Chronic and Early Antiretroviral Therapy Impact Human Immunodeficiency Virus (HIV) Serological Assay Sensitivity, Leading to More False-Negative Test Results in HIV Diagnosis.

This retrospective study evaluated the reactivity of 3 human immunodeficiency virus (HIV) confirmatory assays (INNO-LIA, Geenius, and MP) and 7 HIV rapid tests on samples from 2 different study populations in Belgium. For the early-treated cohort (83 HIV-1 adult patients treated within 3 months after infection), HIV-1 diagnosis was not obtained in at least 1 confirmatory assay in 12.0% (10/83) and in an HIV rapid test in 31.3% (26/83). Confirmation assay sensitivities ranged from 87.5% to 95.2%, whereas rapid test assay sensitivities ranged from 75.9% to 100%. The time to treatment initiation or the length of time on treatment did not have a statistical influence on the probability to obtain a false-negative test result. The fastest reversion was demonstrated after 4 months of treatment. Among the long-term treated cohort (390 HIV-1 patients with ≥ 9 years of undetectable viral load), false-negative test results were found in at least 1 HIV confirmatory assay for 2.1% (8/390) of the patients and in a HIV rapid test for 4.9% (19/390). Confirmation assay sensitivities ranged from 98.1% to 99.5%, whereas rapid test sensitivities ranged from 96.2% to 100%. Longer treatment increased nonreactivity of the HIV rapid tests (P = .033). Undetectable viral load decreases the sensitivities of HIV diagnostic tests, and further monitoring of the performance of serological assays is advised.

Unraveling new molecular players involved in the autoregulation of nodulation in Medicago truncatula.

The number of legume root nodules resulting from a symbiosis with rhizobia is tightly controlled by the plant. Certain members of the CLAVATA3/Embryo Surrounding Region (CLE) peptide family, specifically MtCLE12 and MtCLE13 in Medicago truncatula, act in the systemic autoregulation of nodulation (AON) pathway that negatively regulates the number of nodules. Little is known about the molecular pathways that operate downstream of the AON-related CLE peptides. Here, by means of a transcriptome analysis, we show that roots ectopically expressing MtCLE13 deregulate only a limited number of genes, including three down-regulated genes encoding lysin motif receptor-like kinases (LysM-RLKs), among which are the nodulation factor (NF) receptor NF Perception gene (NFP) and two up-regulated genes, MtTML1 and MtTML2, encoding Too Much Love (TML)-related Kelch-repeat containing F-box proteins. The observed deregulation was specific for the ectopic expression of nodulation-related MtCLE genes and depended on the Super Numeric Nodules (SUNN) AON RLK. Moreover, overexpression and silencing of these two MtTML genes demonstrated that they play a role in the negative regulation of nodule numbers. Hence, the identified MtTML genes are the functional counterpart of the Lotus japonicus TML gene shown to be central in the AON pathway. Additionally, we propose that the down-regulation of a subset of LysM-RLK-encoding genes, among which is NFP, might contribute to the restriction of further nodulation once the first nodules have been formed.

Decision tree for accurate infection timing in individuals newly diagnosed with HIV-1 infection.

BACKGROUND: There is today no gold standard method to accurately define the time passed since infection at HIV diagnosis. Infection timing and incidence measurement is however essential to better monitor the dynamics of local epidemics and the effect of prevention initiatives. METHODS: Three methods for infection timing were evaluated using 237 serial samples from documented seroconversions and 566 cross sectional samples from newly diagnosed patients: identification of antibodies against the HIV p31 protein in INNO-LIA, SediaTM BED CEIA and SediaTM LAg-Avidity EIA. A multi-assay decision tree for infection timing was developed. RESULTS: Clear differences in recency window between BED CEIA, LAg-Avidity EIA and p31 antibody presence were observed with a switch from recent to long term infection a median of 169.5, 108.0 and 64.5 days after collection of the pre-seroconversion sample respectively. BED showed high reliability for identification of long term infections while LAg-Avidity is highly accurate for identification of recent infections. Using BED as initial assay to identify the long term infections and LAg-Avidity as a confirmatory assay for those classified as recent infection by BED, explores the strengths of both while reduces the workload. The short recency window of p31 antibodies allows to discriminate very early from early infections based on this marker. BED recent infection results not confirmed by LAg-Avidity are considered to reflect a period more distant from the infection time. False recency predictions in this group can be minimized by elimination of patients with a CD4 count of less than 100 cells/mm3 or without no p31 antibodies. For 566 cross sectional sample the outcome of the decision tree confirmed the infection timing based on the results of all 3 markers but reduced the overall cost from 13.2 USD to 5.2 USD per sample. CONCLUSIONS: A step-wise multi assay decision tree allows accurate timing of the HIV infection at diagnosis at affordable effort and cost and can be an important new tool in studies analyzing the dynamics of local epidemics or the effects of prevention strategies.

Deep Sequencing of HIV-1 RNA and DNA in Newly Diagnosed Patients with Baseline Drug Resistance Showed No Indications for Hidden Resistance and Is Biased by Strong Interference of Hypermutation.

Deep sequencing of plasma RNA or proviral DNA may be an interesting alternative to population sequencing for the detection of baseline transmitted HIV-1 drug resistance. Using a Roche 454 GS Junior HIV-1 prototype kit, we performed deep sequencing of the HIV-1 protease and reverse transcriptase genes on paired plasma and buffy coat samples from newly diagnosed HIV-1-positive individuals. Selection was based on the outcome of population sequencing and included 12 patients with either a revertant amino acid at codon 215 of the reverse transcriptase or a singleton resistance mutation, 4 patients with multiple resistance mutations, and 4 patients with wild-type virus. Deep sequencing of RNA and DNA detected 6 and 43 mutations, respectively, that were not identified by population sequencing. A subsequently performed hypermutation analysis, however, revealed hypermutation in 61.19% of 3,188 DNA reads with a resistance mutation. The removal of hypermutated reads dropped the number of additional mutations in DNA from 43 to 17. No hypermutation evidence was found in the RNA reads. Five of the 6 additional RNA mutations and all additional DNA mutations, after full exclusion of hypermutation bias, were observed in the 3 individuals with multiple resistance mutations detected by population sequencing. Despite focused selection of patients with T215 revertants or singleton mutations, deep sequencing failed to identify the resistant T215Y/F or M184V or any other resistance mutation, indicating that in most of these cases there is no hidden resistance and that the virus detected at diagnosis by population sequencing is the original infecting variant.

Characteristics and spread to the native population of HIV-1 non-B subtypes in two European countries with high migration rate.

BACKGROUND: Non-B subtypes account for at least 50 % of HIV-1 infections diagnosed in Belgium and Luxembourg. They are considered to be acquired through heterosexual contacts and infect primarily individuals of foreign origin. Information on the extent to which non-B subtypes spread to the local population is incomplete. METHODS: Pol and env gene sequences were collected from 410 non-subtype B infections. Profound subtyping was performed using 5 subtyping tools and sequences of both pol and env. Demographic information, disease markers (viral load, CD4 count) and viral characteristics (co-receptor tropism) were compared between subtypes. Maximum likelihood phylogenetic trees were constructed and examined for clustering. RESULTS: The majority of non-B infections were diagnosed in patients originating from Africa (55.8 %), individuals born in Western Europe represented 30.5 %. Heterosexual transmission was the most frequently reported transmission route (79.9 %), MSM transmission accounted for 12.2 % and was significantly more frequently reported for Western Europeans (25.7 % versus 4.3 % for individuals originating from other regions; p < 0.001). Subtypes A and C and the circulating recombinant forms CRF01_AE and CRF02_AG were the most represented and were included in the comparative analysis. Native Western Europeans were underrepresented for subtype A (14.5 %) and overrepresented for CRF01_AE (38.6 %). The frequency of MSM transmission was the highest for CRF01_AE (18.2 %) and the lowest for subtype A (0 %). No differences in age, gender, viral load or CD4 count were observed. Prevalence of CXCR4-use differed between subtypes but largely depended on the tropism prediction algorithm applied. Indications for novel intersubtype recombinants were found in 20 patients (6.3 %). Phylogenetic analysis revealed only few and small clusters of local transmission but could document one cluster of CRF02_AG transmission among Belgian MSM. CONCLUSIONS: The extent to which non-B subtypes spread in the native Belgian-Luxembourg population is higher than expected, with 30.5 % of the non-B infections diagnosed in native Western Europeans. These infections resulted from hetero- as well as homosexual transmission. Introduction of non-B variants in the local high at risk population of MSM may lead to new sub-epidemics and/or increased genetic variability and is an evolution that needs to be closely monitored.

Markers associated with persisting low-level viraemia under antiretroviral therapy in HIV-1 infection.

OBJECTIVES: To identify host and viral characteristics associated with long-term persisting low-level viraemia (PLLV) under antiretroviral therapy (ART). PATIENTS AND METHODS: Seventy-one ART-treated patients with long-term PLLV (20-250 copies/mL) and 102 control patients with systematically undetectable viral load (VL) were selected retrospectively from ART-treated patients followed at the Ghent HIV reference centre. Host and viral characteristics were compared using univariate and multivariate analyses. RESULTS: Higher plasma VL at therapy initiation (OR 3.52; 95% CI 1.86-6.65; P < 0.001), therapy re-initiation after an interruption (OR 3.94; 95% CI 1.70-9.16; P = 0.001), male gender (OR 4.28; 95% CI 1.40-13.00; P = 0.011), a protease inhibitor-based regimen (OR 2.90; 95% CI 1.20-6.97; P = 0.017) and predicted CCR5 co-receptor tropism (OR 2.53; 95% CI 1.05-6.11; P = 0.039) were independently associated with PLLV. CONCLUSIONS: VL at ART initiation, therapy history, gender, ART regimen and co-receptor tropism were independently associated with PLLV. Gender, therapy history, co-receptor tropism and VL at ART initiation could be valuable predictive markers to identify patients at risk for PLLV.

Role of LONELY GUY genes in indeterminate nodulation on Medicago truncatula.

LONELY GUY (LOG) genes encode cytokinin riboside 5'-monophosphate phosphoribohydrolases and are directly involved in the activation of cytokinins. To assess whether LOG proteins affect the influence of cytokinin on nodulation, we studied two LOG genes of Medicago truncatula. Expression analysis showed that MtLOG1 and MtLOG2 were upregulated during nodulation in a CRE1-dependent manner. Expression was mainly localized in the dividing cells of the nodule primordium. In addition, RNA interference revealed that MtLOG1 is involved in nodule development and that the gene plays a negative role in lateral root development. Ectopic expression of MtLOG1 resulted in a change in cytokinin homeostasis, triggered cytokinin-inducible genes and produced roots with enlarged vascular tissues and shortened primary roots. In addition, those 35S:LOG1 roots also displayed fewer nodules than the wild-type. This inhibition in nodule formation was local, independent of the SUPER NUMERIC NODULES gene, but coincided with an upregulation of the MtCLE13 gene, encoding a CLAVATA3/EMBRYO SURROUNDING REGION peptide. In conclusion, we demonstrate that in M. truncatula LOG proteins might be implicated in nodule primordium development and lateral root formation.

Reverse transcription of plasma-derived HIV-1 RNA generates multiple artifacts through tRNA(Lys-3)-priming.

In vitro reverse transcription of full-length HIV-1 RNA extracted from the blood plasma of people living with HIV-1 remains challenging. Here, we describe the initiation of reverse transcription of plasma-derived viral RNA in the absence of an exogenous primer. Real-time PCR and Sanger sequencing were applied to identify the source and to monitor the outcome of this reaction. Results demonstrated that during purification of viral RNA from plasma, tRNA(Lys-3) is co-extracted in a complex with the viral RNA. In the presence of a reverse transcription enzyme, this tRNA(Lys-3) can induce reverse transcription, a reaction that is not confined to transcription of the 5' end of the viral RNA. A range of cDNA products is generated, most of them indicative for the occurrence of in vitro strand transfer events that involve translocation of cDNA from the 5' end to random positions on the viral RNA. This process results in the formation of cDNAs with large internal deletions. However, near full-length cDNA and cDNA with sequence patterns resembling multiple spliced HIV-1 RNA were also detected. Despite its potential to introduce significant bias in the interpretation of results across various applications, tRNA(Lys-3)-driven reverse transcription has been overlooked thus far. A more in-depth study of this tRNA-driven in vitro reaction may provide new insight into the complex process of in vivo HIV-1 replication.IMPORTANCEThe use of silica-based extraction methods for purifying HIV-1 RNA from viral particles is a common practice, but it involves co-extraction of human tRNA(Lys-3) due to the strong interactions between these molecules. This co-extraction becomes particularly significant when the extracted RNA is used in reverse transcription reactions, as the tRNA(Lys-3) then serves as a primer. Reverse transcription from tRNA(Lys-3) is not confined to cDNA synthesis of the 5' end of the viral RNA but extends across various regions of the viral genome through in vitro strand transfer events. Co-extraction of tRNA(Lys-3) has been overlooked thus far, despite its potential to introduce bias in downstream, reverse transcription-related applications. The observed events in the tRNA(Lys-3)-induced in vitro reverse transcription resemble in vivo replication processes. Therefore, these reactions may offer a unique model to better understand the replication dynamics of HIV-1.

Nodule numbers are governed by interaction between CLE peptides and cytokinin signaling.

CLE peptides are involved in the balance between cell division and differentiation throughout plant development, including nodulation. Previously, two CLE genes of Medicago truncatula, MtCLE12 and MtCLE13, had been identified whose expression correlated with nodule primordium formation and meristem establishment. Gain-of-function analysis indicated that both MtCLE12 and MtCLE13 interact with the SUPER NUMERIC NODULES (SUNN)-dependent auto-regulation of nodulation to control nodule numbers. Here we demonstrate that cytokinin, which is essential for nodule organ formation, regulates MtCLE13 expression. In addition, simultaneous knockdown of MtCLE12 and MtCLE13 resulted in an increase in nodule number, implying that both genes play a role in controlling nodule number. Additionally, a weak link may exist with the ethylene-dependent mechanism that locally controls nodule number.

Wuschel-related homeobox5 gene expression and interaction of CLE peptides with components of the systemic control add two pieces to the puzzle of autoregulation of nodulation.

In legumes, the symbiotic nodules are formed as a result of dedifferentiation and reactivation of cortical root cells. A shoot-acting receptor complex, similar to the Arabidopsis (Arabidopsis thaliana) CLAVATA1 (CLV1)/CLV2 receptor, regulating development of the shoot apical meristem, is involved in autoregulation of nodulation (AON), a mechanism that systemically controls nodule number. The targets of CLV1/CLV2 in the shoot apical meristem, the WUSCHEL (WUS)-RELATED HOMEOBOX (WOX) family transcription factors, have been proposed to be important regulators of apical meristem maintenance and to be expressed in apical meristem "organizers." Here, we focus on the role of the WOX5 transcription factor upon nodulation in Medicago truncatula and pea (Pisum sativum) that form indeterminate nodules. Analysis of temporal WOX5 expression during nodulation with quantitative reverse transcription-polymerase chain reaction and promoter-reporter fusion revealed that the WOX5 gene was expressed during nodule organogenesis, suggesting that WOX genes are common regulators of cell proliferation in different systems. Furthermore, in nodules of supernodulating mutants, defective in AON, WOX5 expression was higher than that in wild-type nodules. Hence, a conserved WUS/WOX-CLV regulatory system might control cell proliferation and differentiation not only in the root and shoot apical meristems but also in nodule meristems. In addition, the link between nodule-derived CLE peptides activating AON in different legumes and components of the AON system was investigated. We demonstrate that the identified AON component, NODULATION3 of pea, might act downstream from or beside the CLE peptides during AON.

Longitudinal patterns of inflammatory mediators after acute HIV infection correlate to intact and total reservoir.

Background: Despite the beneficial effects of antiretroviral therapy (ART) initiation during acute HIV infection (AHI), residual immune activation remains a hallmark of treated HIV infection. Methods: Plasma concentrations of 40 mediators were measured longitudinally in 39 early treated participants of a Belgian AHI cohort (HIV+) and in 21 HIV-negative controls (HIV-). We investigated the association of the inflammatory profile with clinical presentation, plasma viral load, immunological parameters, and in-depth characterization of the HIV reservoir. Results: While levels of most soluble mediators normalized with suppressive ART, we demonstrated the persistence of a pro-inflammatory signature in early treated HIV+ participants in comparison to HIV- controls. Examination of these mediators demonstrated a correlation with their levels during AHI, which seemed to be viremia-driven, and suggested involvement of an activated myeloid compartment, IFN-γ-signaling, and inflammasome-related pathways. Interestingly, some of these pro-inflammatory mediators correlated with a larger reservoir size and slower reservoir decay. In contrast, we also identified soluble mediators which were associated with favorable effects on immunovirological outcomes and reservoir, both during and after AHI. Conclusion: These data highlight how the persistent pro-inflammatory profile observed in early ART treated individuals is shaped during AHI and is intertwined with viral dynamics.

Never too many? How legumes control nodule numbers.

Restricted availability of nitrogen compounds in soils is often a major limiting factor for plant growth and productivity. Legumes circumvent this problem by establishing a symbiosis with soil-borne bacteria, called rhizobia that fix nitrogen for the plant. Nitrogen fixation and nutrient exchange take place in specialized root organs, the nodules, which are formed by a coordinated and controlled process that combines bacterial infection and organ formation. Because nodule formation and nitrogen fixation are energy-consuming processes, legumes develop the minimal number of nodules required to ensure optimal growth. To this end, several mechanisms have evolved that adapt nodule formation and nitrogen fixation to the plant's needs and environmental conditions, such as nitrate availability in the soil. In this review, we give an updated view on the mechanisms that control nodulation.

Prevalence and Evolution of Transmitted Human Immunodeficiency Virus Drug Resistance in Belgium Between 2013 and 2019.

Background: To assess the prevalence and evolution of transmitted drug resistance (TDR) in Belgium, a total of 3708 baseline human immunodeficiency virus (HIV)-1 polymerase sequences from patients diagnosed between 2013 and 2019 were analyzed. Methods: Protease and reverse-transcriptase HIV-1 sequences were collected from the 7 national Aids Reference Laboratories. Subtype determination and drug resistance scoring were performed using the Stanford HIV Drug Resistance Database. Trends over time were assessed using linear regression, and the maximum likelihood approach was used for phylogenetic analysis. Results: A total of 17.9% of the patients showed evidence of TDR resulting in at least low-level resistance to 1 drug (Stanford score ≥15). If only the high-level mutations (Stanford score ≥60) were considered, TDR prevalence dropped to 6.3%. The majority of observed resistance mutations impacted the sensitivity for nonnucleoside reverse-transcriptase inhibitors (NNRTIs) (11.4%), followed by nucleoside reverse-transcriptase inhibitors (6.2%) and protease inhibitors (2.4%). Multiclass resistance was observed in 2.4%. Clustered onward transmission was evidenced for 257 of 635 patients (40.5%), spread over 25 phylogenetic clusters. Conclusions: The TDR prevalence remained stable between 2013 and 2019 and is comparable to the prevalence in other Western European countries. The high frequency of NNRTI mutations requires special attention and follow-up. Phylogenetic analysis provided evidence for local clustered onward transmission of some frequently detected mutations.

Transcriptional and post-transcriptional regulation of a NAC1 transcription factor in Medicago truncatula roots.

• Legume roots develop two types of lateral organs, lateral roots and nodules. Nodules develop as a result of a symbiotic interaction with rhizobia and provide a niche for the bacteria to fix atmospheric nitrogen for the plant. • The Arabidopsis NAC1 transcription factor is involved in lateral root formation, and is regulated post-transcriptionally by miRNA164 and by SINAT5-dependent ubiquitination. We analyzed in Medicago truncatula the role of the closest NAC1 homolog in lateral root formation and in nodulation. • MtNAC1 shows a different expression pattern in response to auxin than its Arabidopsis homolog and no changes in lateral root number or nodulation were observed in plants affected in MtNAC1 expression. In addition, no interaction was found with SINA E3 ligases, suggesting that post-translational regulation of MtNAC1 does not occur in M. truncatula. Similar to what was found in Arabidopsis, a conserved miR164 target site was retrieved in MtNAC1, which reduced protein accumulation of a GFP-miR164 sensor. Furthermore, miR164 and MtNAC1 show an overlapping expression pattern in symbiotic nodules, and overexpression of this miRNA led to a reduction in nodule number. • This work suggests that regulatory pathways controlling a conserved transcription factor are complex and divergent between M. truncatula and Arabidopsis.

CLE peptides control Medicago truncatula nodulation locally and systemically.

The CLAVATA3/embryo-surrounding region (CLE) peptides control the fine balance between proliferation and differentiation in plant development. We studied the role of CLE peptides during indeterminate nodule development and identified 25 MtCLE peptide genes in the Medicago truncatula genome, of which two genes, MtCLE12 and MtCLE13, had nodulation-related expression patterns that were linked to proliferation and differentiation. MtCLE13 expression was up-regulated early in nodule development. A high-to-low expression gradient radiated from the inner toward the outer cortical cell layers in a region defining the incipient nodule. At later stages, MtCLE12 and MtCLE13 were expressed in differentiating nodules and in the apical part of mature, elongated nodules. Functional analysis revealed a putative role for MtCLE12 and MtCLE13 in autoregulation of nodulation, a mechanism that controls the number of nodules and involves systemic signals mediated by a leucine-rich repeat receptor-like kinase, SUNN, which is active in the shoot. When MtCLE12 and MtCLE13 were ectopically expressed in transgenic roots, nodulation was abolished at the level of the nodulation factor signal transduction, and this inhibition involved long-distance signaling. In addition, composite plants with roots ectopically expressing MtCLE12 or MtCLE13 had elongated petioles. This systemic effect was not observed in transgenic roots ectopically expressing MtCLE12 and MtCLE13 in a sunn-1 mutant background, although nodulation was still strongly reduced. These results suggest multiple roles for CLE signaling in nodulation.

Search for nodulation-related CLE genes in the genome of Glycine max.

CLE peptides are potentially involved in nodule organ development and in the autoregulation of nodulation (AON), a systemic process that restricts nodule number. A genome-wide survey of CLE peptide genes in the soybean glycine max genome resulted in the identification of 39 GmCLE genes, the majority of which have not yet been annotated. qRT-PCR analysis indicated two different nodulation-related CLE expression patterns, one linked with nodule primordium development and a new one linked with nodule maturation. Moreover, two GmCLE gene pairs, encoding group-III CLE peptides that were previously shown to be involved in AON, had a transient expression pattern during nodule development, were induced by the essential nodulation hormone cytokinin, and one pair was also slightly induced by the addition of nitrate. Hence, our data support the hypothesis that group-III CLE peptides produced in the nodules are involved in primordium homeostasis and intertwined in activating AON, but not in sustaining it.

High frequency of new recombinant forms in HIV-1 transmission networks demonstrated by full genome sequencing.

The HIV-1 epidemic in Belgium is primarily driven by MSM. In this patient population subtype B predominates but an increasing presence of non-B subtypes has been reported. We aimed to define to what extent the increasing subtype heterogeneity in a high at risk population induces the formation and spread of new recombinant forms. The study focused on transmission networks that reflect the local transmission to an important extent. One hundred and five HIV-1 transmission clusters were identified after phylogenetic analysis of 2849 HIV-1 pol sequences generated for the purpose of baseline drug resistance testing between 2013 and 2017. Of these 105 clusters, 62 extended in size during the last two years and were therefore considered as representing ongoing transmission. These 62 clusters included 774 patients in total. From each cluster between 1 and 3 representative patients were selected for near full-length viral genome sequencing. In total, the full genome sequence of 101 patients was generated. Indications for the presence of a new recombinant form were found for 10 clusters. These 10 clusters represented 105 patients or 13.6% of the patients covered by the study. The findings clearly show that new recombinant strains highly contribute to local transmission, even in an epidemic that is largely MSM and subtype B driven. This is an evolution that needs to be monitored as reshuffling of genome fragments through recombination may influence the transmissibility of the virus and the pathology of the infection. In addition, important changes in the sequence of the viral genome may challenge the performance of tests used for diagnosis, patient monitoring and drug resistance analysis.

Single genome sequencing of near full-length HIV-1 RNA using a limiting dilution approach.

Sequencing very long stretches of the HIV-1 genome can advance studies on virus evolution and in vivo recombination but remains technically challenging. We developed an efficient procedure to sequence near full-length HIV-1 RNA using a two-amplicon approach. The whole genome was successfully amplified for 107 (88%) of 121 plasma samples including samples from patients infected with HIV-1 subtype A1, B, C, D, F1, G, H, CRF01_AE and CRF02_AG. For the 17 samples with a viral load below 1000 c/ml and the 104 samples with a viral load above 1000 c/ml, the amplification efficiency was respectively 53% and 94%. The sensitivity of the method was further evaluated using limiting dilution of RNA extracted from a plasma pool containing an equimolar mixture of three HIV-1 subtypes (B, C and CRF02_AG) and diluted before and after cDNA generation. Both RNA and cDNA dilution showed comparable sensitivity and equal accuracy in reflecting the subtype distribution of the plasma pool. One single event of in vitro recombination was detected amongst the 41 sequences obtained after cDNA dilution but no indications for in vitro recombination were found after RNA dilution. In conclusion, a two-amplicon strategy and limiting dilution of viral RNA followed by reverse transcription, nested PCR and Sanger sequencing, allows near full genome sequencing of individual HIV-1 RNA molecules. This method will be a valuable tool in the study of virus evolution and recombination.

Exploring HIV-1 Transmission Dynamics by Combining Phylogenetic Analysis and Infection Timing.

HIV-1 pol sequences obtained through baseline drug resistance testing of patients newly diagnosed between 2013 and 2017 were analyzed for genetic similarity. For 927 patients the information on genetic similarity was combined with demographic data and with information on the recency of infection. Overall, 48.3% of the patients were genetically linked with 11.4% belonging to a pair and 36.9% involved in a cluster of ≥3 members. The percentage of early diagnosed (≤4 months after infection) was 28.6%. Patients of Belgian origin were more frequently involved in transmission clusters (49.7% compared to 15.3%) and diagnosed earlier (37.4% compared to 12.2%) than patients of Sub-Saharan African origin. Of the infections reported to be locally acquired, 69.5% were linked (14.1% paired and 55.4% in a cluster). Equal parts of early and late diagnosed individuals (59.9% and 52.4%, respectively) were involved in clusters. The identification of a genetically linked individual for the majority of locally infected patients suggests a high rate of diagnosis in this population. Diagnosis however is often delayed for >4 months after infection increasing the opportunities for onward transmission. Prevention of local infection should focus on earlier diagnosis and protection of the still uninfected members of sexual networks with human immunodeficiency virus (HIV)-infected members.

Quantification of total HIV-1 DNA in buffy coat cells, feasibility and potential added value for clinical follow-up of HIV-1 infected patients on ART.

BACKGROUND: Successfully treated HIV-1 infected patients have a sustained undetectable viral RNA load. In these cases the total HIV-1 DNA load may constitute a valuable tool to further follow the overall viral burden. The value of this marker outside of cure research has been rarely studied. OBJECTIVES: To develop a quantitative (q)PCR for total HIV-1 DNA quantification in buffy coat cells and to evaluate the value of this parameter in clinical follow-up. STUDY DESIGN: A qPCR using primers and a probe in the conserved HIV-1 LTR region was adapted for use on DNA extracted from buffy coat cells. Sensitivity, accuracy and reproducibility were evaluated using 8E5 cells and samples from naive and treatment experienced patients. The clinical value of DNA load analysis was assessed by testing 119 longitudinal samples from 9 patients before and after ART initiation and 249 cross sectional samples from therapy-experienced patients. RESULTS: Inter- and intra-assay coefficients of variability were 5.56 and 5.94 (%CV). HIV-1 DNA was detected in 249 of the 263 (94.7%) patients on ART for at least 5 months (median: 53 months; IQR: 28-84 months). The HIV-1 DNA load varied between 0.60 and 3.37 copies/106 blood cells and showed significant correlation with the pre-ART CD4+ T-cell count nadir and peak viral RNA load. ART initiation resulted in a slow and limited decline of the total HIV-1 DNA concentration. CONCLUSIONS: Quantification of total HIV-1 DNA from buffy coat cells is feasible, sensitive and reliable. Although determination of the on-therapy HIV-1 DNA load may be informative, regular testing has limited clinical value because of the very slow evolution.