Reverse transcriptase drug resistance mutations in HIV-1 subtype C infected patients on ART in Karonga District, Malawi

<p>Abstract</p> <p>Background</p> <p>Drug resistance testing before initiation of, or during, antiretroviral therapy (ART) is not routinely performed in resource-limited settings. High levels of viral resistance circulating within the population will have impact on treatment programs by increasing the chances of transmission of resistant strains and treatment failure. Here, we investigate Drug Resistance Mutations (DRMs) from blood samples obtained at regular intervals from patients on ART (Baseline-22 months) in Karonga District, Malawi. One hundred and forty nine reverse transcriptase (RT) consensus sequences were obtained via nested PCR and automated sequencing from blood samples collected at three-month intervals from 75 HIV-1 subtype C infected individuals in the ART programme.</p> <p>Results</p> <p>Fifteen individuals showed DRMs, and in ten individuals DRMs were seen from baseline samples (reported to be ART naïve). Three individuals in whom no DRMs were observed at baseline showed the emergence of DRMs during ART exposure. Four individuals who did show DRMs at baseline showed additional DRMs at subsequent time points, while two individuals showed evidence of DRMs at baseline and either no DRMs, or different DRMs, at later timepoints. Three individuals had immune failure but none appeared to be failing clinically.</p> <p>Conclusion</p> <p>Despite the presence of DRMs to drugs included in the current regimen in some individuals, and immune failure in three, no signs of clinical failure were seen during this study. This cohort will continue to be monitored as part of the Karonga Prevention Study so that the long-term impact of these mutations can be assessed. Documenting proviral population is also important in monitoring the emergence of drug resistance as selective pressure provided by ART compromises the current plasma population, archived viruses can re-emerge</p

HacA-Independent Functions of the ER Stress Sensor IreA Synergize with the Canonical UPR to Influence Virulence Traits in Aspergillus fumigatus

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacAi, or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireAΔ10. Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus

HIV type 1 mutational patterns in HIV type 1 subtype C-infected long-term survivors in Karonga District Malawi: further analysis and correction.

Here we present new sequence data from HIV-1 subtype C-infected long-term survivors (LTS) from Karonga District, Malawi. Gag and env sequence data were produced from nine individuals each of whom has been HIV-1 positive for more than 20 years. We show that the three amino acid deletion in gag p17 previously described from these LTS is not real and was a result of an alignment error. We find that the use of dried blood spots for DNA-based studies is limited after storage for 20 years. We also show some unlikely amino acid changes in env C2-V3 in LTS over time and different patterns of genetic divergence among LTS. Although no clear association between mutations and survival could be shown, amino acid changes that are present in more than one LTS may, in the future, be shown to be important

Emergence of a three codon deletion in gag p17 in HIV type 1 subtype C long-term survivors, and general population spread.

In a population-based study in northern Malawi we investigated HIV-1 subtype C gag and env gene sequences associated with long-term survival. DNA samples were available from 31 individuals surviving between population surveys carried out in the 1980s and 1990s. Most survivors with paired sequences dating from the 1980s and the 1990s had a three codon deletion in the gag p17 region of the sequence retrieved from the sample collected in the 1990s that was not present in the sequence from the same individual dating from the 1980s. This deletion was also not present in any other 1980s sequences from Malawi, but was common in samples collected in Malawi in the 1990s. The deletion is equivalent to the loss of three amino acids in the D helix region of the gag protein, and may be associated with longer survival and onward transmission

Appraising the performance of genotyping tools in the prediction of coreceptor tropism in HIV-1 subtype C viruses

<p>Abstract</p> <p>Background</p> <p>In human immunodeficiency virus type 1 (HIV-1) infection, transmitted viruses generally use the CCR5 chemokine receptor as a coreceptor for host cell entry. In more than 50% of subtype B infections, a switch in coreceptor tropism from CCR5- to CXCR4-use occurs during disease progression. Phenotypic or genotypic approaches can be used to test for the presence of CXCR4-using viral variants in an individual’s viral population that would result in resistance to treatment with CCR5-antagonists. While genotyping approaches for coreceptor-tropism prediction in subtype B are well established and verified, they are less so for subtype C.</p> <p>Methods</p> <p>Here, using a dataset comprising V3 loop sequences from 349 CCR5-using and 56 CXCR4-using HIV-1 subtype C viruses we perform a comparative analysis of the predictive ability of 11 genotypic algorithms in their prediction of coreceptor tropism in subtype C. We calculate the sensitivity and specificity of each of the approaches as well as determining their overall accuracy. By separating the CXCR4-using viruses into CXCR4-exclusive (25 sequences) and dual-tropic (31 sequences) we evaluate the effect of the possible conflicting signal from dual-tropic viruses on the ability of a of the approaches to correctly predict coreceptor phenotype.</p> <p>Results</p> <p>We determined that geno2pheno with a false positive rate of 5% is the best approach for predicting CXCR4-usage in subtype C sequences with an accuracy of 94% (89% sensitivity and 99% specificity). Contrary to what has been reported for subtype B, the optimal approaches for prediction of CXCR4-usage in sequence from viruses that use CXCR4 exclusively, also perform best at predicting CXCR4-use in dual-tropic viral variants.</p> <p>Conclusions</p> <p>The accuracy of genotyping approaches at correctly predicting the coreceptor usage of V3 sequences from subtype C viruses is very high. We suggest that genotyping approaches can be used to test for coreceptor tropism in HIV-1 group M subtype C with a high degree of confidence that they will identify CXCR4-usage in both CXCR4-exclusive and dual tropic variants.</p