Development of a highly sensitive and specific intact proviral DNA assay for HIV-1 subtype B and C.

INTRODUCTION: HIV reservoir quantification is essential for evaluation of HIV curative strategies and may provide valuable insights about reservoir dynamics during antiretroviral therapy. The Intact Proviral DNA Assay (IPDA) provides the unique opportunity to quantify the intact and defective reservoir. The current IPDA is optimized for HIV-1 subtype B, the dominant subtype in resource-rich settings. However, subtype C is dominant in Sub-Saharan Africa, jointly accounting for around 60% of the pandemic. We developed an assay capable of quantifying intact and defective proviral HIV-1 DNA of subtype B and C. METHODS: Primer and probe sequences were strategically positioned at conserved regions in psi and env and adapted to subtype B&C. In silico analysis of 752 subtype B and 697 subtype C near-full length genome sequences (nFGS) was performed to predict  the specificity and sensitivity. Gblocks were used to determine the limit of blank (LoB), limit of detection (LoD), and different annealing temperatures were tested to address impact of sequence variability. RESULTS: The in silico analysis showed that the HIV-1 B&C IPDA correctly identified 100% of the intact subtype B, and 86% of the subtype C sequences. In contrast, the original IPDA identified 86% and 12% of these subtype B and C sequences as intact. Furthermore, the HIV-1 B&C IPDA correctly identified hypermutated (87% and 88%) and other defective sequences (73% and 66%) for subtype B and C with comparable specificity as the original IPDA for subtype B (59% and 63%). Subtype B cis-acting sequences were more frequently identified as intact by the HIV-1 B&C IPDA compared to the original IPDA (39% and 2%). The LoB for intact proviral DNA copies was 0, and the LoD for intact proviral DNA copies was 6 (> 95% certainty) at 60 °C. Quantification of 2-6 copies can be performed with > 80% certainty. Lowering the annealing temperature to 55 °C slightly lowered the specificity but prevented exclusion of samples with single mutations in the primer/probe region. CONCLUSIONS: We developed a robust and sensitive assay for the quantification of intact and defective HIV-1 subtype B and C proviral DNA, making this a suitable tool to monitor the impact of (large-scale) curative interventions.

Strongyloides stercoralis transmission by kidney transplantation in two recipients from a common donor.

Strongyloides stercoralis hyperinfection in an immunocompromised host has a high mortality rate but may initially present with nonspecific pulmonary and gastrointestinal symptoms. Donor-derived S. stercoralis by kidney transplantation is an uncommon diagnosis and difficult to prove. We report two renal allograft recipients on different immunosuppressive maintenance regimens that developed strongyloidiasis after transplantation from the same donor. Recipient 1 presented with a small bowel obstruction. Larvae were demonstrated on a duodenal biopsy and isolated from gastric, pulmonary, and stool samples. Serologic testing for S. stercoralis was negative at a referral laboratory but positive at the Centers for Disease Control. The patient's hospital course was complicated by a hyperinfection syndrome requiring subcutaneous ivermectin due to malabsorption. Recipient 1 survived but the allograft failed. Recipient 2 had larvae detected in stool samples after complaints of diarrhea and was treated. On retrospective testing for S. stercoralis, pretransplant serum collected from the donor and Recipient 1 was positive and negative, respectively. Donor-derived strongyloidiasis by renal transplantation is a preventable disease that may be affected by the immunosuppressive maintenance regimen. Subcutaneous ivermectin is an option in the setting of malabsorption. Finally, routine screening for S. stercoralis infection in donors from endemic areas may prevent future complications.

Routine BK virus surveillance in renal transplantation--a single center's experience.

BACKGROUND: We started a universal screening of all our kidney transplant recipients for BK virus in 2005. This review of our experience includes patients with ≥6 months' posttransplantation follow-up. METHODS: We performed a retrospective chart evaluation of all kidney transplants from January 2005 to February 2010. Urine polymerase chain reaction (PCR) for BK virus was done on all patients starting from 4 weeks after transplantation. If negative, it was repeated monthly for the first 6 months and then every 3-4 months. If the test was positive, a urine and blood BK virus PCR done on the next visit was repeated every 2-4 weeks with a slow reduction in immunosuppression. RESULTS: From January 2005 to February 2010 we performed 173 kidney transplantations with 12 graft losses within the first 6 weeks which were excluded from the analysis. Induction immunosuppression consisted of anti-interleukin-2 receptor antibody (n = 102) or antithymoglobulin (ATG; n = 59). In 112 patients (70%), the urine BK virus PCR remained negative; 18 (11%) only the urine was positive and among an additional 31 (19%) BK virus PCR was positive in blood. There was no difference in incidence according to induction therapy. Delayed graft function was observed among 39 patients (24%); there was no difference in the incidence of BK virus with versus without DGF. The mean time to first detection was shorter with ATG induction (mean, 199 days; median, 90 days; range, 26-1198 days) compared with anti-IL-2 (mean, 321 days; median, 195 days; range, 23-1077 days). Urine-only positivity was first detected from 37 to 1198 days (mean, 366 days; median, 227 days) and blood positivity from 23 to 1069 days (mean, 216 days; median, 90 days). Among BK-positive patients, 26 (53%) were detected within the first 6 months and 38 (76%) within the first year. With reduction in immunosuppression, there was gradual reduction or elimination of positive PCR tests in all cases except one, which resulted in graft failure. CONCLUSIONS: Routine BK virus surveillance is effective; it tends to detect BK virus replication early, allowing reduction of immunosuppression, which results in good outcomes with renal preservation.

Ensembl Genomes: extending Ensembl across the taxonomic space.

Ensembl Genomes (http://www.ensemblgenomes.org) is a new portal offering integrated access to genome-scale data from non-vertebrate species of scientific interest, developed using the Ensembl genome annotation and visualisation platform. Ensembl Genomes consists of five sub-portals (for bacteria, protists, fungi, plants and invertebrate metazoa) designed to complement the availability of vertebrate genomes in Ensembl. Many of the databases supporting the portal have been built in close collaboration with the scientific community, which we consider as essential for maintaining the accuracy and usefulness of the resource. A common set of user interfaces (which include a graphical genome browser, FTP, BLAST search, a query optimised data warehouse, programmatic access, and a Perl API) is provided for all domains. Data types incorporated include annotation of (protein and non-protein coding) genes, cross references to external resources, and high throughput experimental data (e.g. data from large scale studies of gene expression and polymorphism visualised in their genomic context). Additionally, extensive comparative analysis has been performed, both within defined clades and across the wider taxonomy, and sequence alignments and gene trees resulting from this can be accessed through the site.