A study of vaccine-induced immune pressure on breakthrough infections in the Phambili phase 2b HIV-1 vaccine efficacy trial.

INTRODUCTION: The Merck Adenovirus-5 Gag/Pol/Nef HIV-1 subtype-B vaccine evaluated in predominately subtype B epidemic regions (Step Study), while not preventing infection, exerted vaccine-induced immune pressure on HIV-1 breakthrough infections. Here we investigated if the same vaccine exerted immune pressure when tested in the Phambili Phase 2b study in a subtype C epidemic. MATERIALS AND METHODS: A sieve analysis, which compares breakthrough viruses from placebo and vaccine arms, was performed on 277 near full-length genomes generated from 23 vaccine and 20 placebo recipients. Vaccine coverage was estimated by computing the percentage of 9-mers that were exact matches to the vaccine insert. RESULTS: There was significantly greater protein distances from the vaccine immunogen sequence in Gag (p=0.045) and Nef (p=0.021) in viruses infecting vaccine recipients compared to placebo recipients. Twenty-seven putative sites of vaccine-induced pressure were identified (p<0.05) in Gag (n=10), Pol (n=7) and Nef (n=10), although they did not remain significant after adjustment for multiple comparisons. We found the epitope sieve effect in Step was driven by HLA A∗02:01; an allele which was found in low frequency in Phambili participants compared to Step participants. Furthermore, the coverage of the vaccine against subtype C Phambili viruses was 31%, 46% and 14% for Gag, Pol and Nef, respectively, compared to subtype B Step virus coverage of 56%, 61% and 26%, respectively. DISCUSSION: This study presents evidence of sieve effects in Gag and Nef; however could not confirm effects on specific amino acid sites. We propose that this weaker signal of vaccine immune pressure detected in the Phambili study compared to the Step study may have been influenced by differences in host genetics (HLA allele frequency) and reduced impact of vaccine-induced immune responses due to mismatch between the viral subtype in the vaccine and infecting subtypes.

Does participation in an HIV vaccine efficacy trial affect risk behaviour in South Africa?

BACKGROUND: Increased sexual risk behaviour in participants enrolled in HIV prevention trials has been a concern. The HVTN 503/Phambili study, a phase 2B study of the Merck Ad-5 multiclade HIV vaccine in South Africa, suspended enrollment and vaccinations following the results of the Step study. Participants were notified of their treatment allocation and continue to be followed. We investigated changes in risk behaviour over time and assessed the impact of study unblinding. METHODS: 801 participants were enrolled. Risk behaviours were assessed with an interviewer-administered questionnaire at 6-month intervals. We assessed change from enrolment to the first 6-month assessment pre-unblinding and between enrolment and at least 6 months post-unblinding on all participants with comparable data. A one-time unblinding risk perception questionnaire was administered post-unblinding. RESULTS: A decrease in participants reporting unprotected sex was observed in both measured time periods for men and women, with no differences by treatment arm. At 6 months (pre-unblinding), 29.6% of men and 35.8% of women reported changing from unprotected to protected sex (p<0.0001 for each). Men (22%) were more likely than women (14%) to report behaviour change after unblinding (p=0.009). Post-enrolment, 142 (45%) of 313 previously uncircumcised men underwent medical circumcision. 663 participants completed the unblinding questionnaire. More vaccine (24.6%) as compared to placebo recipients (12.0%) agreed that they were more likely to get HIV than most people (p<0.0001), and attributed this to receiving the vaccine. CONCLUSION: We did not find evidence of risk compensation during this clinical trial. Some risk behaviour reductions including male circumcision were noted irrespective of treatment allocation.

Comparison of chlorproguanil-dapsone with sulfadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria in young African children: double-blind randomised controlled trial.

BACKGROUND: Increasing resistance to sulfadoxine-pyrimethamine is leading to a decline in its effectiveness. We aimed to assess the safety profile of chlorproguanil-dapsone (CD), and to compare the safety and efficacy of this drug with that of sulfadoxine-pyrimethamine (SP) as treatment for uncomplicated falciparum malaria. METHODS: We undertook a double-blind, randomised trial in 1850 consecutively recruited children with uncomplicated falciparum malaria, pooling data from five African countries. Analyses were based on all randomised patients with available data. FINDINGS: CD was significantly more efficacious than SP (odds ratio 3.1 [95% CI 2.0-4.8]); 1313 patients (96%) given CD and 306 (89%) given SP achieved acceptable clinical and parasitological response by day 14. Adverse events were reported in 46% and 50% of patients randomised to CD and SP, respectively (treatment difference -4.4%, [95% CI -10.1 to 1.3]). Haemoglobin in the CD group was significantly lower than in the SP group at day 7, a difference of -4 g/L (95% CI -6 to -2). Mean day 14 haemoglobin (measured only for the small number of patients whose day 7 data caused concern) was 94 g/L (92-96) and 97 g/L (92-102) after CD and SP, respectively. Glucose-6-phosphate dehydrogenase deficient patients on CD had greater odds than those on SP of having a fall of 20 g/dL or more in haemoglobin when baseline temperature was high. Methaemoglobinaemia was seen in the CD group (n=320, mean 0.4% [95% CI 0.4-0.4]) before treatment, 4.2% (95% CI 3.8-4.6) (n=301) at day 3, and 0.6% (0.6-0.7) (n=300) at day 7). INTERPRETATION: CD had greater efficacy than SP in Africa and was well tolerated. Haematological adverse effects were more common with CD than with SP and were reversible. CD is a useful alternative where SP is failing due to resistance.

Ocular disease in patients with TB and HIV presenting with fever in Malawi.

AIMS: To investigate ocular disease in Malawian patients with tuberculosis (TB) and HIV in presenting with fever, and to determine if indirect ophthalmoscopy is useful in the diagnosis of mycobacteraemia. METHODS: A prospective study of all adult patients admitted with fever to Queen Elizabeth Central Hospital, Blantyre. All recruited patients had an ophthalmic examination, HIV tests, chest x-ray, sputum examinations, bacterial and mycobacterial blood cultures and malaria slide. RESULTS: 307 patients were recruited; 109 (36%) had TB, including 53 (17%) with mycobacteraemia; 255 (83%) had HIV and 191 (62%) had AIDS. Of the patients with TB 102 (94%) had HIV. Choroidal granulomas were found in four patients, all of whom had AIDS; three had disseminated TB with mycobacteraemia, and one had persistent fever but no other evidence of TB. Among the patients with AIDS, 32 (17%) had retinal microangiopathy manifest by cotton wool spots; one (0.5%) had signs of active cytomegalovirus (CMV) retinitis. The presence of microangiopathy was not related to TB. CONCLUSIONS: In Malawian patients with TB presenting acutely with fever, choroidal granulomas were found in 2.8%, and were concurrent with mycobacteraemia and AIDS. Ophthalmoscopy was not a useful aid in the diagnosis of mycobacteraemia. CMV retinitis is rarely seen in African AIDS patients. This may be due to mortality early in the disease course, or differences in race, HIV sub-type or co-morbidity.

Clinical indicators of mycobacteraemia in adults admitted to hospital in Blantyre.

The aims of the study were to measure the prevalence and outcome of mycobacteraemia in febrile hospitalised adults; to determine what proportion could be identified using routine methods; to assess clinical indicators of mycobacteraemia and the usefulness of a diagnostic trial of anti-TB treatment. We prospectively examined adults with fever or a history of fever admitted to adult medical wards of QECH, Blantyre. All had blood cultured for bacteria and mycobacteria, chest x-ray and sputum smears. M. tuberculosis was the commonest blood isolate, affecting 57 of 344 patients (17%). In 44 (77%) patients with mycobacteraemia, TB was identified using routine investigations; in only 6 (11%) it was not suspected. Strong clinical indicators of mycobacteraemia were anaemia, HIV seropositivity, cough, chronic fever, and a clinical diagnosis of AIDS on the day of admission. Of nine patients selected for a therapeutic trial of TB treatment, six had mycobacteraemia, of whom five died during the trial. Mortality on short course chemotherapy on the TB ward after one month, was similar whether patients had mycobacteramia (21%) or not (32%). TB can be identified with routine methods in most patients with mycobacteraemia. If treated, mycobacteraemia has as good an early outcome as TB without mycobacteraemia. Strengthening of basic facilities is likely to improve detection and treatment of mycobacterial disease.

Chlorproguanil-dapsone versus sulfadoxine-pyrimethamine for sequential episodes of uncomplicated falciparum malaria in Kenya and Malawi: a randomised clinical trial.

BACKGROUND: Chlorproguanil-dapsone exerts lower resistance pressure on Plasmodium falciparum than does sulfadoxine-pyrimethamine, but is rapidly eliminated. We aimed to find out whether chlorproguanil-dapsone results in a higher retreatment rate for malaria than sulfadoxine-pyrimethamine. METHODS: In a randomised trial of paediatric outpatients with uncomplicated falciparum malaria, patients received either chlorproguanil-dapsone or sulfadoxine-pyrimethamine and were followed up for up to 1 year. Sites were in Kenya (n=410) and Malawi (n=500). We used per-protocol analysis to assess the primary outcome of annual malaria incidence. FINDINGS: Drop-outs were 117 of 410 (28.5%) in Kenya, and 342 of 500 (68.4%) in Malawi. Follow-up was for a median of 338 days (IQR 128-360) and 342 days (152-359) in Kilifi (chlorproguanil-dapsone and sulfadoxine-pyrimethamine, respectively), and for 120 days (33-281) and 84 days (26-224) in Blantyre. Mean annual malaria incidence was 2.5 versus 2.1 in Kenya (relative risk 1.16, 95% CI 0.98-1.37), and 2.2 versus 2.8 in Malawi (0.77, 0.63-0.94). 4.3% versus 12.8%, and 5.4% versus 20.1%, of patients were withdrawn for treatment failure in Kenya and Malawi, respectively. In Kenya haemoglobin concentration of 50 g/L or less caused exit in 6.9% of chlorproguanil-dapsone patients and 1.5% of sulfadoxine-pyrimethamine patients, but most anaemia occurred before re-treatment. In Malawi only one patient exited because of anaemia. INTERPRETATION: Despite the rapid elimination of chlorproguanil-dapsone, children treated with this drug did not have a higher incidence of malaria episodes than those treated with sulfadoxine-pyrimethamine. Treatment failure was more common with sulfadoxine-pyrimethamine. Cause of anaemia in Kenya was probably not adverse reaction to chlorproguanil-dapsone, but this observation requires further study.

Ocular disease in patients with tuberculosis and HIV presenting with fever in Africa.

AIMS: To investigate ocular disease in patients with tuberculosis (TB) and HIV in Africa presenting with fever, and to determine if indirect ophthalmoscopy is useful in the diagnosis of mycobacteraemia. METHODS: A prospective study of all adult patients admitted with fever to a large central hospital in Malawi, Africa. All recruited patients had an ophthalmic examination, HIV tests, chest x ray, sputum examinations, bacterial and mycobacterial blood cultures, and malaria slide to observe the presence of parasites. RESULTS: 307 patients were recruited; 109 (36%) had TB, including 53 (17%) with mycobacteraemia; 255 (83%) had HIV and 191 (62%) had AIDS. Of the patients with TB 102 (94%) had HIV. Choroidal granulomas were found in four patients, all of whom had AIDS; three (2.8% of those with TB) had disseminated TB with mycobacteraemia, and one had persistent fever but no other evidence of TB. Among the patients with AIDS, 32 (17%) had microangiopathy manifest by cotton wool spots; one (0.5%) had signs of active cytomegalovirus (CMV) retinitis. The presence of microangiopathy was not related to TB. CONCLUSIONS: In Malawian patients with TB presenting acutely with fever, choroidal granulomas were found in 2.8%, and were concurrent with mycobacteraemia and AIDS. Ophthalmoscopy was not a useful aid in the diagnosis of mycobacteraemia. Cytomegalovirus (CMV) retinitis is rarely seen in African AIDS patients. This may be the result of mortality early in the disease course, or differences in race, HIV subtype, or comorbidity.

Clinical indicators of mycobacteraemia in adults admitted to hospital in Blantyre, Malawi.

SETTING: Adult medical wards of a central hospital in Blantyre, Malawi. OBJECTIVE: To measure the prevalence and outcome of mycobacteraemia in febrile hospitalised adults, and to determine what proportion could be identified using routine methods; to assess clinical indicators of mycobacteraemia, and the usefulness of a diagnostic trial of anti-tuberculosis treatment. DESIGN: We prospectively examined adults admitted with fever or a history of fever. All had blood cultured for bacteria and mycobacteria, chest X-ray and sputum smears. FINDINGS: Mycobacterium tuberculosis was the commonest cause of blood stream infection (BSI), affecting 57 of 344 patients (17%). In 44 (77%) patients with mycobacteraemia, TB was identified using routine investigations; it was not suspected in six (11%). Strong clinical indicators of mycobacteraemia were anaemia, HIV seropositivity, cough, chronic fever and a clinical diagnosis of AIDS on the day of admission. Of nine patients selected for a therapeutic trial of tuberculosis (TB) treatment, six had mycobacteraemia, of whom five died during the trial. Mortality on short-course chemotherapy, on the TB ward after 1 month was similar whether patients had mycobacteremia (21%) or not (32%). CONCLUSION: TB can be identified with routine methods in most patients with mycobacteraemia. If treated, mycobacteraemia has as good an early outcome as TB without mycobacteraemia. Strengthening of basic facilities is likely to improve detection and treatment of mycobacterial disease.

Plasmodium falciparum cross-resistance between trimethoprim and pyrimethamine.

Trimethoprim-sulfamethoxazole has been recommended as part of the standard package of care for people with HIV and AIDS in Africa. A similar antifolate combination, sulfadoxine-pyrimethamine, is now the first-line antimalarial drug in several of the African countries with the highest rates of HIV infection. We present evidence of Plasmodium falciparum cross-resistance between trimethoprim and pyrimethamine at the molecular level. The impact of trimethoprim-sulfamethoxazole on the efficacy of sulfadoxine-pyrimethamine needs to be assessed urgently, and alternative antimalarial treatment should be considered for people on trimethoprim-sulfamethoxazole prophylaxis.

Pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: what next?

Chemotherapy remains the only practicable tool to control falciparum malaria in sub-Saharan Africa, where >90% of the world's burden of malaria mortality and morbidity occurs. Resistance is rapidly eroding the efficacy of chloroquine, and the combination pyrimethamine-sulfadoxine is the most commonly chosen alternative. Resistant populations of Plasmodium falciparum were selected extremely rapidly in Southeast Asia and South America. If this happens in sub-Saharan Africa, it will be a public health disaster because no inexpensive alternative is currently available. This article reviews the molecular mechanisms of this resistance and discusses how to extend the therapeutic life of antifolate drugs.

Rapid selection of Plasmodium falciparum dihydrofolate reductase mutants by pyrimethamine prophylaxis.

A prospective study was conducted to measure the selective effect of pyrimethamine prophylaxis on point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR). A total of 109 Malian children were given pyrimethamine weekly for 5 weeks. P. falciparum infections were analyzed by polymerase chain reaction for DHFR mutations, which were dramatically more frequent among prophylaxis-breakthrough infections than at baseline: the prevalence of Asn-108 rose from 13% to 100%, Ile-51 from 4% to 50%, and Arg-59 from 11% to 90%. Eight persistent infections lacking detectable DHFR mutations at baseline developed multiple mutations within 1 week of the patients' starting pyrimethamine prophylaxis. Microsatellite analysis found no evidence of clonal identity among baseline and breakthrough infections. Analysis of these data demonstrates that under prophylaxis conditions, pyrimethamine is strongly selective for DHFR mutations, which arise extremely rapidly under drug pressure, even when undetectable in the initial infection. These findings have implications for prophylaxis regimens with other antifolate drugs.

P. falciparum dihydrofolate reductase and dihydropteroate synthase mutations: epidemiology and role in clinical resistance to antifolates.

Plasmodium falciparum resistance to the antifolates has arisen rapidly in Asia and South America, and threatens the usefulness of these drugs in Africa. In vitro resistance to the antifolates is determined by mutations in parasite dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS). The role of DHFR and DHPS mutations in therapeutic failure of antifolate antimalarials is less clear. This review summarizes molecular epidemiological surveys, studies of in vivo selection of mutant alleles by drug treatment, and prospective studies of the ability of mutation-specific assays to predict clinical outcomes, and discusses the potential use of these assays for surveillance of resistance.