HIV-1 RNA quantification in CRF02_AG HIV-1 infection: too easy to make mistakes.

The number of patients newly infected by HIV-1 non-B subtypes and circulating recombinant forms (CRFs) is increasing worldwide, including in the western countries. We report on a primary HIV-1 infection in a Caucasian patient. A routine quantitative assay (Nuclisens EasyQ HIV-1 2.0, BioMérieux SA) showed 6,700 HIV-1 RNA copies/ml. A combined antiretroviral therapy (cART) consistent with low baseline HIV-1 RNA was started. Few days later, the analysis performed with REGA HIV-1 Subtyping Tool - Version 3.0 attributed the HIV-1 sequence to the CRF02_AG recombinant form. Therefore, a second real-time PCR assay was performed, using the Versant HIV-1 RNA 1.0 Assay (kPCR) (Siemens HealthCare Diagnostics) which revealed a HIV-1 RNA of 230,000 copies/ml. Consequently, the ongoing cART was potentiated. This case suggests that the wide genetic variability of HIV-1 subtypes may affect the capability of the commonly used assays to detect and accurately quantify HIV-1 RNA in non-B subtypes and CRFs. In presence of CRFs different commercial HIV-1 RNA tests should be performed to find the most reliable for viral load quantification at the diagnosis, because it influences the choice of cART, and during the follow-up. Indeed, international guidelines for HIV-1 infection management suggest to monitor patient' HIV-RNA with the same assay over the course of treatment. As different commercial tests can be performed in the same laboratory with considerable difficulty, the laboratory should select an assay that is suitable not only for the more prevalent strain, but also for less frequent ones that, nevertheless, can occur. Then, knowing and investigating the spread of non-B strains has essential clinical and laboratory implications.

First external quality assurance program of the Italian HLA-B*57:01 Network assessing the performance of clinical virology laboratories in HLA-B*57:01 testing.

BACKGROUND: Since the HLA-B*57:01 allele is strongly associated with abacavir hypersensitivity reaction, testing for the presence of HLA-B*57:01 is mandatory before administration of abacavir. While HLA-B*57:01 testing is usually provided by pharmacogenetics, genetics or blood transfusion services, clinical virology laboratories can be an optimal opportunity for HLA-B*57:01 testing since they receive blood samples for routine HIV monitoring and have the expertise for convenient and less expensive PCR-based point mutation assays. OBJECTIVES: The Italian HLA-B*57:01 Network gathers accredited clinical virology laboratories offering HLA-B*57:01 testing in Italy with the aim to share protocols, test new methods, develop and maintain external quality assurance (EQA) programs. STUDY DESIGN: A panel of 9HLA-B*57:01-positive and 16HLA-B*57:01-negative frozen blood samples were blindly distributed to 10 units including 9 clinical virology laboratories and one reference pharmacology laboratory. Each laboratory was free to use its own routine method for DNA extraction and HLA-B*57:01 testing. RESULTS: DNA was extracted by automated workstations in 6 units and by manual spin columns in 4. Eight units used the Duplicα Real Time HLA-B*57:01 kit by Euroclone and two units used two different PCR homemade protocols. All the 10 units correctly identified all the 25 samples. CONCLUSIONS: The first HLA-B*57:01 EQA program run in Italy showed that clinical virology units are equipped and proficient for providing HLA-B*57:01 testing by inexpensive assays easy to integrate into their routine.

Prevalence of HIV-1 Subtypes and Drug Resistance-Associated Mutations in HIV-1-Positive Treatment-Naive Pregnant Women in Pointe Noire, Republic of the Congo (Kento-Mwana Project).

The Kento-Mwana project was carried out in Pointe Noire, Republic of the Congo, to prevent mother-to-child HIV-1 transmission. To determine the prevalence of different subtypes and transmitted drug resistance-associated mutations, 95 plasma samples were collected at baseline from HIV-1-positive naive pregnant women enrolled in the project during the years 2005-2008. Full protease and partial reverse transcriptase sequencing was performed and 68/95 (71.6%) samples were successfully sequenced. Major mutations to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 4/68 (5.9%), 3/68 (4.4%), and 2/68 (2.9%) samples, respectively. Phylogenetic analysis of HIV-1 isolates showed a high prevalence of unique recombinant forms (24/68, 35%), followed by CRF45_cpx (7/68, 10.3%) and subsubtype A3 and subtype G (6/68 each, 8.8%). Although the prevalence of transmitted drug resistance mutations appears to be currently limited, baseline HIV-1 genotyping is highly advisable in conjunction with antiretroviral therapy scale-up in resource-limited settings to optimize treatment and prevent perinatal transmission.

Genotypic determination of HIV tropism in a cohort of patients perinatally infected with HIV-1 and exposed to antiretroviral therapy.

The aim of this study was to determine the coreceptor tropism by performing genotypic HIV-1 tropism testing in a cohort of patients perinatally infected with HIV-1 and exposed to antiretroviral therapy. Genotypic coreceptor tropism was determined in patients with HIV-1 RNA<100 copies/mL using PBMC samples by gp120 V3 sequencing followed by geno2pheno interpretation (set at a false positive rate [FPR] of 20%) and in patients with ≯100 copies/mL using plasma samples (set at a FPR of 20%), according to European guidelines. Out of 55 patients, 50 had an HIV-1 subtype B strain, and mean (SD) age was 18.2 (4.6) years. The median duration of antiretroviral therapy was 13 years (range, 3-23). Thirty-three (60%) patients harbored the R5 virus. At the time of the testing, the median CD4+ T lymphocyte cell count and percentage were 705 cells/mm3 (474-905) and 32.5% in group R5 and 626 cells/mm3 (450-755) and 31.7% in group X4/D-M, respectively. The nadir of CD4+ T-cell count in groups R5 and X4/D-M were 322 cells/mm3 (230-427) and 340 cells/mm3 (242-356), respectively. These differences were not statistically significant. Fifteen patients had HIV-1 RNA ≯50 copies/mL. The median HIV-1 RNA and HIV-1 DNA were comparable in both groups without a statistical difference. The study provides an overview of the prevalence of coreceptor tropism in a cohort of patients who were vertically infected with HIV-1. The high prevalence of X4/D-M-tropic strains may simply reflect the long-term exposure to HIV.