HIV-1 antiretroviral drug resistance patterns in patients failing NNRTI-based treatment: results from a national survey in South Africa.

BACKGROUND: Routine HIV-1 antiretroviral drug resistance testing for patients failing NNRTI-based regimens is not recommended in resource-limited settings. Therefore, surveys are required to monitor resistance profiles in patients failing ART. METHODS: A cross-sectional survey was conducted amongst patients failing NNRTI-based regimens in the public sector throughout South Africa. Virological failure was defined as two consecutive HIV-1 viral load results >1000 RNA copies/mL. Pol sequences were obtained using RT-PCR and Sanger sequencing and submitted to Stanford HIVdb v7.0.1. RESULTS: A total of 788 sequences were available for analysis. Most patients failed a tenofovir-based NRTI backbone (74.4%) in combination with efavirenz (82.1%) after median treatment duration of 36 months. K103N (48.9%) and V106M (34.9%) were the most common NNRTI mutations. Only one-third of patients retained full susceptibility to second-generation NNRTIs such as etravirine (36.5%) and rilpivirine (27.3%). After M184V/I (82.7%), K65R was the most common NRTI mutation (45.8%). The prevalence of K65R increased to 57.5% in patients failing a tenofovir regimen without prior stavudine exposure. Cross-resistance to NRTIs was often observed, but did not seem to affect the predicted activity of zidovudine as 82.9% of patients remained fully susceptible to this drug. CONCLUSIONS: The introduction of tenofovir-based first-line regimens has dramatically increased the prevalence of K65R mutations in the HIV-1-infected South African population. However, most patients failing tenofovir-based regimens remained fully susceptible to zidovudine. Based on these data, there is currently no need to change either the recommended first- or second-line ART regimens in South Africa.

Prevalence of Antiretroviral Drug Resistance in Patients Who Are Not Responding to Protease Inhibitor-Based Treatment: Results From the First National Survey in South Africa.

Limited data exist on human immunodeficiency virus type 1 (HIV-1) resistance in patients who are not responding to protease inhibitor (PI)-based regimens in resource-limited settings. This study assessed resistance profiles in adults across South Africa who were not responding to PI-based regimens. pol sequencing was undertaken and submitted to the Stanford HIV Drug Resistance Database. At least 1 major PI mutation was detected in 16.4% of 350 participants. A total of 53.4% showed intermediate resistance to darunavir/ritonavir, whereas high-level resistance was not observed. Only 5.2% and 32.8% of participants showed high-level and intermediate resistance to etravirine, respectively. Although the prevalence of major PI mutations was within previously reported ranges, most patients will likely experience virological suppression during receipt of currently available South African third-line regimens.

Viral genetic determinants of nonprogressive HIV type 1 subtype C infection in antiretroviral drug-naive children.

Characterization of HIV-1 from slow progressors is important to facilitate vaccine and antiviral drug development. To identify virus attenuations that may contribute to slower rates of disease progression, the full-length viral genomes from primary isolates of six slow progressing HIV-positive children were sequenced. Proviral DNA was extracted from cocultured peripheral blood mononuclear cells and used to PCR amplify, sequence, and extensively analyze the near full-length genomes and LTR regions. All primary HIV-1 isolates were HIV-1 subtype C throughout their genome, and amino acid (AA) sequence analysis revealed open reading frames for all genes. However, all isolates had at least one unusual gene/protein. For example, isolate LT5 had a 2AA insertion in the Vpr mitochondriotoxic domain. Isolate LT21 contained an additional 5AA in the C-terminus of tat exon 2, while integrase in isolate LT39 had an additional 4AA at the C-terminus. Rev from isolates LT45 and LT46 did not have the characteristic subtype C 16AA truncation, and in addition, had a further 3AA. Furthermore, altered functional domains were noted in several isolates, such as the cAMP-dependent kinase PKA phosphorylation site in Nef (LT5), a Vpr mutation involved in decreased proapoptotic activity (all isolates), and the Nef ExxxLL motif involved in the interaction with AP-1 and AP-2 (LT46). The slower HIV-1 disease progression in these six children may be attributed to altered protein functions. For example, LT46 Nef is unable to bind AP-1 and AP-2 and therefore is inactive on CD4 endocytosis. The biological relevance of these findings requires further investigation.

Phenotypic characterization of HIV type 1 isolates from Ghana.

Viral isolates from 27 HIV-1-infected patients in Ghana, most of whom were symptomatic, were characterized for coreceptor usage using MT-2 and U87.CD4 cells. Irrespective of clinical status, most infections were caused by CCR5-tropic viruses although three CXCR4-tropic viruses were also found. Genotyping was performed by sequencing the gp41 region. Seven viruses clustered with subtype G reference strains, while the remaining 20 viruses clustered within the subtype A reference viruses. Most subtype A isolates clustered loosely with the CRF02_AG viruses and are described as CRF02_AG-like. The V3 loop was sequenced in selected isolates including all isolates capable of using CXCR4. The V3 region of CXCR4-using viruses contained genetic traits characteristic of CXCR4-using subtype B and C viruses, such as increased charge, the presence of positively charged residues at positions 11 and 25, and loss of a predicted glycosylation site. This study supports previous work showing that CRF02_AG is responsible for most HIV-1 infections in Ghana at this time. The predominance of CCR5-using viruses, even in symptomatic patients, suggests that CCR5-blocking strategies may be useful for prevention and treatment of HIV-1 infections in Ghana.

Full-length genome characterization of HIV type 1 subtype C isolates from two slow-progressing perinatally infected siblings in South Africa.

Isolation and characterization of HIV-1 from asymptomatic, slow-progressing individuals are important in studying viral pathogenesis and facilitate the development of vaccines and antivirals. In this study we identified two slow-progressing HIV-1-infected siblings, isolated viruses, and sequenced the full-length genome, to identify virus attenuations that may contribute to their altered rate of disease progression. Proviral DNA from strains 99ZATM10 and 01ZATM45 was isolated from peripheral blood mononuclear cells (PBMC) coculture.Virtually full-length genomes and long terminal repeat (LTR) regions were polymerase chain reaction (PCR) amplified, sequenced, and assembled to generate the complete genomes. Phylogenetic analysis confirmed that both isolates were subtype C throughout their genome. Predicted amino acid sequence analysis for all the HIV-1 proteins showed that both viruses had open reading frames for all genes, and encoded proteins of the expected length, except for the rev gene. The 3' end of rev exon 2 did not have the 16-amino acid (aa) truncation characteristic of subtype C viruses, and in addition, had a three-aa extension (GlyCysCys). Rev is a necessary regulatory factor for HIV expression, and changes in the protein may affect viral replication. These results suggest that slower HIV disease progression in these children may be attributed, at least in part, to an altered Rev protein.

Multiple bacteriocin production by Leuconostoc mesenteroides TA33a and other Leuconostoc/Weissella strains.

Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra. Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography. Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB). Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187. A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread. Lc. carnosum LA54a, W. paramesenteroides LA7a, and Lc. gelidum UAL 187-22 produced all three bacteriocins, whereas W. paramesenteroides OX and Lc. carnosum TA11a produced only leucocin A- and B-type bacteriocins.

Characterization of leucocin B-Ta11a: a bacteriocin from Leuconostoc carnosum Ta11a isolated from meat.

Leuconostoc (Lc.) carnosum Ta11a, isolated from vacuum-packaged processed meats, produced a bacteriocin designated leucocin B-Ta11a. The crude bacteriocin was heat stable and sensitive to proteolytic enzymes, but not to catalase, lysozyme, or chloroform. It was active against Listeria monocytogenes and several lactic acid bacteria. Leucocin B-Ta11a was optimally produced at 25 degrees C in MRS broth at an initial pH of 6.0 or 6.5. An 8.9-MDa plasmid in Leuconostoc carnosum Ta11a hybridized to a 36-mer oligonucleotide probe (JF-1) that was homologous to leucocin A-UAL187. A 4.9-kb Sau3A fragment from a partial digest of the 8.9-MDa plasmid was cloned into pUC118. The 8.1-kb recombinant plasmid (pJF8.1) was used for sequencing and revealed the presence of two open reading frames (ORFs). ORF1 codes for a protein of 61 amino acids comprising a 37-amino-acid bacteriocin that was determined to be the leucocin B-Ta11a structural gene by virtue of its homology to leucocin A-UAL 187 (Hastings et al. 1991. J. Bacteriol 173:7491-7500). The 24-amino-acid N-terminal extension, however, differs from that of leucocin A-UAL187 by seven residues. The predicted protein of the ORF2 has 113 amino acids and is identical with the amino acid sequence of the cognate ORF of the leucocin A-UAL 187 operon.

Antibacterial activity of three Leuconostoc strains isolated from vacuum-packaged processed meats.

One hundred and fifty lactic acid bacteria (LAB) isolated from vacuum-packaged processed meats were screened for antagonistic activity against various food spoilage microorganisms and foodborne pathogens. Nineteen strains produced bacteriocins active against closely related LAB and Listeria strains. Leuconostoc carnosum (LA54a and TA26b) and Leuconostoc mesenteroides subspecies dextranicum (TA33a) produced bacteriocins that were susceptible to proteolytic enzymes, but not to catalase, lysozyme or chloroform. They were heat stable up to 100 degrees C for thirty minutes at pH 2 to 7, and exerted a bacteriolytic effect. Bacteriocin production by all Leuconostoc strains was growth associated, occurring at incubation temperatures of 0 degrees C to 30 degrees C and initial medium pH 4.5 to 7.5. Probing of plasmid DNA from the three Leuconostoc strains with an oligonucleotide probe homologous to the nucleotide sequence of leucocin A-UAL 187 indicated plasmid-mediated bacteriocin production. Homology of the three Leuconostoc bacteriocin-coding genes to the amino-terminal end of the leucocin A-UAL 187 gene from Leuconostoc gelidum UAL 187 is therefore suggested. This evidence implies that all three Leuconostoc strains produce type 2, Listeria active bacteriocins.