Plasma-derived sequences of human immunodeficiency virus type 1 (HIV-1) protease from 1,162 patients (457 drug-naïve patients and 705 patients receiving protease inhibitor [PI]-containing antiretroviral regimens) led to the identification and characterization of 17 novel protease mutations potentially associated with resistance to PIs. Fourteen mutations were positively associated with PIs and significantly correlated in pairs and/or clusters with known PI resistance mutations, suggesting their contribution to PI resistance. In particular, E34Q, K43T, and K55R, which were associated with lopinavir treatment, correlated with mutations associated with lopinavir resistance (E34Q with either L33F or F53L, or K43T with I54A) or clustered with multi-PI resistance mutations (K43T with V82A and I54V or V82A, V32I, and I47V, or K55R with V82A, I54V, and M46I). On the other hand, C95F, which was associated with treatment with saquinavir and indinavir, was highly expressed in clusters with either L90M and I93L or V82A and G48V. K45R and K20T, which were associated with nelfinavir treatment, were specifically associated with D30N and N88D and with L90M, respectively. Structural analysis showed that several correlated positions were within 8 Å of each other, confirming the role of the local environment for interactions among mutations. We also identified three protease mutations (T12A, L63Q, and H69N) whose frequencies significantly decreased in PI-treated patients compared with that in drug-naïve patients. They never showed positive correlations with PI resistance mutations; if anything, H69N showed a negative correlation with the compensatory mutations M36I and L10I. These mutations may prevent the appearance of PI resistance mutations, thus increasing the genetic barrier to PI resistance. Overall, our study contributes to a better definition of protease mutational patterns that regulate PI resistance and strongly suggests that other (novel) mutations beyond those currently known to confer resistance should be taken into account to better predict resistance to antiretroviral drugs.

Non-B HIV subtypes have been estimated to account for 88% of HIV infections in the world. These subtypes are particularly relevant in view of the availability of antiretroviral (ARV) drugs, since subtype-specific mutations are associated with drug-resistance in developing countries. Therefore, the pol gene sequences in HIV-1 isolates were examined from the three distinct groups of 39 infected patients from Ouagadougou in Burkina Faso: 17 patients who had not received any antiretroviral therapy (ART); 16 patients received ART, and 6 HIV-infected children, from infected mothers, received a single Nevirapine dose prophylaxis during birth. HIV-1 pol sequencing was successful for 29 samples. As expected, all patients presented the common (non-B subtype) M36I polymorphism and 26/29 (90%) the K20I mutation. Phylogenetic studies showed high predominance of recombinant HIV-1 strains: CRF06_cpx 16/29 (55.17%), CRF02_AG 9/29 (31.03%), A1 2/29 (6.89%), G 1/29 (3.44%), and CRF09_cpx 1/29 (3.44%). Two twins showed, 6 months after birth, a NNRTI-mutation (Y181C/Y). During the same period, the twin mother presented a different NNRTI-mutation (V106I), thus suggesting that the different blood drug concentration may determine a different drug-resistance pathway. Among 17 non-highly active antiretroviral therapy (HAART) patients, 3/17 (17.64%) presented virus with reverse transcriptase (RT) mutations [V118I: 1/17 patients (5.88%), V179E: 2/17 patients (11.76%)]. 10/17 (58.82%) presented virus with minor protease (PR) mutations [L63P: 5/17 patients (29.41%), V77I: 3/17 patients (17.64%), L10I: 2/17 patients (11.76%)]. 4/17 patients did not show any PR and RT mutations (23.52%). Among six HAART-treated patients, 6/6 and 3/6 had M36I and L63LP protease minor subtypes, respectively; and only two (33.33%) presented virus with K103N mutation. The low prevalence of drug-resistant associated mutations in Burkina Faso is encouraging. However, further studies with a larger cohort with a high non-B subtype prevalence are necessary to optimize ART in developing countries.

Background: Central nervous system (CNS) infections by human polyomaviruses (HPyVs), with the exception of JC (JCPyV), have been poorly studied. Methods: In total, 234 cerebrospinal fluid (CSF) samples were collected from patients affected with neurological disorders. DNA was isolated and subjected to quantitative real-time PCR (Q-PCR) for the detection of six HPyVs: JCPyV, BKPyV, Merkel cell PyV (MCPyV), HPyV6, HPyV7, and HPyV9. Where possible, the molecular characterization of the viral strains was carried out by nested PCR and automated sequencing. Results: JCPyV was detected in 3/234 (1.3%), BKPyV in 15/234 (6.4%), MCPyV in 22/234 (9.4%), and HPyV6 in 1/234 (0.4%) CSF samples. JCPyV was detected at the highest (p < 0.05) mean load (3.7 × 107 copies/mL), followed by BKPyV (1.9 × 106 copies/mL), MCPyV (1.9 × 105 copies/mL), and HPyV6 (3.3 × 104 copies/mL). The noncoding control regions (NCCRs) of the sequenced viral strains were rearranged. Conclusions: HPyVs other than JCPyV were found in the CSF of patients affected with different neurological diseases, probably as bystanders, rather than etiological agents of the disease. However, the fact that they can be latent in the CNS should be considered, especially in immunosuppressed patients.

Our results show that the chances of development of resistance are far lower in M/M than in lymphocytes. This underlines the importance and the peculiar role of M/M as reservoirs of either wild-type or resistant strains in human organs.

Intrahepatic total HBV DNA (it-HBV DNA) level might reflect the size of virus reservoir and correlate with the histological status of the liver. To quantitate it-HBV DNA in a series of 70 liver biopsies obtained from hepatitis B chronic patients, a modified version of the COBASAmpliprep/COBASTaqMan HBV test v2.0 was used for this purpose. The linearity and reproducibility of the modified protocol was tested by quantifying serial dilutions of a full-length HBV containing plasmid and it-HBV DNA from a reference patient. A good linear trend between the expected values and those generated by the assay was observed at different concentrations of both plasmid and reference patient (R = 0.994 and 0.962, respectively). Differences between the values obtained in two independent runs were ≤0.3 log IU for the plasmid and ≤0.6 log IU/mg for the reference patient, showing a high inter-run reproducibility. In the 70 liver biopsies, it-HBV DNA level ranged from 1.4 to 5.4 log IU/mg, with a good linearity and reproducibility between the values obtained in two runs [R = 0.981; median (IQR) difference of it-HBV DNA 0.05 (0.02-0.09) IU/mg]. The modified COBASAmpliprep/COBASTaqMan HBV test v2.0 allows an accurate quantitation of it-HBV DNA. Its determination may have prognostic value and may be a useful tool for the new therapeutic strategies aimed at eradicating the HBV infection.