Our study showed that the majority of the cases were males (67.1%) (Table 1), and were more likely to have VF-M (p = 0.002). Studies conducted in developing countries have shown that male HIV patients are more likely to present to HIV care facilities with advanced disease as compared to their female counterparts [25, 26]. Moreover, advanced HIV/AIDS is a critical predictor for failure on second-line ART [27, 28]. Furthermore, research has shown that males are more prone to virological failure while on second-line ART than females [29, 30], attributable to poor adherence and higher odds of alcohol consumption while on ART [31, 32]. However, our findings are contrasted by a South African study, which showed that 60% of patients who had VF-M were women . That said, our results continue to highlight male HIV positive patients as a key vulnerable population that needs special attention if we are to maintain them on second-line ART over an extended period.
Age and VF-M
Chimbetete et al.  found that older patients (> 24 years of age) were associated with higher odds of VF-M. This could be attributed to long periods of exposure on first-line drugs, some of which are recycled to form part of second-line regimens. Furthermore, long-term exposure to ART is associated with higher odds of non-adherence , which could be the case for older adults. That said, our study did not find any statistical difference between age and VF-M. We recommend more studies to understand the factors behind younger HIV/AIDS patients on second-line ART having lower odds of PI mutations at failure.
HIV/TB coinfection and VF-M
Concomitant tuberculosis treatment, while on second-line ART, was higher among cases (67%) and significantly associated with VF-M (p ≤ 0.004). Our findings are in line with a study conducted by Rossouw et al. 2015, which found that children on tuberculosis treatment while on second-line ART were more likely to have VF-M . This is attributable to factors such as higher pill burden [36, 37], HIV/TB coinfection being associated with advanced HIV/AIDS , and the fact that rifabutin is not readily available in the resource-limited settings as a replacement for Rifampicin which is known to reduce the pharmacokinetic levels of PIs and consequently their efficacy [39, 40]. Our findings highlight VF-M as an outcome of the continued prescription of rifampicin and stress the need for governments in developing countries to adopt rifabutin for HIV/TB co-infected patients. HIV care providers should also provide more personalized attention, counseling, and support to patients concomitantly on TB and second-line ART. This is because they are at a higher risk of VF-M. Furthermore, HIV clinical care specialists have to weigh the options of initiating patients with TB on PIs.
Year of ART initiation and VF-M
Patients who initiated ART after 2001 were less likely to have VF-M as compared to colleagues who were started on ART before. Patients enrolled on ART between 2001–2005, 2006–2010, 2011–2015 had a 96%, 97%, and 93% lower likelihood of VF-M, respectively, as compared to those initiated on ART before 2001. This is attributed to health systems improvements in the provision of HIV/AIDS care services  For example, the introduction of differentiated care models has improved adherence because drugs are taken closer to where patients live. Conversely, before 2001 patients had to return to health facilities within short intervals, which was economically tasking. Relatedly, before 2001, patients on ART had higher pill burdens, which made it hard for them to adhere to treatment  as compared to those post-2001 when the improvement in dose formulations was made to reduce pill burdens. Furthermore, due to high levels of primary resistance to NNRTIs, patients initiated on ART of late are given dolutegravir for first-line ART. For this, patients are likely to stay longer on first-line since it’s a new class of drugs with a high genetic barrier to resistance and can be co-formulated, leading to once-daily dosing with tenofovir/lamivudine .
Type of second-line ART and VF-M
Patients who had “other PIs” saquinavir and nelfinavir had lower odds of VF-M compared to counterparts on lopinavir or atazanavir despite the associated higher pill burden of “other PIs” like saquinavir  and their low genetic barrier to resistance and reduced bioavailability . The existence of this phenomenon needs further exploration. More to this, it is essential to note that in Uganda, due to increased access to HIV care and reliable national supply of lopinavir and boosted atazanavir, which is the recommended PIs for second-line ART, saquinavir is rarely used except for third-line ART.
Significance of the study
To our knowledge, this study is the first endeavor to investigate the factors associated with second-line ART failure with PI mutations. The study was conducted within an HIV care provision setting, which reflects ground reality, and our measures were based on WHO standards for assessing failure on second-line ART. Due to existing data management mechanisms for data validity at JCRC, data used for this study was relatively good and reliable. More importantly, we employed a robust data collection system and trained all staff involved in the data collection and management process to minimize errors and ensure the quality and validity of results.
Our study had a couple of limitations. First, we excluded 69 (45%) files under the “case” arm, and 68 (21%) files under the “control” arm due to data incompleteness, which could have affected the findings. However, we worked with data of over 50% of the whole population of interest, which is generalisable. Second, there might have been original data entry errors since our analysis was solely based on routinely collected project data. Third, our study was conducted within the limitations of case–control studies, with the odds ratios unstable as reflected by the wide confidence intervals. This was minimised by increasing the power of the study. That said, our findings provide a platform for more extensive longitudinal studies to understand further the underlying factors and co-factors for second-line ART failure with PI mutations within low-income settings.