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BackgroundDigital microscopy combined with artificial intelligence (AI) is increasingly being implemented in health care, predominantly in advanced laboratory settings. However, AI-supported digital microscopy could be especially advantageous in primary health care settings, since such methods could improve access to diagnostics via automation and lead to a decreased need for experts on site. To our knowledge, no scoping or systematic review had been published on the use of AI-supported digital microscopy within primary health care laboratories when this scoping review was initiated. A scoping review can guide future research by providing insights to help navigate the challenges of implementing these novel methods in primary health care laboratories. ObjectiveThe objective of this scoping review is to map peer-reviewed studies on AI-supported digital microscopy in primary health care laboratories to generate an overview of the subject. MethodsA systematic search of the databases PubMed, Web of Science, Embase, and IEEE will be conducted. Only peer-reviewed articles in English will be considered, and no limit on publication year will be applied. The concept inclusion criteria in the scoping review include studies that have applied AI-supported digital microscopy with the aim of achieving a diagnosis on the subject level. In addition, the studies must have been performed in the context of primary health care laboratories, as defined by the criteria of not having a pathologist on site and using simple sample preparations. The study selection and data extraction will be performed by 2 independent researchers, and in the case of disagreements, a third researcher will be involved. The results will be presented in a table developed by the researchers, including information on investigated diseases, sample collection, preparation and digitization, AI model used, and results. Furthermore, the results will be described narratively to provide an overview of the studies included. The proposed methodology is in accordance with the JBI methodology for scoping reviews. ResultsThe scoping review was initiated in January 2023, and a protocol was published in the Open Science Framework in January 2024. The protocol was completed in March 2024, and the systematic search will be performed after the protocol has been peer reviewed. The scoping review is expected to be finalized by the end of 2024. ConclusionsA systematic review of studies on AI-supported digital microscopy in primary health care laboratories is anticipated to identify the diseases where these novel methods could be advantageous, along with the shared challenges encountered and approaches taken to address them. International Registered Report Identifier (IRRID)PRR1-10.2196/58149

Background This study aimed to update systematic reviews and meta-analyses on the diagnostic accuracy of postnatal clinical scoring (PCS) methods and foot length (FL) measurement for assessing gestational age (GA) and birth weight in low-income and middle-income countries (LMICs). In addition, the quality of reference standards, including antenatal ultrasound (A-US), last menstrual period (LMP), PCS and newborn weighing scales, was also evaluated.Methods Studies from LMICs published between January 2000 and February 2024 were searched, using databases such as PubMed, Web of Science, Cochrane Library, CINAHL and Scopus. Studies that compared PCS and/or FL with LMP and/or A-US to estimate GA or used calibrated newborn weighing scales for birthweight estimation were included. The risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies-II tool and evaluated the quality of the reference standards. When sufficient data were available, pooled estimates were calculated using random-effects models.Results A total of 50 studies were included. A-US was a reasonable tool for GA assessment if conducted by physicians using fetal biometry and the Hadlock method for GA estimation. LMP was reasonable when women had regular cycles, knew their LMP, were not using contraceptives and LMP data were collected by healthcare providers. When A-US was used as the reference standard, PCS methods estimated GA with a precision of ±2.8 to ±3.2 weeks. FL measurement

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.

Introduction Assessing gestational age accurately is crucial for saving preterm newborns. In low and middle-income countries, such as Pakistan, where access to antenatal ultrasonography (A-USG) is limited, alternative methods are needed. This study evaluated the diagnostic accuracy of foot length (FL) measurement for identifying preterm newborns in rural Pakistan using A-USG as the reference standard.Methods A test validation study was conducted between January and June 2023 in rural Sindh, Pakistan, within the catchment area of the Global Network for Maternal Newborn Health Registry, Thatta. Singleton newborns whose mothers had an A-USG before 20 weeks of gestation were enrolled. A research assistant measured FL three times using a rigid transparent plastic ruler within 48 hours of birth and the average FL was reported. Sensitivity, specificity, positive and negative predictive values (PPV, NPV) and likelihood ratios were calculated. The optimal FL cut-off for the identification of preterm newborns was determined using the Youden Index.Results A total of 336 newborns were included in the final analysis, of whom 75 (22.3%) were born before 37 weeks of gestation. The median gestational age of the newborns was 38.2 weeks, and the median FL was 7.9 cm. The area under the curve was 97.6%. The optimal FL cut-off for identifying preterm newborns was considered as ≤7.6 cm with a sensitivity of 90.8%, specificity of 96.0%, PPV of 86.7% and NPV of 97.3%. A lower cut-off of ≤7.5 cm had a sensitivity of 95.4%, specificity of 84.0%, PPV of 63.1% and NPV of 98.5%.Conclusion In conclusion, this study highlights the utility of FL measurement for identifying preterm newborns in rural settings where A-USG is unavailable before 20 weeks of gestation. Optimal cut-offs of ≤7.6 and ≤7.5 cm provide a simple, cost-effective and reliable tool for clinicians and frontline healthcare providers in rural areas, respectively.Trial registration number NCT05515211.

BackgroundInfections caused by soil-transmitted helminths (STHs) are the most prevalent neglected tropical diseases and result in a major disease burden in low- and middle-income countries, especially in school-aged children. Improved diagnostic methods, especially for light intensity infections, are needed for efficient, control and elimination of STHs as a public health problem, as well as STH management. Image-based artificial intelligence (AI) has shown promise for STH detection in digitized stool samples. However, the diagnostic accuracy of AI-based analysis of entire microscope slides, so called whole-slide images (WSI), has previously not been evaluated on a sample-level in primary healthcare settings in STH endemic countries.Methodology/principal findingsStool samples (n = 1,335) were collected during 2020 from children attending primary schools in Kwale County, Kenya, prepared according to the Kato-Katz method at a local primary healthcare laboratory and digitized with a portable whole-slide microscopy scanner and uploaded via mobile networks to a cloud environment. The digital samples of adequate quality (n = 1,180) were split into a training (n = 388) and test set (n = 792) and a deep-learning system (DLS) developed for detection of STHs. The DLS findings were compared with expert manual microscopy and additional visual assessment of the digital samples in slides with discordant results between the methods. Manual microscopy detected 15 (1.9%) Ascaris lumbricoides, 172 (21.7%) Tricuris trichiura and 140 (17.7%) hookworm (Ancylostoma duodenale or Necator americanus) infections in the test set. Importantly, more than 90% of all STH positive cases represented light intensity infections. With manual microscopy as the reference standard, the sensitivity of the DLS as the index test for detection of A. lumbricoides, T. trichiura and hookworm was 80%, 92% and 76%, respectively. The corresponding specificity was 98%, 90% and 95%. Notably, in 79 samples (10%) classified as negative by manual microscopy for a specific species, STH eggs were detected by the DLS and confirmed correct by visual inspection of the digital samples.Conclusions/significanceAnalysis of digitally scanned stool samples with the DLS provided high diagnostic accuracy for detection of STHs. Importantly, a substantial number of light intensity infections were missed by manual microscopy but detected by the DLS. Thus, analysis of WSIs with image-based AI may provide a future tool for improved detection of STHs in a primary healthcare setting, which in turn could facilitate monitoring and evaluation of control programs.

Abstract Background Reactive case detection (RACD) or testing and treatment of close contacts of recent malaria cases, is commonly practiced in settings approaching malaria elimination, but standard diagnostics have limited sensitivity to detect low level infections. Reactive drug administration (RDA), or presumptive treatment without testing, is an alternative approach, but better understanding regarding community acceptability and operational feasibility are needed. Methods A qualitative study was conducted as part of a two-arm cluster randomized-controlled trial evaluating the effectiveness of RDA targeting high-risk villages and forest workers for reducing Plasmodium vivax and P. falciparum malaria in Thailand. Key informant interviews (KIIs) and focus group discussions (FGDs) were conducted virtually among key public health staff, village health volunteers (VHVs), and household members that implemented or received RDA activities. Transcriptions were reviewed, coded, and managed manually using Dedoose qualitative data analysis software, then underwent qualitative content analysis to identify key themes. Results RDA was well accepted by household members and public health staff that implemented it. RDA participation was driven by fear of contracting malaria, eagerness to receive protection provided by malaria medicines, and the increased access to health care. Concerns were raised about the safety of taking malaria medicines without having an illness, particularly if underlying health conditions existed. Health promotion hospital (HPH) staff implementing RDA noted its operational feasibility, but highlighted difficulty in traveling to remote areas, and requested additional travel resources and hiring more VHVs. Other challenges were highlighted including the need for additional training for VHVs on malaria activities and the inability of HPH staff to conduct RDA due to other health priorities (e.g., Covid-19). More training and practice for VHVs were noted as ways to improve implementation of RDA. Conclusions To maximize uptake of RDA, regular education and sensitization campaigns in collaboration with village leaders on the purpose and rationale of RDA will be critical. To alleviate safety concerns and increase participant safety, a rigorous pharmacovigilance program will be important. To accelerate uptake of RDA, trust between HPH staff and VHVs and the communities they serve must continue to be strengthened to ensure acceptance of the intervention. Trial registration This study was approved by the Committee on Human Research at the University of California San Francisco (19–28,060) and the local Ethics Committee for Research in Human Subjects at Tak Provincial Health office (009/63) and Kanchanaburi Provincial health office (Kor Chor 0032.002/2185). Local authorities and health officers in the provinces, districts, and villages agreed upon and coordinated the implementation of the study. All methods in this study were carried out in accordance with relevant guidelines and regulations.

IntroductionReliable methods for identifying prematurity and low birth weight (LBW) are crucial to ending preventable deaths in newborns. This study explored healthcare providers' (HCPs) knowledge, practice, perceived barriers in assessing gestational age and birth weight and their referral methods for preterm and LBW infants. The study additionally assessed the potential of using a mobile app for the identification and referral decision of preterm and LBW.MethodsThis qualitative descriptive study was conducted in Thatta District, Sindh, Pakistan. Participants, including doctors, nurses, lady health visitors, and midwives, were purposefully selected from a district headquarter hospital, and private providers in the catchment area of Global Network's Maternal and Newborn Health Registry (MNHR). Interviews were conducted using an interview guide after obtaining written informed consent. Audio recordings of the interviews were transcribed and analyzed using NVIVO® software with an inductive approach.ResultsThe HCPs had extensive knowledge about antenatal and postnatal methods for assessing gestational age. They expressed a preference for antenatal ultrasound due to the perceived accuracy, though accept practical barriers including workload, machine malfunctions, and cost. Postnatal assessment using the Ballard score was only undertaken sparingly due to insufficient training and subjectivity. All HCPs preferred electronic weighing scales for birth weight Barriers encountered included weighing scale calibration and battery issues. There was variation in the definition of prematurity and LBW, leading to delays in referral. Limited resources, inadequate education, and negative parent past experiences were barriers to referral. Foot length measurements were not currently being used. While mobile apps are felt to have potential, unreliable electricity supply and internet connectivity are barriers.ConclusionThe HCPs in this study were knowledgeable in terms of potential tools, but acknowledged the logistical and parental barriers to implementation.

Abstract Background The World Health Organization recommends pneumococcal vaccination (PCV) in the first year of life. We investigated pneumococcal serotypes in children with clinical or radiologically confirmed pneumonia and healthy controls prior to PCV13 vaccine introduction in Zanzibar. Methods Children (n = 677) with non-severe acute febrile illness aged 2–59 months presenting to a health centre in Zanzibar, Tanzania April–July 2011 were included. Nasopharyngeal swabs collected at enrolment were analysed by real-time PCR to detect and quantify pneumococcal serotypes in patients (n = 648) and in healthy asymptomatic community controls (n = 161). Children with clinical signs of pneumonia according to the Integrated Management of Childhood illness guidelines (“IMCI pneumonia”) were subjected to a chest-X-ray. Consolidation on chest X-ray was considered “radiological pneumonia”. Results Pneumococcal DNA was detected in the nasopharynx of 562/809 (69%) children (70% in patients and 64% in healthy controls), with no significant difference in proportions between patients with or without presence of fever, malnutrition, IMCI pneumonia or radiological pneumonia. The mean pneumococcal concentration was similar in children with and without radiological pneumonia (Ct value 26.3 versus 27.0, respectively, p = 0.3115). At least one serotype could be determined in 423 (75%) participants positive for pneumococci of which 33% had multiple serotypes detected. A total of 23 different serotypes were identified. One serotype (19F) was more common in children with fever (86/648, 13%) than in healthy controls (12/161, 7%), (p = 0.043). Logistic regression adjusting for age and gender showed that serotype 9A/V [aOR = 10.9 (CI 2.0–60.0, p = 0.006)] and 14 [aOR = 3.9 (CI 1.4–11.0, p = 0.012)] were associated with radiological pneumonia. The serotypes included in the PCV13 vaccine were found in 376 (89%) of the 423 serotype positive participants. Conclusion The PCV13 vaccine introduced in 2012 targets a great majority of the identified serotypes. Infections with multiple serotypes are common. PCR-determined concentrations of pneumococci in nasopharynx were not associated with radiologically confirmed pneumonia. Trial registration Clinicaltrials.gov (NCT01094431).

Abstract Background Primaquine is a pro-drug and its active metabolite is potent against mature Plasmodium falciparum gametocytes. Primaquine is metabolized by a highly polymorphic cytochrome P450 2D6 (CYP2D6) enzyme. Mutations in the gene encoding this enzyme may lead to impaired primaquine activity. This study assessed if 0.25 mg/kg single-dose primaquine is safe and sufficient to reduce transmission of gametocytes in individuals with no, reduced, or increased CYP2D6 enzyme activity. Methods Between June 2019 and January 2020 children aged 1–10 years, attending at Yombo dispensary, Bagamoyo district, with confirmed microcopy-determined uncomplicated P. falciparum malaria were enrolled in the study. The enrolled patients were treated with a standard artemether-lumefantrine regimen plus 0.25 mg/kg single-dose primaquine and followed up for 28 days for clinical and laboratory assessment. Primaquine was administered with the first dose of artemether-lumefantrine. Safety assessment involved direct questioning and recording of the nature and incidence of clinical signs and symptoms, and measurement of haemoglobin (Hb) concentration. Blood samples collected from 100 patients were used for assessment of post-treatment infectiousness on day 7 using mosquito membrane feeding assays. Molecular methods were used to determine CYP2D6 and glucose-6-phosphate dehydrogenase (G6PD) status. The primary outcome was the safety of 0.25 mg/kg single-dose primaquine based on CYP2D6 status. Results In total, 157 children [median age 6.4 (Interquartile range 4.0–8.2) years] were recruited, of whom 21.0% (33/157) and 12.7% (20/157) had reduced CYP2D6 and deficient G6PD activity, respectively. Day 3 mean absolute Hb concentration reduction was 1.50 g/dL [95% confidence interval (CI) 1.10–1.90] and 1.51 g/dL (95% CI 1.31–1.71) in reduced and normal CYP2D6 patients, respectively (t = 0.012, p = 0.990). The day 3 mean absolute Hb concentration reduction in G6PD deficient, G6PD normal and heterozygous female was 1.82 g/dL (95% CI 1.32–2.32), 1.48 g/dL (95% CI 1.30–1.67) and 1.47 g/dL (95% CI 0.76–2.18), respectively (F = 0.838, p = 0.435). Sixteen percent (16/98) of the patients each infected at least one mosquito on day 7, and of these, 10.0% (2/20) and 17.9% (14/78) had reduced and normal CYP2D6 enzyme activity, respectively (x 2 = 0.736, p = 0.513). Conclusion Single-dose 0.25 mg/kg primaquine was safe and sufficient for reducing transmission of P. falciparum gametocytes regardless of CYP2D6 or G6PD status. Trial registration Study registration number: NCT03352843.

Abstract Background Bartonella spp. are emerging pathogens transmitted by arthropod vectors, possibly including ticks. We have investigated signs of bartonellosis in Swedish patients with presumed tick-bite exposure and symptom duration of at least 6 months. Methods Serological testing for Bartonella henselae and Bartonella quintana was performed in 224 patients. Symptoms, tick exposure, evidence of co-infection and previous treatments were evaluated. Seropositive patients were compared to a matched group (twofold larger and negative serology) from the same study cohort. Results Seroprevalence was 7% for B. henselae and 1% for B. quintana, with one patient testing positive to both agents. Tick bites were reported by 63% of the patients in the seropositive group and 88% in the seronegative group and presumed tick exposure was more common in the seronegative group. Animal contact was equally common in both groups, along with reported symptoms. The most common symptoms were fatigue, muscular symptoms, arthralgia and cognitive symptoms. Exposure to co-infections was evenly distributed in the seropositive and seronegative groups. Conclusions Antibodies to Bartonella were more common in this cohort of patients than in cohorts of healthy Swedish blood donors in previous studies but lower than those in blood donors from southern Europe. Positive Bartonella serology was not linked to any specific symptom, nor to (suspected) tick-bite exposure. Graphical abstract

Objectives: This study aimed to analyze any reported antibiotic use for children aged

Abstract Background The anti-malarial drug, amodiaquine, a commonly used, long-acting partner drug in artemisinin-based combination therapy, is metabolized to active desethyl-amodiaquine (DEAQ) by cytochrome P450 2C8 (CYP2C8). The CYP2C8 gene carries several polymorphisms including the more frequent minor alleles, CYP2C8*2 and CYP2C8*3. These minor alleles have been associated with decreased enzymatic activity, slowing the amodiaquine biotransformation towards DEAQ. This study aimed to assess the influence of these CYP2C8 polymorphisms on the efficacy and tolerability of artesunate–amodiaquine (AS–AQ) treatment for uncomplicated Plasmodium falciparum malaria in Zanzibar. Methods Dried blood spots on filter paper were collected from 618 children enrolled in two randomized clinical trials comparing AS–AQ and artemether-lumefantrine in 2002–2005 in Zanzibar. Study participant were under five years of age with uncomplicated falciparum malaria. Human CYP2C8*2 and CYP2C8*3 genotype frequencies were determined by PCR-restriction fragment length polymorphism. Statistical associations between CYP2C8*2 and/or CYP2C8*3 allele carriers and treatment outcome or occurrence of adverse events were assessed by Fisher’s exact test. Results The allele frequencies of CYP2C8*2 and CYP2C8*3 were 17.5 % (95 % CI 15.4–19.7) and 2.7 % (95 % CI 1.8–3.7), respectively. There was no significant difference in the proportion of subjects carrying either CYP2C8*2 or CYP2C8*3 alleles amongst those with re-infections (44.1 %; 95 % CI 33.8–54.8) or those with recrudescent infections (48.3 %; 95 % CI 29.4–67.5), compared to those with an adequate clinical and parasitological response (36.7 %; 95 % CI 30.0-43.9) (P = 0.25 and P = 0.31, respectively). However, patients carrying either CYP2C8*2 or CYP2C8*3 alleles were significantly associated with an increased occurrence of non-serious adverse events, when compared with CYP2C8 *1/*1 wild type homozygotes (44.9 %; 95 % CI 36.1–54.0 vs. 28.1 %; 95 % CI 21.9–35.0, respectively; P = 0.003). Conclusions CYP2C8 genotypes did not influence treatment efficacy directly, but the tolerability to AS–AQ may be reduced in subjects carrying the CYP2C8*2 and CYP2C8*3 alleles. The importance of this non-negligible association with regard to amodiaquine-based malaria chemotherapy warrants further investigation.

Abstract Background Peer supervision improves health care delivery by health workers. However, in rural Uganda, self-supervision is what is prescribed for licensed private drug sellers by statutory guidelines. Evidence shows that self-supervision encourages inappropriate treatment of children less than 5 years of age by private drug sellers. This study constructed a model for an appropriate peer supervisor to augment the self-supervision currently practiced by drug sellers at district level in rural Uganda. Methods In this qualitative study, six Key informant interviews were held with inspectors while ten focus group discussions were conducted with 130 drug sellers. Data analysis was informed by the Kathy Charmaz constructive approach to grounded theory. Atlas ti.7 software package was used for data management. Results A model with four dimensions defining an appropriate peer supervisor was developed. The dimensions included; incentives, clearly defined roles, mediation and role model peer supervisor. While all dimensions were regarded as being important, all participants interviewed agreed that incentives for peer supervisors were the most crucial. Overall, an appropriate peer supervisor was described as being exemplary to other drug sellers, operated within a defined framework, well facilitated to do their role and a good go-between drug sellers and government inspectors. Conclusion Four central contributions advance literature by the model developed by our study. First, the model fills a supervision gap for rural private drug sellers. Second, it highlights the need for terms of reference for peer supervisors. Third, it describes who an appropriate peer supervisor should be. Lastly, it elucidates the kind of resources needed for peer supervision.

Artemisinin-based combination therapies (ACTs) are first-line treatments for uncomplicated Plasmodium falciparum malaria. ACT resistance is spreading in Asia but not yet in Africa. Reduced effects of ACT partner drugs have been reported but with little information regarding widely used artesunate/amodiaquine (ASAQ). We studied its efficacy in Zanzibar after 14 years as first-line treatment directly by an in vivo, single-armed trial and indirectly by prevalences of different genotypes in the P. falciparum chloroquine-resistance transporter, multidrug-resistance 1, and Kelch 13 propeller domain genes. In vivo efficacy was higher during 2017 (100%; 95% CI 97.4%–100%) than during 2002–2005 (94.7%; 95% CI 91.9%–96.7%) (p = 0.003). Molecular findings showed no artemisinin resistance–associated genotypes and major increases in genotypes associated with high sensitivity/efficacy for amodiaquine than before ASAQ was introduced. Thus, the efficacy of ASAQ is maintained and appears to be increased after long-term use in contrast to what is observed for other ACTs used in Africa.

Abstract Background Artesunate plus sulfadoxine–pyrimethamine (ASP) is first-line treatment for uncomplicated Plasmodium falciparum malaria in most of India, except for six North-eastern provinces where treatment failure rates were high. In Ujjain, central India, the frequency of mutations associated with increased drug tolerance, but not overt resistance to sulfadoxine and pyrimethamine were 9% and > 80%, respectively, in 2009 and 2010, just prior to the introduction of ASP. The frequency of drug resistance associated mutations in Ujjain in 2015–2016 after 3–4 years of ASP use, are reported. Methods Blood samples from patients with P. falciparum mono-infection verified by microscopy were collected on filter-paper at all nine major pathology laboratories in Ujjain city. Codons pfdhfr 16–185, pfdhps 436–632 and K13 407–689 were identified by sequencing. Pfcrt K76T and pfmdr1 N86Y were identified by restriction fragment length polymorphism. Results Sulfadoxine–pyrimethamine resistance-associated pfdhfr 108 N and 59R alleles were found in 100/104 (96%) and 87/91 (96%) samples, respectively. Pfdhps 437G was found in 10/105 (10%) samples. Double mutant pfdhfr 59R + 108 N were found in 75/81 (93%) samples. Triple mutant pfdhfr 59R + 108 N and pfdhps 437G were found in 6/78 (8%) samples. Chloroquine-resistance-associated pfcrt 76T was found in 102/102 (100%). Pfmdr1 N86 and 86Y were identified in 83/115 (72%) and 32/115 (28%) samples, respectively. Conclusion The frequency of P. falciparum with reduced susceptibility to sulfadoxine–pyrimethamine remained high, but did not appear to have increased significantly since the introduction of ASP. No polymorphisms in K13 associated with decreased artemisinin susceptibility were found. ASP probably remained effective, supporting continued ASP use.

Abstract Background Extended artemisinin-based combination therapy (ACT) for treatment of uncomplicated Plasmodium falciparum malaria with already existing drug regimens, such as artemether-lumefantrine, might be effective in tackling the emerging ACT resistance. However, given the history of cardiotoxicity among anti-malarial drugs structurally similar to lumefantrine, the potential effect of extended artemether-lumefantrine treatment on the electrocardiographic (ECG) QTc interval is of high concern. Methods Male and non-pregnant females aged 1–65 years, diagnosed with uncomplicated P. falciparum malaria in Bagamoyo district, Tanzania, were randomized into two arms. The intervention arm received an extended, i.e. 6-day, course of artemether-lumefantrine and an additional single low-dose primaquine (0.25 mg/kg) administered together with the last artemether-lumefantrine dose. The control arm received the standard weight-based 3-day course. ECGs were performed at day 0 and 4–5 h after the last dose at day 5. QT intervals were read manually using the tangent method and automatically. Bazett’s (QTcB) and Fridericia’s (QTcF) formulae were used for correction for heart rate. Descriptive statistics were used to calculate baseline characteristics and the number of supra-thresholds QTc intervals (QTc prolongation > 500, change in QTc interval (ΔQTc) > 60 ms). The mean change in QTc interval in and between the two arms was compared using the paired t-test and independent samples t-test, respectively. Results A total of 195 patients were enrolled, 103 and 92 in the intervention and control arm, respectively. No patient experienced QTc intervals > 500 ms on day 5 by both formulae. Patients with ΔQTc > 60 ms, for QTcF were 6/103 (5.8%) vs 2/92 (2.2%) and for QTcB 2/103 (1.9%) vs 1/92 (1.1%) in the intervention and control arms, respectively. The mean difference in ΔQTc interval was statistically significant between the two arms with both correction formulae, 11.4 ms (95% CI 2.7–20.0, p = 0.010) and 13.4 ms (95% CI 5.3–21.5, p = 0.001), for QTcB and QTcF, respectively. Conclusion The extended 6-day course of artemether-lumefantrine did not reveal clinically relevant QTc prolonging effects. However, significant QTcF prolongation and presence of patients with supra-threshold QTc values observed in the intervention arm underscore the importance of further monitoring of QTc parameters in extended artemether-lumefantrine treatment. Trial registration ClinicalTrials.gov, NCT03241901. Registered July 27, 2017. https://clinicaltrials.gov/show/NCT03241901

Abstract Background Support supervision improves performance outcomes among health workers. However, the national professional guidelines for new licenses and renewal for Class C drug shops in Uganda prescribe self-supervision of licensed private drug sellers. Without support supervision, inappropriate treatment of malaria, pneumonia and diarrhoea among children under 5 years of age continues unabated. This study assessed experiences of drug sellers and peer supervisors at the end of a peer supervision intervention in Luuka District in East Central Uganda. Methods Eight in-depth interviews (IDIs) were held with peer supervisors while five focus group discussions (FGDs) were conducted among registered drug sellers at the end of the peer supervision intervention. The study assessed experiences and challenges of peer supervisors and drug sellers regarding peer supervision. Transcripts were imported into Atlas.ti 7 qualitative data management software where they were analysed using thematic content analysis. Results Initially, peer supervisors were disliked and regarded by drug sellers as another extension of drug inspectors. However, with time a good relationship was established between drug sellers and peer supervisors leading to regular, predictable and supportive peer supervision. This increased confidence of drug sellers in using respiratory timers and rapid diagnostic tests in diagnosing pneumonia symptoms and uncomplicated malaria, respectively, among children under 5 years. There was also an improvement in completing the sick child register which was used for self-assessment by drug sellers. The drug shop association was mentioned as a place where peer supervision should be anchored since it was a one-stop centre for sharing experiences and continuous professional development. Drug sellers proposed including community health workers in monthly drug shop association meetings so that they may also gain from the associated benefits. Untimely completion of the sick child registers by drug sellers and inadequate financial resources were the main peer supervision challenges mentioned. Conclusion Drug sellers benefitted from peer supervision by developing a good relationship with peer supervisors. This relationship guaranteed reliable and predictable supervision ultimately leading to improved treatment practices. There is need to explore the minimum resources needed for peer supervision of drug sellers to further inform practice and policy.

Abstract Background Artemisinin-based combination therapy (ACT) resistant Plasmodium falciparum represents an increasing threat to Africa. Extended ACT regimens from standard 3 to 6 days may represent a means to prevent its development and potential spread in Africa. Methods Standard 3-day treatment with artemether–lumefantrine (control) was compared to extended 6-day treatment and single low-dose primaquine (intervention); in a randomized controlled, parallel group, superiority clinical trial of patients aged 1–65 years with microscopy confirmed uncomplicated P. falciparum malaria, enrolled in Bagamoyo district, Tanzania. The study evaluated parasite clearance, including proportion of PCR detectable P. falciparum on days 5 and 7 (primary endpoint), cure rate, post-treatment prophylaxis, safety and tolerability. Clinical, and laboratory assessments, including ECG were conducted during 42 days of follow-up. Blood samples were collected for parasite detection (by microscopy and PCR), molecular genotyping and pharmacokinetic analyses. Kaplan–Meier survival analyses were done for both parasite clearance and recurrence. Results A total of 280 patients were enrolled, 141 and 139 in the control and intervention arm, respectively, of whom 121 completed 42 days follow-up in each arm. There was no difference in proportion of PCR positivity across the arms at day 5 (80/130 (61.5%) vs 89/134 (66.4%), p = 0.44), or day 7 (71/129 (55.0%) vs 70/134 (52.2%), p = 0.71). Day 42 microscopy determined cure rates (PCR adjusted) were 97.4% (100/103) and 98.3% (110/112), p = 0.65, in the control and intervention arm, respectively. Microscopy determined crude recurrent parasitaemia during follow-up was 21/121 (17.4%) in the control and 14/121 (11.6%) in the intervention arm, p = 0.20, and it took 34 days and 42 days in the respective arms for 90% of the patients to remain without recurrent parasitaemia. Lumefantrine exposure was significantly higher in intervention arm from D3 to D42, but cardiac, biochemical and haematological safety was high and similar in both arms. Conclusion Extended 6-day artemether–lumefantrine treatment and a single low-dose of primaquine was not superior to standard 3-day treatment for ACT sensitive P. falciparum infections but, importantly, equally efficacious and safe. Thus, extended artemether–lumefantrine treatment may be considered as a future treatment regimen for ACT resistant P. falciparum, to prolong the therapeutic lifespan of ACT in Africa. Trial registration ClinicalTrials.gov, NCT03241901. Registered July 27, 2017 https://clinicaltrials.gov/show/NCT03241901

Abstract Background Tanzania’s Zanzibar archipelago has made significant gains in malaria control over the last decade and is a target for malaria elimination. Despite consistent implementation of effective tools since 2002, elimination has not been achieved. Importation of parasites from outside of the archipelago is thought to be an important cause of malaria’s persistence, but this paradigm has not been studied using modern genetic tools. Methods Whole-genome sequencing (WGS) was used to investigate the impact of importation, employing population genetic analyses of Plasmodium falciparum isolates from both the archipelago and mainland Tanzania. Ancestry, levels of genetic diversity and differentiation, patterns of relatedness, and patterns of selection between these two populations were assessed by leveraging recent advances in deconvolution of genomes from polyclonal malaria infections. Results Significant decreases in the effective population sizes were inferred in both populations that coincide with a period of decreasing malaria transmission in Tanzania. Identity by descent analysis showed that parasites in the two populations shared long segments of their genomes, on the order of 5 cM, suggesting shared ancestry within the last 10 generations. Even with limited sampling, two of isolates between the mainland and Zanzibar were identified that are related at the expected level of half-siblings, consistent with recent importation. Conclusions These findings suggest that importation plays an important role for malaria incidence on Zanzibar and demonstrate the value of genomic approaches for identifying corridors of parasite movement to the island.

BackgroundCurrent diagnostics for patients with lingering symptoms categorized as post-treatment Lyme disease syndrome (PTLDS) have their limitations and may be difficult to interpret. The aim of this exploratory study was to evaluate the feasibility of protein biomarker profiling as a diagnostic platform for this category of patients and to compare these results with similarly obtained results from a group of patients with acute neuroborreliosis.Methods and findingsTwo groups of patient cohorts (Cohort 1 and 2) were analyzed for biomarkers in serum and cerebrospinal fluid (CSF); the results were used for group-level comparison. Cohort 1 comprised 158 adult patients selected from 224 previously diagnosed patients, who between October 2015 and December 2018, after referral, were enrolled and structurally investigated based on defined inclusion criteria. They displayed similar lingering symptoms, with a duration of at least 6 months, after presumed previous tick-borne infection (TBI) and are fully described in a previously published study originating from the Center for Vector-borne Infections (CVI), Uppsala University Hospital, Sweden. Cohort 2, comprised 30 patients diagnosed at Uppsala University Hospital between 2016 and 2019 with laboratory-confirmed acute neuroborreliosis. Their proteomic results, based on serum and CSF analyses, were compared with the 158 patients in Cohort 1. The expression and the concentration of potential biomarkers in each patient's serum and CSF samples were measured based on two multiplex protein panels enabling simultaneous analysis of 92 inflammatory and neurology biomarkers. The PTLDS patient subgroup showed no nominally significant proteins compared to the other CVI patients in Cohort 1. However, CVI patients with signs of inflammation, which were evenly distributed in Cohort 1, showed 16 significantly (p ConclusionsIn this first comprehensive inflammatory and neurological biomarker profile study no differences in biomarker profiles were detected between patients with PTLDS and patients with similar persisting symptoms but who did not meet the PTLDS criteria, regardless of whether laboratory verified previous exposure to Borrelia or other TBI's were present. However, the expressed markers differed from those found in patients with confirmed acute neuroborreliosis, which does not support the view that PTLDS reflects an ongoing Borrelia infection. Further studies are needed to understand and assess the usefulness of biosignatures of patients with PTLDS before they can be applied in a clinical setting.

Objectives In many resource-limited health systems, point-of-care tests (POCTs) are the only means for clinical patient sample analyses. However, the speed and simplicity of POCTs also makes their use appealing to clinicians in high-income countries (HICs), despite greater laboratory accessibility. Although also part of the clinical routine in HICs, clinician perceptions of the utility of POCTs are relatively unknown in such settings as compared with others. In a Swedish paediatric emergency department (PED) where POCT use is routine, we aimed to characterise healthcare providers’ perspectives on the clinical utility of POCTs and explore their implementation in the local setting; to discuss and compare such perspectives, to those reported in other settings; and finally, to gather requests for ideal novel POCTs.Design Qualitative focus group discussions study. A data-driven content analysis approach was used for analysis.Setting The PED of a secondary paediatric hospital in Stockholm, Sweden.Participants Twenty-four healthcare providers clinically active at the PED were enrolled in six focus groups.Results A range of POCTs was routinely used. The emerging theme Utility of our POCT use is double-edged illustrated the perceived utility of POCTs. While POCT services were considered to have clinical and social value, the local routine for their use was named to distract clinicians from the care for patients. Requests were made for ideal POCTs and their implementation.Conclusion Despite their clinical integration, deficient implementation routines limit the benefits of POCT services to this well-resourced paediatric clinic. As such deficiencies are shared with other settings, it is suggested that some characteristics of POCTs and of their utility are less related to resource level and more to policy deficiency. To address this, we propose the appointment of skilled laboratory personnel as ambassadors to hospital clinics offering POCT services, to ensure higher utility of such services.

Background: Though Maytenus senegalensis is one of the medicinal plants widely used in traditional medicine to treat infectious and inflammatory diseases in Africa, there is a lack of safety data regarding its use. Therefore, the study aimed to asselss the safety and tolerability of the antimalarial herbal remedy M. senegalensis. Material and Methods: The study design was an open-label, single-arm, dose-escalation. Twelve eligible male healthy Tanzanians aged 18 to 45 years were enrolled in four study dose groups. Volunteers’ safety and tolerability post-investigational-product administration were monitored on days 0 to 7,14, and 56. Results: There were no deaths or serious adverse events in any of the study groups, nor any adverse events that resulted in premature discontinuation. The significant mean changes observed in WBC (p = 0.003), Neutrophils (p = 0.02), Lymphocytes (p = 0.001), Eosinophils (p = 0.009), Alanine aminotransferase (p = 0.002), Creatinine (p = 0.03) and Total bilirubin (p = 0.004) laboratory parameters were not associated with any signs of toxicity or clinical symptoms. Conclusions: M. senegalensis was demonstrated to be safe and tolerable when administered at a dose of 800 mg every eight hours a day for four days. This study design may be adapted to evaluate other herbal remedies.

Abstract Rationale, aims, and objectives Appropriate treatment of pediatric fever in rural areas remains a challenge and maybe partly due to inadequate supervision of licensed drug sellers. This study assessed the effectiveness of peer‐supervision among drug sellers on the appropriate treatment of pneumonia symptoms, uncomplicated malaria, and non‐bloody diarrhea among children less than 5 years of age in the intervention (Luuka) and comparison (Buyende) districts, in East‐Central Uganda. Methods Data on pneumonia symptoms, uncomplicated malaria, and non‐bloody diarrhea among children less than 5 years of age was abstracted from drug shop sick child registers over a 12‐month period; 6 months before and 6 months after the introduction of peer‐supervision. Interrupted time series were applied to determine the effectiveness of the peer‐supervision intervention on the appropriate treatment of pneumonia, uncomplicated malaria, and non‐bloody diarrhea among children less than 5 years of age attending drug shops in East Central Uganda. Results The proportion of children treated appropriately for pneumonia symptoms was 10.84% (P

BackgroundPersistent symptoms attributed to presumed tick-bite exposure constitute an unresolved medical controversy. We evaluated whether Swedish adults who met the criteria for post-treatment Lyme disease syndrome (PTLDS) exhibited characteristics distinguishable from adults who did not, but who displayed similar symptoms and disease course after suspected previous tick-bite infection (TBI).Methods and findingsDuring 2015-2018, 255 patients-referred to the Centre for Vector-borne Infections, Uppsala University Hospital, Sweden with symptoms lasting longer than six months-were recruited. Of this group, 224 completed the study. Each patient was examined by an infectious disease specialist and, besides a full medical history, underwent a panel of blood and cerebrospinal fluid laboratory tests including hematological, biochemical, microbiological and immunological analyses, and the RAND-36 scale to measure quality of life. For analysis purposes, patients were divided into five subgroups, of which one represented PTLDS. According to serological results indicating TBI and documented/ reported objective signs of Lyme disease, 85 (38%) patients fulfilled the criteria for PTLDS and were compared with the other 139 (62%) serologically classified patients. In the PTLDS group, erythema chronicum migrans (ECM) was documented/reported in 86% of patients, previous neuroborreliosis in 15%, and acrodermatitis chronica atroficans (ACA) in 3.5%. However, there were no significant differences regarding symptoms, laboratory results or disease course between patients with PTLDS and those without laboratory evidence of Borrelia exposition. Most reported symptoms were fatigue-related (70%), musculoskeletal (79%), neurological (82%) and neurocognitive (57%). Tick bites were recalled by 74%. The RAND-36 score was significantly below that of the general Swedish population. Signs of immunological/inflammatory reactivity with myositis antibodies were detected in 20% of patients, fibrinogen levels were moderately increased in 21% and elevated rheumatoid factor in 6%.ConclusionsThe PTLDS group did not differ exclusively in any respect from the other subgroups, which either lacked previously documented/reported evidence of borreliosis or even lacked detectable serological signs of exposure to Lyme disease. The results suggest that symptoms often categorized as Chronic-Lyme-Disease (CLD) in the general debate, cannot be uniquely linked to Lyme disease. However, approximately 20% of the total group of patients showed signs of autoimmunity. Further studies are needed to elucidate the underlying causes and mechanisms of PTLDS and there is reason to consider a multifactorial approach.

Background Regulatory inspection of private drug shops in Uganda is a mandate of the Ministry of Health carried out by the National Drug Authority. This study evaluated how this mandate is being carried out at national, district, and drug shop levels. Specifically, perspectives on how the inspection is done, who does it, and challenges faced were sought from inspectors and drug sellers. Methods Six key informant interviews (KIIs) were held with inspectors at the national and district level, while eight focus group discussions (FGDs) were conducted among nursing assistants, and two FGDs were held with nurses. The study appraised current methods of inspecting drug sellers against national professional guidelines for licensing and renewal of class C drug shops in Uganda. Transcripts were managed using Atlas ti version 7 (ATLAS.ti GmbH, Berlin) data management software where the thematic content analysis was done. Results Five themes emerged from the study: authoritarian inspection, delegated inspection, licensing, training, and bribes. Under authoritarian inspection, drug sellers decried the high handedness used by inspectors when found with expired or no license at all. For delegated inspection, drug sellers said that sometimes, inspectors send health assistants and sub-county chiefs for inspection visits. This cadre of people is not recognized by law as inspectors. Inspectors trained drug sellers on how to organize their drug shops better and how to use new technologies such as rapid diagnostic tests (RDTs) in diagnosing malaria. Bribes were talked about mostly by nursing assistants who purported that inspectors were not interested in inspection per se but collecting illicit payments from them. Inspectors said that the facilitation they received from the central government were inadequate for a routine inspection. Conclusion The current method of inspecting drug sellers is harsh and instills fear among drug sellers. There is a need to establish a well-recognized structure of inspection as well as establish channels of dialogue between inspectors and drug sellers if meaningful compliance is to be achieved. The government also needs to enhance both human and financial resources if meaningful inspection of drug sellers is to take place.

Summary: Background: Global assessments of antibiotic consumption have relied on pharmaceutical sales data that do not measure individual-level use, and are often unreliable or unavailable for low-income and middle-income countries (LMICs). To help fill this evidence gap, we compiled data from national surveys in LMICs in 2005–17 reporting antibiotic use for sick children under the age of 5 years. Methods: Based on 132 Demographic and Health Surveys and Multiple Indicator Cluster Surveys from 73 LMICs, we analysed trends in reported antibiotic use among children under 5 years of age with fever, diarrhoea, or cough with fast or difficult breathing by WHO region, World Bank income classification, and symptom complaint. A logit transformation was used to estimate the outcome using a linear Bayesian regression model. The model included country-level socioeconomic, disease incidence, and health system covariates to generate estimates for country-years with missing values. Findings: Across LMICs, reported antibiotic use among sick children under 5 years of age increased from 36·8% (uncertainty interval [UI] 28·8–44·7) in 2005 to 43·1% (33·2–50·5) in 2017. Low-income countries had the greatest relative increase; in these countries, reported antibiotic use for sick children under 5 years of age rose 34% during the study period, from 29·6% (21·2–41·1) in 2005 to 39·5% (32·9–47·6) in 2017, although it remained the lowest of any income group throughout the study period. Interpretation: We found a limited but steady increase in reported antibiotic use for sick children under 5 years of age across LMICs in 2005–17, although overlapping UIs complicate interpretation. The increase was largely driven by gains in low-income countries. Our study expands the evidence base from LMICs, where strengthening antibiotic consumption and resistance surveillance is a global health priority. Funding: Uppsala Antibiotic Centre, Uppsala University, Uppsala University Hospital, Makerere University, Gothenburg University.

Background Despite advances in vaccination and case management, pneumonia remains the single largest contributor to early child mortality worldwide. Zinc has immune-enhancing properties, but its role in adjunctive treatment of pneumonia in low-income and middle-income countries (LMICs) is controversial and research still active.Methods Systematic review and meta-analysis of randomised controlled trials of zinc and placebo in pneumonia in children aged 2 to 60 months in LMICs. Databases included MEDLINE, the Cochrane Library, EMBASE, LILACS, SciELO, the WHO portal, Scopus, Google Scholar and ClinicalTrials.gov. Inclusion criteria included accepted signs of pneumonia and clear measure of outcome. Risk of bias was independently assessed by two authors. ORs with 95% CI were used for calculating the pooled estimate of dichotomous outcomes including treatment failure and mortality. Time to recovery was expressed as HRs. Sensitivity analyses considering risk of bias and subgroup analyses for pneumonia severity were performed.Results We identified 11 trials published between 2004 and 2019 fulfilling the a priori defined criteria, 7 from South Asia and 3 from Africa and 1 from South America. Proportional treatment failure was comparable in both zinc and placebo groups when analysed for all patients (OR 0.95 (95% CI 0.80 to 1.14)) and only for those with severe pneumonia (OR 0.93 (95% CI 0.75 to 1.14)). No difference was seen in mortality between zinc and placebo groups (OR 0.64 (95% CI 0.31 to 1.31)). Time to recovery from severe pneumonia did not differ between the treatment and control groups for patients with severe pneumonia (HR 1.01 (95% CI 0.89 to 1.14)). Removal of four studies with high risk of bias made no difference to the conclusions.Conclusion There is no evidence that adjunctive zinc treatment improves recovery from pneumonia in children in LMICs.Trial registration number CRD42019141602.

BackgroundMalaria remains a major global health problem with a need for improved field-usable diagnostic tests. We have developed a portable, low-cost digital microscope scanner, capable of both brightfield and fluorescence imaging. Here, we used the instrument to digitize blood smears, and applied deep learning (DL) algorithms to detect Plasmodium falciparum parasites.MethodsThin blood smears (n = 125) were collected from patients with microscopy-confirmed P. falciparum infections in rural Tanzania, prior to and after initiation of artemisinin-based combination therapy. The samples were stained using the 4',6-diamidino-2-phenylindole fluorogen and digitized using the prototype microscope scanner. Two DL algorithms were trained to detect malaria parasites in the samples, and results compared to the visual assessment of both the digitized samples, and the Giemsa-stained thick smears.ResultsDetection of P. falciparum parasites in the digitized thin blood smears was possible both by visual assessment and by DL-based analysis with a strong correlation in results (r = 0.99, p < 0.01). A moderately strong correlation was observed between the DL-based thin smear analysis and the visual thick smear-analysis (r = 0.74, p < 0.01). Low levels of parasites were detected by DL-based analysis on day three following treatment initiation, but a small number of fluorescent signals were detected also in microscopy-negative samples.ConclusionQuantification of P. falciparum parasites in DAPI-stained thin smears is feasible using DL-supported, point-of-care digital microscopy, with a high correlation to visual assessment of samples. Fluorescent signals from artefacts in samples with low infection levels represented the main challenge for the digital analysis, thus highlighting the importance of minimizing sample contaminations. The proposed method could support malaria diagnostics and monitoring of treatment response through automated quantification of parasitaemia and is likely to be applicable also for diagnostics of other Plasmodium species and other infectious diseases.

Abstract Background Mass drug administration (MDA) has the potential to interrupt malaria transmission and has been suggested as a tool for malaria elimination in low-endemic settings. This study aimed to determine the effectiveness and safety of two rounds of MDA in Zanzibar, a pre-elimination setting. Methods A cluster randomised controlled trial was conducted in 16 areas considered as malaria hotspots, with an annual parasite index of > 0.8%. The areas were randomised to eight intervention and eight control clusters. The intervention included two rounds of MDA with dihydroartemisinin-piperaquine and single low-dose primaquine 4 weeks apart in May–June 2016. Primary and secondary outcomes were cumulative confirmed malaria case incidences 6 months post-MDA and parasite prevalences determined by PCR 3 months post-MDA. Additional outcomes included intervention coverage, treatment adherence, occurrence of adverse events, and cumulative incidences 3, 12, and 16 months post-MDA. Results Intervention coverage was 91.0% (9959/10944) and 87.7% (9355/10666) in the first and second rounds, respectively; self-reported adherence was 82.0% (881/1136) and 93.7% (985/1196). Adverse events were reported in 11.6% (147/1268) and 3.2% (37/1143) of post-MDA survey respondents after both rounds respectively. No serious adverse event was reported. No difference in cumulative malaria case incidence was observed between the control and intervention arms 6 months post-MDA (4.2 and 3.9 per 1000 population; p = 0.94). Neither was there a difference in PCR-determined parasite prevalences 3 months post-MDA (1.4% and 1.7%; OR = 1.0, p = 0.94), although having received at least the first MDA was associated with reduced odds of malaria infection (aOR = 0.35; p = 0.02). Among confirmed malaria cases at health facilities, 26.0% and 26.3% reported recent travel outside Zanzibar in the intervention and control shehias (aOR ≥ 85; p ≤ 0.001). Conclusions MDA was implemented with high coverage, adherence, and tolerability. Despite this, no significant impact on transmission was observed. The findings suggest that two rounds of MDA in a single year may not be sufficient for a sustained impact on transmission in a pre-elimination setting, especially when the MDA impact is restricted by imported malaria. Importantly, this study adds to the limited evidence for the use of MDA in low transmission settings in sub-Saharan Africa. Trial registration ClinicalTrials.gov, NCT02721186 (registration date: March 29, 2016)

Abstract Background Malaria rapid diagnostic tests (RDTs) available as dipsticks or strips, are simple to perform, easily interpretable and do not require electricity nor infrastructural investment. Correct interpretation of and compliance with the RDT results is a challenge to drug sellers. Thus, drug seller interpretation of RDT strips was compared with laboratory scientist re-reading, and PCR analysis of Plasmodium DNA extracted from RDT nitrocellulose strips and fast transient analysis (FTA) cards. Malaria RDT cassettes were also assessed as a potential source of Plasmodium DNA. Methods A total of 212 children aged between 2 and 60 months, 199 of whom had complete records at two study drug shops in south western Uganda participated in the study. Duplicate 5 μL samples of capillary blood were picked from the 212 children, dispensed onto the sample well of the CareStart™ Pf-HRP2 RDT cassette and a FTA, Whatman™ 3MM filter paper in parallel. The RDT strip was interpreted by the drug seller within 15–20 min, visually re-read centrally by laboratory scientist and from it; Plasmodium DNA was recovered and detected by PCR, and compared with FTA recovered P. falciparum DNA PCR detection. Results Malaria positive samples were 62/199 (31.2%, 95% CI 24.9, 38.3) by drug seller interpretation of RDT strip, 59/212 (27.8%, 95% CI 22.2, 34.3) by laboratory scientist, 55/212 (25.9%, 95% CI 20.0, 32.6) by RDT nitrocellulose strip PCR and 64/212 (30.2%, 95% CI 24.4, 37.7). The overall agreement between the drug seller interpretation and laboratory scientist re-reading of the RDT strip was 93.0% with kappa value of 0.84 (95% CI 0.75, 0.92). The drug seller compliance with the reported RDT results was 92.5%. The performance of the three diagnostic strategies compared with FTA-PCR as the gold standard had sensitivity between 76.6 and 86.9%, specificity above 90%, positive predictive values ranging from 79.0 to 89.8% and negative predictive values above 90%. Conclusion Drug sellers can use RDTs in field conditions and achieve acceptable accuracy for malaria diagnosis, and they comply with the RDT results. Plasmodium DNA can be recovered from RDT nitrocellulose strips even in the context of drug shops. Future malaria surveillance and diagnostic quality control studies with RDT cassette as a source of Plasmodium DNA are recommended.

Dihydroartemisinin/piperaquine (DHA/PPQ) is increasingly deployed as an antimalaria drug in Africa. We report the detection in Mali of Plasmodium falciparum infections carrying plasmepsin 2 duplications (associated with piperaquine resistance) in 7/65 recurrent infections within 2 months after DHA/PPQ treatment. These findings raise concerns about the long-term efficacy of DHA/PPQ treatment in Africa.

(1) Immunization with pneumococcal conjugate vaccines has decreased the burden of community-acquired pneumonia (CAP) in children and likely led to a shift in CAP etiology. (2) The Trial of Respiratory infections in children for ENhanced Diagnostics (TREND) enrolled children 1-59 months with clinical CAP according to the World Health Organization (WHO) criteria at Sachs’ Children and Youth Hospital, Stockholm, Sweden. Children with rhonchi and indrawing underwent “bronchodilator challenge”. C-reactive protein and nasopharyngeal PCR detecting 20 respiratory pathogens, were collected from all children. Etiology was defined according to an a priori defined algorithm based on microbiological, biochemical, and radiological findings. (3) Of 327 enrolled children, 107 (32%) required hospitalization; 91 (28%) received antibiotic treatment; 77 (24%) had a chest X-ray performed; and 60 (18%) responded to bronchodilator challenge. 243 (74%) episodes were classified as viral, 11 (3%) as mixed viral-bacterial, five (2%) as bacterial, two (0.6%) as atypical bacterial and 66 (20%) as undetermined etiology. After exclusion of children responding to bronchodilator challenge, the proportion of bacterial and mixed viral-bacterial etiology was 1% and 4%, respectively. (4) The novel TREND etiology algorithm classified the majority of clinical CAP episodes as of viral etiology, whereas bacterial etiology was uncommon. Defining CAP in children

BackgroundThe fixed dose combination of artemether-lumefantrine (AL) is the most widely used treatment for uncomplicated Plasmodium falciparum malaria. Relatively lower cure rates and lumefantrine levels have been reported in young children and in pregnant women during their second and third trimester. The aim of this study was to investigate the pharmacokinetic and pharmacodynamic properties of lumefantrine and the pharmacokinetic properties of its metabolite, desbutyl-lumefantrine, in order to inform optimal dosing regimens in all patient populations.Methods and findingsA search in PubMed, Embase, ClinicalTrials.gov, Google Scholar, conference proceedings, and the WorldWide Antimalarial Resistance Network (WWARN) pharmacology database identified 31 relevant clinical studies published between 1 January 1990 and 31 December 2012, with 4,546 patients in whom lumefantrine concentrations were measured. Under the auspices of WWARN, relevant individual concentration-time data, clinical covariates, and outcome data from 4,122 patients were made available and pooled for the meta-analysis. The developed lumefantrine population pharmacokinetic model was used for dose optimisation through in silico simulations. Venous plasma lumefantrine concentrations 7 days after starting standard AL treatment were 24.2% and 13.4% lower in children weighing ConclusionsOur findings suggest that revised AL dosing regimens for young children and pregnant women would improve drug exposure but would require longer or more complex schedules. These dosing regimens should be evaluated in prospective clinical studies to determine whether they would improve cure rates, demonstrate adequate safety, and thereby prolong the useful therapeutic life of this valuable antimalarial treatment.

Background: Microscopy remains the gold standard in the diagnosis of neglected tropical diseases. As resource limited, rural areas often lack laboratory equipment and trained personnel, new diagnostic techniques are needed. Low-cost, point-of-care imaging devices show potential in the diagnosis of these diseases. Novel, digital image analysis algorithms can be utilized to automate sample analysis. Objective: Evaluation of the imaging performance of a miniature digital microscopy scanner for the diagnosis of soil-transmitted helminths and Schistosoma haematobium, and training of a deep learning-based image analysis algorithm for automated detection of soil-transmitted helminths in the captured images. Methods: A total of 13 iodine-stained stool samples containing Ascaris lumbricoides, Trichuris trichiura and hookworm eggs and 4 urine samples containing Schistosoma haematobium were digitized using a reference whole slide-scanner and the mobile microscopy scanner. Parasites in the images were identified by visual examination and by analysis with a deep learning-based image analysis algorithm in the stool samples. Results were compared between the digital and visual analysis of the images showing helminth eggs. Results: Parasite identification by visual analysis of digital slides captured with the mobile microscope was feasible for all analyzed parasites. Although the spatial resolution of the reference slide-scanner is higher, the resolution of the mobile microscope is sufficient for reliable identification and classification of all parasites studied. Digital image analysis of stool sample images captured with the mobile microscope showed high sensitivity for detection of all helminths studied (range of sensitivity = 83.3–100%) in the test set (n = 217) of manually labeled helminth eggs. Conclusions: In this proof-of-concept study, the imaging performance of a mobile, digital microscope was sufficient for visual detection of soil-transmitted helminths and Schistosoma haematobium. Furthermore, we show that deep learning-based image analysis can be utilized for the automated detection and classification of helminths in the captured images.

BACKGROUND:New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. METHODS:HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015. RESULTS:The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95%CI 1.3-2.4) and 0.7% (95%CI 0.4-1.1), respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0-55.8) and the specificity was 99.9% (CI95% 99.8-100). For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/μL, range 0.2-770) and HTP-LAMP negative (1.4 p/μL, range 0.1-7) samples (p = 0.088). Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly. CONCLUSIONS:Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination.

BACKGROUND:Point-of-care (POC) tests have become increasingly available and more widely used in recent years. They have been of particular importance to low-income settings, enabling them with clinical capacities that had previously been limited. POC testing programs hold a great potential for significant improvement in low-income health systems. However, as most POC tests are developed in high-income countries, disengagement between developers and end-users inhibit their full potential. This study explores perceptions of POC test end-users in a low-income setting, aiming to support the development of novel POC tests for low-income countries. METHODS:A qualitative study was conducted in Mbarara District, Southwestern Uganda, in October 2014. Fifty health care workers were included in seven focus groups, comprising midwives, laboratory technicians, clinical and medical officers, junior and senior nurses, and medical doctors. Discussions were audio-recorded and transcribed verbatim. Transcripts were coded through a data-driven approach for qualitative content analysis. RESULTS:Nineteen different POC tests were identified as currently being in use. While participants displayed being widely accustomed to and appreciative of the use of POC tests, they also assessed the use and characteristics of current tests as imperfect. An ideal POC test was characterized as being adapted to local conditions, thoughtfully implemented in the specific health system, and capable of improving the care of patients. Tests for specific medical conditions were requested. Opinions differed with regard to the ideal distribution of POC tests in the local health system. CONCLUSION:POC tests are commonly used and greatly appreciated in this study setting. However, there are dissatisfactions with current POC tests and their use. To maximize benefit, stakeholders need to include end-user perspectives in the development and implementation of POC tests. Insights from this study will influence our ongoing efforts to develop POC tests that will be particularly usable in low-income settings.

BACKGROUND:Despite the fact that a large proportion of children with fever in Africa present at primary health care facilities, few studies have been designed to specifically study the causes of uncomplicated childhood febrile illness at this level of care, especially in areas like Zanzibar that has recently undergone a dramatic change from high to low malaria transmission. METHODS:We prospectively studied the aetiology of febrile illness in 677 children aged 2-59 months with acute uncomplicated fever managed by IMCI (Integrated Management of Childhood Illness) guidelines in Zanzibar, using point-of-care tests, urine culture, blood-PCR, chest X-ray (CXR) of IMCI-pneumonia classified patients, and multiple quantitative (q)PCR investigations of nasopharyngeal (NPH) (all patients) and rectal (GE) swabs (diarrhoea patients). For comparison, we also performed NPH and GE qPCR analyses in 167 healthy community controls. Final fever diagnoses were retrospectively established based on all clinical and laboratory data. Clinical outcome was assessed during a 14-day follow-up. The utility of IMCI for identifying infections presumed to require antibiotics was evaluated. FINDINGS:NPH-qPCR and GE-qPCR detected ≥1 pathogen in 657/672 (98%) and 153/164 (93%) of patients and 158/166 (95%) and 144/165 (87%) of controls, respectively. Overall, 57% (387/677) had IMCI-pneumonia, but only 12% (42/342) had CXR-confirmed pneumonia. Two patients were positive for Plasmodium falciparum. Respiratory syncytial virus (24.5%), influenza A/B (22.3%), rhinovirus (10.5%) and group-A streptococci (6.4%), CXR-confirmed pneumonia (6.2%), Shigella (4.3%) were the most common viral and bacterial fever diagnoses, respectively. Blood-PCR conducted in a sub-group of patients (n = 83) without defined fever diagnosis was negative for rickettsiae, chikungunya, dengue, Rift Valley fever and West Nile viruses. Antibiotics were prescribed to 500 (74%) patients, but only 152 (22%) had an infection retrospectively considered to require antibiotics. Clinical outcome was generally good. However, two children died. Only 68 (11%) patients remained febrile on day 3 and three of them had verified fever on day 14. An additional 29 (4.5%) children had fever relapse on day 14. Regression analysis determined C-reactive Protein (CRP) as the only independent variable significantly associated with CXR-confirmed pneumonia. CONCLUSIONS:This is the first study on uncomplicated febrile illness in African children that both applied a comprehensive laboratory panel and a healthy control group. A majority of patients had viral respiratory tract infection. Pathogens were frequently detected by qPCR also in asymptomatic children, demonstrating the importance of incorporating controls in fever aetiology studies. The precision of IMCI for identifying infections requiring antibiotics was low.

A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR) targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale) for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl) for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5-10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as 'final positive' if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5-100%) and a specificity of 99.9% (2910/2912; 95%CI 99.7-100%) when compared against 'final positive' samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of low-density Plasmodium infections in malaria pre-elimination settings.

IntroductionMicroscopy is the gold standard for diagnosis of malaria, however, manual evaluation of blood films is highly dependent on skilled personnel in a time-consuming, error-prone and repetitive process. In this study we propose a method using computer vision detection and visualization of only the diagnostically most relevant sample regions in digitized blood smears.MethodsGiemsa-stained thin blood films with P. falciparum ring-stage trophozoites (n = 27) and uninfected controls (n = 20) were digitally scanned with an oil immersion objective (0.1 µm/pixel) to capture approximately 50,000 erythrocytes per sample. Parasite candidate regions were identified based on color and object size, followed by extraction of image features (local binary patterns, local contrast and Scale-invariant feature transform descriptors) used as input to a support vector machine classifier. The classifier was trained on digital slides from ten patients and validated on six samples.ResultsThe diagnostic accuracy was tested on 31 samples (19 infected and 12 controls). From each digitized area of a blood smear, a panel with the 128 most probable parasite candidate regions was generated. Two expert microscopists were asked to visually inspect the panel on a tablet computer and to judge whether the patient was infected with P. falciparum. The method achieved a diagnostic sensitivity and specificity of 95% and 100% as well as 90% and 100% for the two readers respectively using the diagnostic tool. Parasitemia was separately calculated by the automated system and the correlation coefficient between manual and automated parasitemia counts was 0.97.ConclusionWe developed a decision support system for detecting malaria parasites using a computer vision algorithm combined with visualization of sample areas with the highest probability of malaria infection. The system provides a novel method for blood smear screening with a significantly reduced need for visual examination and has a potential to increase the throughput in malaria diagnostics.

Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots.Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6-782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94-99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0-4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1-98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2-99.9) and 76.9% (95%CI 46.2-95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1-100) in both study groups.Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low-density malaria infections in pre-elimination settings.

We assessed if histidine-rich-protein-2 (HRP2) based rapid diagnostic test (RDT) remains an efficient tool for Plasmodium falciparum case detection among fever patients in Zanzibar and if primary health care workers continue to adhere to RDT results in the new epidemiological context of low malaria transmission. Further, we evaluated the performance of RDT within the newly adopted integrated management of childhood illness (IMCI) algorithm in Zanzibar.We enrolled 3890 patients aged ≥ 2 months with uncomplicated febrile illness in this health facility based observational study conducted in 12 primary health care facilities in Zanzibar, between May-July 2010. One patient had an inconclusive RDT result. Overall 121/3889 (3.1%) patients were RDT positive. The highest RDT positivity rate, 32/528 (6.1%), was found in children aged 5-14 years. RDT sensitivity and specificity against PCR was 76.5% (95% CI 69.0-83.9%) and 99.9% (95% CI 99.7-100%), and against blood smear microscopy 78.6% (95% CI 70.8-85.1%) and 99.7% (95% CI 99.6-99.9%), respectively. All RDT positive, but only 3/3768 RDT negative patients received anti-malarial treatment. Adherence to RDT results was thus 3887/3889 (99.9%). RDT performed well in the IMCI algorithm with equally high adherence among children

Early diagnosis and prompt, effective treatment of uncomplicated malaria is critical to prevent severe disease, death and malaria transmission. We assessed the impact of rapid malaria diagnostic tests (RDTs) by community health workers (CHWs) on provision of artemisinin-based combination therapy (ACT) and health outcome in fever patients.Twenty-two CHWs from five villages in Kibaha District, a high-malaria transmission area in Coast Region, Tanzania, were trained to manage uncomplicated malaria using RDT aided diagnosis or clinical diagnosis (CD) only. Each CHW was randomly assigned to use either RDT or CD the first week and thereafter alternating weekly. Primary outcome was provision of ACT and main secondary outcomes were referral rates and health status by days 3 and 7. The CHWs enrolled 2930 fever patients during five months of whom 1988 (67.8%) presented within 24 hours of fever onset. ACT was provided to 775 of 1457 (53.2%) patients during RDT weeks and to 1422 of 1473 (96.5%) patients during CD weeks (Odds Ratio (OR) 0.039, 95% CI 0.029-0.053). The CHWs adhered to the RDT results in 1411 of 1457 (96.8%, 95% CI 95.8-97.6) patients. More patients were referred on inclusion day during RDT weeks (10.0%) compared to CD weeks (1.6%). Referral during days 1-7 and perceived non-recovery on days 3 and 7 were also more common after RDT aided diagnosis. However, no fatal or severe malaria occurred among 682 patients in the RDT group who were not treated with ACT, supporting the safety of withholding ACT to RDT negative patients.RDTs in the hands of CHWs may safely improve early and well-targeted ACT treatment in malaria patients at community level in Africa.ClinicalTrials.gov NCT00301015.

The use of rapid diagnostic tests (RDTs) for Plasmodium falciparum malaria is being suggested to improve diagnostic efficiency in peripheral health care settings in Africa. Such improved diagnostics are critical to minimize overuse and thereby delay development of resistance to artemisinin-based combination therapies (ACTs). Our objective was to study the influence of RDT-aided malaria diagnosis on drug prescriptions, health outcomes, and costs in primary health care settings.We conducted a cross-over validation clinical trial in four primary health care units in Zanzibar. Patients of all ages with reported fever in the previous 48 hours were eligible and allocated alternate weeks to RDT-aided malaria diagnosis or symptom-based clinical diagnosis (CD) alone. Follow-up was 14 days. ACT was to be prescribed to patients diagnosed with malaria in both groups. Statistical analyses with multilevel modelling were performed. A total of 1,887 patients were enrolled February through August 2005. RDT was associated with lower prescription rates of antimalarial treatment than CD alone, 361/1005 (36%) compared with 752/882 (85%) (odds ratio [OR] 0.04, 95% confidence interval [CI] 0.03-0.05, p

BackgroundThe Roll Back Malaria strategy recommends a combination of interventions for malaria control. Zanzibar implemented artemisinin-based combination therapy (ACT) for uncomplicated malaria in late 2003 and long-lasting insecticidal nets (LLINs) from early 2006. ACT is provided free of charge to all malaria patients, while LLINs are distributed free to children under age 5 y ("under five") and pregnant women. We investigated temporal trends in Plasmodium falciparum prevalence and malaria-related health parameters following the implementation of these two malaria control interventions in Zanzibar.Methods and findingsCross-sectional clinical and parasitological surveys in children under the age of 14 y were conducted in North A District in May 2003, 2005, and 2006. Survey data were analyzed in a logistic regression model and adjusted for complex sampling design and potential confounders. Records from all 13 public health facilities in North A District were analyzed for malaria-related outpatient visits and admissions. Mortality and demographic data were obtained from District Commissioner's Office. P. falciparum prevalence decreased in children under five between 2003 and 2006; using 2003 as the reference year, odds ratios (ORs) and 95% confidence intervals (CIs) were, for 2005, 0.55 (0.28-1.08), and for 2006, 0.03 (0.00-0.27); p for trend < 0.001. Between 2002 and 2005 crude under-five, infant (under age 1 y), and child (aged 1-4 y) mortality decreased by 52%, 33%, and 71%, respectively. Similarly, malaria-related admissions, blood transfusions, and malaria-attributed mortality decreased significantly by 77%, 67% and 75%, respectively, between 2002 and 2005 in children under five. Climatic conditions favorable for malaria transmission persisted throughout the observational period.ConclusionsFollowing deployment of ACT in Zanzibar 2003, malaria-associated morbidity and mortality decreased dramatically within two years. Additional distribution of LLINs in early 2006 resulted in a 10-fold reduction of malaria parasite prevalence. The results indicate that the Millennium Development Goals of reducing mortality in children under five and alleviating the burden of malaria are achievable in tropical Africa with high coverage of combined malaria control interventions.