Background Cotrimoxazole (CTX) prophylaxis is recommended by the World Health Organization (WHO) for HIV-1-infected individuals in settings with high infectious disease prevalence. The WHO 2006 guidelines were developed prior to the scale-up of antiretroviral therapy (ART). The threshold for CTX discontinuation following ART is undefined in resource-limited settings. Methods and Findings Between 1 February 2012 and 30 September 2013, we conducted an unblinded non-inferiority randomized controlled trial of CTX prophylaxis cessation versus continuation among HIV-1-infected adults on ART for ≥18 mo with CD4 count > 350 cells/mm3 in a malaria-endemic region in Kenya. Participants were randomized and followed up at 3-mo intervals for 12 mo. The primary endpoint was a composite of morbidity (malaria, pneumonia, and diarrhea) and mortality. Incidence rate ratios (IRRs) were estimated using Poisson regression. Among 538 ART-treated adults screened, 500 were enrolled and randomized, 250 per arm. Median age was 40 y, 361 (72%) were women, and 442 (88%) reported insecticide-treated bednet use. Combined morbidity/mortality was significantly higher in the CTX discontinuation arm (IRR = 2.27, 95% CI 1.52–3.38; p < 0.001), driven by malaria morbidity. There were 34 cases of malaria, with 33 in the CTX discontinuation arm (IRR = 33.02, 95% CI 4.52–241.02; p = 0.001). Diarrhea and pneumonia rates did not differ significantly between arms (IRR = 1.36, 95% CI 0.82–2.27, and IRR = 1.43, 95% CI 0.54–3.75, respectively). Study limitations include a lack of placebo and a lower incidence of morbidity events than expected. Conclusions CTX discontinuation among ART-treated, immune-reconstituted adults in a malaria-endemic region resulted in increased incidence of malaria but not pneumonia or diarrhea. Malaria endemicity may be the most relevant factor to consider in the decision to stop CTX after ART-induced immune reconstitution in regions with high infectious disease prevalence. These data support the 2014 WHO CTX guidelines. Trial registration ClinicalTrials.gov NCT01425073, In a randomized controlled non-inferiority trial, Christina Polyak and colleagues assess whether CTX prophylaxis remains important among HIV+ adults on ART in a malaria-endemic setting., Editors' Summary Background AIDS has killed 39 million people over the past three decades, and 35 million people (mostly living in resource-limited countries) are currently infected with HIV, the virus that causes AIDS. HIV, which is usually transmitted through unprotected sex with an infected individual, gradually destroys CD4 lymphocytes and other immune system cells. Because destruction of the immune system leaves HIV-infected individuals susceptible to “opportunistic” infections, early in the AIDS epidemic, most HIV-positive individuals died within ten years of infection. In 1996, effective antiretroviral therapy (ART)—drug regimens that stop HIV replicating and allow the immune system to recover its ability to fight infections—became available. For people living in affluent countries, HIV/AIDS became a chronic condition, but because ART was expensive, HIV/AIDS remained fatal in resource-limited countries. In 2003, the international community began to work towards achieving universal access to ART. Now, at least a third of people living with HIV have access to ART, and the global mortality (death) rate from HIV/AIDS is falling. Why Was This Study Done? To prevent opportunistic infections, the World Health Organization (WHO) recommends cotrimoxazole (CTX) prophylaxis for HIV-infected individuals. CTX is a combination of two antimicrobial drugs that is active against a range of bacterial, fungal, and parasitic infections; prophylactic antimicrobials are taken to prevent infection. CTX decreases morbidity (illness) and mortality among HIV-infected individuals primarily by reducing the rates of malaria, pneumonia, diarrhea, and severe bacterial infections. The 2006 WHO guidelines for CTX prophylaxis recommend that, in resource-limited countries, HIV-infected patients whose CD4 count is ≤350 cells/mm3 blood should take CTX and that, in settings with a high prevalence of HIV infection and limited health infrastructure, all HIV-infected adults should take CTX. However, these guidelines were developed before access to ART was scaled-up, and CTX prophylaxis has risks (for example, drug toxicity) as well as benefits. In this unblinded non-inferiority randomized controlled trial, the researchers investigate the effects of CTX discontinuation among ART-treated HIV-infected adults in Kenya. A randomized controlled trial compares the outcomes of individuals randomly assigned to different treatments; in an unblinded trial, everyone involved in the trial knows who is taking which treatment; a non-inferiority trial investigates whether one treatment is not worse than another treatment. What Did the Researchers Do and Find? The researchers enrolled 500 HIV-infected adults living in a malaria-endemic region of Kenya (an area where malaria is always present) who had been treated with ART for ≥18 months, who had a CD4 count of >350 cells/mm3, and who were taking CTX. Half the participants continued to take CTX; the remainder stopped taking CTX. After 12 months of follow-up, the combined rate of morbidity (malaria, pneumonia, and diarrhea) and mortality was significantly higher in the CTX discontinuation arm than in the CTX continuation arm (a significant difference is one that is unlikely to have happened by chance). The difference in this primary outcome between the trial arms was driven by malaria morbidity—there were 33 cases of malaria in the CTX discontinuation arm but only one case in the CTX continuation arm. By contrast, the rates of pneumonia and diarrhea did not differ significantly between the trial arms. What Do These Findings Mean? Because the difference between the two trial arms in terms of the primary endpoint was greater than the researchers’ predefined non-inferiority limit, these findings suggest that CTX discontinuation is inferior to CTX continuation among ART-treated, immune-reconstituted HIV-infected adults living in a malaria-endemic region. The accuracy of these findings may be affected by the lack of blinding—the clinicians’ care decisions and the patients’ threshold for seeking care may have been influenced by their knowledge of their trial arm assignment. Overall, however, these findings suggest that malaria endemicity should be considered when making decisions about whether to stop CTX after ART-driven immune reconstitution. Indeed, in December 2014, following the release of preliminary data from this trial and the completion of an independent trial on CTX discontinuation, WHO issued supplemental guidelines on CTX prophylaxis. These state that CTX prophylaxis can be discontinued for HIV-infected adults who are clinically stable on ART and have evidence of immune recovery and viral suppression but recommend that CTX prophylaxis be continued regardless of CD4 cell count or HIV/AIDS clinical stage in settings where malaria is endemic and/or severe bacterial infections are common. Additional Information This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001934. Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS NAM/aidsmap provides basic information about HIV/AIDS, summaries of recent research findings on HIV care and treatment, information on opportunistic infections and on CTX prophylaxis, and personal stories about living with HIV/AIDS Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on HIV/AIDS in Kenya, on ART, and on opportunistic infections and CTX prophylaxis: Avert also provides personal stories about living with HIV/AIDS WHO provides information on all aspects of HIV/AIDS, including its 2013 guidelines on using ART to treat HIV infection and its December 2014 supplement to these guidelines, which contains updated recommendations on CTX prophylaxis; a factsheet about CTX prophylaxis is also available The 2015 World AIDS Day Report provides up-to-date information about the AIDS epidemic, including progress towards universal access to ART More information about this trial is available