BackgroundEarly recognition of antiretroviral therapy (ART) failure in resource limited settings is a challenge given the limited laboratory facilities and trained personnel. This study aimed at describing the incidence, risk factors and the resistance associated mutations (RAMs) of first line treatment failure among HIV-1-infected children attending the Joint Clinical Research Centre (JCRC), Kampala, Uganda.MethodsA retrospective cohort of 701 children who had been initiated on ART between January 2004 and September 2009 at the JCRC was studied. Data of children aged 6 months up to 18 years who had been started on ART for at least 6 months was extracted from the clinic charts. The children who failed the first-line ART were taken as cases and those who did not fail as the controls. Data was analysed using STATA version10.ResultsOf 701 children, 240(34%) failed on first line ART (cases) and 461(66%) did not fail (controls). The overall median time (IQR) to first line ART failure was 26.4 (18.9 – 39.1) months. The factors associated with treatment failure were poor adherence [(OR = 10, 95 CI: 6.4 – 16.7) p < 0.001], exposure to single dose nevirapine (sdNVP) [(OR = 4.2, 95% CI:1.8-9.4), p = 0.005] and a NVP containing regimen [(OR = 2.2,95% CI:1.4-3.6), p < 0.001]. Of 109 genotypic resistance profiles analyzed, the commonest non nucleoside reverse transcriptase inhibitor (NNRTI) resistance associated mutations (RAM) were: K103N (59; 54%)), Y181C (36; 27%)) and G190A (26; 24%)) while the commonest nucleoside reverse transcriptase inhibitor (NRTI) RAM was the M184V (89; 81%). Thymidine analogue- mutations (TAMs) were detected in 20% of patients.ConclusionsOne in three children on first-line ART are likely to develop virological treatment failure after the first 24 months of therapy. Poor adherence to ART, a NVP based first-line regimen, prior exposure to sdNVP were associated with treatment failure.

Overall immunological and virologic ART responses were similar in children in both cohorts. Poorer CD4 recovery in more immunosuppressed Kampala children and blunted growth responses likely reflect higher background malnutrition and infection rates in Uganda, suggesting the need for earlier HIV diagnosis, nutritional support, cotrimoxazole prophylaxis, and ART.

Tuberculosis (TB) remains a significant, yet under-recognized cause of death in the pediatric population, with a WHO estimate of 1 million new cases of childhood TB in 2016 resulting in 250,000 deaths. Diagnosis is notoriously difficult; manifestations are protean due to the high proportion of cases of extra-pulmonary TB in children, and logistical problems exist in obtaining suitable specimens. These issues are compounded by the paucibacillary nature of disease with the result that an estimated 96% of pediatric TB-associated mortality occurs prior to commencing anti-tuberculous treatment. Further development of sensitive, rapid diagnostic tests and their incorporation into diagnostic algorithms is vital in this population, and central to the WHO End-TB strategy. Initial gains were made with the expansion of nucleic acid amplification technology, particularly the introduction of the GeneXpert fully-automated PCR Xpert MTB/Rif assay in 2010, and more recently, the Xpert MTB/Rif Ultra (Ultra) assay in 2017. Ultra provides increased analytical sensitivity when compared with the initial Xpert assay in vitro ; a finding now also supported by six clinical studies to date, two of which included pediatric samples. Here, we review the published evidence for the performance of Ultra in TB diagnosis in children, as well as studies in adults with paucibacillary disease providing results relevant to the pediatric population. Following on from this, we speculate upon future directions for Ultra, with focus on its potential use with alternative diagnostic specimens, which may be of particular utility in children.

Enterocytozoon bieneusi is clinically the most significant of the microsporidia in humans, causing chronic diarrhea wasting and cholangitis in individuals with human immunodeficiency virus infection and AIDS. Little progress on this infection has been made because of the inability to propagate E. bieneusi in vitro and in vivo, which limits the source of parasite spores to the stools of infected human patients. Given the size and shape of the E. bieneusi spores (1.1 to 1.6 by 0.7 to 1.0 m) and the lack of specific immune reagents, the identification and purification of large quantities of spores from feces are technically challenging. Consequently, diagnosis relies entirely on PCR, a labor-intensive approach that requires highly skilled personnel. We describe a method for the purification of E. bieneusi spores from human stools and the production of rabbit-specific antisera. Spores were purified by a combination of isopycnic Percoll gradient centrifugation and continuous sucrose gradient centrifugation. Specific polyclonal antibodies raised in mice and rabbits reacted by indirect immunofluorescence with E. bieneusi but not with Encephalitozoon spp., Candida albicans, Staphylococcus aureus, Escherichia coli, or other forms present in human stools.Enterocytozoon bieneusi is a widespread enteric microsporidium which probably infects most mammals. The infection was first identified two decades ago in individuals with AIDS with severe symptoms of chronic diarrhea and wasting (10) and has remained a significant pathogen in this subpopulation ever since (5,6,18). E. bieneusi is recognized as the most common and clinically significant organism among the microsporidia which infect humans who have immunodeficiencies (18,21) or who are receiving immunosuppressive therapy (13,22). E. bieneusi is only rarely symptomatic in immunocompetent individuals, possibly contributing to traveler's diarrhea (11). It is an intracellular microorganism which appears to infect the epithelium of the upper small intestine and the hepatobiliary tract (26), causing chronic diarrhea and cholangitis. The number of E. bieneusi cases has diminished considerably in developed countries due to the use of antiretroviral therapy in people with human immunodeficiency virus infection and AIDS (12). The number of cases, however, in such individuals in developing countries, where antiretroviral therapy is either not available or not affordable, remains very high (4, 14, 23).Scientific progress on E. bieneusi has been slow, largely because of a lack of in vitro and in vivo models for parasite propagation and laboratory investigations. While E. bieneusi did appear to infect cells in culture, the spore yields were low and continuous culture could not be maintained (25). Natural infections of immunologically competent and immunodeficient macaques have also been reported, and the distribution of infection and lesions are similar to those seen in infected humans (8,9,16,17,20). In macaques infected with simian immunodeficiency virus, lesions associated with E. bie...