negative Ethiopians have lower CD4+ T-cell counts compared to the Dutch population and in addition they have chronically activated immune systems, possibly as a result of the highly prevalent intestinal parasitic infections as well as other infections. HIV positive Ethiopians are mainly infected with HIV-1 subtype C, which can be subdivided in 2 subtypes, both of which entered Ethiopia in the early 1980's. There are considerable differences between Ethiopians and Dutch in terms of biomedical parameters relevant for HIV infection progression; these justify further efforts in future scientific research. The emphasis for this should be on robust and applicable laboratory methods, research in the field of HIV vaccine trials and information transfer to the various partners combating HIV infection/aids in Ethiopia.

[Ethiopia-Netherlands AIDS research project]. / 'Ethiopia-Netherlands AIDS research project'.

The 'Ethiopia-Netherlands AIDS Research Project' (ENARP), started in 1994, is a long-term collaboration between AIDS researchers in Amsterdam and the Ethiopian Health and Nutrition Research Institute in Addis Ababa. The ENARP's primary objectives include conducting studies on HIV and AIDS in Ethiopia, especially by means of some large-scale prospective cohort studies, training Ethiopian scientists in PhD programmes in epidemiology, immunology and virology and establishing a reference laboratory for HIV and AIDS in Ethiopia and neighbouring countries. External funding for ENARP amounts to 32 million Dutch guilders for two periods of four years and is being provided by the Dutch Government. ENARP is the largest third world biomedical project supported by the Dutch Government. In 2000 two Ethiopian students obtained their doctorates from the University of Amsterdam. Five new PhD students commenced their training in 1999. ENARP hopes to set up HIV-1 vaccine phase I and phase II trials in the near future.

Timing of the introduction into Ethiopia of subcluster C' of HIV type 1 subtype C.

Viruses circulating in Ethiopia during the 1990s cluster with main subtype C, but a significant subcluster, C', was noted in multiple analyses. This subcluster of subtype C(C') was in a fifty-fifty equilibrium with the main subtype C (Abebe et al., AIDS Res Hum Retroviruses 2000;16:1909-1914). To analyze genetic diversification within the subcluster of HIV-1 subtype C designated C' in the course of the epidemic in Ethiopia, we analyzed 165 env gp120 V3 sequences obtained between 1988 and 1999. We observed a highly significant positive correlation between sampling years of individual sequences and their synonymous distances to the reconstructed common ancestor of the HIV-1 subtype C' subcluster. The extrapolation of the regression line of synonymous distances back to the date when no synonymous heterogeneity was present among the Ethiopian HIV-1 C' population allowed us to estimate 1982 (95% CI, 1980-1983) as the year of the onset of HIV-1 C' genetic diversification and expansion in Ethiopia. These results are in agreement with retrospective epidemiological and serological data, which demonstrated the absence of an HIV-1 epidemic in the Ethiopian population before the 1980s.

No difference in in vitro susceptibility to HIV type 1 between high-risk HIV-negative Ethiopian commercial sex workers and low-risk control subjects.

Host factors such as increased beta-chemokine production, HIV-1 coreceptor expression level, and HIV-1 coreceptor polymorphism have been thought to influence susceptibility to HIV-1 infection. To determine the protective role of these factors in Ethiopians who remained HIV-1 uninfected, despite multiple high-risk sexual exposures, we studied 21 Ethiopian women who had been employed as commercial sex workers (CSWs) for five or more years. The HIV-1-resistant CSWs were compared with low-risk age-matched female controls who had a comparable CD4+ cell percentage and mean fluorescence intensity (MFI). Genetic polymorphism in the CCR5, CCR2b, or SDF-1 genes appeared not to be associated with resistance in the Ethiopian CSWs. Expression levels of CCR5 and CXCR4 on naive, memory, and total CD4+ T cells tended to be higher in the resistant CSWs, while the production of beta-chemokines RANTES, MIP-1alpha, and MIP-1beta by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) was lower compared with low-risk HIV-1 negative controls. In vitro susceptibility of PHA-stimulated PBMCs to primary, CCR5-restricted, Ethiopian HIV-1 isolates was comparable between resistant CSWs and low-risk controls. In vitro susceptibility was positively correlated to CD4+ cell mean fluorescence intensity and negatively correlated to CCR5 expression levels, suggesting that infection of PBMCs was primarily dependent on expression levels of CD4 and that CCR5 expression, above a certain threshold, did not further increase susceptibility. Our results show that coreceptor polymorphism, coreceptor expression levels, beta-chemokine production, and cellular resistance to in vitro HIV-1 infection are not associated with protection in high-risk HIV-1-negative Ethiopian CSWs.

Plasma levels of viro-immunological markers in HIV-infected and non-infected Ethiopians: correlation with cell surface activation markers.

Cross-sectional studies were conducted to measure soluble viral and immunological markers in plasma in order to determine the prognostic value of these markers for HIV disease progression in Ethiopians and to see their association with cell surface markers in HIV-1-infected and noninfected Ethiopians. Whole blood samples were collected from 52 HIV-1-negative Ethiopians, 32 HIV-1-positive Ethiopians with absolute CD4(+) T-cell count >200/microl whole blood and no AIDS defining conditions, and 39 HIV-positive Ethiopians with CD4(+) T-cell count <200/microl and/or AIDS defining conditions. Plasma levels of b(2)-microglobulin (b(2)m), soluble CD27 (sCD27), soluble tumor necrosis factor alpha receptor type II (sTNFR-II), IgG, IgA, IGE, and IL12 were elevated in HIV-1-infected individuals. The plasma levels of sTNFR-II, sCD27, b(2)m, IL12, and IgG were inversely correlated with numbers of CD4(+) T-cells, the proportion of naïve (CD45RA(+)CD27(+)) CD8(+) T-cells, and the proportion of CD8(+) T-cells expressing CD28 (CD8(+)CD28(+)) were positively correlated with the proportions of activated (HLA-DR(+)CD38(+)) CD4(+) T-cells, as well as activated (HLA-DR(+)CD38(+)) CD8(+) T-cells. A strong positive correlation was also observed when soluble immune markers were compared to each other. Multivariate regression analyses of soluble markers with numbers of CD4(+) T-cells showed that sCD27 is the best independent marker for CD4(+) T-cell decline in the HIV-1-infected Ethiopians. Our results indicate that measurement of soluble immune markers, which is relatively easy to perform, could be a good alternative to the quantification of T-cell subsets for monitoring HIV-1 disease progression in places where there is no facility for flow cytometric measurements.

Identification of a genetic subcluster of HIV type 1 subtype C (C') widespread in Ethiopia.

Others and we have previously shown that subtype C is the predominant HIV-1 subtype and the major cause of AIDS in Ethiopia. The present study shows that subtype C in Ethiopia has a genetic subcluster, designated C', has not increased in frequency, or spread geographically, over the period 1988 (%C' = 23/53) to 1996-1997 (%C' = 26/50). There is no association of the HIV-1 subtype C or subcluster C' with geographic location, time of sample collection, or risk group in Ethiopia. Of 105 randomly collected samples representing 7 different towns in Ethiopia, all but 2 (1 subtype A from Addis Ababa, 1997 and 1 subtype D from Dessie, 1996) belong to subtype C.

Increased cell division but not thymic dysfunction rapidly affects the T-cell receptor excision circle content of the naive T cell population in HIV-1 infection.

Recent thymic emigrants can be identified by T cell receptor excision circles (TRECs) formed during T-cell receptor rearrangement. Decreasing numbers of TRECs have been observed with aging and in human immunodeficiency virus (HIV)-1 infected individuals, suggesting thymic impairment. Here, we show that in healthy individuals, declining thymic output will affect the TREC content only when accompanied by naive T-cell division. The rapid decline in TRECs observed during HIV-1 infection and the increase following HAART are better explained not by thymic impairment, but by changes in peripheral T-cell division rates. Our data indicate that TREC content in healthy individuals is only indirectly related to thymic output, and in HIV-1 infection is mainly affected by immune activation.

Nonradioactive techniques for measurement of in vitro T-cell proliferation: alternatives to the [(3)H]thymidine incorporation assay.

T-cell proliferation is an important in vitro parameter of in vivo immune function and has been used as a prognostic marker of human immunodeficiency virus type 1 (HIV-1) disease progression. The proliferative capacity of T cells in response to various stimuli is commonly determined by a radioactive assay based on incorporation of [(3)H]thymidine ([(3)H]TdR) into newly generated DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, two alternative methods were tested and compared to the [(3)H]TdR assay as a "gold standard." As an alternative, T-cell proliferation was measured by flow cytometric assessment of CD38 expression on T cells and by an enzyme-linked immunosorbent assay (ELISA) based on bromo-2'-deoxyuridine (BrdU) incorporation. Peripheral blood mononuclear cells (PBMCs), either in whole blood or Ficoll-Isopaque separated, from a total of 26 HIV-1-positive and 18 HIV-1-negative Dutch individuals were stimulated with CD3 monoclonal antibody (MAb) alone, a combination of CD3 and CD28 MAbs, or phytohemagglutinin. BrdU incorporation after 3 days of stimulation with a combination of CD3 and CD28 MAbs correlated excellently with the [(3)H]TdR incorporation in both study groups (HIV-1 positives, r = 0.96; HIV-1 negatives, r = 0.83). A significant correlation of absolute numbers of T cells expressing CD38 with [(3)H]TdR incorporation, both in HIV-1-positive (r = 0.96) and HIV-1-negative (r = 0.84) individuals, was also observed under these conditions. The results of this study indicate that determination of both the number of CD38-positive T cells and BrdU incorporation can be used as alternative techniques to measure the in vitro T-cell proliferative capacity. The measurement of CD38 expression on T cells provides the additional possibility to further characterize the proliferating T-cell subsets for expression of other surface markers.