Alexandre Macedo de Oliveira

Background: Resistance to anti-malarial drugs is associated with polymorphisms in target genes and surveillance for these molecular markers is important to detect the emergence of mutations associated with drug resistance and signal recovering sensitivity to anti-malarials previously used. Methods: The presence of polymorphisms in genes associated with Plasmodium falciparum resistance to chloroquine and sulfadoxine-pyrimethamine was evaluated by Sanger sequencing, in 85 P. falciparum day of enrollment samples from a therapeutic efficacy study of artemether–lumefantrine conducted in 2018–2019 in Quibdo, Colombia. Samples were genotyped to assess mutations in pfcrt (codons 72–76), pfdhfr (codons 51, 59, 108, and 164), and pfdhps genes (codons 436, 437, 540, and 581). Further, the genetic diversity of infections using seven neutral microsatellites (NMSs) (C2M34, C3M69, Poly α, TA1, TA109, 2490, and PfPK2) was assessed. Results: All isolates carried mutant alleles for pfcrt (K76T and N75E), and for pfdhfr (N51I and S108N), while for pfdhps, mutations were observed only for codon A437G (32/73, 43.8%). Fifty samples (58.8%) showed a complete neutral microsatellites (NMS) profile. The low mean number of alleles (2 ± 0.57) per locus and mean expected heterozygosity (0.17 ± 0.03) showed a reduced genetic diversity. NMS multilocus genotypes (MMG) were built and nine MMG were identified. Conclusions: Overall, these findings confirm the fixation of chloroquine and pyrimethamine-resistant alleles already described in the literature, implying that these drugs are not currently appropriate for use in Colombia. In contrast, mutations in the pfdhps gene were only observed at codon 437, an indication that full resistance to sulfadoxine has not been achieved in Choco. MMGs found matched the clonal lineage E variant 1 previously reported in northwestern Colombia.

Background: Vector control for malaria prevention relies most often on the use of insecticide-treated bed net (ITNs) and indoor residual spraying. Little is known about the longevity of long-lasting insecticidal nets (LLINs) in the Americas. The physical integrity and insecticide retention of LLINs over time were monitored after a bed net distribution campaign to assess community practices around LLIN care and use in Waspam, northeastern Nicaragua. Methods: At least 30 nets were collected at 6, 12, 24, and 36 months post distribution. Physical integrity was measured by counting holes and classifying nets into categories (good, damaged, and too torn) depending on a proportionate hole index (pHI). Insecticide bioefficacy was assessed using cone bioassays, and insecticide content measured using a cyanopyrethroid field test (CFT). Results: At 6 months, 87.3 % of LLINs were in good physical condition, while by 36 months this decreased to 20.6 %, with 38.2 % considered ‘too torn.’ The median pHI increased from 7 at the 6-month time point to 480.5 by 36 months. After 36 months of use, median mortality in cone bioassays was 2 % (range: 0–6 %) compared to 16 % (range: 2–70 %) at 6 months. There was a decrease in the level of deltamethrin detected on the surface of the LLINs with 100 % of tested LLINs tested at 12 months and 24 months crossing the threshold for being considered a failed net by CFT. Conclusions: This first comprehensive analysis of LLIN durability in Central America revealed rapid loss of chemical bioefficacy and progressive physical damage over a 36-month period. Use of these findings to guide future LLIN interventions in malaria elimination settings in Nicaragua, and potentially elsewhere in the Americas, could help optimize the successful implementation of vector control strategies.

Artemether–lumefantrine (AL) is the first-line treatment for uncomplicated Plasmodium falciparum infection in Colombia. To assess AL efficacy for uncomplicated falciparum malaria in Quibdo, Choco, Colombia, we conducted a 28-day therapeutic efficacy study (TES) following the WHO guidelines. From July 2018 to February 2019, febrile patients aged 5–65 years with microscopy-confirmed P. falciparum mono-infection and asexual parasite density of 250–100,000 parasites/µL were enrolled and treated with a supervised 3-day course of AL. The primary endpoint was adequate clinical and parasitological response (ACPR) on day 28. We attempted to use polymerase chain reaction (PCR) genotyping to differentiate reinfection and recrudescence, and conducted genetic testing for antimalarial resistance–associated genes. Eighty-eight patients consented and were enrolled; four were lost to follow-up or missed treatment doses. Therefore, 84 (95.5%) participants reached a valid endpoint: treatment failure or ACPR. No patient remained microscopy positive for malaria on day 3, evidence of delayed parasite clearance and artemisinin resistance. One patient had recurrent infection (12 parasites/µL) on day 28. Uncorrected ACPR rate was 98.8% (83/84) (95% CI: 93.5–100%). The recurrent infection sample did not amplify during molecular testing, giving a PCR-corrected ACPR of 100% (83/83) (95% CI: 95.7–100%). No P. falciparum kelch 13 polymorphisms associated with artemisinin resistance were identified. Our results support high AL efficacy for falciparum malaria in Choco. Because of the time required to conduct TESs in low-endemic settings, it is important to consider complementary alternatives to monitor antimalarial efficacy and resistance.

We evaluated the therapeutic efficacy of artemether–lumefantrine (AL) fixed-dose combination to treat uncomplicated Plasmodium falciparum malaria in Cruzeiro do Sul, Acre State, in the Amazon region of Brazil. Between December 2015 and May 2016, we enrolled 79 patients, 5–79 years old with fever or history of fever in the previous 48 hours and P. falciparum monoinfection confirmed by microscopy. Attempts were made to provide direct observation or phone reminders for all six doses of AL, and patients were followed-up for 28 days. AL was well tolerated, with no adverse events causing treatment interruption. All but one of the 74 patients who completed the 28-day follow-up had an adequate clinical and parasitologic response = 98.6% (95% CI: 93.2-100%). We could not amplify the one isolate of the case with recurrent infection to differentiate between recrudescence and reinfection. Five (6.3%) patients demonstrated persistent asexual parasitemia on Day 3, but none met definition for early treatment failure. We found no mutations in selected kelch13 gene domains, known to be associated with artemisinin resistance in P. falciparum isolates from Day 0. These results strongly support the continued use of AL as a first-line therapy for uncomplicated P. falciparum malaria in Acre. Routine monitoring of in vivo drug efficacy coupled with molecular surveillance of drug resistance markers remains critical.

We evaluated the efficacy of chloroquine and primaquine on uncomplicated Plasmodium vivax malaria in Cruzeiro do Sul, Brazil, in 2014. Patients ≥ 5 years of age with either fever or history of fever, and laboratory-confirmed P. vivax monoinfection received chloroquine (total dose = 25 mg/kg) and primaquine (total dose = 3.5 mg/kg), and were followed up for 168 days (24 weeks). We used microsatellite genotyping to differentiate recurrent infections caused by heterologous parasites from those caused by homologous ones. No new P. vivax episode occurred by Day 28 among 119 enrolled patients, leading to Day 28, with adequate clinical and parasitological response (ACPR) of 100% (95% confidence interval [CI] = 96.7–100%). Twenty-eight P. vivax episodes occurred by Day 168, with uncorrected ACPR of 69.9% (95% CI = 59.5–79.0%). Fifteen of these episodes were caused by either homologous haplotypes or haplotypes that could not be determined. Excluding the 13 recurrent episodes caused by heterologous parasites, Day 168 microsatellite-corrected ACPR was estimated at 81.2% (95% CI = 71.0–89.1%). Chloroquine and primaquine remain efficacious to treat acute uncomplicated P. vivax infection, but moderate recurrence rates were observed within 24 weeks of follow-up.

Recent studies have demonstrated the deletion of the histidine-rich protein 2 (PfHRP2) gene (pfhrp2) in field isolates of Plasmodium falciparum, which could result in false negative test results when PfHRP2-based rapid diagnostic tests (RDTs) are used for malaria diagnosis. Although primary diagnosis of malaria in Honduras is determined based on microscopy, RDTs may be useful in remote areas. In this study, it was investigated whether there are deletions of the pfhrp2, pfhrp3 and their respective flanking genes in 68 P. falciparum parasite isolates collected from the city of Puerto Lempira, Honduras. In addition, further investigation considered the possible correlation between parasite population structure and the distribution of these gene deletions by genotyping seven neutral microsatellites. Sixty-eight samples used in this study, which were obtained from a previous chloroquine efficacy study, were utilized in the analysis. All samples were genotyped for pfhrp2, pfhrp3 and flanking genes by PCR. The samples were then genotyped for seven neutral microsatellites in order to determine the parasite population structure in Puerto Lempira at the time of sample collection. It was found that all samples were positive for pfhrp2 and its flanking genes on chromosome 8. However, only 50% of the samples were positive for pfhrp3 and its neighboring genes while the rest were either pfhrp3-negative only or had deleted a combination of pfhrp3 and its neighbouring genes on chromosome 13. Population structure analysis predicted that there are at least two distinct parasite population clusters in this sample population. It was also determined that a greater proportion of parasites with pfhrp3-(and flanking gene) deletions belonged to one cluster compared to the other. The findings indicate that the P. falciparum parasite population in the municipality of Puerto Lempira maintains the pfhrp2 gene and that PfHRP2-based RDTs could be considered for use in this region; however continued monitoring of parasite population will be useful to detect any parasites with deletions of pfhrp2.

Background: Honduras is a tropical country with more than 70% of its population living at risk of being infected with either Plasmodium vivax or Plasmodium falciparum. Laboratory diagnosis is a very important factor for adequate treatment and management of malaria. In Honduras, malaria is diagnosed by both, microscopy and rapid diagnostic tests and to date, no molecular methods have been implemented for routine diagnosis. However, since mixed infections, and asymptomatic and low-parasitaemic cases are difficult to detect by light microscopy alone, identifying appropriate molecular tools for diagnostic applications in Honduras deserves further study. The present study investigated the utility of different molecular tests for the diagnosis of malaria in Honduras. Methods: A total of 138 blood samples collected as part of a clinical trial to assess the efficacy of chloroquine were used: 69 microscopically confirmed P. falciparum positive samples obtained on the day of enrolment and 69 follow-up samples obtained 28 days after chloroquine treatment and shown to be malaria negative by microscopy. Sensitivity and specificity of microscopy was compared to an 18 s ribosomal RNA gene-based nested PCR, two single-PCR reactions designed to detect Plasmodium falciparum infections, one single-PCR to detect Plasmodium vivax infections, and one multiplex one-step PCR reaction to detect both parasite species. Results: Of the 69 microscopically positive P. falciparum samples, 68 were confirmed to be P. falciparum-positive by two of the molecular tests used. The one sample not detected as P. falciparum by any of the molecular tests was shown to be P. vivax-positive by a reference molecular test indicating a misdiagnosis by microscopy. The reference molecular test detected five cases of P. vivax/P. falciparum mixed infections, which were not recognized by microscopy as mixed infections. Only two of these mixed infections were recognized by a multiplex test while a P. vivax-specific polymerase chain reaction (PCR) detected three of them. In addition, one of the day 28 samples, previously determined to be malaria negative by microscopy, was shown to be P. vivax-positive by three of the molecular tests specific for this parasite. Conclusions: Molecular tests are valuable tools for the confirmation of Plasmodium species and in detecting mixed infections in malaria endemic regions.

We conducted a health facility-based survey to estimate the prevalence of malaria among febrile patients at health facilities (HFs) in Maputo City. Patients answered a questionnaire on malaria risk factors and underwent malaria testing. A malaria case was defined as a positive result for malaria by microscopy in a patient with fever or history of fever in the previous 24 hours. Among 706 patients with complete information, 111 (15.7%) cases were identified: 105 were positive for Plasmodium falciparum only, two for Plasmodium ovale only, and four for both P. falciparum and P. ovale. Fever documented at study enrollment, age ≥ 5 years, rural HF, and travel outside Maputo City were statistically significantly associated with malaria by multivariate analysis. We found a high prevalence of laboratory-confirmed malaria among febrile patients in Maputo City. Further studies are needed to relate these findings with mosquito density to better support malaria prevention and control.

We evaluated the efficacy and effectiveness of mefloquine (MQ) plus artesunate (AS) to treat patients with uncomplicated malaria in the Peruvian Amazon Basin in April 2005–March 2006. Patients ≥ 1 year of age with fever (axillary temperature ≥ 37.5°C) or history of fever and Plasmodium falciparum monoinfection were included. Patients received antimalarial treatment with MQ (12.5 mg/kg/day for two days) and AS (4.0 mg/kg/day for three days) either by directly observed therapy or without directly observed therapy. After a 28-day follow-up, treatment efficacy and effectiveness were assessed on the basis of clinical and parasitologic outcomes. Ninety-six patients were enrolled in each study group; nine patients were lost to follow-up. All patients, except for one in the observed group, demonstrated adequate clinical and parasitologic response; none had detectable parasitemia on day 3. The efficacy of MQ + AS efficacy was 98.9% (95% confidence interval = 94.1–100.0%) and the effectiveness was 100.0% (95% confidence interval = 95.9–100.0%). Our study shows that MQ + AS is highly efficacious in the Peruvian Amazon.

Insecticide-treated bed nets (ITNs) are an efficacious intervention for malaria prevention. During a national immunization campaign in Mozambique, vouchers, which were to be redeemed at a later date for free ITNs, were distributed in Manica and Sofala provinces. A survey to evaluate ITN ownership and usage post-campaign was conducted. Four districts in each province and four enumeration areas (EAs) in each district were selected using probability proportional to size. Within each EA, 32 households (HHs) were selected using a simple random sample. Interviews to assess ownership and usage were conducted in each of the selected HHs using personal digital assistants. Valid interviews were completed for 947 (92.5%) (440 in Manica and 507 in Sofala) of the 1,024 selected HHs. Among participating HHs, 65.0% in Manica and 63.1% in Sofala reported that at least one child under five years of age slept in the house the previous night. HH ownership of at least one bed net of any kind was 20.6% (95% confidence interval [CI]: 7.9%-43.6%) and 35.6% (95% CI: 27.8%-44.3%) pre-campaign; and 55.1% (95% CI: 43.6%-66.1%) and 59.6 (95% CI: 42.4%-74.7%) post-campaign in Manica and Sofala, respectively. Post-campaign HH ownership of at least one ITN was 50.2% (95% CI: 41.8%-58.5%) for both provinces combined. In addition, 60.3% (95% CI: 50.6%-69.2%) of children under five years of age slept under an ITN the previous night. This ITN distribution increased bed net ownership and usage rates. Integration of ITN distribution with immunization campaigns presents an opportunity for reaching malaria control targets and should continue to be considered.