Introduction: HAART reduces the risk of mother to child transmission, morbidity plus mortality among HIV-infected pregnant and breastfeeding women. To optimize these outcomes, pregnant and breastfeeding women need to be virologically suppressed while on HAART. However, there is paucity of data regarding viral load testing and virologic status among these women in Mayuge District and Uganda as a whole. General objective: To determine the trend of viral load testing and virologic non-suppression plus associated factors among HIV-infected among pregnant or breastfeeding women in the PMTCT program in Mayuge district in order to improve outcomes of PMTCT program in Uganda. Methods: This was a cross sectional study design with concurrent mixed methods. Quantitative data was abstracted from records and qualitative data collected through in-depth interviews. Data of 575 eligible women was entered into Epi-data version 3.1 and analyzed using STATA version 13.0. Fourteen (14) in-depth interviews were conducted for both virologically suppressed and non-suppressed pregnant or breastfeeding mothers. Quarterly proportions of first viral load testing and virologic nonsuppression were calculated to get trends of each outcome. Factors associated with virologic nonsuppression were assessed for at both bivariate and multivariable level using logistic regression with their 95% confidence intervals and P-values. Qualitative data was analyzed manually using thematic analysis. Results: A total of 575 records of eligible women were reviewed; median age was 28 (IQR: 23, 32) median gestational age at enrolment was 23.0 weeks (IQR 23.0, 28.0) and 77.6% were multiparous at enrolment into PMTCT Program. Overall, 65.4% (95% CI: 61.5 – 69.3) of the women had a first Viral load test since entry into PMTCT Program with VL testing highest between October 2016 and December 2016 (94.7%) and lowest in the period between April 2017 and June 2017 (51.3%). There was a significant decline in trend of first viral load testing over time (p < 0.001). Overall virologic nonsuppression proportion was 11.2%, the highest proportion of virologic non-suppression (25%) was registered between October 2016 and December 2016 and the lowest (4.8%) was among women enrolled from April 2018-June 2018. Being on ART for more than 24 months ART [aOR=2.99; 95% CI (1.15 – 7.79)] was significantly associated with higher odds of virologic non-suppression whereas multiparous women [aOR=0.32; 95%CI (0.13 – 0.78) ], were less likely to be virologically nonsuppressed. Poor adherence was the single mentioned reason for virologic non-suppression by the women. From the lived experiences of both suppressed and non-suppressed women, adherence to ARVs and patient knowledge/information regarding virologic suppression emerged as major themes. Barriers to ARV adherence included fear to start ARVs, forgetfulness and long distances from the health facilities. Information on virologic suppression was limited among both the suppressed and nonsuppressed women. Conclusion and recommendation: There was a declining trend of first viral load testing but virologic non-suppression remained above the acceptable 10% among women in PMTCT program in Mayuge district. Predictors of virologic non-suppression were parity and duration on ART. Fear to start ARVs, forgetfulness, long distances and lack of food were the barriers to ARV drug adherence and all women had limited information regarding virologic suppression. We recommend strengthening of monitoring and evaluation of viral load testing, interventions aimed at improving adherence and use of Community Health Extension Workers (CHEWs).. Furthermore, health workers need to communicate consistent and accurate information to patients regarding virologic suppression.

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