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.

Epidemiologic Utility of a Framework for Partition Number Selection When Dissecting Hierarchically Clustered Genetic Data Evaluated on the Intestinal Parasite Cyclospora cayetanensis.

Comparing parasite genotypes to inform parasitic disease outbreak investigations involves computation of genetic distances that are typically analyzed by hierarchical clustering to identify related isolates, indicating a common source. A limitation of hierarchical clustering is that hierarchical clusters are not discrete; they are nested. Consequently, small groups of similar isolates exist within larger groups that get progressively larger as relationships become increasingly distant. Investigators must dissect hierarchical trees at a partition number ensuring grouped isolates belong to the same strain; a process typically performed subjectively, introducing bias into resultant groupings. We describe an unbiased, probabilistic framework for partition number selection that ensures partitions comprise isolates that are statistically likely to belong to the same strain. We computed distances and established a normalized distribution of background distances that we used to demarcate a threshold below which the closeness of relationships is unlikely to be random. Distances are hierarchically clustered and the dendrogram dissected at a partition number where most within-partition distances fall below the threshold. We evaluated this framework by partitioning 1,137 clustered Cyclospora cayetanensis genotypes, including 552 isolates epidemiologically linked to various outbreaks. The framework was 91% sensitive and 100% specific in assigning epidemiologically linked isolates to the same partition.

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.

Evaluation of various distance computation methods for construction of haplotype-based phylogenies from large MLST datasets.

Multi-locus sequence typing (MLST) is widely used to investigate genetic relationships among eukaryotic taxa, including parasitic pathogens. MLST analysis workflows typically involve construction of alignment-based phylogenetic trees - i.e., where tree structures are computed from nucleotide differences observed in a multiple sequence alignment (MSA). Notably, alignment-based phylogenetic methods require that all isolates/taxa are represented by a single sequence. When multiple loci are sequenced these sequences may be concatenated to produce one tree that includes information from all loci. Alignment-based phylogenetic techniques are robust and widely used yet possess some shortcomings, including how heterozygous sites are handled, intolerance for missing data (i.e., partial genotypes), and differences in the way insertions-deletions (indels) are scored/treated during tree construction. In certain contexts, 'haplotype-based' methods may represent a viable alternative to alignment-based techniques, as they do not possess the aforementioned limitations. This is namely because haplotype-based methods assess genetic similarity based on numbers of shared (i.e., intersecting) haplotypes as opposed to similarities in nucleotide composition observed in an MSA. For haplotype-based comparisons, choosing an appropriate distance statistic is fundamental, and several statistics are available to choose from. However, a comprehensive assessment of various available statistics for their ability to produce a robust haplotype-based phylogenetic reconstruction has not yet been performed. We evaluated seven distance statistics by applying them to extant MLST datasets from the gastrointestinal parasite Cyclospora cayetanensis and two species of pathogenic nematode of the genus Strongyloides. We compare the genetic relationships identified using each statistic to epidemiologic, geographic, and host metadata. We show that Barratt's heuristic definition of genetic distance was the most robust among the statistics evaluated. Consequently, it is proposed that Barratt's heuristic represents a useful approach for use in the context of challenging MLST datasets possessing features (i.e., high heterozygosity, partial genotypes, and indel or repeat-based polymorphisms) that confound or preclude the use of alignment-based methods.

Coronavirus Disease 2019 (COVID-19) in Americans Aboard the Diamond Princess Cruise Ship.

BACKGROUND: The Diamond Princess cruise ship was the site of a large outbreak of coronavirus disease 2019 (COVID-19). Of 437 Americans and their travel companions on the ship, 114 (26%) tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: We interviewed 229 American passengers and crew after disembarkation following a ship-based quarantine to identify risk factors for infection and characterize transmission onboard the ship. RESULTS: The attack rate for passengers in single-person cabins or without infected cabinmates was 18% (58/329), compared with 63% (27/43) for those sharing a cabin with an asymptomatic infected cabinmate, and 81% (25/31) for those with a symptomatic infected cabinmate. Whole genome sequences from specimens from passengers who shared cabins clustered together. Of 66 SARS-CoV-2-positive American travelers with complete symptom information, 14 (21%) were asymptomatic while on the ship. Among SARS-CoV-2-positive Americans, 10 (9%) required intensive care, of whom 7 were ≥70 years. CONCLUSIONS: Our findings highlight the high risk of SARS-CoV-2 transmission on cruise ships. High rates of SARS-CoV-2 positivity in cabinmates of individuals with asymptomatic infections suggest that triage by symptom status in shared quarters is insufficient to halt transmission. A high rate of intensive care unit admission among older individuals complicates the prospect of future cruise travel during the pandemic, given typical cruise passenger demographics. The magnitude and severe outcomes of this outbreak were major factors contributing to the Centers for Disease Control and Prevention's decision to halt cruise ship travel in US waters in March 2020.

Plasmodium falciparum kelch 13 Mutations, 9 Countries in Africa, 2014-2018.

The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014-2018. Of the 2,865 samples successfully sequenced before treatment (day of enrollment) and on the day of treatment failure, 29 (1.0%) samples contained 11 unique nonsynonymous mutations and 83 (2.9%) samples contained 27 unique synonymous mutations. Two samples from Kenya contained the S522C mutation, which has been associated with delayed parasite clearance; however, no samples contained validated or candidate artemisinin-resistance mutations.

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.

Conventional and High-Sensitivity Malaria Rapid Diagnostic Test Performance in 2 Transmission Settings: Haiti 2017.

Accurate malaria diagnosis is foundational for control and elimination, and Haiti relies on histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) identifying Plasmodium falciparum in clinical and community settings. In 2017, 1 household and 2 easy-access group surveys tested all participants (N = 32 506) by conventional and high-sensitivity RDTs. A subset of blood samples (n = 1154) was laboratory tested for HRP2 by bead-based immunoassay and for P. falciparum 18S rDNA by photo-induced electron transfer polymerase chain reaction. Both RDT types detected low concentrations of HRP2 with sensitivity estimates between 2.6 ng/mL and 14.6 ng/mL. Compared to the predicate HRP2 laboratory assay, RDT sensitivity ranged from 86.3% to 96.0% between tests and settings, and specificity from 90.0% to 99.6%. In the household survey, the high-sensitivity RDT provided a significantly higher number of positive tests, but this represented a very small proportion (<0.2%) of all participants. These data show that a high-sensitivity RDT may have limited utility in a malaria elimination setting like Haiti.

Public Health Responses to COVID-19 Outbreaks on Cruise Ships - Worldwide, February-March 2020.

An estimated 30 million passengers are transported on 272 cruise ships worldwide each year* (1). Cruise ships bring diverse populations into proximity for many days, facilitating transmission of respiratory illness (2). SARS-CoV-2, the virus that causes coronavirus disease (COVID-19) was first identified in Wuhan, China, in December 2019 and has since spread worldwide to at least 187 countries and territories. Widespread COVID-19 transmission on cruise ships has been reported as well (3). Passengers on certain cruise ship voyages might be aged ≥65 years, which places them at greater risk for severe consequences of SARS-CoV-2 infection (4). During February-March 2020, COVID-19 outbreaks associated with three cruise ship voyages have caused more than 800 laboratory-confirmed cases among passengers and crew, including 10 deaths. Transmission occurred across multiple voyages of several ships. This report describes public health responses to COVID-19 outbreaks on these ships. COVID-19 on cruise ships poses a risk for rapid spread of disease, causing outbreaks in a vulnerable population, and aggressive efforts are required to contain spread. All persons should defer all cruise travel worldwide during the COVID-19 pandemic.

Efficacy and safety of artesunate-amodiaquine and artemether-lumefantrine and prevalence of molecular markers associated with resistance, Guinea: an open-label two-arm randomised controlled trial.

BACKGROUND: Anti-malarial resistance is a threat to recent gains in malaria control. This study aimed to assess the efficacy and safety of artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) in the management of uncomplicated malaria and to measure the prevalence of molecular markers of resistance of Plasmodium falciparum in sentinel sites in Maferinyah and Labé Health Districts in Guinea in 2016. METHODS: This was a two-arm randomised controlled trial of the efficacy of AL and ASAQ among children aged 6-59 months with uncomplicated Plasmodium falciparum malaria in two sites. Children were followed for 28 days to assess clinical and parasitological response. The primary outcome was the Kaplan-Meier estimate of Day 28 (D28) efficacy after correction by microsatellite-genotyping. Pre-treatment (D0) and day of failure samples were assayed for molecular markers of resistance in the pfk13 and pfmdr1 genes. RESULTS: A total of 421 participants were included with 211 participants in the Maferinyah site and 210 in Labé. No early treatment failure was observed in any study arms. However, 22 (5.3%) participants developed a late treatment failure (8 in the ASAQ arm and 14 in the AL arm), which were further classified as 2 recrudescences and 20 reinfections. The Kaplan-Meier estimate of the corrected efficacy at D28 was 100% for both AL and ASAQ in Maferinyah site and 99% (95% Confidence Interval: 97.2-100%) for ASAQ and 99% (97.1-100%) for AL in Labé. The majority of successfully analysed D0 (98%, 380/389) and all day of failure (100%, 22/22) samples were wild type for pfk13. All 9 observed pfk13 mutations were polymorphisms not associated with artemisinin resistance. The NFD haplotype was the predominant haplotype in both D0 (197/362, 54%) and day of failure samples (11/18, 61%) successfully analysed for pfmdr1. CONCLUSION: This study observed high efficacy and safety of both ASAQ and AL in Guinea, providing evidence for their continued use to treat uncomplicated malaria. Continued monitoring of ACT efficacy and safety and molecular makers of resistance in Guinea is important to detect emergence of parasite resistance and to inform evidence-based malaria treatment policies.

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.

Assessing Performance of HRP2 Antigen Detection for Malaria Diagnosis in Mozambique.

Rapid diagnostic tests (RDTs) that detect the Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) antigen are the primary methods for malaria diagnosis in Mozambique. However, these tests do not detect infections with non-falciparum malaria or Pfhrp2- and Pfhrp3-deleted P. falciparum parasites. To assess the appropriateness of conventional PfHRP2-only RDTs for malaria diagnosis in Mozambique, samples collected during a health facility survey conducted in three provinces of Mozambique were screened using antigen detection methods and further characterized by molecular techniques. Samples from 1,861 outpatients of all ages and symptoms attending 117 randomly selected public health facilities in 2018 were analyzed with an ultrasensitive bead-based immunoassay for the presence of PfHRP2, pan-Plasmodium aldolase (pAldo), and pan-Plasmodium lactate dehydrogenase (pLDH). The presence of PfHRP2 in patient blood detected using the bead-based assay was compared to the results of PfHRP2-based RDTs performed during the routine health facility consult and during the survey reexamination at the exit interview. Samples with discordant antigen profiles (negative for PfHRP2 but positive for pAldo and/or pLDH) were further characterized by photoinduced electron transfer PCR (PET-PCR). Using the bead-based laboratory assay as the gold standard, the sensitivities of the conventional RDTs administered during the routine health facility consult and the exit interview were 90% and 83%, respectively, and the specificities were 91% and 97%, respectively. Of 710 samples positive for at least one antigen, 704 (99.2%) were positive for PfHRP2. Six (0.8% of total) discordant samples lacked PfHRP2 but were positive for pAldo and/or pLDH; 3 of these (0.4% of total) were Plasmodium ovale monoinfections or coinfections where P. ovale was the dominant species. The remaining 3 discordant samples were negative by PET-PCR. The sensitivity and specificity of the conventional RDTs performed in the routine health facility consults and survey exit interviews were acceptable, and there was no evidence of Pfhrp2- and Pfhrp3-deleted parasites. Monoinfections with non-falciparum malaria species comprised <1% of the total malaria infections. Nearly all malaria antigen-positive patients had detectable PfHRP2, confirming that this antigen remains an appropriate malaria diagnostic target in the surveyed provinces.

Multiplex malaria antigen detection by bead-based assay and molecular confirmation by PCR shows no evidence of Pfhrp2 and Pfhrp3 deletion in Haiti.

BACKGROUND: The Plasmodium falciparum parasite is the only human malaria that produces the histidine-rich protein 2 and 3 (HRP2/3) antigens. Currently, HRP2/3 are widely used in malaria rapid diagnostic tests (RDTs), but several global reports have recently emerged showing genetic deletion of one or both of these antigens in parasites. Deletion of these antigens could pose a major concern for P. falciparum diagnosis in Haiti which currently uses RDTs based solely on the detection of the HRP2/3 antigens. METHODS: From September 2012 through February 2014, dried blood spots (DBS) were collected in Haiti from 9317 febrile patients presenting to 17 health facilities in 5 departments throughout the country as part of a bed net intervention study. All DBS from RDT positive persons and a random sampling of DBS from RDT negative persons were assayed for P. falciparum DNA by nested and PET-PCR (n = 2695 total). All PCR positive samples (n = 331) and a subset of PCR negative samples (n = 95) were assayed for three malaria antigens by a multiplex bead assay: pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and HRP2/3. Any samples positive for P. falciparum DNA, but negative for HRP2/3 antigens were tested by nested PCR for Pfhrp2 and Pfhrp3 gene deletions. RESULTS: Of 2695 DBS tested for Plasmodium DNA, 345 (12.8%) were originally found to be positive for P. falciparum DNA; 331 of these had DBS available for antigen detection. Of these, 266 (80.4%) were positive for pAldo, 221 (66.8%) positive for pLDH, and 324 (97.9%) were positive for HRP2/3 antigens. Seven samples (2.1%) positive for P. falciparum DNA were not positive for any of the three antigens by the bead assay, and were investigated for potential Pfhrp2/3 gene deletion by PCR. These samples either successfully amplified Pfhrp2/3 genes or were at an estimated parasite density too low for sufficient DNA to perform successful genotyping. CONCLUSIONS: Malaria positive samples in multiple Haitian sites were found to contain the HRP2/3 antigens, and no evidence was found of Pfhrp2/3 deletions. Malaria RDTs based on the detection of the HRP2/3 antigens remain a reliable P. falciparum diagnostic tool as Haiti works towards malaria elimination.

Quality of malaria services offered in public health facilities in three provinces of Mozambique: a cross-sectional study.

BACKGROUND: Fever associated with malaria is the leading cause of health care-seeking in Mozambique, yet there is limited evidence on the quality of malaria case management. This study evaluated the quality of malaria service provision offered in public health facilities in Mozambique. METHODS: A cross-sectional assessment was conducted in April-May 2018 in three provinces of Mozambique: Maputo Province (low malaria burden), Cabo Delgado (high), and Zambézia (high). The study included all secondary and tertiary facilities and a random sample of primary facilities in each province. Data collection included exit interviews and re-examinations of 20 randomly selected outpatient service patients, interviews with up to five health care providers and the health facility director, a stockroom inventory and routine data abstraction. RESULTS: A total of 319 health care providers and 1840 patients from 117 health facilities were included. Of these, 1325 patients (72%) had suspected malaria (fever/history of fever) and 550 (30%) had febrile, confirmed malaria with the highest burden in Cabo Delgado (43%), followed by Zambézia (34%) and Maputo Province (2%). Appropriate management of malaria cases, defined as testing malaria suspects and treating confirmed cases with the correct dose of anti-malarial, was highest in Zambézia and Cabo Delgado where 52% (95% CI 42-62) and 49% (42-57) of febrile malaria cases were appropriately managed, respectively. Only 14% (5-34) of febrile cases in Maputo Province were appropriately managed. The biggest gap in the malaria case management pathway was failure to test febrile patients, with only 46% of patients with this indication tested for malaria in Maputo Province. Additionally, anti-malarial treatment of patients with a negative malaria test result was common, ranging from 8% (2-23) in Maputo Province to 22% (14-32) of patients with a negative test in Zambézia. Only 58-62% of patients prescribed an anti-malarial correctly recited dosing instructions. Provider training and malaria knowledge was low outside of Zambézia and supervision rates were low in all provinces. Factors associated with correct case management varied by province and included patient age, facility type, treatment and testing availability, supervision, and training. CONCLUSION: These findings underscore the need to strengthen provider testing of all patients with fever, provider adherence to negative test results, and effective counselling of patients across epidemiological settings in Mozambique.

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.

Adherence to Ebola-specific malaria case management guidelines at health facilities in Guinea during the West African Ebola epidemic.

BACKGROUND: Malaria case management in the context of the 2014-2016 West African Ebola virus disease (EVD) epidemic was complicated by a similar initial clinical presentation of the two diseases. In September 2014, the World Health Organization (WHO) released recommendations titled, "Guidance on temporary malaria control measures in Ebola-affected countries", which aimed at reducing the risk of EVD transmission and improving malaria outcomes. This guidance recommended malaria diagnostic testing of fever cases only if adequate personal protective equipment (PPE) was available, defined as examination gloves, face shield, disposable gown, boots, and head cover; otherwise presumptive anti-malarial treatment was recommended. The extent to which health workers adhered to these guidelines in affected countries has not been assessed. METHODS: A cross-sectional survey was conducted in 118 health units in Guinea in November 2014 to produce a representative and probabilistic sample of health facilities and patients. Adherence to the EVD-specific malaria case management guidelines during the height of the EVD epidemic was assessed. Associations between case management practices and possible determinants were calculated using multivariate logistic regression, controlling for expected confounders and the complex sample design. RESULTS: Most (78%) facilities reported availability of examination gloves, but adequate PPE was available at only 27% of facilities. Only 28% of febrile patients received correct malaria case management per the WHO temporary malaria case management guidelines. The most common error was diagnostic testing in the absence of adequate PPE (45% of febrile patients), followed by no presumptive treatment in the absence of adequate PPE (14%). Having had a report of an EVD case at a health facility and health worker-reported participation in EVD-specific malaria trainings were associated with lower odds of diagnostic testing and higher odds of presumptive treatment. CONCLUSIONS: Adherence to guidance on malaria case management in EVD-affected countries was low at the height of the EVD epidemic in Guinea, and there was substantial malaria diagnostic testing in the absence of adequate PPE, which could have contributed to increased EVD transmission in the healthcare setting. Conversely, low presumptive treatment when diagnostic tests were not performed may have led to additional morbidity and mortality among malaria positive patients. National malaria control programs may consider preparing contingency plans for future implementation of temporary changes to malaria case management guidelines to facilitate uptake by health workers. Additional training on standard and transmission-based precautions should help health workers understand how to protect themselves in the face of emerging and unknown pathogens.

Evaluating the quality of routinely reported data on malaria commodity stocks in Guinea, 2014-2016.

BACKGROUND: Ensuring malaria commodity availability at health facilities is a cornerstone of malaria control. Since 2013, the Guinea National Malaria Control Programme has been routinely collecting data on stock levels of key malaria commodities through a monthly routine malaria information system (RMIS). In parallel, biannual end-user verification (EUV) surveys have also assessed malaria commodity availability at a subset of health facilities, potentially representing a duplication of efforts. METHODS: Data on 12 malaria commodity stock levels verified during four EUV surveys conducted between 2014 and 2016 was compared to data for the corresponding months submitted by the same health facilities through the RMIS. The sensitivity and specificity of the RMIS in detecting stock-outs was calculated, as was the percent difference between average stock levels reported through the two systems. RESULTS: Of the 171 health facilities visited during the four EUV surveys, 129 (75%) had data available in the RMIS. Of 351 commodity stock-outs observed during the EUV in the sampled reporting health facilities, 256 (73%) were also signaled through the corresponding RMIS reports. When the presence of malaria commodity stocks was confirmed during the EUV surveys, the RMIS also reported available stock 87% (677/775) of the time. For all commodities, the median percent difference in average stock levels between the EUV and RMIS was 4% (interquartile range - 7 to 27%). CONCLUSION: The concordance between stock levels reported through the RMIS and those verified during the EUV visits provides certain evidence that RMIS data can inform quantification and procurement decisions. However, lower than acceptable rates of reporting and incomplete detection of stock-outs from facilities that do report suggest that further systems strengthening is needed to improve RMIS reporting completeness and data quality.

Specificity of the IgG antibody response to Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale MSP119 subunit proteins in multiplexed serologic assays.

BACKGROUND: Multiplex bead assays (MBA) that measure IgG antibodies to the carboxy-terminal 19-kDa sub-unit of the merozoite surface protein 1 (MSP119) are currently used to determine malaria seroprevalence in human populations living in areas with both stable and unstable transmission. However, the species specificities of the IgG antibody responses to the malaria MSP119 antigens have not been extensively characterized using MBA. METHODS: Recombinant Plasmodium falciparum (3D7), Plasmodium malariae (China I), Plasmodium ovale (Nigeria I), and Plasmodium vivax (Belem) MSP119 proteins were covalently coupled to beads for MBA. Threshold cut-off values for the assays were estimated using sera from US citizens with no history of foreign travel and by receiver operator characteristic curve analysis using diagnostic samples. Banked sera from experimentally infected chimpanzees, sera from humans from low transmission regions of Haiti and Cambodia (N = 12), and elutions from blood spots from humans selected from a high transmission region of Mozambique (N = 20) were used to develop an antigen competition MBA for antibody cross-reactivity studies. A sub-set of samples was further characterized using antibody capture/elution MBA, IgG subclass determination, and antibody avidity measurement. RESULTS: Total IgG antibody responses in experimentally infected chimpanzees were species specific and could be completely suppressed by homologous competitor protein at a concentration of 10 µg/ml. Eleven of 12 samples from the low transmission regions and 12 of 20 samples from the high transmission area had antibody responses that were completely species specific. For 7 additional samples, the P. falciparum MSP119 responses were species specific, but various levels of incomplete heterologous competition were observed for the non-P. falciparum assays. A pan-malaria MSP119 cross-reactive antibody response was observed in elutions of blood spots from two 20-30 years old Mozambique donors. The antibody response from one of these two donors had low avidity and skewed almost entirely to the IgG3 subclass. CONCLUSIONS: Even when P. falciparum, P. malariae, P. ovale, and P. vivax are co-endemic in a high transmission setting, most antibody responses to MSP119 antigens are species-specific and are likely indicative of previous infection history. True pan-malaria cross-reactive responses were found to occur rarely.

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.