Over the 18-month period, 203 patients had suspected treatment failure on the basis of clinical and immunological criteria and had viral load performed. Of these, 88 patients were suppressed (HIV RNA <400 copies/ml), six had HIV RNA between 400–1000 copies/ml, three had previous protease inhibitor exposure, and 10 had insufficient stored sample. The 96 remaining patients served as the basis for this evaluation. Among these 96 patients, most were identified by CD4 cell count decline (87) or new or progressive WHO stage 4 conditions (16) or both. Ninety-two patients received d4T/3TC/NVP as initial therapy, and four patients had received ZDV/3TC dual therapy prior to initiation of d4T/3TC/NVP. Thirty patients had ZDV substituted for d4T, and nine had EFV substituted for NVP because of toxicity. The median CD4 cell count, HIV RNA, and time on ART were 68 cells/μl, 52 374 copies/ml [interquartile range (IQR) 16913–138259], and 36.5 months (range 8–127 months), and 50% were women (). Patients who were on ZDV at the time of failure evaluation had longer durations of ART treatment (48.6 vs. 34.7 months, P <0.001) with a median of 27 months (IQR 10–42) of d4T use prior to switch and a median ZDV treatment duration of 14.9 months (IQR 5.4–23).
Characteristics of clinical/immunological failure patients with confirmed virological failure (HIV-1 RNA >1000 copies/ml) (n = 96).
Of the 96 samples, two did not amplify, leaving 94 samples that could be evaluated by genotyping. All samples were subtype C. The median number of resistance mutations (any class) was five (IQR 3–7) with a range of 0–11. Five samples had no mutations identified (). NNRTI mutations occurred in 93% of the samples. Two patients had HIV with only NNRTI mutations. The median number of NNRTI mutations was two (range 0–3), and of those with at least one NNRTI mutation (n =87), 39% had one, 52% had two, and 8% had three mutations. The most frequent NNRTI mutations were Y181C (55%), G190A (30%), K103N (28%), K101E (15%), Y188L (8%), V106M (7%), Y181I (6%), K103N/S (2%), P225H (1%), G190S (1%), and G190E (1%). Among patients with exposure to NVP but not to EFV (n =85), the most common mutations were Y181C (55%) and G190A (26%). Among those with exposure to both NVP and EFV (n =9), K103N (33%) and G190A (33%) mutations were the most common. Y181C was significantly more common among NVP-only-exposed patients (55 vs. 11%, P =0.012).
Patterns of mutations seen among patients with antiretroviral therapy failure in Malawi (n = 94). Full NRTI and NNRTI resistance profiles available online on journal website.
The M184Vor M184I mutation was present in virus from 77 patients (81%), although never as the only mutation. Fifteen patients (16%) had virus with M184V and NNRTI mutations only.
The most common mutation pattern was M184V and NNRTI mutations with one or more TAMs, which occurred in 56% of patients. Of the patients with TAM-containing virus (n =53), 28% had one, 28% had two, and 44% had three or more TAMs. The most frequent TAMs were T215 F/Y (73%), D67N (53%), K70R (36%), M41L (36%), K219 Q/E (23%), and L210W (23%). Patients who had ever received ZDV had more TAMs (1.8 vs. 1.2 mutations, P =0.055) and were more likely to have at least three TAMs (19% for d4T vs. 36% for ZDV, P =0.09). In spite of no known exposure to TDF, 23% (22/94) of patients acquired virus with the K70E (n =4) or K65R mutations (n =18). Eighteen patients developed virus with the Q151M mutation, and in 15 patients, the Q151M mutation was associated with either K65R or K70E. One patient developed a T69 insertion. In total, 16 patients had pan-nucleoside-resistant virus, on the basis of the presence of the K65R or K70E mutation in association with the Q151M complex or the presence of a 69 insertion. Compared with those with ZDV exposure, the emergence of the K65R, Q151M, and K70E mutations was more common in those exposed only to d4T: K65R (24 vs. 7%, P =0.05), K70E (6 vs. 0%, P =0.183), and Q151M (24 vs. 7%, P =0.05).
No patient with a CD4 cell count more than 200 cells/μl or HIV-1 RNA less than 10 000 copies/ml had pan-nucleoside-resistant virus. One-third of patients with HIV-1 RNA less than 10 000 copies/ml had TAMs. Only two of 30 patients who had ever received ZDV developed either K65R/K70E or pan-nucleoside resistance mutations.
Univariate and multivariate analyses were performed to look for associations with the emergence of pan-nucleoside resistance, at least three TAMS, or either K65R or K70E. A CD4 cell count of less than 100 cells/μl was strongly associated with emergence of K65R or K70E, pan-nucleoside resistance, and the emergence of three or more TAMs (). However, associations with ZDVuse were not consistently in the same direction. ZDV use was strongly protective for the emergence of K65R or K70E. ZDV use was protective for the emergence of pan-nucleoside resistance in the univariate analysis resistance, but the trend was no longer significant in the multivariate analysis [odds ratio (OR) 0.13, 95% confidence interval (CI) 0.02–1.07]. Conversely, ZDV was associated with an increased risk for emergence of three or more TAMs in the multivariate analysis (OR 3.4, 95% CI 1.7–10.97).
Univariate and multivariate logistic modeling of three potential outcomes for emergence of resistance among patients failing antiretroviral therapy based on clinical or immunological criteria in Malawi.
Phenotypic drug susceptibility was determined for 70 samples with complex genotypes. Given the selection criteria, nearly all samples had high-level NNRTI resistance (96% for NVP and 83% for EFV) and 3TC resistance (97%). Substantial phenotypic resistance was demonstrated for most other NRTI agents (). The majority of viruses (61%) were fully susceptible to TDF, whereas fewer were fully susceptible to ZDV (37%), d4T (37%), ABC (33%), or DDI (1%). Partial susceptibility of the virus isolates was noted for TDF (33%), DDI (29%), and ABC (20%). Consistent with the genotypic findings, those with ZDV exposure were more likely to retain full susceptibility to TDF (82 vs. 56%, P =0.05).
Fold change in 50% inhibitory concentration for individual nonnucleoside reverse transcriptase inhibitor and nucleoside reverse transcriptase inhibitor agents
We evaluated the expected activity of NRTI combinations recommended by the WHO considering only the phenotype results () (n =70). Three NRTI combinations were evaluated, including ZDV/3TC/TDF, FTC/TDF, and DDI/ABC, taking advantage of the clinical cut-off available for TDF, ABC, and DDI (). includes an overall assessment of activity, including only fully sensitive NRTI or sensitive NRTI and those with partial susceptibility. Overall, the three-NRTI combination provided the greatest likelihood of one or two active NRTIs in either analysis, although 30 and 6% would have no active NRTI option depending on consideration of partial susceptibility (). Notably, the ABC/DDI combination performed poorly in either assessment, with neither NRTI active in 67% in the more stringent analysis and 46% when partial susceptibility was considered.
Resistance profiles according to combinations chosen.
Overall nucleoside reverse transcriptase inhibitor combination activity including either fully sensitive agents or agents with full sensitivity plus partial susceptibility.