The immune response to sub-clinical mastitis is impaired in HIV-infected women.

BACKGROUND: Subclinical mastitis (SCM) is relatively common in lactating women and may be associated with HIV shedding in breast milk. The potential association between HIV infection and breast milk immunologic factors and immune response to SCM needs to be addressed. METHODS: In this cross-sectional study, SCM (Na/K ratio > 1) was tested in 165 mature breast milk samples collected from 40 HIV-infected women who didn't transmit HIV to their child by breastfeeding and 43 HIV-uninfected women enrolled in an interventional cohort in South-Africa (Vertical Transmission Study). The level of 33 immune markers related to Th1/Th2 related response, inflammation and bacterial exposure were compared in ART-naive HIV-infected versus HIV-uninfected women. The associations between HIV infection and SCM on the concentration of immune factors were tested separately by Wilcoxon rank-sum test and corrected for false discovery rate. To control for potential confounder effects and take into account the clustering of breast milk samples from a single woman, multivariate mixed linear models adjusted on child age at the time of sampling were performed for each immune factor. RESULTS: Subclinical mastitis was detected in 15 (37.5%) HIV-infected women and 10 (23.3%) HIV-uninfected women. In the absence of SCM, the breast milk levels of IP-10 and MIG were higher and IL1-RA lower in HIV-infected women than in HIV-uninfected women (respectively p < 0.001, p = 0.001, p = 0.045). In HIV-uninfected women, SCM was characterized by a robust immune response with higher concentrations of a broad panel of Th1 and inflammatory related immune markers than in samples without SCM. By contrast, in HIV-infected women a limited number of immune markers were increased and lower increases were observed in samples with SCM than without SCM. CONCLUSION: HIV infection in ART-naïve women was associated with elevated breast milk levels of IP-10 and MIG, which areTh1-related cytokines induced by IFN-γ. During SCM, a lower and narrower immune response was observed in HIV-infected than HIV-uninfected women, suggesting that HIV infection affects the capacity of the mammary gland to respond to SCM.

Pivotal role of HIV and EBV replication in the long-term persistence of monoclonal gammopathy in patients on antiretroviral therapy.

A high prevalence of monoclonal gammopathy (MG) has been observed in HIV-infected patients. We explored the conditions associated with long-term persistence of serum monoclonal protein (M protein) in HIV-infected patients on antiretroviral therapy (ART). Of 21 patients with MG, M protein disappeared in 12 patients (58%) over 5 years of ART. Higher level of serum γ-globulin and higher percentages of circulating plasmablasts and plasma cells were observed in patients with persistent MG compared with patients with transient MG. MG persistence was associated with the cumulative time of detectable plasma HIV RNA after ART initiation, detection of Epstein-Barr virus (EBV) DNA in plasma, and a high level of EBV DNA in B cells. Poor control of HIV replication and detectable EBV replication in plasma were both associated with long-term MG persistence in patients on ART. In the case of viral control, MG associated with HIV infection is usually transient.

Comparison of EBV DNA viral load in whole blood, plasma, B-cells and B-cell culture supernatant.

Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P < 0.0001), but no significant correlation existed between EBV DNA levels in whole blood and enriched B-cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios.

Generating open-source 3D phytoplankton models by integrating photogrammetry with scanning electron microscopy.

The community structure and ecological function of marine ecosystems are critically dependent on phytoplankton. However, our understanding of phytoplankton is limited due to the lack of detailed information on their morphology. To address this gap, we developed a framework that combines scanning electron microscopy (SEM) with photogrammetry to create realistic 3D (three-dimensional) models of phytoplankton. The workflow of this framework is demonstrated using two marine algal species, one dinoflagellate Prorocentrum micans and one diatom Halamphora sp. The resulting 3D models are made openly available and allow users to interact with phytoplankton and their complex structures virtually (digitally) and tangibly (3D printing). They also allow for surface area and biovolume calculations of phytoplankton, as well as the exploration of their light scattering properties, which are both important for ecosystem modeling. Additionally, by presenting these models to the public, it bridges the gap between scientific inquiry and education, promoting broader awareness on the importance of phytoplankton.

Elevated concentrations of milk ß2-microglobulin are associated with increased risk of breastfeeding transmission of HIV-1 (Vertical Transmission Study).

There is increasing evidence to support a relationship between human immunodeficiency virus (HIV-1) transmission through breastfeeding and milk host factors. We analyzed skim milk proteome to further determine the contribution of host factors to the risk of mother-to-child transmission of HIV-1. Quantitative mass spectrometry analysis was performed on nine case-control pairs of HIV+ transmitter/nontransmitter mothers, and specific biochemical assays on two selected proteins were assessed in an independent validation set of 127 samples. 33 identified proteins were differentially expressed between HIV+ transmitter and nontransmitter mothers. Among them, ß2-microglobulin was significantly higher in the maternal transmitter than in the nontransmitter groups (p value = 0.0007), and S100A9 was significantly higher in the early maternal transmitter cases (before 4 months of age) compared with the nontransmitters (p value = 0.004). ß2-Microglobulin correlated with milk and plasma HIV viral load and CD4+ cell count, whereas S100A9 correlated with the estimated timing of infection of the infant through breastfeeding. Finally, ß2-microglobulin concentration in milk could accurately predict the risk of HIV-1 postnatal transmission by breastfeeding (p value < 0.0001, log-rank test). In conclusion, milk ß2-microglobulin and S100A9 are host factors that are found to be associated with mother-to-child transmission of HIV-1.

Human papillomavirus seropositivity and subsequent risk of HIV acquisition in rural South African women.

OBJECTIVE: This study aimed to provide a population-based estimate of human papillomavirus (HPV) seropositivity for women in a rural African context and to evaluate the impact of HPV serostatus on subsequent acquisition of HIV outside a clinical setting. DESIGN: A random sample of women participating in a longitudinal, population-based HIV survey combined with a case-control study. METHODS: Blood samples of women participating in a single round of population-based HIV surveillance (N = 1049) in a rural South African population were used to measure vaccine-preventable HPV seropositivity (types 6, 11, 16, and 18) in the general population in 2010. Using results from the repeat HIV surveys, a case-control analysis was then performed comparing HPV sero-status in samples taken from HIV sero-converting women (prior to infection with HIV) against samples from HIV-uninfected, sexually-active controls matched 1:1 according to 5-year age band (377:377). Unconditional multivariable logistic regression with multiple imputations was used to control for sociodemographic and behavioral variables associated with HIV acquisition. RESULTS: Human papillomavirus seropositivity in the population-based sample of women was 20.8% (95% confidence interval [CI], 18.3-23.4), and HIV prevalence was 27.6% (95% CI, 24.9-30.4). In the case-control analysis, allowing for variables known to be associated with HIV incidence, HPV seropositivity was associated with nearly 2.5 times the odds of subsequent acquisition of HIV (adjusted odds ratio, 2.33 [95% CI, 1.61-3.39]; P < 0.001). CONCLUSIONS: These results suggest that HPV vaccination before or soon after sexual debut could lower HIV infection risk. Randomized trials that quantify the impact of HPV vaccination in girls on the risk of acquiring HIV are urgently required.

Cumulative exposure to cell-free HIV in breast milk, rather than feeding pattern per se, identifies postnatally infected infants.

BACKGROUND: We quantified the relationship between human immunodeficiency virus (HIV) RNA shedding in breast milk, cumulative RNA exposure, and postnatal transmission, relating timing of infection in the infant to estimated total volume of milk exposure. METHODS: Nested case-control study of 36 infants of HIV-infected mothers. Case patients were infants who acquired HIV infection through breastfeeding from age 6 through 28 weeks, and control subjects were uninfected infants matched on age at obtainment of a breast milk sample. Mothers and infants received peripartum single-dose nevirapine prophylaxis. Feeding data were collected daily; breast milk samples were collected and infant anthropometry was performed at 6 weeks and monthly thereafter. Volume of milk ingested was estimated using infant weight and feeding pattern. RESULTS: Before HIV acquisition in case patients, feeding pattern (exclusive breastfeeding; median duration, 65 vs 70 days; P = .6) and daily milk intake (mean volume, 638 vs 637 mL; P = .97) did not differ significantly between case patients and control subjects. Case mothers were more likely to shed virus (64% vs 9% always, 22% vs 20.5% intermittently, 14% vs 70.5% never shed; overall, P < .001). Case patients ingested ~15 times more HIV-1 RNA particles than did control subjects (196.5 vs 13 × 106 copies; P < .001). Allowing for maternal antenatal CD4 cell count and plasma HIV-1 load, child sex and duration of mixed breastfeeding, the association between HIV RNA exposure and infection remained statistically significant (P < .001). CONCLUSIONS: Postnatal acquisition of HIV-1 is more strongly associated with cumulative exposure to cell-free particles in breast milk than with feeding mode. Reducing breast milk viral load through antiretroviral therapy to mother or child can further decrease postnatal transmission in exclusively breastfed infants.

Tetra-kis(8-quinolinolato-κN,O)hafnium(IV) dimethyl-formamide solvate monohydrate.

In the title compound, [Hf(C(9)H(6)NO)]·C(3)H(7)NO·H(2)O, the hafnium(IV) atom is coordinated by four 8-quinolinolate (Ox) ligands, forming a slightly distorted square-anti-prismatic coordination polyhedron. The crystal packing is controlled by O-H⋯O and C-H⋯O hydrogen-bonding inter-actions and π-π inter-actions between quinoline ligands of neighbouring mol-ecules. The inter-planar distances vary between 3.150 (1) and 3.251 (2) Å, while centroid-centroid distances vary from 3.589 (1) to 4.1531 (1) Å.

Genotypic and phenotypic characterization of viral isolates from HIV-1 subtype C-infected children with slow and rapid disease progression.

The genotypes and biological phenotypes of HIV-1 isolates obtained from 40 perinatally infected children in South Africa were analyzed. This included 15 infants who had HIV-related symptoms, most of whom died within 2 years of birth (rapid progressors), and 25 children who survived between 4 and 9 years with varying signs of disease (slow progressors). Heteroduplex mobility assays and sequence analysis confirmed that within the env and gag regions, all isolates were HIV-1 subtype C. Viral isolates from 14 of the 15 rapid progressors used the CCR5 coreceptor, whereas 1 (02ZARP1) used both the CXCR4 and CCR5 coreceptors. Among the 25 slow progressors, 22 isolates used CCR5 only, 2 used CXCR4 only, and 1 used both CCR5 and CXCR4. Two of the slow-progressing children who harbored CXCR4-using viruses had AIDS. All four CXCR4-using viruses had genotypic changes in the V3 region previously shown to be associated with CXCR4 usage. This cross-sectional study shows that HIV-1 subtype C viruses from both rapid- and slow-progressing perinatally infected children used predominantly CCR5. Similar to adults, CXCR4 usage was uncommon among HIV-1 subtype C isolates from pediatric infections.

Cytomegalovirus, and possibly Epstein-Barr virus, shedding in breast milk is associated with HIV-1 transmission by breastfeeding.

OBJECTIVE: Postnatal HIV-1 mother-to-child transmission (MTCT) occurs in spite of antiretroviral therapy. Co-infections in breast milk with cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are associated with increased HIV-1 shedding in this compartment. We investigated CMV levels and EBV detection in breast milk as potential risk factors for MTCT of HIV-1 via breastfeeding. METHODS: Cell-free HIV-1 RNA, cell-associated HIV-1 DNA, CMV and EBV DNA were quantified in breast milk from 62 HIV-infected mothers and proven postnatal MTCT of HIV-1 via breastfeeding. Controls were 62 HIV-positive mothers with HIV-uninfected infants. RESULTS: Median (interquartile range) CMV DNA viral load was significantly higher in cases [88,044 (18,586-233,904)] than in controls [11,167 (3221-31,152)] copies/10 breast milk cells (P < 0.001). Breast milk CMV DNA level correlated positively with breast milk HIV-1 RNA level in cases and controls. EBV DNA was detectable in a higher proportion of breast milk samples of cases (37.1%) than controls (16.1%; P = 0.009). HIV-1 MTCT was strongly associated with HIV-1 RNA shedding in breast milk and plasma. In multivariable analysis, every 1 log10 increase in breast milk CMV DNA was associated with a significant 2.5-fold greater odds of MTCT of HIV-1, independent of breast milk and plasma HIV-1 levels; the nearly three-fold increased risk of HIV-1 MTCT with breast milk EBV DNA detection did not reach significance. CONCLUSION: We provide the first evidence of an independent association between CMV in breast milk, and postnatal MTCT of HIV-1. This association could fuel persistent shedding of HIV-1 in breast milk in women receiving antiretroviral therapy. EBV DNA detection in breast milk may also be associated with MTCT of HIV-1, but only marginally so.

Evidence of long-lived founder virus in mother-to-child HIV transmission.

Exposure of the infant's gut to cell-associated and cell-free HIV-1 trafficking in breast milk (BM) remains a primary cause of mother-to-child transmission (MTCT). The mammary gland represents a unique environment for HIV-1 replication and host-virus interplay. We aimed to explore the origin of the virus transmitted during breastfeeding, and the link with quasi-species found in acellular and cellular fractions of breast-milk (BM) and in maternal plasma. The C2-V5 region of the env gene was amplified, cloned and sequenced from the RNA and DNA of BM, the RNA from the mother's plasma (PLA) and the DNA from infant's dried blood spot (DBS) in 11 post-natal mother-infant pairs. Sequences were assembled in Geneious, aligned in ClustalX, manually edited in SeAL and phylogenetic reconstruction was undertaken in PhyML and MrBayes. We estimated the timing of transmission (ETT) and reconstructed the time for the most recent common ancestor (TMRCA) of the infant in BEAST. Transmission of single quasi-species was observed in 9 of 11 cases. Phylogenetic analysis illustrated a BM transmission event by cell-free virus in 4 cases, and by cell-associated virus in 2 cases but could not be identified in the remaining 5 cases. Molecular clock estimates, of the infant ETT and TMRCA, corresponded well with the timing of transmission estimated by sequential infant DNA PCR in 10 of 11 children. The TMRCA of BM variants were estimated to emerge during gestation in 8 cases. We hypothesize that in the remaining cases, the breast was seeded with a long-lived lineage latently infecting resting T-cells. Our analysis illustrated the role of DNA and RNA virus in MTCT. We postulate that DNA archived viruses stem from latently infected quiescent T-cells within breast tissue and MTCT can be expected to continue, albeit at low levels, should interventions not effectively target these cells.

Performance of two real-time PCR assays for hepatitis B virus DNA detection and quantitation.

In-house developed real-time PCR (qPCR) techniques could be useful conjunctives to the management of hepatitis B virus (HBV) infection in resource-limited settings with high prevalence. Two qPCR assays (qPCR1 and qPCR2), based on primers/probes targeting conserved regions of the X and S genes of HBV respectively, were evaluated using clinical samples of varying HBV genotypes, and compared to the commercial Roche Cobas AmpliPrep/Cobas TaqMan HBV Test v2.0. The lower detection limit (LDL) was established at 104 IU/ml for qPCR1, and 91 IU/ml for qPCR2. Good agreement and correlation were obtained between the Roche assay and both qPCR assays (r = 0.834 for qPCR1; and r = 0.870 for qPCR2). Differences in HBV DNA load of > 0.5 Log10 IU/ml between the Roche and the qPCR assays were found in 49/122 samples of qPCR1, and 35/122 samples of qPCR2. qPCR1 tended to underestimate HBV DNA quantity in samples with a low viral load and overestimate HBV DNA concentration in samples with a high viral load when compared to the Roche test. Both molecular tools that were developed, used on an open real-time PCR system, were reliable for HBV DNA detection and quantitation. The qPCR2 performed better than the qPCR1 and had the additional advantage of various HBV genotype detection and quantitation. This low cost quantitative HBV DNA PCR assay may be an alternative solution when implementing national programmes to diagnose, monitor and treat HBV infection in low- to middle-income countries where testing for HBV DNA is not available in governmental health programmes.

An affordable HIV-1 drug resistance monitoring method for resource limited settings.

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Population-based CD4 counts in a rural area in South Africa with high HIV prevalence and high antiretroviral treatment coverage.

BACKGROUND: Little is known about the variability of CD4 counts in the general population of sub-Saharan Africa countries affected by the HIV epidemic. We investigated factors associated with CD4 counts in a rural area in South Africa with high HIV prevalence and high antiretroviral treatment (ART) coverage. METHODS: CD4 counts, health status, body mass index (BMI), demographic characteristics and HIV status were assessed in 4990 adult resident participants of a demographic surveillance in rural KwaZulu-Natal in South Africa; antiretroviral treatment duration was obtained from a linked clinical database. Multivariable regression analysis, overall and stratified by HIV status, was performed with CD4 count levels as outcome. RESULTS: Median CD4 counts were significantly higher in women than in men overall (714 vs. 630 cells/µl, p<0.0001), both in HIV-uninfected (833 vs. 683 cells/µl, p<0.0001) and HIV-infected adults (384.5 vs. 333 cells/µl, p<0.0001). In multivariable regression analysis, women had 19.4% (95% confidence interval (CI) 16.1-22.9) higher CD4 counts than men, controlling for age, HIV status, urban/rural residence, household wealth, education, BMI, self-reported tuberculosis, high blood pressure, other chronic illnesses and sample processing delay. At ART initiation, HIV-infected adults had 21.7% (95% CI 14.6-28.2) lower CD4 counts than treatment-naive individuals; CD4 counts were estimated to increase by 9.2% (95% CI 6.2-12.4) per year of treatment. CONCLUSIONS: CD4 counts are primarily determined by sex in HIV-uninfected adults, and by sex, age and duration of antiretroviral treatment in HIV-infected adults. Lower CD4 counts at ART initiation in men could be a consequence of lower CD4 cell counts before HIV acquisition.

HIV-1 reservoirs in breast milk and challenges to elimination of breast-feeding transmission of HIV-1.

By compensating for the relative immaturity of the neonatal immune system, breast milk and breast-feeding prevent deaths in children. Nevertheless, transmission of HIV-1 through breast-feeding is responsible for more than half of new pediatric HIV infections. Recent studies of possible HIV-1 reservoirs in breast milk shed new light on features that influence HIV-1 transmission through breast-feeding. The particular characteristics of breast milk CD4(+) T cells that distinguish them from circulating blood lymphocytes (high frequency of cell activation and expression of memory and mucosal homing markers) facilitate the establishment of HIV-1 replication. Breast milk also contains a plethora of factors with anti-infectious, immunomodulatory, or anti-inflammatory properties that can regulate both viral replication and infant susceptibility. In addition, CD8(+) T lymphocytes, macrophages, and epithelial cells in breast milk can alter the dynamics of HIV-1 transmission. Even during efficient antiretroviral therapy, a residual stable, CD4(+) T cell-associated reservoir of HIV-1 is persistently present in breast milk, a likely source of infection. Only prophylactic treatment in infants--ideally with a long-acting drug, administered for the entire duration of breast-feeding--is likely to protect HIV-exposed babies against all forms of HIV transmission from breast milk, including cell-to-cell viral transfer.

Cell-free (RNA) and cell-associated (DNA) HIV-1 and postnatal transmission through breastfeeding.

INTRODUCTION: Transmission through breastfeeding remains important for mother-to-child transmission (MTCT) in resource-limited settings. We quantify the relationship between cell-free (RNA) and cell-associated (DNA) shedding of HIV-1 virus in breastmilk and the risk of postnatal HIV-1 transmission in the first 6 months postpartum. MATERIALS AND METHODS: Thirty-six HIV-positive mothers who transmitted HIV-1 by breastfeeding were matched to 36 non-transmitting HIV-1 infected mothers in a case-control study nested in a cohort of HIV-infected women. RNA and DNA were quantified in the same breastmilk sample taken at 6 weeks and 6 months. Cox regression analysis assessed the association between cell-free and cell-associated virus levels and risk of postnatal HIV-1 transmission. RESULTS: There were higher median levels of cell-free than cell-associated HIV-1 virus (per ml) in breastmilk at 6 weeks and 6 months. Multivariably, adjusting for antenatal CD4 count and maternal plasma viral load, at 6 weeks, each 10-fold increase in cell-free or cell-associated levels (per ml) was significantly associated with HIV-1 transmission but stronger for cell-associated than cell-free levels [2.47 (95% CI 1.33-4.59) vs. aHR 1.52 (95% CI, 1.17-1.96), respectively]. At 6 months, cell-free and cell-associated levels (per ml) in breastmilk remained significantly associated with HIV-1 transmission but was stronger for cell-free than cell-associated levels [aHR 2.53 (95% CI 1.64-3.92) vs. 1.73 (95% CI 0.94-3.19), respectively]. CONCLUSIONS: The findings suggest that cell-associated virus level (per ml) is more important for early postpartum HIV-1 transmission (at 6 weeks) than cell-free virus. As cell-associated virus levels have been consistently detected in breastmilk despite antiretroviral therapy, this highlights a potential challenge for resource-limited settings to achieve the UNAIDS goal for 2015 of eliminating vertical transmission. More studies would further knowledge on mechanisms of HIV-1 transmission and help develop more effective drugs during lactation.

Current challenges to viral load testing in the context of emerging genetic diversity of HIV-1.

INTRODUCTION: One of the major characteristics of HIV-1 is its extreme genetic diversity. A key factor in assessing the sensitivity of a molecular-based assay measuring HIV-1 RNA viral load (VL) in plasma is its ability to detect/quantify all (or most of) relevant HIV-1 genetic subtype/recombinant forms accurately. AREAS COVERED: This review provides an overview of the current commercially available quantitative real-time assays (the Abbott RealTime HIV-1, Roche TaqMan HIV-1 versions 1.0 and 2.0, BioMérieux Nuclisens EasyQ HIV-1, Siemens VERSANT HIV-1 RNA 1.0 kinetic PCR, and Biocentric Generic HIV Viral Load assays). For each assay, studies from 2005 to 2010 assessing the impact of HIV-1 genetic diversity on the reliability of HIV-1 RNA quantification are described. EXPERT OPINION: In light of HIV-1 genetic diversity, a general recommendation to favor one test over the other cannot categorically be made. Larger field evaluations of HIV-1 RNA assays should be conducted in areas where HIV-1 genetic diversity is the highest. The large-scale implementation of HIV-1 VL testing is urgently required in the developing world to change HIV infection from a likely death sentence into a manageable chronic infection, as done in Northern countries.

Dried blood spot HIV-1 RNA quantification using open real-time systems in South Africa and Burkina Faso.

There is an urgent need to assess the accuracy/feasibility of using dried blood spots (DBS) for monitoring of HIV-1 viral load in resource-limited settings. A total of 892 DBS from HIV-1-positive pregnant women and their neonates enrolled in the Kesho Bora prevention of mother-to-child transmission trial conducted in Durban (South Africa) and Bobo-Dioulasso (Burkina Faso) between May 2005 and July 2008 were tested for HIV-1 RNA. The combination Nuclisens extraction method (BioMérieux)/Generic HIV Viral Load assay (Biocentric) was performed using one DBS (in Durban) versus 2 DBS (in Bobo-Dioulasso) on 2 distinct open real-time polymerase chain reaction instruments. DBS HIV-1 RNA results were compared with plasma HIV-1 RNA and HIV serology results used as the gold standards. The limits of detection of assays on DBS were 3100 and 1550 copies per milliliter in Durban and Bobo-Dioulasso, respectively. DBS HIV-1 RNA values correlated significantly with plasma levels (n = 327; R = 0.7351) and were uniformly distributed according to duration of DBS storage at -20°C (median duration, 280 days). For early infant diagnosis, the sensitivity and specificity were 100% (95% confidence interval: 97.2 to 100.0 and 96.5 to 100.0, respectively). HIV-1 viral load kinetics in DNase-pretreated DBS were similar to those obtained in plasma specimens among 13 patients receiving antiretroviral treatment. HIV-1 RNA findings from serial infant DBS collected prospectively (n = 164) showed 100% concordance with HIV serology at 18 months of life. Our findings strongly advocate the implementation of DBS HIV-1 RNA testing in remote areas from low-income and middle-income countries.

Comparison of the Generic HIV Viral Load assay with the Amplicor HIV-1 monitor v1.5 and Nuclisens HIV-1 EasyQ v1.2 techniques for plasma HIV-1 RNA quantitation of non-B subtypes: the Kesho Bora preparatory study.

The implementation of cost effective HIV-1 RNA quantitation assays in resource-poor settings is of paramount importance for monitoring HV-1 infection. A study comparing the analytical performance of three HIV-1 RNA assays (Generic HIV Viral Load, Amplicor v1.5 and Nuclisens EasyQ v1.2) was performed on 160 plasma samples from 160 consecutive antiretroviral treatment naive HIV-1-infected pregnant women assessed for eligibility in the Kesho Bora trial aimed at prevention of mother-to-child transmission of HIV-1 in three African countries (Burkina Faso, Kenya and South Africa). Correlation and agreement of results of the three assays were assessed for plasma HIV-1 RNA quantitation in specimens harbouring mainly sub-subtype A1, subtype C, and circulating recombinant form (CRF) 02_AG and CRF06_cpx. Good degrees of correlation and agreement were observed between these HIV-1 RNA assays. However, nine (9/160, 5.6%) strains detectable with the Generic HIV Viral Load assay were not detected by either the Amplicor (n=7) or EasyQ (n=2) test. One strain (0.6%) was missed with the Generic HIV Viral Load assay. Further, concordantly positive plasma samples harbouring CRF02_AG and CRF06_cpx yielded significantly higher HIV-1 RNA concentrations when tested by Generic HIV Viral Load, as compared to Amplicor v1.5 (mean differences, +0.33 and +0.67 log(10) copies/ml; P=0.0004 and P=0.002, respectively). The Generic HIV Viral Load assay accurately quantified the majority of the non-B HIV-1 subtypes assessed in this study. Due to its low cost (approximately 10 US $/test), this assay performed with open real-time PCR instruments is now used routinely in the Kesho Bora trial and may be recommended in other African settings.

HIV incidence in rural South Africa: comparison of estimates from longitudinal surveillance and cross-sectional cBED assay testing.

BACKGROUND: The BED IgG-Capture Enzyme Immunoassay (cBED assay), a test of recent HIV infection, has been used to estimate HIV incidence in cross-sectional HIV surveys. However, there has been concern that the assay overestimates HIV incidence to an unknown extent because it falsely classifies some individuals with non-recent HIV infections as recently infected. We used data from a longitudinal HIV surveillance in rural South Africa to measure the fraction of people with non-recent HIV infection who are falsely classified as recently HIV-infected by the cBED assay (the long-term false-positive ratio (FPR)) and compared cBED assay-based HIV incidence estimates to longitudinally measured HIV incidence. METHODOLOGY/PRINCIPAL FINDINGS: We measured the long-term FPR in individuals with two positive HIV tests (in the HIV surveillance, 2003-2006) more than 306 days apart (sample size n = 1,065). We implemented four different formulae to calculate HIV incidence using cBED assay testing (n = 11,755) and obtained confidence intervals (CIs) by directly calculating the central 95(th) percentile of incidence values. We observed 4,869 individuals over 7,685 person-years for longitudinal HIV incidence estimation. The long-term FPR was 0.0169 (95% CI 0.0100-0.0266). Using this FPR, the cross-sectional cBED-based HIV incidence estimates (per 100 people per year) varied between 3.03 (95% CI 2.44-3.63) and 3.19 (95% CI 2.57-3.82), depending on the incidence formula. Using a long-term FPR of 0.0560 based on previous studies, HIV incidence estimates varied between 0.65 (95% CI 0.00-1.32) and 0.71 (95% CI 0.00-1.43). The longitudinally measured HIV incidence was 3.09 per 100 people per year (95% CI 2.69-3.52), after adjustment to the sex-age distribution of the sample used in cBED assay-based estimation. CONCLUSIONS/SIGNIFICANCE: In a rural community in South Africa with high HIV prevalence, the long-term FPR of the cBED assay is substantially lower than previous estimates. The cBED assay performs well in HIV incidence estimation if the locally measured long-term FPR is used, but significantly underestimates incidence when a FPR estimate based on previous studies in other settings is used.