Enhancing HIV Treatment Access and Outcomes Amongst HIV Infected Children and Adolescents in Resource Limited Settings.

Introduction Increasing access to HIV-related care and treatment for children aged 0-18 years in resource-limited settings is an urgent global priority. In 2011-2012 the percentage increase in children accessing antiretroviral therapy was approximately half that of adults (11 vs. 21 %). We propose a model for increasing access to, and retention in, paediatric HIV care and treatment in resource-limited settings. Methods Following a rapid appraisal of recent literature seven main challenges in paediatric HIV-related care and treatment were identified: (1) lack of regular, integrated, ongoing HIV-related diagnosis; (2) weak facility-based systems for tracking and retention in care; (3) interrupted availability of dried blood spot cards (expiration/stock outs); (4) poor quality control of rapid HIV testing; (5) supply-related gaps at health facility-laboratory interface; (6) poor uptake of HIV testing, possibly relating to a fatalistic belief about HIV infection; (7) community-associated reasons e.g. non-disclosure and weak systems for social support, resulting in poor retention in care. Results To increase sustained access to paediatric HIV-related care and treatment, regular updating of Policies, review of inter-sectoral Plans (at facility and community levels) and evaluation of Programme implementation and impact (at national, subnational, facility and community levels) are non-negotiable critical elements. Additionally we recommend the intensified implementation of seven main interventions: (1) update or refresher messaging for health care staff and simple messaging for key staff at early childhood development centres and schools; (2) contact tracing, disclosure and retention monitoring; (3) paying particular attention to infant dried blood spot (DBS) stock control; (4) regular quality assurance of rapid HIV testing procedures; (5) workshops/meetings/dialogues between health facilities and laboratories to resolve transport-related gaps and to facilitate return of results to facilities; (6) community leader and health worker advocacy at creches, schools, religious centres to increase uptake of HIV testing and dispel fatalistic beliefs about HIV; (7) use of mobile communication technology (m-health) and peer/community supporters to maintain contact with patients. Discussion and Conclusion We propose that this package of facility, community and family-orientated interventions are needed to change the trajectory of the paediatric HIV epidemic and its associated patterns of morbidity and mortality, thus achieving the double dividend of improving HIV-free survival.

Cutting Edge: Unusual NK cell responses to HIV-1 peptides are associated with protection against maternal-infant transmission of HIV-1.

Most infants exposed to HIV-1 in utero and at delivery do not acquire infection. We show that mothers and infants who have CD3-negative cells that respond to HIV-1 peptides are substantially less likely to transmit and acquire infection, respectively. The CD3-negative cells, shown to be NK cells, respond with remarkable specificity and high magnitude to HIV-1 peptides from Env (envelope) and Reg (regulatory) protein regions, as measured by a whole blood intracellular cytokine assay only in the context of HIV-1 infection or exposure. These findings identify an important new measure of protective immunity to HIV-1 that highlights the importance of innate immunity in preventing the establishment of HIV-1 infection.

Natural killer cells that respond to human immunodeficiency virus type 1 (HIV­1) peptides are associated with control of HIV­1 infection.

Human immunodeficiency virus (HIV)-specific natural killer (CD3- cells), CD4, and CD8 T cellular responses were determined in 79 HIV­1-infected women in response to HIV­1 peptide pools (Gag, Pol, Nef, Reg, and Env) with use of a whole­blood intracellular cytokine staining assay that measures interferon-γ and/or interleukin-2. HIV­specific CD3- cell responses to any region (Env and Reg predominantly targeted) were associated with lower viral load (P = .031) and higher CD4 T cell count (P = .015). Env­specific CD3- cell responses were stronger in women who had both Gag CD4 and CD8 T cell responses and, in turn, was associated with lower viral load (P = .005). CD3- cell responders had significantly higher representation of CD4 T cell responses to Env and Reg (P = .012 and P = .015, respectively) and higher magnitudes of CD4 T cell responses (P = .017 and P = .037, respectively) than did nonresponders. Peptide­specific natural killer cells are associated with markers of less severe disease progression among HIV­1-infected women (lower viral load and higher CD4 T cell count) and with stronger HIV­specific T cell responses.

Evaluation of seven rapid HIV tests to detect HIV-exposure and seroreversion during infancy.

BACKGROUND: Failure to determine the HIV status of all pregnant women impedes progress in preventing and treating paediatric HIV because vertically exposed infants are not identified for prophylaxis, early HIV diagnosis and care. OBJECTIVES: To assess the performance of rapid HIV tests in comparison to a laboratory-based HIV ELISA test for determining HIV-exposure and excluding HIV infection during infancy. STUDY DESIGN: Seven rapid HIV tests were evaluated on 2266 stored samples from 116 HIV-exposed infants of known HIV status at four ages during infancy. The HIV ELISA for each sample was the standard against which rapid results were assessed to establish HIV-exposure. RESULTS: Rapid tests did not perform uniformly during infancy. For detecting HIV-exposure the sensitivity of most rapid tests to 3 months of age approached that of an HIV ELISA however only Determine maintained this sensitivity (99.7%) throughout infancy. For excluding HIV infection (i.e. for correctly identifying HIV-uninfected infants) the specificity of all rapid tests except Determine exceeded that of the HIV ELISA from 7 months of age. CONCLUSIONS: The use of rapid tests in infancy could improve identification and care of HIV-exposed infants. Further evaluation under field conditions is required before rapid tests can be incorporated into evidence-based diagnostic algorithms.

African infants' CCL3 gene copies influence perinatal HIV transmission in the absence of maternal nevirapine.

BACKGROUND: Individuals with more copies of CCL3L1 (CCR5 ligand) than their population median have been found to be less susceptible to HIV infection. We investigated whether maternal or infant CCL3L1 gene copy numbers are associated with perinatal HIV transmission when single-dose nevirapine is given for prevention. METHOD: A nested case-control study was undertaken combining data from four cohorts including 849 HIV-infected mothers and their infants followed prospectively in Johannesburg, South Africa. Access to antiretroviral drugs for the prevention of perinatal transmission differed across the cohorts. Maternal and infant CCL3L1 gene copy numbers per diploid genome (pdg) were determined by real-time polymerase chain reaction for 79 out of 83 transmitting pairs ( approximately 10% transmission rate) and 235 randomly selected non-transmitting pairs. RESULTS: Higher numbers of infant, but not maternal, CCL3L1 gene copies were associated with reduced HIV transmission (P = 0.004) overall, but the association was attenuated if mothers took single-dose nevirapine or if the maternal viral load was low. Maternal nevirapine was also associated with reduced spontaneously released CCL3 (P = 0.007) and phytohemagglutinin-stimulated CCL3 (P = 0.005) production in cord blood mononuclear cells from uninfected infants. CONCLUSION: We observed a strong association between higher infant CCL3L1 gene copies and reduced susceptibility to HIV in the absence of maternal nevirapine. We also observed a reduction in newborn CCL3 production with nevirapine exposure. Taken together, we hypothesize that nevirapine may have direct or indirect effects that partly modify the role of the CCR5 ligand CCL3 in HIV transmission, obscuring the relationship between this genetic marker and perinatal HIV transmission.

Polymerase chain reaction for diagnosis of human immunodeficiency virus infection in infancy in low resource settings.

BACKGROUND: Diagnosis of human immunodeficiency virus (HIV) is essential for accessing treatment. Current HIV diagnostic protocols for infants require adaptation and validation before they can be implemented in the developing world. The timing and type of HIV assays will be dictated by country-specific circumstances and experience from similar settings. The performance of an HIV-1 DNA polymerase chain reaction (PCR) test, and in particular a single test at 6 weeks of age, in diagnosing HIV subtype C infection acquired in utero or peripartum was assessed. METHODS: A retrospective review of 1825 Amplicor HIV-1 DNA PCR version 1.5 tests performed between 2000 and 2004 in 2 laboratories in Johannesburg, South Africa on 769 effectively non-breast-fed infants from 3 clinically well characterized cohorts was undertaken. The HIV status of each infant was used as the standard against which the HIV PCR results were compared. RESULTS: The overall sensitivity and specificity of the HIV PCR test were 99.3 and 99.5% respectively. A single test was 98.8% sensitive and 99.4% specific in the 627 infants tested at 6 weeks of age (58 HIV-infected and 569 HIV-uninfected). Repeat testing of all positive HIV PCR tests minimized false positive results. CONCLUSIONS: In resource-poor settings where HIV PCR testing in an environment of good laboratory practice is feasible, a single 6-week HIV DNA PCR test can increase identification of HIV-infected children substantially from current levels. Further operational research on how best to implement and monitor such a diagnostic protocol in specific local settings, especially in breast-fed infants, is necessary.

The performance of 5 rapid HIV tests using whole blood in infants and children: selecting a test to achieve the clinical objective.

BACKGROUND: Rapid tests have the potential to improve the identification of HIV-infected children in resource-limited settings. However, they remain underutilized because of a lack of data on their performance in the field using whole blood specimens. This study aimed to assess the accuracy of rapid tests for detecting HIV exposure, excluding HIV infection in HIV-exposed infants, and diagnosing HIV infection in children older than 18 months of age. METHODS: Five rapid tests (First Response, Pareekshak, Determine, Smart Check, and Insti) were performed using whole blood from children enrolled in a multisite, cross-sectional study in South Africa. HIV enzyme-linked immunosorbent assay and DNA polymerase chain reaction results defined HIV exposure and infection, respectively, and were the standards used for comparison. RESULTS: Of the 851 children enrolled, 186 (21.9%) were infected with HIV. For detecting HIV exposure, Determine demonstrated the highest sensitivity of 99.3% (95% confidence interval, 98.0-99.8) in early infancy, but sensitivity declined with age as seroreversion occurred. After 8 months of age, all tests except First Response excluded HIV infection in 82% to 100% of HIV-uninfected infants and, in conjunction with a clinical assessment, did not miss any HIV-infected children. Insti was the only test that detected all HIV-infected infants, albeit on the smallest number of samples. The performance of all rapid tests in children older than 18 months of age was similar to that in adults. CONCLUSIONS: Determine was the only rapid test that had a high enough sensitivity for detecting HIV exposure in early infancy, but it identified seroreversion later in life than the other tests. Insti warrants further investigation for both indications.

KIR-HLA and maternal-infant HIV-1 transmission in sub-Saharan Africa.

Numerous studies have suggested a role for natural killer (NK) cells in attenuation of HIV-1 disease progression via recognition by killer-cell immunoglobulin-like receptors (KIRs) of specific HLA class I molecules. The role of KIR and HLA class I has not been addressed in the context of maternal-infant HIV-1 transmission. KIR and HLA class I B and C genes from 224 HIV-1-infected mothers and 222 infants (72 infected and 150 uninfected) from South Africa were characterized. Although a number of significant associations were determined in both the total group and in the nevirapine (NVP) exposed group, the most significant findings involved KIR2DL2 and KIR2DL3 and HLA-C. KIR2DL2/KIR2DL3 was underrepresented in intrapartum (IP)-transmitting mothers compared to non-transmitting (NT) mothers (P = 0.008) and remained significant (P = 0.036) after correction for maternal viral load (MVL). Homozygosity for KIR2DL3 alone and in combination with HLA-C allotype heterozygosity (C1C2) was elevated in IP-transmitting mothers compared to NT mothers (P = 0.034 and P = 0.01 respectively), and after MVL correction (P = 0.033 and P = 0.027, respectively). In infants, KIR2DL3 in combination with its HLA-C1 ligand (C1) as well as homozygosity for KIR2DL3 with C1C2, were both found to be underrepresented in infected infants compared to exposed uninfected infants in the total group (P = 0.06 and P = 0.038, respectively) and in the sub-group of infants whose mothers received NVP (P = 0.007 and P = 0.03, respectively). These associations were stronger post MVL adjustment (total group: P = 0.02 and P = 0.009, respectively; NVP group: P = 0.004 and P = 0.02, respectively). Upon stratification according to low and high MVL, all significant associations fell within the low MVL group, suggesting that with low viral load, the effects of genotype can be more easily detected. In conclusion this study has identified a number of significant associations that suggest an important role for NK cells in maternal-to-infant HIV-1 transmission.

Natural killer cell responses to HIV-1 peptides are associated with more activating KIR genes and HLA-C genes of the C1 allotype.

BACKGROUND: What characterizes individuals whose natural killer (NK) cells are able to respond to HIV-1 peptides is not known. METHODS: The association between NK cell responses and KIR gene profiles and HLA-B and HLA-C alleles was investigated among 76 HIV-1-infected women in South Africa previously categorized as responders (n = 39) or nonresponders (n = 37) to HIV-1 peptide pools in a whole blood intracellular cytokine assay. Viral load was significantly lower and CD4 T-cell counts higher among responders compared with nonresponders (P = 0.023 and P = 0.030, respectively). RESULTS: Possession of one HLA-C1 allele associated with increased magnitude of NK cell responses to Env (P = 0.031) and significantly decreased viral load (P = 0.027) compared with its absence. There was a trend to increased possession of KIR2DL3+HLA-C1 in responders (71.8% vs 51.4%, P = 0.098) and decreased possession of KIR2DL3/2DL3+C2C2 (2.6% vs 16.2%, P = 0.053). A total of 64.1% of responders versus 32.4% of nonresponders had 13 or more KIR genes (P = 0.0067). Notably, the 13-KIR gene containing the Bx21 genotype (has eight inhibitory and three activating genes KIR2DS2, 2DS4, 2DS5) showed substantially higher representation among the responders (28.2% vs 2.6%, P = 0.001). A significantly higher proportion of responders had both KIR2DS2 and KIR2DS5 compared with either gene alone (72.4% vs 37%; P = 0.015). At least one HLA-C1 allele together with 13 or more KIR genes was associated with NK cell responsiveness (48.7% vs 13.5%; P = 0.001). CONCLUSION: NK cell responses to HIV-1 peptides are more likely to occur among individuals with a genotype supporting a more activating NK cell phenotype and who possess at least one HLA-C1 allele.

Oral fluid tests for screening of human immunodeficiency virus-exposed infants.

In high human immunodeficiency virus (HIV) prevalence settings, routine screening of infants attending immunization visits could improve identification of HIV-exposed infants to receive an early diagnosis and appropriate interventions. This first assessment of 2 rapid oral fluid HIV tests in early infancy demonstrates a sensitivity of <90% for detection of HIV-exposure resulting in failure to detect at least 1 in 10 HIV-infected infants.

Identification of human immunodeficiency virus-1 specific CD8+ and CD4+ T cell responses in perinatally-infected infants and their mothers.

BACKGROUND: There are few data describing the specificity, breadth and magnitude of T cell responses to HIV-1 in infancy. METHODS: HIV-specific CD8+ and CD4+ T cell responses to peptide pools representing Gag, Env, Pol, Nef and the regulatory regions (Reg) were simultaneously measured in 18 perinatally-infected infants and 14 of their chronically-infected mothers, using a whole blood interleukin-2 and interferon-gamma flow cytometric intracellular cytokine staining assay. RESULTS: HIV-specific CD8+ T cell responses were detected in all the infants aged 6 weeks and older (range 0.1-6.62%) and their mothers (range 0.1-4.89%). HIV-specific CD4+ T cell responses were detected in 33% of the infants (range 0.11-0.54%) and 73% of the mothers (range 0.16-0.84). CD8+ T cell responses in the mothers were almost equally spread between the variable (Nef, Reg and Env) and conserved proteins (Gag and Pol). Conversely, CD8+ T cell responses to the more variable proteins dominated in the perinatally-infected infants comprising 74% of the total response. Interestingly, mothers and infants shared responses to at least one peptide pool, whereas only one mother-infant pair shared a peptide pool targeted by CD4+ T cells. Two in-utero-infected infants tested at birth had CD8+ T cell responses, and one of them had an Env-specific CD4 T cell response. CONCLUSION: Our observations that HIV-specific CD8+ and CD4+ T cell responses can be detected in perinatally-infected infants from 6 weeks of age and that CD8+ T cell responses predominantly target the variable proteins have important implications for HIV vaccine design.

Evaluation of the ultrasensitive human immunodeficiency virus type 1 (HIV-1) p24 antigen assay performed on dried blood spots for diagnosis of HIV-1 infection in infants.

The diagnostic accuracy of the modified p24 antigen assay performed on pediatric dried blood spots was evaluated. Samples analyzed within 6 weeks of collection yielded no false-positive results (specificity, 100%) and few false-negative results (sensitivity, 96.5% to 98.3%). Laboratory services with limited resources should assess this option for routine infant diagnosis.

Host CCL3L1 gene copy number in relation to HIV-1-specific CD4+ and CD8+ T-cell responses and viral load in South African women.

HIV-specific T-cell responses play an important role in control of infection. Because CCL3 has immune modulatory and antiviral activities, we hypothesized that host CCL3 genotype (CCL3L1 gene duplications) would influence the development of effective HIV-specific immune responses. Copy numbers of CCL3L1 were determined for 71 HIV-infected women, and HIV-specific CD4 and CD8 T-cell responses to overlapping peptide pools spanning the HIV-1 subtype C genome were simultaneously measured by an interferon-gamma and interleukin-2 whole-blood flow cytometric assay. Host CCL3L1 copy number correlated negatively with viral load (r=-0.239, P=0.045), as did magnitudes of Gag CD4 (r=-0.362, P=0.002) and CD8 (r=-0.261, P=0.028) T-cell responses. Patients with a Gag CD4 response (P=0.002) or dominant Gag CD8 (P=0.006) response had significantly lower viral loads than those whose dominant response targeted another region of the genome, whereas a dominant Nef-specific CD8 T-cell response was associated with higher HIV viral load. CCL3L1 copy number greater than or equal to the population median of 5 was significantly associated with increased magnitude of CD4 Gag responses (P=0.017), and women who had CD4 and CD8 Gag-specific responses had significantly lower viral loads (P=0.004) and higher CCL3L1 copy number (P=0.015) than those women with only CD8 Gag-specific responses.

Evaluation of dried whole blood spots obtained by heel or finger stick as an alternative to venous blood for diagnosis of human immunodeficiency virus type 1 infection in vertically exposed infants in the routine diagnostic laboratory.

The diagnostic accuracy of the Roche Amplicor human immunodeficiency virus type 1 DNA PCR assay (version 1.5) on DNA extracted from pediatric heel prick dried blood spots using Roche MagNA Pure nucleic acid purification technology was evaluated. The methodologies transfer successfully from the labor-intensive research laboratory to the high-throughput automated routine laboratory.

Ultrasensitive human immunodeficiency virus type 1 p24 antigen assay modified for use on dried whole-blood spots as a reliable, affordable test for infant diagnosis.

The ultrasensitive human immunodeficiency virus (HIV) p24 antigen assay was modified for use on pediatric dried whole-blood spots on Whatman no. 1 filter paper. The modified assay was found to be reliable and accurate, making it an affordable tool for pediatric HIV diagnosis in developing countries.