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.

An improved framework for detecting discrete epidemiologically meaningful partitions in hierarchically clustered genetic data.

Motivation: Hierarchical clustering of microbial genotypes has the limitation that hierarchical clusters are nested, where smaller groups of related isolates exist within larger groups that get progressively larger as relationships become increasingly distant. In an epidemiologic context, investigators must dissect hierarchical trees into discrete groupings that are epidemiologically meaningful. We recently described a statistical framework (Method A) for dissecting hierarchical trees that attempts to minimize investigator bias. Here, we apply a modified version of that framework (Method B) to a hierarchical tree constructed from 2111 genotypes of the foodborne parasite Cyclospora, including 639 genotypes linked to epidemiologically defined outbreaks. To evaluate Method B's performance, we examined the concordance between these epidemiologically defined groupings and the genetic partitions identified. We also used the same epidemiologic clusters to evaluate the performance of Method A, plus two tree-dissection methods (cutreeHybrid and cutreeDynamic) available within the Dynamic Tree Cut R package, in addition to the TreeCluster method and PARNAS. Results: Compared to the other methods, Method B, TreeCluster, and PARNAS were the most accurate (99.4%) in identifying genetic groups that reflected the epidemiologic groupings, noting that TreeCluster and PARNAS performed identically on our dataset. CutreeHybrid identified groups reflecting patterns in the wider Cyclospora population structure but lacked finer, strain-level discrimination (Simpson's D: cutreeHybrid=0.785). CutreeDynamic displayed good strain discrimination (Simpson's D = 0.933), though lacked sensitivity (77%). At two different threshold/radius settings TreeCluster/PARNAS displayed similar utility to Method B. However, Method B computes a tree-dissection threshold automatically, and the threshold/radius settings used when executing TreeCluster/PARNAS here were computed using Method B. Using a TreeCluster threshold of 0.045 as recommended in the TreeCluster documentation, epidemiologic utility dropped markedly below that of Method B. Availability and implementation: Relevant code and data are publicly available. Source code (Method B) and instructions for its use are available here: https://github.com/Joel-Barratt/Hierarchical-tree-dissection-framework.

Targeted and whole-genome sequencing reveal a north-south divide in P. falciparum drug resistance markers and genetic structure in Mozambique.

Mozambique is one of the four African countries which account for over half of all malaria deaths worldwide, yet little is known about the parasite genetic structure in that country. We performed P. falciparum amplicon and whole genome sequencing on 2251 malaria-infected blood samples collected in 2015 and 2018 in seven provinces of Mozambique to genotype antimalarial resistance markers and interrogate parasite population structure using genome-wide microhaplotyes. Here we show that the only resistance-associated markers observed at frequencies above 5% were pfmdr1-184F (59%), pfdhfr-51I/59 R/108 N (99%) and pfdhps-437G/540E (89%). The frequency of pfdhfr/pfdhps quintuple mutants associated with sulfadoxine-pyrimethamine resistance increased from 80% in 2015 to 89% in 2018 (p < 0.001), with a lower expected heterozygosity and higher relatedness of microhaplotypes surrounding pfdhps mutants than wild-type parasites suggestive of recent selection. pfdhfr/pfdhps quintuple mutants also increased from 72% in the north to 95% in the south (2018; p < 0.001). This resistance gradient was accompanied by a concentration of mutations at pfdhps-436 (17%) in the north, a south-to-north increase in the genetic complexity of P. falciparum infections (p = 0.001) and a microhaplotype signature of regional differentiation. The parasite population structure identified here offers insights to guide antimalarial interventions and epidemiological surveys.

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.

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.

Risk Factors for Ebola Virus Persistence in Semen of Survivors in Liberia.

BACKGROUND: Long-term persistence of Ebola virus (EBOV) in immunologically privileged sites has been implicated in recent outbreaks of Ebola virus disease (EVD) in Guinea and the Democratic Republic of Congo. This study was designed to understand how the acute course of EVD, convalescence, and host immune and genetic factors may play a role in prolonged viral persistence in semen. METHODS: A cohort of 131 male EVD survivors in Liberia were enrolled in a case-case study. "Early clearers" were defined as those with 2 consecutive negative EBOV semen test results by real-time reverse-transcription polymerase chain reaction (rRT-PCR) ≥2 weeks apart within 1 year after discharge from the Ebola treatment unit or acute EVD. "Late clearers" had detectable EBOV RNA by rRT-PCR >1 year after discharge from the Ebola treatment unit or acute EVD. Retrospective histories of their EVD clinical course were collected by questionnaire, followed by complete physical examinations and blood work. RESULTS: Compared with early clearers, late clearers were older (median, 42.5 years; P < .001) and experienced fewer severe clinical symptoms (median 2, P = .006). Late clearers had more lens opacifications (odds ratio, 3.9 [95% confidence interval, 1.1-13.3]; P = .03), after accounting for age, higher total serum immunoglobulin G3 (IgG3) titers (P = .005), and increased expression of the HLA-C*03:04 allele (0.14 [.02-.70]; P = .007). CONCLUSIONS: Older age, decreased illness severity, elevated total serum IgG3 and HLA-C*03:04 allele expression may be risk factors for the persistence of EBOV in the semen of EVD survivors. EBOV persistence in semen may also be associated with its persistence in other immunologically protected sites, such as the eye.

Performance of antigen detection for HRP2-based malaria rapid diagnostic tests in community surveys: Tanzania, July-November 2017.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) based on the detection of the Plasmodium falciparum histidine-rich protein 2 (HRP2) antigen are widely used for detection of active infection with this parasite and are the only practical malaria diagnostic test in some endemic settings. External validation of RDT results from field surveys can confirm appropriate RDT performance. METHODS: A community-based cross-sectional survey was conducted between July and November 2017 enrolling participants of all ages in households from 15 villages in four border regions of Tanzania: Geita, Kigoma, Mtwara and Ruvuma. All participants had an RDT performed in the field and provided a blood sample for later laboratory multiplex antigen detection of HRP2. In assessing the continuous HRP2 levels in participant blood versus RDT result, dose-response logistic regression provided quantitative estimates for HRP2 limit of detection (LOD). RESULTS: From the 15 study villages, 6941 persons were enrolled that had a RDT at time of enrollment and provided a DBS for later laboratory antigen detection. RDT positive prevalence for the HRP2 band by village ranged from 20.0 to 43.6%, but the magnitude of this prevalence did not have an effect on the estimated LOD of RDTs utilized in different villages. Overall, HRP2 single-target tests had a lower LOD at the 95% probability of positive RDT (4.3 ng/mL; 95% CI 3.4-5.4) when compared to pLDH/HRP2 dual target tests (5.4 ng/mL; 4.5-6.3), though this difference was not significant. With the exception of one village, all other 14 villages (93.3%) showed RDT LOD estimates at 90% probability of positive RDT between 0.5 and 12.0 ng/mL. CONCLUSIONS: Both HRP2-only and pLDH/HRP2 combo RDTs utilized in a 2017 Tanzania cross-sectional survey of border regions generally performed well, and reliably detected HRP2 antigen in the low ng/mL range. Though single target tests had lower levels of HRP2 detection, both tests were within similar ranges among the 15 villages. Comparison of quantitative HRP2 detection limits among study sites can help interpret RDT testing results when generating population prevalence estimates for malaria infection.

Novel application of one-step pooled molecular testing and maximum likelihood approaches to estimate the prevalence of malaria parasitaemia among rapid diagnostic test negative samples in western Kenya.

BACKGROUND: Detection of malaria parasitaemia in samples that are negative by rapid diagnostic tests (RDTs) requires resource-intensive molecular tools. While pooled testing using a two-step strategy provides a cost-saving alternative to the gold standard of individual sample testing, statistical adjustments are needed to improve accuracy of prevalence estimates for a single step pooled testing strategy. METHODS: A random sample of 4670 malaria RDT negative dried blood spot samples were selected from a mass testing and treatment trial in Asembo, Gem, and Karemo, western Kenya. Samples were tested for malaria individually and in pools of five, 934 pools, by one-step quantitative polymerase chain reaction (qPCR). Maximum likelihood approaches were used to estimate subpatent parasitaemia (RDT-negative, qPCR-positive) prevalence by pooling, assuming poolwise sensitivity and specificity was either 100% (strategy A) or imperfect (strategy B). To improve and illustrate the practicality of this estimation approach, a validation study was constructed from pools allocated at random into main (734 pools) and validation (200 pools) subsets. Prevalence was estimated using strategies A and B and an inverse-variance weighted estimator and estimates were weighted to account for differential sampling rates by area. RESULTS: The prevalence of subpatent parasitaemia was 14.5% (95% CI 13.6-15.3%) by individual qPCR, 9.5% (95% CI (8.5-10.5%) by strategy A, and 13.9% (95% CI 12.6-15.2%) by strategy B. In the validation study, the prevalence by individual qPCR was 13.5% (95% CI 12.4-14.7%) in the main subset, 8.9% (95% CI 7.9-9.9%) by strategy A, 11.4% (95% CI 9.9-12.9%) by strategy B, and 12.8% (95% CI 11.2-14.3%) using inverse-variance weighted estimator from poolwise validation. Pooling, including a 20% validation subset, reduced costs by 52% compared to individual testing. CONCLUSIONS: Compared to individual testing, a one-step pooled testing strategy with an internal validation subset can provide accurate prevalence estimates of PCR-positivity among RDT-negatives at a lower cost.

Predicting Plasmodium falciparum infection status in blood using a multiplexed bead-based antigen detection assay and machine learning approaches.

BACKGROUND: Plasmodium blood-stage infections can be identified by assaying for protein products expressed by the parasites. While the binary result of an antigen test is sufficient for a clinical result, greater nuance can be gathered for malaria infection status based on quantitative and sensitive detection of Plasmodium antigens and machine learning analytical approaches. METHODS: Three independent malaria studies performed in Angola and Haiti enrolled persons at health facilities and collected a blood sample. Presence and parasite density of P. falciparum infection was determined by microscopy for a study in Angola in 2015 (n = 193), by qRT-PCR for a 2016 study in Angola (n = 208), and by qPCR for a 2012-2013 Haiti study (n = 425). All samples also had bead-based detection and quantification of three Plasmodium antigens: pAldolase, pLDH, and HRP2. Decision trees and principal component analysis (PCA) were conducted in attempt to categorize P. falciparum parasitemia density status based on continuous antigen concentrations. RESULTS: Conditional inference trees were trained using the known P. falciparum infection status and corresponding antigen concentrations, and PCR infection status was predicted with accuracies ranging from 73-96%, while level of parasite density was predicted with accuracies ranging from 59-72%. Multiple decision nodes were created for both pAldolase and HRP2 antigens. For all datasets, dichotomous infectious status was more accurately predicted when compared to categorization of different levels of parasite densities. PCA was able to account for a high level of variance (>80%), and distinct clustering was found in both dichotomous and categorical infection status. CONCLUSIONS: This pilot study offers a proof-of-principle of the utility of machine learning approaches to assess P. falciparum infection status based on continuous concentrations of multiple Plasmodium antigens.

Antibody dynamics in children with first or repeat Plasmodium falciparum infections.

Immunoglobulin (Ig) production during and after infection with Plasmodium parasites is one of the greatest adaptive immune defenses the human host has against this parasite. Infection with P. falciparum has been shown to induce different B cell maturation responses dependent upon the age of the patient, number of previous exposures, and severity of the disease. Described here are dynamics of Ig responses to a panel of 32 P. falciparum antigens by patients followed for 42 days and classified individuals as showing characteristics of an apparent first P. falciparum infection (naïve) or a repeat exposure (non-naïve). Six parameters were modeled to characterize the dynamics of IgM, IgG1, IgG3, and IgA for these two exposure groups with differences assessed among Ig isotypes/subclasses and unique antigens. Naïve patients had significantly longer periods of time to reach peak Ig titer (range 4-7 days longer) and lower maximum Ig titers when compared with non-naïve patients. Modeled time to seronegativity was significantly higher in non-naïve patients for IgM and IgA, but not for the two IgG subclasses. IgG1 responses to Rh2030, HSP40, and PfAMA1 were at the highest levels for non-naïve participants and may be used to predict previous or nascent exposure by themselves. The analyses presented here demonstrate the differences in the development of the Ig response to P. falciparum if the infection represents a boosting response or a primary exposure. Consistency in Ig isotype/subclasses estimates and specific data for P. falciparum antigens can better guide interpretation of seroepidemiological data among symptomatic persons.

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.

Adaptation to a Multiplex Bead Assay and Seroprevalence to Rift Valley Fever N Protein: Nampula Province, Mozambique, 2013-2014.

Rift Valley fever virus (RVFV) is endemic in sub-Saharan Africa (SSA), with outbreaks reported in the Arabian Peninsula and throughout SSA. The natural reservoir for RVFV are ruminants, with livestock populations exceeding 50% exposure rates in some areas of SSA. Transmission to humans can occur through exposure to infected livestock products or multiple species of mosquito vectors. In 2013 and 2014, cross-sectional surveys occurred in two districts of Nacala-a-Velha and Mecubúri in northern Mozambique, and participants provided blood samples for later serological assays. IgG against the N protein of RVFV was detected through multiplex bead assay (MBA). Of the 2,278 persons enrolled between the two surveys and study sites, 181 (7.9%, 95% confidence interval (CI): 6.9%-9.1%) were found to be IgG seropositive with increasing seroprevalence with older age and significantly higher seroprevalence in Nacala-a-Velha (10.5%, 8.8%-12.5%) versus Mecubúri (5.7%, 4.5%-7.1%). Seroprevalence estimates were not significantly different between the 2013 and 2014 surveys. Significant spatial clustering of IgG positive persons were consistent among surveys and within the two districts, pointing toward the consistency of serology data for making population-level assumptions regarding RVFV seroprevalence. A subset of persons (n = 539) provided samples for both the 2013 and 2014 surveys, and a low percentage (0.81%) of these were found to seroconvert between these two surveys. Including the RVFV N protein in an MBA antigen panel could assist elucidate RVFV exposure in SSA. IMPORTANCE Due to sporadic transmission, human contact with Rift Valley Fever Virus (RVFV) is difficult to ascertain at a population level. Detection of antibodies against RVFV antigens assist in estimating exposure as antibodies remain in the host long after the virus has been cleared. In this study, we show that antibodies against RVFV N protein can be detected from dried blood spot (DBS) samples being assayed by multiplex bead assay. DBS from two districts in northern Mozambique were tested for IgG against the N protein, and 7.9% of all enrolled persons were seropositive. Older persons, males, and persons residing closer to the coast had higher RVFV N protein seroprevalence. Spatial clustering of IgG positive persons was noted in both districts. These results show low exposure rates to RVFV in these two northern districts in Mozambique, and the ability to perform serology for the RVFV N protein from dried blood samples.

Missed Plasmodium ovale Infections Among Symptomatic Persons in Angola, Mozambique, and Ethiopia.

The majority of symptomatic malaria in sub-Saharan Africa is caused by Plasmodium falciparum. Infection with Plasmodium ovale is often not recorded and not considered clinically relevant. Here, we describe 8 cases of P ovale infection from 3 African countries-all of which were misdiagnosed at the presenting health facility.

The use of a chimeric antigen for Plasmodium falciparum and P. vivax seroprevalence estimates from community surveys in Ethiopia and Costa Rica.

BACKGROUND: In low-transmission settings, accurate estimates of malaria transmission are needed to inform elimination targets. Detection of antimalarial antibodies provides exposure history, but previous studies have mainly relied on species-specific antigens. The use of chimeric antigens that include epitopes from multiple species of malaria parasites in population-based serological surveys could provide data for exposure to multiple Plasmodium species circulating in an area. Here, the utility of P. vivax/P. falciparum chimeric antigen for assessing serological responses was evaluated in Ethiopia, an endemic country for all four human malarias, and Costa Rica, where P. falciparum has been eliminated with reports of sporadic P. vivax cases. METHODS: A multiplex bead-based assay was used to determine the seroprevalence of IgG antibodies against a chimeric malaria antigen (PvRMC-MSP1) from blood samples collected from household surveys in Ethiopia in 2015 (n = 7,077) and Costa Rica in 2015 (n = 851). Targets specific for P. falciparum (PfMSP1) and P. vivax (PvMSP1) were also included in the serological panel. Seroprevalence in the population and seroconversion rates were compared among the three IgG targets. RESULTS: Seroprevalence in Costa Rica was 3.6% for PfMSP1, 41.5% for PvMSP1 and 46.7% for PvRMC-MSP1. In Ethiopia, seroprevalence was 27.6% for PfMSP1, 21.4% for PvMSP1, and 32.6% for PvRMC-MSP1. IgG levels in seropositive individuals were consistently higher for PvRMC-MSP1 when compared to PvMSP1 in both studies. Seroconversion rates were 0.023 for PvMSP1 and 0.03 for PvRMC-MSP1 in Costa Rica. In Ethiopia, seroconversion rates were 0.050 for PfMSP1, 0.044 for PvMSP1 and 0.106 for PvRMC-MSP1. CONCLUSIONS: Our data indicate that chimeric antigen PvRMC-MSP1 is able to capture antibodies to multiple epitopes from both prior P. falciparum and P. vivax infections, and suitable chimeric antigens can be considered for use in serosurveys with appropriate validation.

Investigation of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions and performance of a rapid diagnostic test for identifying asymptomatic malaria infection in northern Ethiopia, 2015.

BACKGROUND: Rapid diagnostic tests (RDTs) are widely used for malaria diagnosis of both symptomatic and asymptomatic infections. Although RDTs are a reliable and practical diagnostic tool, the sensitivity of histidine-rich protein 2 (HRP2)-based RDTs can be reduced if pfhrp2 or pfhrp3 (pfhrp2/3) gene deletions exist in the Plasmodium falciparum parasite population. This study evaluated dried blood spot (DBS) samples collected from a national household survey to investigate the presence of pfhrp2/3 deletions and the performance of the RDT used in the cross-sectional survey in a low transmission setting. METHODS: The 2015 Ethiopia Malaria Indicator Survey tested household members by RDT and collected DBS samples. DBS (n = 2648) from three regions in northern Ethiopia were tested by multiplex bead-based antigen detection assay after completion of the survey. The multiplex assay detected pan-Plasmodium lactate dehydrogenase (LDH), pAldolase, and HRP2 antigens in samples. Samples suspected for pfhrp2/3 gene deletions (pLDH and/or pAldolase positive but low or absent HRP2) were further investigated by molecular assays for gene deletions. Antigen results were also compared to each individual's RDT results. Dose-response logistic regression models were fit to estimate RDT level of detection (LOD) antigen concentrations at which 50, 75, 90, and 95% of the RDTs returned a positive result during this survey. RESULTS: Out of 2,648 samples assayed, 29 were positive for pLDH or pAldolase antigens but low or absent for HRP2 signal, and 15 of these samples (51.7%) were successfully genotyped for pfhrp2/3. Of these 15 P. falciparum infections, eight showed single deletions in pfhrp3, one showed a single pfhrp2 deletion, and six were pfhrp2/3 double-deletions. Six pfhrp2 deletions were observed in Tigray and one in Amhara. Twenty-five were positive for HRP2 by the survey RDT while the more sensitive bead assay detected 30 HRP2-positive samples. A lower concentration of HRP2 antigen generated a positive test result by RDT compared to pLDH (95% LOD: 16.9 ng/mL vs. 319.2 ng/mL, respectively). CONCLUSIONS: There is evidence of dual pfhrp2/3 gene deletions in the Tigray and Amhara regions of Ethiopia in 2015. As the prevalence of malaria was very low (< 2%), it is difficult to make strong conclusions on RDT performance, but these results challenge the utility of biomarkers in household surveys in very low transmission settings.

Active surveillance and early detection of community transmission of SARS-CoV-2 Mu variant (B.1.621) in the Brazilian Amazon.

Through active surveillance and contact tracing from outpatients, we aimed to identify and characterize SARS-CoV-2 variants circulating in Porto Velho-Rondônia, a city in the Brazilian Amazon. As part of a prospective cohort, we gathered information from 2,506 individuals among COVID-19 patients and household contacts. Epidemiological data, nasopharyngeal swabs, and blood samples were collected from all participants. Nasopharyngeal swabs were tested for antigen rapid diagnostic test and reverse transcription-polymerase chain reaction (RT-PCR) followed by genomic sequencing. Blood samples underwent ELISA testing for IgA, IgG, and IgM antibody levels. From 757 specimens sequenced, three were identified as Mu variant, none of the individuals carrying this variant had a travel history in the previous 15 days before diagnosis. One case was asymptomatic and two presented mild symptoms. Two infected individuals from different households caring viruses with additional amino acid substitutions ORF7a P45L and ORF1a T1055A compared to the Mu virus reference sequence. One patient presented IgG levels. Our results highlight that genomic surveillance for SARS-CoV-2 variants can assist in detecting the emergency of SARS-CoV-2 variants in the community, before its identification in other parts of the country.

Cluster of SARS-CoV-2 Gamma Variant Infections, Parintins, Brazil, March 2021.

High case counts after the Gamma (P. 1) variant of severe acute respiratory syndrome coronavirus 2 emerged in Brazil raised concerns that previously infected persons might become reinfected. Investigation of a cluster of coronavirus disease cases in Parintins, in the Brazilian Amazon, suggested household transmission but did not identify high rates of reinfection.