Point-of-care C-reactive protein measurement by community health workers safely reduces antimicrobial use among children with respiratory illness in rural Uganda: A stepped wedge cluster randomized trial.

BACKGROUND: Acute respiratory illness (ARI) is one of the most common reasons children receive antibiotic treatment. Measurement of C-reaction protein (CRP) has been shown to reduce unnecessary antibiotic use among children with ARI in a range of clinical settings. In many resource-constrained contexts, patients seek care outside the formal health sector, often from lay community health workers (CHWs). This study's objective was to determine the impact of CRP measurement on antibiotic use among children presenting with febrile ARI to CHW in Uganda. METHODS AND FINDINGS: We conducted a cross-sectional, stepped wedge cluster randomized trial in 15 villages in Bugoye subcounty comparing a clinical algorithm that included CRP measurement by CHW to guide antibiotic treatment (STAR Sick Child Job Aid [SCJA]; intervention condition) with the Integrated Community Care Management (iCCM) SCJA currently in use by CHW in the region (control condition). Villages were stratified into 3 strata by altitude, distance to the clinic, and size; in each stratum, the 5 villages were randomly assigned to one of 5 treatment sequences. Children aged 2 months to 5 years presenting to CHW with fever and cough were eligible. CHW conducted follow-up assessments 7 days after the initial visit. Our primary outcome was the proportion of children who were given or prescribed an antibiotic at the initial visit. Our secondary outcomes were (1) persistent fever on day 7; (2) development of prespecified danger signs; (3) unexpected visits to the CHW; (4) hospitalizations; (5) deaths; (6) lack of perceived improvement per the child's caregiver on day 7; and (7) clinical failure, a composite outcome of persistence of fever on day 7, development of danger signs, hospitalization, or death. The 65 participating CHW enrolled 1,280 children, 1,220 (95.3%) of whom had sufficient data. Approximately 48% (587/1,220) and 52% (633/1,220) were enrolled during control (iCCM SCJA) and intervention periods (STAR SCJA), respectively. The observed percentage of children who were given or prescribed antibiotics at the initial visit was 91.8% (539/587) in the control periods as compared to 70.8% (448/633) during the intervention periods (adjusted prevalence difference -24.6%, 95% CI: -36.1%, -13.1%). The odds of antibiotic prescription by the CHW were over 80% lower in the intervention as compared to the control periods (OR 0.18, 95% CI: 0.06 to 0.49). The frequency of clinical failure (iCCM SCJA 3.9% (23/585) v. STAR SCJA 1.8% (11/630); OR 0.41, 95% CI: 0.09, 1.83) and lack of perceived improvement by the caregiver (iCCM SCJA 2.1% (12/584) v. STAR SCJA 3.5% (22/627); OR 1.49, 95% CI: 0.37, 6.52) was similar. There were no unexpected visits or deaths in either group within the follow-up period. CONCLUSIONS: Incorporating CRP measurement into iCCM algorithms for evaluation of children with febrile ARI by CHW in rural Uganda decreased antibiotic use. There is evidence that this decrease was not associated with worse clinical outcomes, although the number of adverse events was low. These findings support expanded access to simple, point-of-care diagnostics to improve antibiotic stewardship in rural, resource-constrained settings where individuals with limited medical training provide a substantial proportion of care. TRIAL REGISTRATION: ClinicalTrials.gov NCT05294510. The study was reviewed and approved by the University of North Carolina Institutional Review Board (#18-2803), Mbarara University of Science and Technology Research Ethics Committee (14/03-19), and Uganda National Council on Science and Technology (HS 2631).

Prevalence of mutations associated with artemisinin partial resistance and sulfadoxine-pyrimethamine resistance in 13 regions in Tanzania in 2021: a cross-sectional survey.

BACKGROUND: The emergence of the artemisinin partial resistance (ART-R) mutation in the Plasmodium falciparum kelch13 gene (k13), Arg561His, in Rwanda and the regional presence of polymorphisms affecting sulfadoxine-pyrimethamine have raised concern in neighbouring Tanzania. The goal of this study was to assess the status of antimalarial resistance in Tanzania, with a focus on the border with Rwanda, to understand the distribution of the Arg561His mutation, partner drug resistance, and resistance to chemoprevention drugs. METHODS: In this cross-sectional survey, capillary dried blood spots were collected from malaria positive asymptomatic individuals in the community and symptomatic individuals in health facilities aged 6 months and older, in 13 regions of mainland Tanzania from Jan 31 to June 26, 2021. Exclusion criteria included residence of the areas other than the target sites, presenting to the health facility for care and treatment of conditions other than malaria, and not providing informed consent. Samples were assessed for antimalarial resistance polymorphisms and genetic relatedness using molecular inversion probes targeting P falciparum and short-read whole-genome sequencing. The primary outcome was the prevalence of molecular markers of antimalarial resistance at the region level, as well as at the district level in Kagera, a region in the northwest of the country at the border with Rwanda. FINDINGS: 6855 (88·1%) of 7782 capillary dried blood spot samples collected were successfully genotyped. The overall prevalence of k13 Arg561His in Kagera was 7·7% (90% CI 6·0-9·4; 50 of 649), with the highest prevalence in the districts near the Rwandan border (22·8% [31 of 136] in Karagwe, 14·4% [17 of 118]) in Kyerwa, and 1·4% [two of 144] in Ngara). k13 Arg561His was uncommon in the other regions. Haplotype analysis suggested that some of these parasites are related to isolates collected in Rwanda in 2015, supporting regional spread of Arg561His. However, a novel k13 Arg561His haplotype was observed, potentially indicating a second origin in the region. Other validated k13 resistance polymorphisms (one Arg622Ile and two Ala675Val isolates) were also identified. A region of prevalent dihydrofolate reductase Ile164Leu mutation, associated with sulfadoxine-pyrimethamine resistance, was also identified in Kagera (15·2% [12·6-17·8%]; 80 of 526). The mutant crt Lys76Thr mutation, associated with chloroquine and amodiaquine resistance, was uncommon, occurring only in 75 of 2861 genotyped isolates, whereases the wild-type mdr1 Asn86Tyr allele, associated with reduced sensitivity to lumefantrine, was found in 99·7% (3819 of 3830) of samples countrywide. INTERPRETATION: These findings show that the k13 Arg561His mutation is common in northwest Tanzania and that multiple emergences of ART-R, similar as to what was seen in southeast Asia, have occurred. Mutations associated with high levels of sulfadoxine-pyrimethamine resistance are common. These results raise concerns about the long-term efficacy of artemisinin and chemoprevention antimalarials in the region. Understanding how multiple emergences interact with drivers of regional spread is essential for combating ART-R in Africa. FUNDING: This study was funded by the Bill & Melinda Gates Foundation and the National Institutes of Health.

Evidence of artemisinin partial resistance in northwestern Tanzania: clinical and molecular markers of resistance.

BACKGROUND: In 2021, nationwide malaria molecular surveillance revealed a high prevalence of a validated artemisinin resistance marker, the kelch13 (k13) Arg561His mutation, in the Kagera region of northwestern Tanzania. We aimed to investigate the efficacy of artemether-lumefantrine and artesunate-amodiaquine and to confirm the presence of artemisinin partial resistance (ART-R) in the Karagwe district of this region. METHODS: This single-arm, therapeutic efficacy study was carried out at the Bukangara dispensary in the Karagwe district of the Kagera region in northwestern Tanzania. Eligible participants were aged between 6 months and 120 months, had confirmed Plasmodium falciparum asexual parasitaemia, and met other inclusion criteria according to WHO's standard protocol. Participants were enrolled, treated sequentially with either artemether-lumefantrine or artesunate-amodiaquine, and assessed clinically and parasitologically for 28 days as per WHO protocol. Parasitaemia was measured every 8 h until day 3, on day 7, and then during weekly follow-up visits until day 28. Mutations in the k13 gene and extended haplotypes with the mutations were analysed, and comparisons were made with previous samples collected in the same region of Kagera and in Rwanda and southeast Asia. The primary endpoint was PCR-corrected cure rate. FINDINGS: Between April 29 and Sept 1, 2022, 343 patients were screened, of whom 176 were enrolled: 88 in each treatment group. The PCR-corrected cure rate was 98% (95% CI 91-100) in the artemether-lumefantrine group and 100% (96-100) in the artesunate-amodiaquine group. Persistent parasitaemia on day 3 occurred in 11 (13%) of 88 patients in the artemether-lumefantrine group and 17 (19%) of 88 patients in the artesunate-amodiaquine group. Arg561His mutations on day 0 and parasitaemia on day 3 were reported in eight (9%) of 87 patients in the artemether-lumefantrine group and ten (12%) of 86 patients in the artesunate-amodiaquine group. The median parasite clearance half-life in patients harbouring parasites with Arg561His mutation was 6·1 h in the artemether-lumefantrine group and 6·0 h in the artesunate-amodiaquine group. Parasites with the Arg561His mutation were not similar to those from southeast Asia and Rwanda but had similar haplotypes to parasites reported in the same Tanzanian region of Kagera in 2021. INTERPRETATION: This study confirms the presence of ART-R in Tanzania, although artemether-lumefantrine and artesunate-amodiaquine showed high efficacy. A context-specific response strategy and vigilance to detect the reduced efficacy of current antimalarial treatments and ART-R in other parts of the country are urgently needed. FUNDING: The Bill & Melinda Gates Foundation and the US National Institutes of Health.

Clinical and genomic diversity of Treponema pallidum subspecies pallidum to inform vaccine research: an international, molecular epidemiology study.

BACKGROUND: The increase in syphilis rates worldwide necessitates development of a vaccine with global efficacy. We aimed to explore Treponema pallidum subspecies pallidum (TPA) molecular epidemiology essential for vaccine research by analysing clinical data and specimens from early syphilis patients using whole-genome sequencing (WGS) and publicly available WGS data. METHODS: In this multicentre, cross-sectional, molecular epidemiology study, we enrolled patients with primary, secondary, or early latent syphilis from clinics in China, Colombia, Malawi, and the USA between Nov 28, 2019, and May 27, 2022. Participants aged 18 years or older with laboratory confirmation of syphilis by direct detection methods or serological testing, or both, were included. Patients were excluded from enrolment if they were unwilling or unable to give informed consent, did not understand the study purpose or nature of their participation, or received antibiotics active against syphilis in the past 30 days. TPA detection and WGS were conducted on lesion swabs, skin biopsies, skin scrapings, whole blood, or rabbit-passaged isolates. We compared our WGS data to publicly available genomes and analysed TPA populations to identify mutations associated with lineage and geography. FINDINGS: We screened 2802 patients and enrolled 233 participants, of whom 77 (33%) had primary syphilis, 154 (66%) had secondary syphilis, and two (1%) had early latent syphilis. The median age of participants was 28 years (IQR 22-35); 154 (66%) participants were cisgender men, 77 (33%) were cisgender women, and two (1%) were transgender women. Of the cisgender men, 66 (43%) identified as gay, bisexual, or other sexuality. Among all participants, 56 (24%) had HIV co-infection. WGS data from 113 participants showed a predominance of SS14-lineage strains with geographical clustering. Phylogenomic analyses confirmed that Nichols-lineage strains were more genetically diverse than SS14-lineage strains and clustered into more distinct subclades. Differences in single nucleotide variants (SNVs) were evident by TPA lineage and geography. Mapping of highly differentiated SNVs to three-dimensional protein models showed population-specific substitutions, some in outer membrane proteins (OMPs) of interest. INTERPRETATION: Our study substantiates the global diversity of TPA strains. Additional analyses to explore TPA OMP variability within strains is vital for vaccine development and understanding syphilis pathogenesis on a population level. FUNDING: US National Institutes of Health National Institute for Allergy and Infectious Disease, the Bill & Melinda Gates Foundation, Connecticut Children's, and the Czech Republic National Institute of Virology and Bacteriology.

High-Throughput Genotyping of Plasmodium vivax in the Peruvian Amazon via Molecular Inversion Probes.

Plasmodium vivax transmission occurs throughout the tropics and is an emerging threat in areas of Plasmodium falciparum decline, causing relapse infections that complicate treatment and control. Targeted sequencing for P. falciparum has been widely deployed to detect population structure and the geographic spread of antimalarial and diagnostic resistance. However, there are fewer such tools for P. vivax . Leveraging global variation data, we designed four molecular inversion probe (MIP) genotyping panels targeting geographically differentiating SNPs, neutral SNPs, putative antimalarial resistance genes, and vaccine candidate genes. We deployed these MIP panels on 866 infections from the Peruvian Amazon and identified transmission networks with clonality (IBD>0.99), copy number variation in Pvdbp and multiple Pvrbps , fixation of putative antimalarial resistance, and balancing selection in 13 vaccine candidate genes. Our MIP panels are the broadest genotyping panel currently available and are poised for successful deployment in other regions of P. vivax transmission.

Strong isolation by distance and evidence of population microstructure reflect ongoing Plasmodium falciparum transmission in Zanzibar.

Background: The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania and continued local transmission. Methods: To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo district on the coastal mainland from 2016 to 2018. Results: Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to the rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low-level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Conclusions: Our data support importation as a main source of genetic diversity and contribution to the parasite population in Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive to malaria reemergence due to susceptible hosts and competent vectors. Funding: This research was funded by the National Institutes of Health, grants R01AI121558, R01AI137395, R01AI155730, F30AI143172, and K24AI134990. Funding was also contributed from the Swedish Research Council, Erling-Persson Family Foundation, and the Yang Fund. RV acknowledges funding from the MRC Centre for Global Infectious Disease Analysis (reference MR/R015600/1), jointly funded by the UK Medical Research Council (MRC) and the UK Foreign, Commonwealth & Development Office (FCDO), under the MRC/FCDO Concordat agreement and is also part of the EDCTP2 program supported by the European Union. RV also acknowledges funding by Community Jameel.

Temporal genomic analysis of Plasmodium falciparum reveals increased prevalence of mutations associated with delayed clearance following treatment with artemisinin-lumefantrine in Choma District, Southern Province, Zambia.

The emergence of antimalarial drug resistance is an impediment to malaria control and elimination in Africa. Analysis of temporal trends in molecular markers of resistance is critical to inform policy makers and guide malaria treatment guidelines. In a low and seasonal transmission region of southern Zambia, we successfully genotyped 85.5% (389/455) of Plasmodium falciparum samples collected between 2013-2018 from 8 spatially clustered health centres using molecular inversion probes (MIPs) targeting key drug resistance genes. Aside from one sample carrying K13 R622I, none of the isolates carried other World Health Organization-validated or candidate artemisinin partial resistance (ART-R) mutations in K13. However, 13% (CI, 9.6-17.2) of isolates had the AP2MU S160N mutation, which has been associated with delayed clearance following artemisinin combination therapy in Africa. This mutation increased in prevalence between 2015-2018 and bears a genomic signature of selection. During this time period, there was an increase in the MDR1 NFD haplotype that is associated with reduced susceptibility to lumefantrine. Sulfadoxine-pyrimethamine polymorphisms were near fixation. While validated ART-R mutations are rare, a mutation associated with slow parasite clearance in Africa appears to be under selection in southern Zambia.

Chloroquine has shown high therapeutic efficacy against uncomplicated Plasmodium vivax malaria in southern Ethiopia: seven decades after its introduction.

BACKGROUND: Plasmodium vivax malaria is a leading cause of morbidity in Ethiopia. The first-line treatment for P. vivax is chloroquine (CQ) and primaquine (PQ), but there have been local reports of CQ resistance. A clinical study was conducted to determine the efficacy of CQ for the treatment of P. vivax malaria in southern Ethiopia. METHODS: In 2021, patients with P. vivax mono-infection and uncomplicated malaria were enrolled and treated with 25 mg/kg CQ for 3 consecutive days. Patients were followed for 28 days according to WHO guidelines. The data were analysed using per-protocol (PP) and Kaplan‒Meier (K‒M) analyses to estimate the risk of recurrent P. vivax parasitaemia on day 28. RESULTS: A total of 88 patients were enrolled, 78 (88.6%) of whom completed the 28 days of follow-up. Overall, 76 (97.4%) patients had adequate clinical and parasitological responses, and two patients had late parasitological failures. The initial therapeutic response was rapid, with 100% clearance of asexual parasitaemia within 48 h. CONCLUSION: Despite previous reports of declining chloroquine efficacy against P. vivax, CQ retains high therapeutic efficacy in southern Ethiopia, supporting the current national treatment guidelines. Ongoing clinical monitoring of CQ efficacy supported by advanced molecular methods is warranted to inform national surveillance and ensure optimal treatment guidelines.

Close Proximity to Mining Is Associated with Increased Prevalence of the Drug Resistance-Associated Mutation dhps540E in Eastern Democratic Republic of the Congo.

Increasing sulfadoxine-pyrimethamine (SP) resistance in the Democratic Republic of the Congo (DRC) has threatened its use for prevention of malaria in one of the most malarious countries in the world. Using geographic information on mining operations in the DRC and genetic data on SP drug resistance markers from the 2013-2014 Demographic and Health Surveys, we evaluated associations between close residence to mining and the presence of mutations conferring resistance to sulfadoxine. Close residential proximity to mining was associated with increased prevalence odds ratio (POR) of the dhps540E mutation (POR: 2.11, 95% uncertainty interval: 1.15-3.96) with adjustments for confounding variables and space. Our findings indicate that exposure to mining is associated with increased presence of an antimalarial drug resistance haplotype that threatens effective use of SP for vulnerable populations. Areas actively engaged in mining could be considered for interventions to reduce the spread of emerging drug resistance in the DRC.

Genetic polymorphism and evidence of signatures of selection in the Plasmodium falciparum circumsporozoite protein gene in Tanzanian regions with different malaria endemicity.

BACKGROUND: In 2021 and 2023, the World Health Organization approved RTS,S/AS01 and R21/Matrix M malaria vaccines, respectively, for routine immunization of children in African countries with moderate to high transmission. These vaccines are made of Plasmodium falciparum circumsporozoite protein (PfCSP), but polymorphisms in the gene raise concerns regarding strain-specific responses and the long-term efficacy of these vaccines. This study assessed the Pfcsp genetic diversity, population structure and signatures of selection among parasites from areas of different malaria transmission intensities in Mainland Tanzania, to generate baseline data before the introduction of the malaria vaccines in the country. METHODS: The analysis involved 589 whole genome sequences generated by and as part of the MalariaGEN Community Project. The samples were collected between 2013 and January 2015 from five regions of Mainland Tanzania: Morogoro and Tanga (Muheza) (moderate transmission areas), and Kagera (Muleba), Lindi (Nachingwea), and Kigoma (Ujiji) (high transmission areas). Wright's inbreeding coefficient (Fws), Wright's fixation index (FST), principal component analysis, nucleotide diversity, and Tajima's D were used to assess within-host parasite diversity, population structure and natural selection. RESULTS: Based on Fws (< 0.95), there was high polyclonality (ranging from 69.23% in Nachingwea to 56.9% in Muheza). No population structure was detected in the Pfcsp gene in the five regions (mean FST = 0.0068). The average nucleotide diversity (π), nucleotide differentiation (K) and haplotype diversity (Hd) in the five regions were 4.19, 0.973 and 0.0035, respectively. The C-terminal region of Pfcsp showed high nucleotide diversity at Th2R and Th3R regions. Positive values for the Tajima's D were observed in the Th2R and Th3R regions consistent with balancing selection. The Pfcsp C-terminal sequences revealed 50 different haplotypes (H_1 to H_50), with only 2% of sequences matching the 3D7 strain haplotype (H_50). Conversely, with the NF54 strain, the Pfcsp C-terminal sequences revealed 49 different haplotypes (H_1 to H_49), with only 0.4% of the sequences matching the NF54 strain (Hap_49). CONCLUSIONS: The findings demonstrate high diversity of the Pfcsp gene with limited population differentiation. The Pfcsp gene showed positive Tajima's D values, consistent with balancing selection for variants within Th2R and Th3R regions. The study observed differences between the intended haplotypes incorporated into the design of RTS,S and R21 vaccines and those present in natural parasite populations. Therefore, additional research is warranted, incorporating other regions and more recent data to comprehensively assess trends in genetic diversity within this important gene. Such insights will inform the choice of alleles to be included in the future vaccines.

Trimethoprim-Sulfamethoxazole Plus Azithromycin to Prevent Malaria and Sexually Transmitted Infections in Pregnant Women With HIV (PREMISE): A Randomized, Double-Masked, Placebo-Controlled, Phase IIB Clinical Trial.

Background: This trial tested the effectiveness of a novel regimen to prevent malaria and sexually transmitted infections (STIs) among pregnant women with HIV in Cameroon. Our hypothesis was that the addition of azithromycin (AZ) to standard daily trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis would reduce malaria and STI infection rates at delivery. Methods: Pregnant women with HIV at gestational age <28 weeks were randomized to adjunctive monthly oral AZ 1 g daily or placebo for 3 days and both groups received daily standard oral TMP-SMX through delivery. Primary outcomes were (1) positive peripheral malaria infection by microscopy or polymerase chain reaction and (2) composite bacterial genital STI (Chlamydia trachomatis, Neisseria gonorrhoeae, or syphilis) at delivery. Relative risk and 95% confidence intervals were estimated using 2 × 2 tables with significance as P < .05. Results: Pregnant women with HIV (n = 308) were enrolled between March 2018 and August 2020: 155 women were randomized to TMP-SMX-AZ and 153 women to TMP-SMX-placebo. Groups were similar at baseline and loss to follow up was 3.2%. There was no difference in the proportion with malaria (16.3% in TMP-SMX-AZ vs 13.2% in TMP-SMX; relative risk, 1.24 [95% confidence interval, .71-2.16]) or STI at delivery (4.2% in TMP-SMX-AZ vs 5.8% in TMP-SMX; relative risk, 0.72 [95% confidence interval, .26-2.03]). Adverse birth outcomes were not significantly different, albeit lower in the TMP-SMX-AZ arm (preterm delivery 6.7% vs 10.7% [P = .3]; low birthweight 3.4% vs 5.4% [P = .6]). Conclusions: The addition of monthly azithromycin to daily TMP-SMX prophylaxis in pregnant women living with HIV in Cameroon did not reduce the risk of malaria or bacterial STI at delivery.

Microheterogeneity of Transmission Shapes Submicroscopic Malaria Carriage in Coastal Tanzania.

BACKGROUND: Asymptomatic carriage of malaria parasites persists even as malaria transmission declines. Low-density infections are often submicroscopic, not detected with rapid diagnostic tests (RDTs) or microscopy but detectable by polymerase chain reaction (PCR). METHODS: To characterize submicroscopic Plasmodium falciparum carriage in an area of declining malaria transmission, asymptomatic persons >5 years of age in rural Bagamoyo District, Tanzania, were screened using RDT, microscopy, and PCR. We investigated the size of the submicroscopic reservoir of infection across villages, determined factors associated with submicroscopic carriage, and assessed the natural history of submicroscopic malaria over 4 weeks. RESULTS: Among 6076 participants, P. falciparum prevalences by RDT, microscopy, and PCR were 9%, 9%, and 28%, respectively, with roughly two-thirds of PCR-positive individuals harboring submicroscopic infection. Adult status, female sex, dry season months, screened windows, and bed net use were associated with submicroscopic carriage. Among 15 villages encompassing 80% of participants, the proportion of submicroscopic carriers increased with decreasing village-level malaria prevalence. Over 4 weeks, 23% of submicroscopic carriers (61 of 266) became RDT positive, with half exhibiting symptoms, while half (133 of 266) were no longer parasitemic at the end of 4 weeks. Progression to RDT-positive patent malaria occurred more frequently in villages with higher malaria prevalence. CONCLUSIONS: Microheterogeneity in transmission observed at the village level appears to affect both the size of the submicroscopic reservoir and the likelihood of submicroscopic carriers developing patent malaria in coastal Tanzania.

Expansion of artemisinin partial resistance mutations and lack of histidine rich protein-2 and -3 deletions in Plasmodium falciparum infections from Rukara, Rwanda.

BACKGROUND: Emerging artemisinin partial resistance and diagnostic resistance are a threat to malaria control in Africa. Plasmodium falciparum kelch13 (k13) propeller-domain mutations that confer artemisinin partial resistance have emerged in Africa. k13-561H was initially described at a frequency of 7.4% from Masaka in 2014-2015, but not present in nearby Rukara. By 2018, 19.6% of isolates in Masaka and 22% of isolates in Rukara contained the mutation. Longitudinal monitoring is essential to inform control efforts. In Rukara, an assessment was conducted to evaluate recent k13-561H prevalence changes, as well as other key mutations. Prevalence of hrp2/3 deletions was also assessed. METHODS: Samples collected in Rukara in 2021 were genotyped for key artemisinin and partner drug resistance mutations using molecular inversion probe assays and for hrp2/3 deletions using qPCR. RESULTS: Clinically validated k13 artemisinin partial resistance mutations continue to increase in prevalence with the overall level of mutant infections reaching 32% in Rwanda. The increase appears to be due to the rapid emergence of k13-675V (6.4%, 6/94 infections), previously not observed, rather than continued expansion of 561H (23.5% 20/85). Mutations to partner drugs and other anti-malarials were variable, with high levels of multidrug resistance 1 (mdr1) N86 (95.5%) associated with lumefantrine decreased susceptibility and dihydrofolate reductase (dhfr) 164L (24.7%) associated with a high level of antifolate resistance, but low levels of amodiaquine resistance polymorphisms with chloroquine resistance transporter (crt) 76T: at 6.1% prevalence. No hrp2 or hrp3 gene deletions associated with diagnostic resistance were found. CONCLUSIONS: Increasing prevalence of artemisinin partial resistance due to k13-561H and the rapid expansion of k13-675V is concerning for the longevity of artemisinin effectiveness in the region. False negative RDT results do not appear to be an issue with no hrp2 or hpr3 deletions detected. Continued molecular surveillance in this region and surrounding areas is needed to follow artemisinin partial resistance and provide early detection of partner drug resistance, which would likely compromise control and increase malaria morbidity and mortality in East Africa.

Impact of Sulfadoxine-Pyrimethamine and Dihydroartemisinin-Piperaquine as Intermittent Preventive Treatment in Pregnancy on Stool Antimicrobial Resistance Gene Abundance.

Increasing antimicrobial resistance (AMR) is a global public health emergency. Although chemoprevention has improved malaria-related pregnancy outcomes, the downstream effects on AMR have not been characterized. We compared the abundance of 10 AMR genes in stool samples from pregnant women receiving sulfadoxine-pyrimethamine (SP) as intermittent preventive treatment against malaria in pregnancy (IPTp) to that in samples from women receiving dihydroartemisinin-piperaquine (DP) for IPTp. All participants had at least one AMR gene at baseline. Mean quantities of the antifolate gene dfrA17 were increased after two or more doses of SP (mean difference = 1.6, 95% CI: 0.4-2.7, P = 0.008). Antimicrobial resistance gene abundance tended to increase from baseline in SP recipients compared with a downward trend in the DP group. Overall, IPTp-SP had minimal effects on the abundance of antifolate resistance genes (except for dfrA17), potentially owing to a high starting prevalence. However, the trend toward increasing AMR in SP recipients warrants further studies.

High frequency of artemisinin partial resistance mutations in the great lake region revealed through rapid pooled deep sequencing.

In Africa, the first Plasmodium falciparum Kelch13 (K13) artemisinin partial resistance mutation 561H was first detected and validated in Rwanda. Surveillance to better define the extent of the emergence in Rwanda and neighboring countries as other mutations arise in East Africa is critical. We employ a novel scheme of liquid blood drop preservation combined with pooled sequencing to provide a cost-effective rapid assessment of resistance mutation frequencies at multiple collection sites across Rwanda and neighboring countries. Malaria-positive samples (n=5,465) were collected from 39 health facilities in Rwanda, Uganda, Tanzania, and the Democratic Republic of the Congo (DRC) between May 2022 and March 2023 and sequenced in 199 pools. In Rwanda, K13 561H and 675V were detected in 90% and 65% of sites with an average frequency of 19.0% (0-54.5%) and 5.0% (0-35.5%), respectively. In Tanzania, 561H had high frequency in multiple sites while it was absent from the DRC although 675V was seen at low frequency. Conceringly candidate mutations were observed: 441L, 449A, and 469F co-occurred with validated mutations suggesting they are arising under the same pressures. Other resistance markers associated with artemether-lumefantrine are common: P. falciparum multidrug resistance protein 1 N86 at 98.0% and 184F at 47.0% (0-94.3%) and P. falciparum chloroquine resistance transporter 76T at 14.7% (0-58.6%). Additionally, sulfadoxine-pyrimethamine-associated mutations show high frequencies. Overall, K13 mutations are rapidly expanding in the region further endangering control efforts with the potential of engendering partner drug resistance.

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients enrolled at 100 health facilities throughout Tanzania: February to July 2021.

Plasmodium falciparum with the histidine rich protein 2 gene (pfhrp2) deleted from its genome can escape diagnosis by HRP2-based rapid diagnostic tests (HRP2-RDTs). The World Health Organization (WHO) recommends switching to a non-HRP2 RDT for P. falciparum clinical case diagnosis when pfhrp2 deletion prevalence causes ≥ 5% of RDTs to return false negative results. Tanzania is a country of heterogenous P. falciparum transmission, with some regions approaching elimination and others at varying levels of control. In concordance with the current recommended WHO pfhrp2 deletion surveillance strategy, 100 health facilities encompassing 10 regions of Tanzania enrolled malaria-suspected patients between February and July 2021. Of 7863 persons of all ages enrolled and providing RDT result and blood sample, 3777 (48.0%) were positive by the national RDT testing for Plasmodium lactate dehydrogenase (pLDH) and/or HRP2. A second RDT testing specifically for the P. falciparum LDH (Pf-pLDH) antigen found 95 persons (2.5% of all RDT positives) were positive, though negative by the national RDT for HRP2, and were selected for pfhrp2 and pfhrp3 (pfhrp2/3) genotyping. Multiplex antigen detection by laboratory bead assay found 135/7847 (1.7%) of all blood samples positive for Plasmodium antigens but very low or no HRP2, and these were selected for genotyping as well. Of the samples selected for genotyping based on RDT or laboratory multiplex result, 158 were P. falciparum DNA positive, and 140 had sufficient DNA to be genotyped for pfhrp2/3. Most of these (125/140) were found to be pfhrp2+/pfhrp3+, with smaller numbers deleted for only pfhrp2 (n = 9) or only pfhrp3 (n = 6). No dual pfhrp2/3 deleted parasites were observed. This survey found that parasites with these gene deletions are rare in Tanzania, and estimated that 0.24% (95% confidence interval: 0.08% to 0.39%) of false-negative HRP2-RDTs for symptomatic persons were due to pfhrp2 deletions in this 2021 Tanzania survey. These data provide evidence for HRP2-based diagnostics as currently accurate for P. falciparum diagnosis in Tanzania.

An intervention to improve lumbar puncture rates for meningitis surveillance in children at four secondary health facilities in Malawi: A before/after analysis.

OBJECTIVES: A lumbar puncture (LP) procedure plays a key role in meningitis diagnosis. In Malawi and other sub-Saharan African countries, LP completion rates are sometimes poor, making meningitis surveillance challenging. Our objective was to measure LP rates following an intervention to improve these during a sentinel hospital meningitis surveillance exercise in Malawi. METHODS: We conducted a before/after intervention analysis among under-five children admitted to paediatric wards at four secondary health facilities in Malawi. We used local and World Health Organization (WHO) guidelines to determine indications for LP, as these are widely used in low- and middle-income countries (LMIC). The intervention comprised of refresher trainings for facility staff on LP indications and procedure, use of automated reminders to perform LP in real time in the wards, with an electronic data management system, and addition of surveillance-specific clinical officers to support existing health facility staff with performing LPs. Due to the low numbers in the before/after analysis, we also performed a during/after analysis to supplement the findings. RESULTS: A total of 13,375 under-five children were hospitalised over the 21 months window for this analysis. The LP rate was 10.4% (12/115) and 60.4% (32/53) in the before/after analysis, respectively, and 43.8% (441/1006) and 72.5% (424/599) in the supplemental during/after analysis, respectively. In our intervention-specific analysis among the three individual components, there were improvements in the LP rate by 48% (p < 0.001) following the introduction of surveillance-specific clinical officers, 10% (p < 0.001) following the introduction of automated reminders to perform an LP and 13% following refresher training. CONCLUSIONS: This analysis demonstrated a rise in LP rates following our intervention. This intervention package may be considered for planning future facility-based meningitis surveillances in similar low-resource settings.

Evaluation of Malaria Rapid Diagnostic Test Performance and pfhrp2 Deletion in Tanzania School Surveys, 2017.

As part of malaria nationwide monitoring and evaluation initiatives, there is an increasing trend of incorporating malaria rapid diagnostic tests (mRDTs) in surveys conducted within primary schools to detect malaria parasites. However, mRDTs based on the detection of histidine-rich protein 2 (HRP2) are known to yield false-positive results due to persistent antigenemia, and false-negative results may result from low parasitemia or Plasmodium falciparum hrp2/3 gene deletion. We evaluated diagnostic performance of an HRP2 and pan-parasite lactate dehydrogenase (HRP2/pLDH) mRDT against polymerase chain reaction (PCR) for detection of P. falciparum among 17,051 primary school-age children from eight regions of Tanzania in 2017. According to PCR, the prevalence of P. falciparum was 19.2% (95% CI: 18.6-19.8). Using PCR as reference, the sensitivity and specificity of mRDT was 76.2% (95% CI: 74.7-77.7) and 93.9% (95% CI: 93.5-94.3), respectively. Test agreement was lowest in low transmission areas, where true-positive mRDTs were outnumbered by false-negatives due to low parasitemia. Discordant samples (mRDT-negative but PCR-positive) were screened for pfhrp2/3 deletion by real-time PCR. Among those with a parasite density sufficient for analysis, pfhrp2 deletion was confirmed in 60 samples, whereas pfhrp3 deletion was confirmed in two samples; one sample had both pfhrp2 and pfhrp3 deletions. The majority of samples with gene deletions were detected in the high-transmission Kagera region. Compared with mRDTs, PCR and other molecular methods offer increased sensitivity and are not affected by pfhrp2/3 deletions, making them a useful supplement to mRDTs in schools and other epidemiological surveys.

Prevalence of non-falciparum malaria infections among asymptomatic individuals in four regions of Mainland Tanzania.

BACKGROUND: Recent studies point to the need to incorporate the detection of non-falciparum species into malaria surveillance activities in sub-Saharan Africa, where 95% of the world's malaria cases occur. Although malaria caused by infection with Plasmodium falciparum is typically more severe than malaria caused by the non-falciparum Plasmodium species P. malariae, P. ovale spp. and P. vivax, the latter may be more challenging to diagnose, treat, control and ultimately eliminate. The prevalence of non-falciparum species throughout sub-Saharan Africa is poorly defined. Tanzania has geographical heterogeneity in transmission levels but an overall high malaria burden. METHODS: To estimate the prevalence of malaria species in Mainland Tanzania, we randomly selected 1428 samples from 6005 asymptomatic isolates collected in previous cross-sectional community surveys across four regions and analyzed these by quantitative PCR to detect and identify the Plasmodium species. RESULTS: Plasmodium falciparum was the most prevalent species in all samples, with P. malariae and P. ovale spp. detected at a lower prevalence (< 5%) in all four regions; P. vivax was not detected in any sample. CONCLUSIONS: The results of this study indicate that malaria elimination efforts in Tanzania will need to account for and enhance surveillance of these non-falciparum species.