Therapeutic response to four artemisinin-based combination therapies in Angola, 2021.

Monitoring antimalarial efficacy is important to detect the emergence of parasite drug resistance. Angola conducts in vivo therapeutic efficacy studies (TESs) every 2 years in its fixed sentinel sites in Benguela, Lunda Sul, and Zaire provinces. Children with uncomplicated Plasmodium falciparum malaria were treated with artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ), dihydroartemisinin-piperaquine (DP), or artesunate-pyronaridine (ASPY) and followed for 28 (AL and ASAQ) or 42 days (DP and ASPY) to assess clinical and parasitological response to treatment. Two drugs were sequentially assessed in each site in February-July 2021. The primary indicator was the Kaplan-Meier estimate of the PCR-corrected efficacy at the end of the follow-up period. A total of 622 patients were enrolled in the study and 590 (95%) participants reached a study endpoint. By day 3, ≥98% of participants were slide-negative in all study sites and arms. After PCR correction, day 28 AL efficacy was 88.0% (95% CI: 82%-95%) in Zaire and 94.7% (95% CI: 90%-99%) in Lunda Sul. For ASAQ, day 28 efficacy was 92.0% (95% CI: 87%-98%) in Zaire and 100% in Lunda Sul. Corrected day 42 efficacy was 99.6% (95% CI: 99%-100%) for ASPY and 98.3% (95% CI: 96%-100%) for DP in Benguela. High day 3 clearance rates suggest no clinical evidence of artemisinin resistance. This was the fourth of five rounds of TES in Angola showing a corrected AL efficacy <90% in a site. For Zaire, AL has had an efficacy <90% in 2013, 2015, and 2021. ASAQ, DP, and ASPY are appropriate choices as artemisinin-based combination therapies in Angola.

Molecular Markers of Sulfadoxine-Pyrimethamine Resistance in Samples from Children with Uncomplicated Plasmodium falciparum at Three Sites in Angola in 2019.

Sulfadoxine-pyrimethamine (SP) is used for prevention of malaria in pregnant women in Angola. We sequenced the Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes, implicated in SP resistance, in samples collected during a 2019 study of artemisinin-based combination therapy efficacy in Benguela, Lunda Sul, and Zaire provinces. A total of 90 day 0 and day of failure samples were individually sequenced, while 508 day 0 samples from participants without recurrent parasitemia were pooled after DNA extraction into 61 pools. The N51I, C59R, and S108N pfdhfr mutations and A437G pfdhps mutations were present at high proportions in all provinces (weighted allele frequencies, 62% to 100%). The K540E pfdhps mutation was present at lower proportions (10% to 14%). The A581G pfdhps mutation was only observed in Zaire, at a 4.6% estimated prevalence. The I431V and A613S mutations were also only observed in Zaire, at a prevalence of 2.8% to 2.9%. The most common (27% to 66%) reconstructed haplotype in all three provinces was the canonical quadruple pfdhfr pfdhps mutant. The canonical quintuple mutant was absent in Lunda Sul and Benguela and present in 7.9% of samples in Zaire. A single canonical sextuple (2.6%) mutant was observed in Zaire Province. Proportions of the pfdhps K540E and A581G mutations were well below the World Health Organization thresholds for meaningful SP resistance (prevalence of 95% for K540E and 10% for A581G). Samples from therapeutic efficacy studies represent a convenient source of samples for monitoring SP resistance markers.

Malaria in Angola: recent progress, challenges and future opportunities using parasite demography studies.

Over the past two decades, a considerable expansion of malaria interventions has occurred at the national level in Angola, together with cross-border initiatives and regional efforts in southern Africa. Currently, Angola aims to consolidate malaria control and to accelerate the transition from control to pre-elimination, along with other country members of the Elimination 8 initiative. However, the tremendous heterogeneity in malaria prevalence among Angolan provinces, as well as internal population movements and migration across borders, represent major challenges for the Angolan National Malaria Control Programme. This review aims to contribute to the understanding of factors underlying the complex malaria situation in Angola and to encourage future research studies on transmission dynamics and population structure of Plasmodium falciparum, important areas to complement host epidemiological information and to help reenergize the goal of malaria elimination in the country.

Continued Low Efficacy of Artemether-Lumefantrine in Angola in 2019.

Biennial therapeutic efficacy monitoring is a crucial activity for ensuring the efficacy of currently used artemisinin-based combination therapy in Angola. Children with acute uncomplicated Plasmodium falciparum infection in sentinel sites in the Benguela, Zaire, and Lunda Sul Provinces were treated with artemether-lumefantrine (AL) or artesunate-amodiaquine (ASAQ) and monitored for 28 days to assess clinical and parasitological responses. Molecular correction was performed using seven microsatellite markers. Samples from treatment failures were genotyped for the pfk13, pfcrt, and pfmdr1 genes. Day 3 clearance rates were ≥95% in all arms. Uncorrected day 28 Kaplan-Meier efficacy estimates ranged from 84.2 to 90.1% for the AL arms and 84.7 to 100% for the ASAQ arms. Corrected day 28 estimates were 87.6% (95% confidence interval [CI], 81 to 95%) for the AL arm in Lunda Sul, 92.2% (95% CI, 87 to 98%) for AL in Zaire, 95.6% (95% CI, 91 to 100%) for ASAQ in Zaire, 98.4% (95% CI, 96 to 100%) for AL in Benguela, and 100% for ASAQ in Benguela and Lunda Sul. All 103 analyzed samples had wild-type pfk13 sequences. The 76T pfcrt allele was found in most (92%; 11/12) ASAQ late-failure samples but in only 16% (4/25) of AL failure samples. The N86 pfmdr1 allele was found in 97% (34/35) of treatment failures. The AL efficacy in Lunda Sul was below the 90% World Health Organization threshold, the third time in four rounds that this threshold was crossed for an AL arm in Angola. In contrast, the observed ASAQ efficacy has not been below 95% to date in Angola, including this latest round.

Malaria Test, Treat and Track policy implementation in Angola: a retrospective study to assess the progress achieved after 4 years of programme implementation.

BACKGROUND: Malaria is one of the main causes of death in Angola, particularly among children under 5 years of age. An essential means to improve the situation is with strong malaria case management; this includes diagnosing suspected patients with a confirmatory test, either with a rapid diagnostic test (RDT) or microscopy, prompt and correct treatment with artemisinin-based combination therapy (ACT), and proper case registration (track). In 2011, the United States President's Malaria Initiative (PMI) launched a country-wide programme to improve malaria case management through the provision of regular training and supervision at different levels of health care provision. An evaluation of malaria testing, treatment and registration practices in eight provinces, and at health facilities of various capacities, across Angola was conducted to assess progress of the national programme implementation. METHODS: A retrospective assessment analysed data collected during supervision visits to health facilities conducted between 2012 and 2016 in 8 provinces in Angola. The supervision tool used data collected for malaria knowledge, testing, treatment and case registration practices among health workers as well as health facilities stock outs from different levels of health care delivery. Contingency tables with Pearson chi-squared (χ2) tests were used to identify factors associated with "knowledge", "test", "treat" and "track." Multivariable logistic regression models were used to assess factors associated with the defined outcomes. RESULTS: A total of 7156 supervisions were conducted between September 2012 and July 2016. The overall knowledge, testing, treatment and tracking practices among health care workers (HCWs) increased significantly from 2013 to 2016. Health care workers in 2016 were 3.3 times (95% CI: 2.7-3.9) as likely to have a higher knowledge about malaria case management as in 2013 (p < 0.01), 7.4 (95% CI: 6.1-9.0) times as likely to test more suspected cases (p < 0.01), 10.9 (95% CI: 8.6-13.6) times as likely to treat more confirmed cases (p < 0.01) and 3.7 (95% CI: 3.2-4.4) times as likely to report more accurately in the same period (p < 0.01). DISCUSSION: Improvements demonstrated in knowledge about malaria case management, testing with RDT and treatment with artemisinin-based combinations among HCWs is likely associated with malaria case management trainings and supportive supervisions. Gaps in testing and treatment practices are associated with RDT and ACT medicines stock outs in health facilities. Tracking of malaria cases still poses a major challenge, despite training and supervision. Hospitals consistently performed better compared to other health facilities against all parameters assessed; likely due to a better profile of HCWs. CONCLUSION: Significant progress in malaria case management in eight provinces Angola was achieved in the period of 2013-2016. Continued training and supportive supervision is essential to sustain gains and close existing gaps in malaria case management and reporting in Angola.

Attrition, physical integrity and insecticidal activity of long-lasting insecticidal nets in sub-Saharan Africa and modelling of their impact on vectorial capacity.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the primary malaria prevention and control intervention in many parts of sub-Saharan Africa. While LLINs are expected to last at least 3 years under normal use conditions, they can lose effectiveness because they fall out of use, are discarded, repurposed, physically damaged, or lose insecticidal activity. The contributions of these different interrelated factors to durability of nets and their protection against malaria have been unclear. METHODS: Starting in 2009, LLIN durability studies were conducted in seven countries in Africa over 5 years. WHO-recommended measures of attrition, LLIN use, insecticidal activity, and physical integrity were recorded for eight different net brands. These data were combined with analyses of experimental hut data on feeding inhibition and killing effects of LLINs on both susceptible and pyrethroid resistant malaria vectors to estimate the protection against malaria transmission-in terms of vectorial capacity (VC)-provided by each net cohort over time. Impact on VC was then compared in hypothetical scenarios where one durability outcome measure was set at the best possible level while keeping the others at the observed levels. RESULTS: There was more variability in decay of protection over time by country than by net brand for three measures of durability (ratios of variance components 4.6, 4.4, and 1.8 times for LLIN survival, use, and integrity, respectively). In some countries, LLIN attrition was slow, but use declined rapidly. Non-use of LLINs generally had more effect on LLIN impact on VC than did attrition, hole formation, or insecticide loss. CONCLUSIONS: There is much more variation in LLIN durability among countries than among net brands. Low levels of use may have a larger impact on effectiveness than does variation in attrition or LLIN degradation. The estimated entomological effects of chemical decay are relatively small, with physical decay probably more important as a driver of attrition and non-use than as a direct cause of loss of effect. Efforts to maximize LLIN impact in operational settings should focus on increasing LLIN usage, including through improvements in LLIN physical integrity. Further research is needed to understand household decisions related to LLIN use, including the influence of net durability and the presence of other nets in the household.

Screening for Pfhrp2/3-Deleted Plasmodium falciparum, Non-falciparum, and Low-Density Malaria Infections by a Multiplex Antigen Assay.

Background: Detection of Plasmodium antigens provides evidence of malaria infection status and is the basis for most malaria diagnosis. Methods: We developed a sensitive bead-based multiplex assay for laboratory use, which simultaneously detects pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and P. falciparum histidine-rich protein 2 (PfHRP2) antigens. The assay was validated against purified recombinant antigens, monospecies malaria infections, and noninfected blood samples. To test against samples collected in an endemic setting, Angolan outpatient samples (n = 1267) were assayed. Results: Of 466 Angolan samples positive for at least 1 antigen, the most common antigen profiles were PfHRP2+/pAldo+/pLDH+ (167, 36%), PfHRP2+/pAldo-/pLDH- (163, 35%), and PfHRP2+/pAldo+/pLDH- (129, 28%). Antigen profile was predictive of polymerase chain reaction (PCR) positivity and parasite density. Eight Angolan samples (1.7%) had no or very low PfHRP2 but were positive for 1 or both of the other antigens. PCR analysis confirmed 3 (0.6%) were P. ovale infections and 2 (0.4%) represented P. falciparum parasites lacking Pfhrp2 and/or Pfhrp3. Conclusions: These are the first reports of Pfhrp2/3 deletion mutants in Angola. High-throughput multiplex antigen detection can inexpensively screen for low-density P. falciparum, non-falciparum, and Pfhrp2/3-deleted parasites to provide population-level antigen estimates and identify specimens requiring further molecular characterization.

Performance of Antigen Concentration Thresholds for Attributing Fever to Malaria among Outpatients in Angola.

The density of malaria parasites is a key determinant of whether an infected individual develops fever. While the pyrogenic threshold for malaria parasite density has been well studied, there are no analogous data on the antigen levels associated with fever during infection. Samples from 797 afebrile and 457 febrile outpatients from two provinces in Angola with known concentrations of histidine-rich protein 2 (HRP2), aldolase, and lactate dehydrogenase (LDH) antigens were analyzed by Bayesian latent class modeling to attribute malarial etiology to the fevers and to estimate the sensitivity and specificity of different antigen thresholds for detection of malaria fevers. Among patients with aldolase or LDH levels detectable with a bead-based assay, the concentrations of these two antigens did not differ between afebrile and febrile patients. In contrast, the concentrations of HRP2 were substantially higher in febrile HRP2-positive patients than in afebrile HRP2-positive patients. When HRP2 concentrations were considered, the malaria-attributable fractions of fever cases were 0.092 in Huambo Province and 0.39 in Uíge Province. Diagnostic tests detecting HRP2 with limits of detection (LODs) in the range of 3,000 to 10,000 pg/µl would provide ideal sensitivity and specificity for determination of malarial etiology among febrile persons.

Posttreatment HRP2 Clearance in Patients with Uncomplicated Plasmodium falciparum Malaria.

Background: The response to antimalarial treatment is assessed using serial microscopy. New techniques for accurate measurement of the Plasmodium falciparum histidine-rich protein 2 (HRP2) antigen have allowed for monitoring of the antigen concentration over time, offering a potential alternative for assessing treatment response. Methods: Posttreatment HRP2 concentrations were measured in samples obtained longitudinally from 537 individuals with P. falciparum malaria who were participating in efficacy trials in Angola, Tanzania, and Senegal. The HRP2 half-life was estimated using a first-order kinetics clearance model. The association between the HRP2 concentration 3 days after treatment and recrudescence of infection was assessed. Results: Despite substantial variation in HRP2 concentrations among participants at baseline, concentrations consistently showed a first-order exponential decline. The median half-life of HRP2 was estimated to be 4.5 days (interquartile range [IQR], 3.3-6.6 days) in Angola, 4.7 days (IQR, 4.0-5.9 days) in Tanzania, and 3.0 days (IQR, 2.1-4.5 days) in Senegal. The day 3 HRP2 concentration was predictive of eventual recrudescence, with an area under the receiver operating characteristic curve of 0.86 (95% confidence interval, .73-.99). Conclusions: Consistent HRP2 clearance dynamics following successful antimalarial treatment imply a common underlying mechanism of biological clearance. Patients who ultimately did not respond to treatment did not exhibit this same pattern of clearance, even in the absence of other indications of inadequate response to treatment.

Prevalence of molecular markers of artemisinin and lumefantrine resistance among patients with uncomplicated Plasmodium falciparum malaria in three provinces in Angola, 2015.

BACKGROUND: Artemisinin-based combination therapy is the first-line anti-malarial treatment for uncomplicated Plasmodium falciparum infection in Angola. To date, the prevalence of polymorphisms in the pfk13 gene, associated with artemisinin resistance, and pfmdr1, associated with lumefantrine resistance, have not been systematically studied in Angola. METHODS: DNA was isolated from pretreatment and late treatment failure dried blood spots collected during the 2015 round of therapeutic efficacy studies in Benguela, Lunda Sul, and Zaire Provinces in Angola. The pfk13 propeller domain and pfmdr1 gene were sequenced and analysed for polymorphisms. Pfmdr1 copy number variation was assessed using a real-time PCR method. The association between pfmdr1 and pfk13 mutations and treatment failure was investigated. RESULTS: The majority of pretreatment (99%, 466/469) and all late treatment failure (100%, 50/50) samples were wild type for pfk13. Three of the pretreatment samples (1%) carried the A578S mutation commonly observed in Africa and not associated with artemisinin resistance. All 543 pretreatment and day of late treatment failure samples successfully analysed for pfmdr1 copy number variation carried one copy of pfmdr1. The NYD haplotype was the predominant pfmdr1 haplotype, present in 63% (308/491) of pretreatment samples, followed by NFD, which was present in 32% (157/491) of pretreatment samples. The pfmdr1 N86 allele was overrepresented in day of late treatment failure samples from participants receiving artemether-lumefantrine (p value 0.03). CONCLUSIONS: The pretreatment parasites in patients participating in therapeutic efficacy studies in 2015 in Angola's three sentinel sites showed genetic evidence of susceptibility to artemisinins, consistent with clinical outcome data showing greater than 99% day 3 clearance rates. The lack of increased pfmdr1 copy number is consistent with previous reports from sub-Saharan Africa. Although pfmdr1 NYD and NFD haplotypes were overrepresented in artemether-lumefantrine late treatment failure samples, their role as markers of resistance was unclear given that these haplotypes were also present in the majority of successfully treated patients in the artemether-lumefantrine treatment arms.

Efficacy and safety of artemether-lumefantrine, artesunate-amodiaquine, and dihydroartemisinin-piperaquine for the treatment of uncomplicated Plasmodium falciparum malaria in three provinces in Angola, 2017.

BACKGROUND: The Angolan government recommends three artemisinin-based combinations for the treatment of uncomplicated Plasmodium falciparum malaria: artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ), and dihydroartemisinin-piperaquine (DP). Due to the threat of emerging anti-malarial drug resistance, it is important to periodically monitor the efficacy of artemisinin-based combination therapy (ACT). This study evaluated these medications' therapeutic efficacy in Benguela, Lunda Sul, and Zaire Provinces. METHODS: Enrollment occurred between March and July 2017. Study participants were children with P. falciparum monoinfection from each provincial capital. Participants received a 3-day course of a quality-assured artemisinin-based combination and were monitored for 28 (AL and ASAQ arms) or 42 days (DP arm). Each ACT was assessed in two provinces. The primary study endpoints were: (1) follow-up without complications and (2) failure to respond to treatment or development of recurrent P. falciparum infection. Parasites from each patient experiencing recurrent infection were genotyped to differentiate new infection from recrudescence of persistent parasitaemia. These parasites were also analysed for molecular markers associated with ACT resistance. RESULTS: Of 608 children enrolled in the study, 540 (89%) reached a primary study endpoint. Parasitaemia was cleared within 3 days of medication administration in all participants, and no early treatment failures were observed. After exclusion of reinfections, the corrected efficacy of AL was 96% (91-100%, 95% confidence interval) in Zaire and 97% (93-100%) in Lunda Sul. The corrected efficacy of ASAQ was 100% (97-100%) in Benguela and 93% (88-99%) in Zaire. The corrected efficacy of DP was 100% (96-100%) in Benguela and 100% in Lunda Sul. No mutations associated with artemisinin resistance were identified in the pfk13 gene in the 38 cases of recurrent P. falciparum infection. All 33 treatment failures in the AL and ASAQ arms carried pfmdr1 or pfcrt mutations associated with lumefantrine and amodiaquine resistance, respectively, on day of failure. CONCLUSIONS: AL, ASAQ, and DP continue to be efficacious against P. falciparum malaria in these provinces of Angola. Rapid parasite clearance and the absence of genetic evidence of artemisinin resistance are consistent with full susceptibility to artemisinin derivatives. Periodic monitoring of in vivo drug efficacy remains a priority routine activity for Angola.

Efficacy of artemether-lumefantrine, artesunate-amodiaquine, and dihydroartemisinin-piperaquine for treatment of uncomplicated Plasmodium falciparum malaria in Angola, 2015.

BACKGROUND: Recent anti-malarial resistance monitoring in Angola has shown efficacy of artemether-lumefantrine (AL) in certain sites approaching the key 90% lower limit of efficacy recommended for artemisinin-based combination therapy. In addition, a controversial case of malaria unresponsive to artemisinins was reported in a patient infected in Lunda Sul Province in 2013. METHODS: During January-June 2015, investigators monitored the clinical and parasitological response of children with uncomplicated Plasmodium falciparum infection treated with AL, artesunate-amodiaquine (ASAQ), or dihydroartemisinin-piperaquine (DP). The study comprised two treatment arms in each of three provinces: Benguela (AL, ASAQ), Zaire (AL, DP), and Lunda Sul (ASAQ, DP). Samples from treatment failures were analysed for molecular markers of resistance for artemisinin (K13) and lumefantrine (pfmdr1). RESULTS: A total of 467 children reached a study endpoint. Fifty-four treatment failures were observed: four early treatment failures, 40 re-infections and ten recrudescences. Excluding re-infections, the 28-day microsatellite-corrected efficacy was 96.3% (95% CI 91-100) for AL in Benguela, 99.9% (95-100) for ASAQ in Benguela, 88.1% (81-95) for AL in Zaire, and 100% for ASAQ in Lunda Sul. For DP, the 42-day corrected efficacy was 98.8% (96-100) in Zaire and 100% in Lunda Sul. All treatment failures were wild type for K13, but all AL treatment failures had pfmdr1 haplotypes associated with decreased lumefantrine susceptibility. CONCLUSIONS: No evidence was found to corroborate the specific allegation of artemisinin resistance in Lunda Sul. The efficacy below 90% of AL in Zaire matches findings from 2013 from the same site. Further monitoring, particularly including measurement of lumefantrine blood levels, is recommended.

Evaluating malaria case management at public health facilities in two provinces in Angola.

BACKGROUND: Malaria accounts for the largest portion of healthcare demand in Angola. A pillar of malaria control in Angola is the appropriate management of malaria illness, including testing of suspect cases with rapid diagnostic tests (RDTs) and treatment of confirmed cases with artemisinin-based combination therapy (ACT). Periodic systematic evaluations of malaria case management are recommended to measure health facility readiness and adherence to national case management guidelines. METHODS: Cross-sectional health facility surveys were performed in low-transmission Huambo and high-transmission Uíge Provinces in early 2016. In each province, 45 health facilities were randomly selected from among all public health facilities stratified by level of care. Survey teams performed inventories of malaria commodities and conducted exit interviews and re-examinations, including RDT testing, of a random selection of all patients completing outpatient consultations. Key health facility readiness and case management indicators were calculated adjusting for the cluster sampling design and utilization. RESULTS: Availability of RDTs or microscopy on the day of the survey was 71% (54-83) in Huambo and 85% (67-94) in Uíge. At least one unit dose pack of one formulation of an ACT (usually artemether-lumefantrine) was available in 83% (66-92) of health facilities in Huambo and 79% (61-90) of health facilities in Uíge. Testing rates of suspect malaria cases in Huambo were 30% (23-38) versus 69% (53-81) in Uíge. Overall, 28% (13-49) of patients with uncomplicated malaria, as determined during the re-examination, were appropriately treated with an ACT with the correct dose in Huambo, compared to 60% (42-75) in Uíge. Incorrect case management of suspect malaria cases was associated with lack of healthcare worker training in Huambo and ACT stock-outs in Uíge. CONCLUSIONS: The results reveal important differences between provinces. Despite similar availability of testing and ACT, testing and treatment rates were lower in Huambo compared to Uíge. A majority of true malaria cases seeking care in health facilities in Huambo were not appropriately treated with anti-malarials, highlighting the importance of continued training and supervision of healthcare workers in malaria case management, particularly in areas with decreased malaria transmission.

Molecular epidemiology of rotavirus in four provinces of Angola before vaccine introduction.

Angola is a sub-Saharan country in southern Africa highly affected by diarrhoeal disease with limited epidemiological data regarding etiologic agents. This study was performed during 2012-2013, prior to rotavirus vaccine introduction, with the objective to detect and characterize the rotavirus strains circulating in four provinces of the country: Huambo, Luanda, Zaire, and Cabinda. A high rotavirus detection rate (35%, 117/334) was observed. G1 was the most common G-genotype (83.6%), whereas P[8] (50.9%) followed by P[6] (38.8%) were the most common P-types. G1P[8] was identified as the predominant combination (50%), followed by the unusual G1P[6] (29.3%). Strains such G2P[4], G8P[6], G9P[6], and G12P[6] were also found in lower frequencies (5.2-1.7%). The P[6] strains did not cluster in the phylogenetic trees according to their geographic origin or even the corresponding G-genotype, suggesting a limited number of recent introductions and extensive reassortment events. Our results represent the first report on rotavirus genotype profiles in Angola, showing a wide circulation of the unusual genotype G1P[6], and underline the importance of RV surveillance after the vaccine introduction. J. Med. Virol. 88:1511-1520, 2016. © 2016 Wiley Periodicals, Inc.

Low-grade sulfadoxine-pyrimethamine resistance in Plasmodium falciparum parasites from Lubango, Angola.

BACKGROUND: Malaria is a major parasitic disease, affecting millions of people in endemic areas. Plasmodium falciparum parasites are responsible for the most severe cases and its resistance to anti-malarial drugs is notorious. This is a possible obstacle to the effectiveness of intermittent preventive treatment (IPT) based on sulfadoxine-pyrimethamine (SP) cures administrated to pregnant women (IPTp) during their pregnancy. As this intervention is recommended in Angola since 2006, it has assessed, in this country, the molecular profiles in P. falciparum dhfr and dhps, two polymorphic genes associated to pyrimethamine and sulfadoxine resistance, respectively. METHODS: Blood samples from 52 falciparum patients were collected in Lubango, Angola and pfdhfr and pfdhps polymorphisms were analysed using nested-PCR and DNA sequencing. RESULTS: In the pfdhfr gene, the 108N mutation was almost fixed (98 %), followed by 59R (63 %), 51I (46 %), 50R and 164L (2 %, respectively). No 16V/S mutations were found. The most common double mutant genotype was CNRN (59 + 108; 46 %), followed by CICN (51 + 108; 29 %) whereas IRN (51 + 59 + 108; 15 %), CNRNVL (59 + 108 + 164; 2 %) and RICN (50 + 51 + 108; 2 %) triple mutant genotypes were detected. Investigations of the pfdhps gene showed that the 437G mutation was the most prevalent (97 %). Only two and one samples disclosed the 540E (7 %) and the 436A (3 %), respectively. Single mutant SGKAA (437; 86 %) was higher than SGEAA (437 + 540; 7 %) or AGKAA (436 + 437; 3 %) double mutants genotypes. No polymorphism was detected at codons 581G and 613T/S. Combining pfdhfr and pfdhps alleles two triple mutant haplotypes (double mutant in dhfr and single mutant in dhps) were observed: the ACICNVI/SGKAA in 14 (56 %) samples and the ACNRNVI/SGKAA in five (20 %) samples. One quadruple mutant haplotype was detected (ACIRNVI/SGKAA) in six (24 %) P. falciparum samples. No quintuple pfdhfr-pfdhps mutant was noted. CONCLUSION: pfdhfr and pfdhps gene mutations in isolates from Lubango are suggestive of a low-grade SP resistance and IPT for pregnant women and infant based on SP treatment could be effective. Routine molecular studies targeting polymorphism in these two genes need to be routinely conducted at country level.

Efficacy of artemether-lumefantrine and dihydroartemisinin-piperaquine for treatment of uncomplicated malaria in children in Zaire and Uíge Provinces, angola.

The development of resistance to antimalarials is a major challenge for global malaria control. Artemisinin-based combination therapies, the newest class of antimalarials, are used worldwide but there have been reports of artemisinin resistance in Southeast Asia. In February through May 2013, we conducted open-label, nonrandomized therapeutic efficacy studies of artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) in Zaire and Uíge Provinces in northern Angola. The parasitological and clinical responses to treatment in children with uncomplicated Plasmodium falciparum monoinfection were measured over 28 days, and the main outcome was a PCR-corrected adequate clinical and parasitological response (ACPR) proportion on day 28. Parasites from treatment failures were analyzed for the presence of putative molecular markers of resistance to lumefantrine and artemisinins, including the recently identified mutations in the K13 propeller gene. In the 320 children finishing the study, 25 treatment failures were observed: 24 in the AL arms and 1 in the DP arm. The PCR-corrected ACPR proportions on day 28 for AL were 88% (95% confidence interval [CI], 78 to 95%) in Zaire and 97% (91 to 100%) in Uíge. For DP, the proportions were 100% (95 to 100%) in Zaire, and 100% (96 to 100%) in Uíge. None of the treatment failures had molecular evidence of artemisinin resistance. In contrast, 91% of AL late-treatment failures had markers associated with lumefantrine resistance on the day of failure. The absence of molecular markers for artemisinin resistance and the observed efficacies of both drug combinations suggest no evidence of artemisinin resistance in northern Angola. There is evidence of increased lumefantrine resistance in Zaire, which should continue to be monitored.

Molecular epidemiology of drug-resistant Plasmodium falciparum in Benguela province, Angola.

BACKGROUND: The malaria situation has been worsening in Angola, partly due to armed conflict until the recent past and drug-resistant Plasmodium falciparum. Malaria transmission is heterogeneous within the country, and data on drug-resistant malaria in different parts of the country are incomplete. The aim of the present study was to evaluate resistance to 4-aminoquinolines and antifolate drugs in P. falciparum isolates collected in Benguela province, central Angola, using molecular markers. METHODS: Fingerprick capillary blood was collected from asymptomatic children aged less than 15 years old during a household survey in and around Balombo town in 2010-2011. Samples were screened for P. falciparum by nested PCR. Molecular markers (P. falciparum dihydrofolate reductase [pfdhfr], P. falciparum dihydropteroate synthase [pfdhps], P. falciparum chloroquine resistance transporter [pfcrt], and P. falciparum multidrug-resistance gene 1 [pfmdr1]) were sequenced to determine the key codons associated with drug resistance. RESULTS: A total of 60 blood samples were positive for P. falciparum. Most isolates with successful PCR amplification had mutant pfdhfr alleles, with either double mutant AICNI (69%) or triple mutant AIRNI (21%) haplotypes. A16V, S108T, and I164L substitutions were not found. Many of the isolates were carriers of either SGKAA (60%) or AGKAA (27%) pfdhps haplotype. K540E substitution was absent. There were only two pfcrt haplotypes: wild-type CVMNK (11%) and mutant CVIET (89%). Wild-type pfmdr1 NYSND haplotype was found in 19% of the isolates, whereas single mutant pfmdr1 YYSND and NFSND haplotypes occurred in 48% and 11%, respectively. Double mutant pfmdr1 haplotypes (YFSND and YYSNY) occurred rarely. CONCLUSIONS: The results suggest that the high prevalence of mutant pfcrt CVIET haplotype is in agreement with low clinical efficacy of chloroquine observed in earlier studies and that the double pfdhfr mutant AICNI and single pfdhps mutant SGKAA are currently the predominant haplotypes associated with antifolate resistance in Benguela province. The hallmark of clinical resistance observed in East Africa, i.e. triple pfdhfr mutant haplotype (AIRNI) and double pfdhps mutant haplotype (SGEAA), was absent. These molecular findings need to be further evaluated in parallel with clinical studies, in particular with the efficacy of intermittent preventive treatment using sulphadoxine-pyrimethamine in pregnant women and artesunate-amodiaquine for uncomplicated malaria.

Impact of a training course on the quality of malaria diagnosis by microscopy in Angola.

BACKGROUND: In Angola, malaria is an endemic disease having a major impact on the economy. The WHO recommends testing for all suspected malaria cases, to avoid the presumptive treatment of this disease. In malaria endemic regions laboratory technicians must be very comfortable with microscopy, the golden standard for malaria diagnosis, to avoid the incorrect diagnosis. The improper use of medication promotes drug resistance and undesirable side effects. The present study aims to assess the impact of a three-day refresher course on the knowledge of technicians, quality of blood smears preparation and accuracy of microscopy malaria diagnosis, using qPCR as reference method. METHODS: This study was implemented in laboratories from three hospitals in different provinces of Angola: Bengo, Benguela and Luanda. In each laboratory samples were collected before and after the training course (slide with thin and thick blood smears, a dried blood spot and a form). The impact of the intervention was evaluated through a written test, the quality of slide preparation and the performance of microscopy. RESULTS: It was found a significant increase on the written test median score, from 52.5% to 65.0%. A total of 973 slides were analysed to evaluate the quality of thick and thin blood smears. Considering all laboratories there was a significant increase in quality of thick and thin blood smears. To determine the performance of microscopy using qPCR as the reference method we used 1,028 samples. Benguela presented the highest values for specificity, 92.9% and 98.8% pre and post-course, respectively and for sensitivity the best pre-course was Benguela (75.9%) and post-course Luanda (75.0%). However, no significant increase in sensitivity and specificity after the training course was registered in any laboratory analysed. DISCUSSION: The findings of this study support the need of continuous refresher training for microscopists and other laboratory staff. The laboratories should have a quality control programme to supervise the diagnosis and also to assess the periodicity of new training. However, other variables needed to be considered to have a correct malaria diagnosis, such as adequate equipment and reagents for staining and visualization, good working conditions, motivated and qualified personnel.

Assessment of the yellow fever outbreak in Angola from December 2015 through December 2016: A retrospective study.

Background and Aims: The acute tropical infectious disease known as yellow fever (YF) is caused by an arbovirus and is characterized by fever, jaundice, hemorrhage, headache, muscle pain, nausea, vomiting, and fatigue. Angola experienced a yellow fever virus (YFV) outbreak that was documented in December 2015. However, little is known about the outcome of this outbreak. We aimed to demonstrate epidemic features and lessons learned during the YF epidemic in Angola. Methods: A total of 4618 blood samples from suspected YF cases were sent to the Instituto Nacional de Investigação em Saúde (INIS), a national referral and public health laboratory, between December 5, 2015, and December 23, 2016. Sample analyses were conducted using enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) assays. Blood samples were sent from 16 out of the 18 provinces of Angola. Results: We detected 884 (19.1%) cases that were positive for ELISA, which were confirmed by RT-PCR assay. Considering the positive cases, the incidence among male patients was around three times higher (n = 223; 10.9%) than in female patients (n = 59; 2.6%) in the 20-29 age group, followed by the age group 10-19 with n = 211 (6.8%) in males versus n = 108 (3.3%) in females; and the age group 30-39 had n = 68 (4.8%) in males versus n = 28 (1.8%) in females. The other groups had an incidence below 3.0%. The case fatality ratio for YF was in young adults in the age group 20-29 with n = 39 cases, followed by the age group 10-19 with n = 16 cases, and finally the age group 0-9 with n = 13 cases. The other age groups had several deaths by YF below 10 cases. Conclusions: This study demonstrates features of the YF epidemic that occurred in Angola. Also, it demonstrates that YF causes deaths in young people but is preventable by high vaccine coverage. Thus, public health laboratory surveillance must be strengthened to reduce the possibility of emerging and re-emerging human infections.