Treatment Failure in a UK Malaria Patient Harboring Genetically Variant Plasmodium falciparum From Uganda With Reduced In Vitro Susceptibility to Artemisinin and Lumefantrine.

BACKGROUND: Recent cases of clinical failure in malaria patients in the United Kingdom (UK) treated with artemether-lumefantrine have implications for malaria chemotherapy worldwide. METHODS: Parasites were isolated from an index case of confirmed Plasmodium falciparum treatment failure after standard treatment, and from comparable travel-acquired UK malaria cases. Drug susceptibility in vitro and genotypes at 6 resistance-associated loci were determined for all parasite isolates and compared with clinical outcomes for each parasite donor. RESULTS: A traveler, who returned to the UK from Uganda in 2022 with Plasmodium falciparum malaria, twice failed treatment with full courses of artemether-lumefantrine. Parasites from the patient exhibited significantly reduced susceptibility to artemisinin (ring-stage survival, 17.3% [95% confidence interval {CI}, 13.6%-21.1%]; P < .0001) and lumefantrine (effective concentration preventing 50% of growth = 259.4 nM [95% CI, 130.6-388.2 nM]; P = .001). Parasite genotyping identified an allele of pfk13 encoding both the A675V variant in the Pfk13 propeller domain and a novel L145V nonpropeller variant. In vitro susceptibility testing of 6 other P. falciparum lines of Ugandan origin identified reduced susceptibility to artemisinin and lumefantrine in 1 additional line, also from a 2022 treatment failure case. These parasites did not harbor a pfk13 propeller domain variant but rather the novel nonpropeller variant T349I. Variant alleles of pfubp1, pfap2mu, and pfcoronin were also identified among the 7 parasite lines. CONCLUSIONS: We confirm, in a documented case of artemether-lumefantrine treatment failure imported from Uganda, the presence of pfk13 mutations encoding L145V and A675V. Parasites with reduced susceptibility to both artemisinin and lumefantrine may be emerging in Uganda.

Medical management of cystic echinococcosis.

PURPOSE OF REVIEW: Cystic echinococcosis is a neglected zoonosis for which humans are dead end hosts. It is not only widely distributed in sheep rearing areas of low-income and middle-income countries but also has a significant presence in wealthy countries, for example, in Europe. It results in considerable morbidity, and its current management is far from optimal. Medical management is with a benzimidazole, with the addition of praziquantel under some circumstances. RECENT FINDINGS: Interest in mebendazole as an anticancer drug has stimulated research into new drug formulations to improve bioavailability and possibly reduce inter-individual variability in in-vivo drug levels, which may help its activity against cystic echinococcosis. Further evidence to support administration of albendazole with a fatty meal has been provided. GlaxoSmithKilne (GSK) has agreed to extend its albendazole donation programme to include echinococcosis. The search for new drugs has focussed on natural products, such as essential oils and on repurposing of existing drugs licensed for human use against other conditions. SUMMARY: The medical treatment of cystic echinococcosis remains sorely neglected, with no new drugs for almost 40 years. We need a better understanding of how to use the drugs we do have, whilst seeking new ones. Drug repurposing may be the best pathway.

Where Have All the Diagnostic Morphological Parasitologists Gone?

Advances in laboratory techniques have revolutionized parasitology diagnostics over the past several decades. Widespread implementation of rapid antigen detection tests has greatly expanded access to tests for global parasitic threats such as malaria, while next-generation amplification and sequencing methods allow for sensitive and specific detection of human and animal parasites in complex specimen matrices. Recently, the introduction of multiplex panels for human gastrointestinal infections has enhanced the identification of common intestinal protozoa in feces along with bacterial and viral pathogens. Despite the benefits provided by novel diagnostics, increased reliance on nonmicroscopy-based methods has contributed to the progressive, widespread loss of morphology expertise for parasite identification. Loss of microscopy and morphology skills has the potential to negatively impact patient care, public health, and epidemiology. Molecular- and antigen-based diagnostics are not available for all parasites and may not be suitable for all specimen types and clinical settings. Furthermore, inadequate morphology experience may lead to missed and inaccurate diagnoses and erroneous descriptions of new human parasitic diseases. This commentary highlights the need to maintain expert microscopy and morphological parasitology diagnostic skills within the medical and scientific community. We proposed that light microscopy remains an important part of training and practice in the diagnosis of parasitic diseases and that efforts should be made to train the next generation of morphological parasitologists before the requisite knowledge, skills, and capacity for this complex and important mode of diagnosis are lost. In summary, the widespread, progressive loss of morphology expertise for parasite identification negatively impacts patient care, public health, and epidemiology.

Surveillance of leishmaniasis cases from 15 European centres, 2014 to 2019: a retrospective analysis.

BackgroundSurveillance of human leishmaniasis in Europe is mostly limited to country-specific information from autochthonous infections in the southern part. As at the end of 2021, no integrated analysis has been performed for cases seen across centres in different European countries.AimTo provide a broad perspective on autochthonous and imported leishmaniasis cases in endemic and non-endemic countries in Europe.MethodsWe retrospectively collected records from cutaneous, mucosal and visceral leishmaniasis cases diagnosed in 15 centres between 2014 and 2019. Centres were located in 11 countries: Belgium, France, Germany, Italy, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Data on country of infection, reason for travelling, infecting species, age and sex were analysed.ResultsWe obtained diagnostic files from 1,142 cases, of which 76%, 21% and 3% had cutaneous, visceral, and mucosal disease, respectively. Of these, 68% were men, and 32% women, with the median age of 37 years (range: 0-90) at diagnosis. Visceral leishmaniasis was mainly acquired in Europe (88%; 167/190), while cutaneous leishmaniasis was primarily imported from outside Europe (77%; 575/749). Sixty-two percent of cutaneous leishmaniasis cases from outside Europe were from the Old World, and 38% from the New World. Geographic species distribution largely confirmed known epidemiology, with notable exceptions.ConclusionsOur study confirms previous reports regarding geographic origin, species, and traveller subgroups importing leishmaniasis into Europe. We demonstrate the importance of pooling species typing data from many centres, even from areas where the aetiology is presumably known, to monitor changing epidemiology.

Performance of a fully-automated system on a WHO malaria microscopy evaluation slide set.

BACKGROUND: Manual microscopy remains a widely-used tool for malaria diagnosis and clinical studies, but it has inconsistent quality in the field due to variability in training and field practices. Automated diagnostic systems based on machine learning hold promise to improve quality and reproducibility of field microscopy. The World Health Organization (WHO) has designed a 55-slide set (WHO 55) for their External Competence Assessment of Malaria Microscopists (ECAMM) programme, which can also serve as a valuable benchmark for automated systems. The performance of a fully-automated malaria diagnostic system, EasyScan GO, on a WHO 55 slide set was evaluated. METHODS: The WHO 55 slide set is designed to evaluate microscopist competence in three areas of malaria diagnosis using Giemsa-stained blood films, focused on crucial field needs: malaria parasite detection, malaria parasite species identification (ID), and malaria parasite quantitation. The EasyScan GO is a fully-automated system that combines scanning of Giemsa-stained blood films with assessment algorithms to deliver malaria diagnoses. This system was tested on a WHO 55 slide set. RESULTS: The EasyScan GO achieved 94.3 % detection accuracy, 82.9 % species ID accuracy, and 50 % quantitation accuracy, corresponding to WHO microscopy competence Levels 1, 2, and 1, respectively. This is, to our knowledge, the best performance of a fully-automated system on a WHO 55 set. CONCLUSIONS: EasyScan GO's expert ratings in detection and quantitation on the WHO 55 slide set point towards its potential value in drug efficacy use-cases, as well as in some case management situations with less stringent species ID needs. Improved runtime may enable use in general case management settings.

Predictors of aetiology and outcomes of acute gastrointestinal illness in returning travellers: a retrospective cohort analysis.

BACKGROUND: Gastrointestinal illness is a major cause of morbidity in travellers and is a common reason for presentation to healthcare services on return. Whilst the aetiology of imported gastrointestinal disease is predominantly infectious, outcomes are variable due to a range of phenomena such as post-infectious irritable bowel syndrome, drug resistance and occult pathology (both infectious and non-infectious). Previous studies have focussed on predictors of aetiology of gastrointestinal disease in travellers; we present a retrospective study combining both aetiological and early outcome data in a large cohort of returned travellers. METHOD: We identified 1450 patients who attended our post-travel walk-in clinic with gastrointestinal symptoms between 2010 and 2016. Demographic, travel, clinical and laboratory data was collected through case note review. Logistic regression analysis to examine correlates of aetiology and outcome were performed in R (CRAN Project 2017). RESULTS: Of 1450 patients in our cohort 153 reported bloody diarrhoea and 1081 (74.6%) reported non-bloody diarrhoea. A definitive microbiological diagnosis was made in 310 (20.8%) of which 137 (9.4%) had a parasite identified and 111 (7.7%) had a bacterial cause identified. Factors associated with a parasitological diagnosis included history of travel to South Asia (aOR = 2.55; 95%CI 1.75-3.70, p < 0.0001) and absence of bloody diarrhoea (aOR = 0.22; 95%CI 0.066-0.53, p < 0.005). Factors associated with a bacteriological diagnosis included male gender (aOR = 1.69; 95%CI 1.10-2.62, p < 0.05), an age < 37 years on presentation (aOR = 2.04; 95%CI 1.25-3.43, p < 0.01), white cells on stool microscopy (aOR = 3.52; 95%CI 2.09-5.86, p < 0.0001) and a C-reactive protein level of >5iu/dL (aOR = 4.68; 95%CI 2.91-7.72, p < 0.0001). The majority (1235/1450, 82.6%) reported full symptomatic resolution by the first follow up visit; factors associated with lack of symptomatic resolution included female gender (aOR = 1.45 95%CI 1.06-1.99, p < 0.05), dysenteric diarrhoea (aOR = 2.14 (95%CI 1.38-3.25, p < 0.0005) and elevated peripheral leukocyte count (aOR = 1.58 95%CI 1.02-2.40, p < 0.05). CONCLUSIONS: In a cohort of returned travellers, we were able to identify multiple factors that are correlated with both aetiology and outcome of imported gastrointestinal syndromes. We predict these data will be valuable in the development of diagnostic and therapeutic pathways for patients with imported gastrointestinal infections.

Neurological deterioration in a patient with HIV-associated cryptococcal meningitis initially improving on antifungal treatment: a case report of coincidental racemose neurocysticercosis.

BACKGROUND: Managing HIV-associated cryptococcal meningitis (CM) can become challenging in the presence of concurrent unusual central nervous system infections. CASE PRESENTATION: A 58-year old HIV infected woman new ART starter, who was being treated effectively for cryptococcal meningitis, represented with worsening of neurological symptoms. Brain MRI revealed a multicystic lesion in the left temporal lobe. Anti-fungal treatment was escalated for a suspected cryptococcoma, but post-mortem CSF serological test confirmed racemose neurocysticercosis. CONCLUSION: Patients with HIV-associated CM are highly immunocompromised and may have multiple pathologies simultaneously. In endemic countries, neurocysticercosis should be considered in the differential diagnosis where there is central nervous system deterioration despite effective therapy for CM.

Epidemiology and surveillance of human (neuro)cysticercosis in Europe: is enhanced surveillance required?

OBJECTIVES: To report on relevant national surveillance systems of (N)CC and taeniasis (the infection with the adult tapeworm) in the European Union/European Economic Area and to assess the magnitude of (N)CC occurrence by retrieving information on cases for the period 2000-2016. METHODS: (N)CC cases were retrieved via national reporting systems, a systematic literature search, contact with clinicians and a search for relevant 'International Statistical Classification of Diseases and Related Health Problems' (ICD)-based data. RESULTS: Mandatory notification systems for (N)CC were found in Hungary, Iceland and Poland. Ten cases were reported in Poland and none in Hungary and Iceland. Through the systematic literature review and information given by clinicians, 263 individual and 721 aggregated (N)CC cases from 19 European countries were identified. ICD-based data were obtained from five countries. From 2000 to 2016, a total of 3489 cases (N)CC cases were coded: 832 in Italy, eight in Latvia, 357 in Portugal, 2116 in Spain and 176 in Sweden. CONCLUSION: Despite being classified as a possible eradicable disease, (N)CC is still diagnosed across Europe, yet its true extent and impact remain unclear.

OBJECTIFS: Rapporter sur les systèmes nationaux de surveillance pertinents de la (neuro)cysticercose (N)CC et de la téniase (infection par le ténia adulte) dans l'Union européenne/l'Espace économique européen, et évaluer l'ampleur de l'occurrence de la (N)CC en reprenant des informations sur les cas durant la période 2000-2016. MÉTHODES: Les cas de (N)CC ont été repris à partir des systèmes nationaux de notification, une recherche systématique de la littérature, des contacts avec des cliniciens et une recherche de données pertinentes basées sur la 'Classification Statistique Internationale des Maladies et Problèmes de Santé Connexes' (ICD). RÉSULTATS: Des systèmes de notification obligatoires pour la (N)CC ont été trouvés en Hongrie, en Islande et en Pologne. Dix cas ont été rapportés en Pologne et aucun en Hongrie et en Islande. Grâce à la revue systématique de la littérature et aux informations fournies par les cliniciens, 263 cas individuels et 721 cas agrégés de (N)CC de 19 pays européens ont été identifiés. Des données ICD ont été obtenues de cinq pays. De 2000 à 2016, un total de 3489 cas de (N)CC ont été codés: 832 en Italie, 8 en Lettonie, 357 au Portugal, 2116 en Espagne et 176 en Suède. CONCLUSION: Bien qu'elle soit classée comme une maladie pouvant être éradiquée, la (N)CC est toujours diagnostiquée à travers l'Europe, mais sa véritable ampleur et son impact restent incertains .

Impact of Plasmodium falciparum gene deletions on malaria rapid diagnostic test performance.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) have greatly improved access to diagnosis in endemic countries. Most RDTs detect Plasmodium falciparum histidine-rich protein 2 (HRP2), but their sensitivity is seriously threatened by the emergence of pfhrp2-deleted parasites. RDTs detecting P. falciparum or pan-lactate dehydrogenase (Pf- or pan-LDH) provide alternatives. The objective of this study was to systematically assess the performance of malaria RDTs against well-characterized pfhrp2-deleted P. falciparum parasites. METHODS: Thirty-two RDTs were tested against 100 wild-type clinical isolates (200 parasites/µL), and 40 samples from 10 culture-adapted and clinical isolates of pfhrp2-deleted parasites. Wild-type and pfhrp2-deleted parasites had comparable Pf-LDH concentrations. Pf-LDH-detecting RDTs were also tested against 18 clinical isolates at higher density (2,000 parasites/µL) lacking both pfhrp2 and pfhrp3. RESULTS: RDT positivity against pfhrp2-deleted parasites was highest (> 94%) for the two pan-LDH-only RDTs. The positivity rate for the nine Pf-LDH-detecting RDTs varied widely, with similar median positivity between double-deleted (pfhrp2/3 negative; 63.9%) and single-deleted (pfhrp2-negative/pfhrp3-positive; 59.1%) parasites, both lower than against wild-type P. falciparum (93.8%). Median positivity for HRP2-detecting RDTs against 22 single-deleted parasites was 69.9 and 35.2% for HRP2-only and HRP2-combination RDTs, respectively, compared to 96.0 and 92.5% for wild-type parasites. Eight of nine Pf-LDH RDTs detected all clinical, double-deleted samples at 2,000 parasites/µL. CONCLUSIONS: The pan-LDH-only RDTs evaluated performed well. Performance of Pf-LDH-detecting RDTs against wild-type P. falciparum does not necessarily predict performance against pfhrp2-deleted parasites. Furthermore, many, but not all HRP2-based RDTs, detect pfhrp2-negative/pfhrp3-positive samples, with implications for the HRP2-based RDT screening approach for detection and surveillance of HRP2-negative parasites.

WHO malaria nucleic acid amplification test external quality assessment scheme: results of distribution programmes one to three.

BACKGROUND: The World Health Organization (WHO) recommends parasite-based diagnosis of malaria. In recent years, there has been surge in the use of various kinds of nucleic-acid amplification based tests (NAATs) for detection and identification of Plasmodium spp. to support clinical care in high-resource settings and clinical and epidemiological research worldwide. However, these tests are not without challenges, including lack (or limited use) of standards and lack of reproducibility, due in part to variation in protocols amongst laboratories. Therefore, there is a need for rigorous quality control, including a robust external quality assessment (EQA) scheme targeted towards malaria NAATs. To this effect, the WHO Global Malaria Programme worked with the UK National External Quality Assessment Scheme (UK NEQAS) Parasitology and with technical experts to launch a global NAAT EQA scheme in January 2017. METHODS: Panels of NAAT EQA specimens containing five major species of human-infecting Plasmodium at various parasite concentrations and negative samples were created in lyophilized blood (LB) and dried blood spot (DBS) formats. Two distributions per year were sent, containing five LB and five DBS specimens. Samples were tested and validated by six expert referee laboratories prior to distribution. Between 37 and 45 laboratories participated in each distribution and submitted results using the online submission portal of UK NEQAS. Participants were scored based on their laboratory's stated capacity to identify Plasmodium species, and individual laboratory reports were sent which included performance comparison with anonymized peers. RESULTS: Analysis of the first three distributions revealed that the factors that most significantly affected performance were sample format (DBS vs LB), species and parasite density, while laboratory location and the reported methodology used (type of nucleic acid extraction, amplification, or DNA vs RNA target) did not significantly affect performance. Referee laboratories performed better than non-referee laboratories. CONCLUSIONS: Globally, malaria NAAT assays now inform a range of clinical, epidemiological and research investigations. EQA schemes offer a way for laboratories to assess and improve their performance, which is critical to safeguarding the reliability of data and diagnoses especially in situations where various NAAT methodologies and protocols are in use.

World Gastroenterology Organisation Global Guidelines: Management of Strongyloidiasis February 2018-Compact Version>.

Strongyloides stercoralis is a soil-transmitted helminth, but it has a unique life cycle that can be completed in the human host, in a process known as autoinfection. Worldwide, the burden of disease is substantial (300 to 400 million infections). Strongyloidiasis is mainly prevalent in the tropics and subtropics, but there is as yet no global public health strategy for controlling the parasite.

A review of the WHO malaria rapid diagnostic test product testing programme (2008-2018): performance, procurement and policy.

Malaria rapid diagnostic tests (RDTs) emerged in the early 1990s into largely unregulated markets, and uncertain field performance was a major concern for the acceptance of tests for malaria case management. This, combined with the need to guide procurement decisions of UN agencies and WHO Member States, led to the creation of an independent, internationally coordinated RDT evaluation programme aiming to provide comparative performance data of commercially available RDTs. Products were assessed against Plasmodium falciparum and Plasmodium vivax samples diluted to two densities, along with malaria-negative samples from healthy individuals, and from people with immunological abnormalities or non-malarial infections. Three measures were established as indicators of performance, (i) panel detection score (PDS) determined against low density panels prepared from P. falciparum and P. vivax wild-type samples, (ii) false positive rate, and (iii) invalid rate, and minimum criteria defined. Over eight rounds of the programme, 332 products were tested. Between Rounds 1 and 8, substantial improvements were seen in all performance measures. The number of products meeting all criteria increased from 26.8% (11/41) in Round 1, to 79.4% (27/34) in Round 8. While products submitted to further evaluation rounds under compulsory re-testing did not show improvement, those voluntarily resubmitted showed significant increases in P. falciparum (p = 0.002) and P. vivax PDS (p < 0.001), with more products meeting the criteria upon re-testing. Through this programme, the differentiation of products based on comparative performance, combined with policy changes has been influential in the acceptance of malaria RDTs as a case-management tool, enabling a policy of parasite-based diagnosis prior to treatment. Publication of product testing results has produced a transparent market allowing users and procurers to clearly identify appropriate products for their situation, and could form a model for introduction of other, broad-scale diagnostics.

Disseminated cerebral hydatid disease (multiple intracranial echinococcosis).

Intracranial echinococcosis is relatively uncommon and usually occurs in the context of echinococcal lesions elsewhere in the body, mostly liver and lung. Multiple intracranial lesions can result from rupture of an initial single intracranial cyst (in cystic echinococcosis) or from dissemination of systemic disease of the lung, liver or heart (cystic and alveolar echinococcosis). The two main subtypes, cystic and alveolar echinococcosis, present differently and have distinct imaging features in the brain. We discuss the presentation, imaging findings and clinical course of three cases (two cystic and one alveolar) of intracranial echinococcal disease in adults.

Visceral Leishmaniasis in Traveler to Guyana Caused by Leishmania siamensis, London, UK.

The parasite Leishmania siamensis is a zoonotic agent of leishmaniasis; infection in animals has been documented in Europe and the United States. Reported authochthonous human infections have been limited to Thailand. We report a case of human visceral Leishmania siamensis infection acquired in Guyana, suggesting colonization in South America.

Geographical and temporal trends and seasonal relapse in Plasmodium ovale spp. and Plasmodium malariae infections imported to the UK between 1987 and 2015.

BACKGROUND: Plasmodium ovale spp. and P. malariae cause illness in endemic regions and returning travellers. Far less is known about these species than P. falciparum and P. vivax. METHODS: The UK national surveillance data, collected 1987 to 2015, were collated with the International Passenger Survey and climatic data to determine geographical, temporal and seasonal trends of imported P. ovale spp. and P. malariae infection. RESULTS: Of 52,242 notified cases of malaria, 6.04% (3157) were caused by P. ovale spp. and 1.61% (841) by P. malariae; mortality was 0.03% (1) and 0.12% (1), respectively. Almost all travellers acquired infection in West or East Africa. Infection rate per travel episode fell fivefold during the study period. The median latency of P. malariae and P. ovale spp. was 18 and 76 days, respectively; delayed presentation occurred with both species. The latency of P. ovale spp. infection imported from West Africa was significantly shorter in those arriving in the UK during the West African peak malarial season compared to those arriving outside it (44 days vs 94 days, p < 0.0001), implying that relapse synchronises with the period of high malarial transmission. This trend was not seen in P. ovale spp. imported from East Africa nor in P. malariae. CONCLUSION: In West Africa, where malaria transmission is highly seasonal, P. ovale spp. may have evolved to relapse during the malarial high transmission season. This has public health implications. Deaths are very rare, supporting current guidelines emphasising outpatient treatment. However, late presentations do occur.

The inverted cup device for blood transfer on malaria RDTs: ease of use, acceptability and safety in routine use by health workers in Nigeria.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) are becoming widely adopted for case management at community level. However, reports and anecdotal observations indicate that the blood transfer step poses a significant challenge to many users. This study sought to evaluate the inverted cup device in the hands of health workers in everyday clinical practice, in comparison with the plastic pipette, and to determine the volume accuracy of the device made of a lower-cost plastic. METHODS: The volume accuracy of inverted cup devices made of two plastics, PMMA and SBC, was compared by transferring blood 150 times onto filter paper and comparing the blood spot areas with those produced by 20 reference transfers with a calibrated micropipette. The ease of use, safety and acceptability of the inverted cup device and the pipette were evaluated by 50 health workers in Nigeria. Observations were recorded on pre-designed questionnaires, by the health workers themselves and by trained observers. Focus group discussions were also conducted. RESULTS: The volume accuracy assessment showed that the device made from the low-cost material (SBC) delivered a more accurate volume (mean 5.4 µL, SD 0.48 µL, range 4.5-7.0 µL) than the PMMA device (mean 5.9 µL, SD 0.48 µL, range 4.9-7.2 µL). The observational evaluation demonstrated that the inverted cup device performed better than the pipette in all aspects, e.g. higher proportions of health workers achieved successful blood collection (96%, vs. 66%), transfer of the required blood volume (90%, vs. 58%), and blood deposit without any loss (95%, vs. 50%). Majority of health workers also considered it' very easy' to use (81%),'very appropriate' for everyday use (78%), and 50% of them reported that it was their preferred BTD. CONCLUSIONS: The good volume accuracy and high acceptability of the inverted cup device shown in this study, along with observed ease of use and safety in hands of health workers, further strengthens prior findings which demonstrated its higher accuracy as compared with other BTDs in a laboratory setting. Altogether, these studies suggest that the inverted cup device should replace other types of devices for use in day-to-day malaria diagnosis with RDTs.

A new highly sensitive enzyme-linked immunosorbent assay for the detection of Plasmodium falciparum histidine-rich protein 2 in whole blood.

BACKGROUND: The detection of submicroscopic infections in low prevalence settings has become an increasingly important challenge for malaria elimination strategies. The current field rapid diagnostic tests (RDTs) for Plasmodium falciparum malaria are inadequate to detect low-density infections. Therefore, there is a need to develop more sensitive field diagnostic tools. In parallel, a highly sensitive laboratory reference assay will be essential to evaluate new diagnostic tools. Recently, the highly sensitive Alere™ Malaria Ag P.f ELISA (HS ELISA) was developed to detect P. falciparum histidine-rich protein 2 (HRP2) in clinical whole blood specimens. In this study, the analytical and clinical performance of the HS ELISA was determined using recombinant P. falciparum HRP2, P. falciparum native culture parasites, and archived highly pedigreed clinical whole blood specimens from Karen village, Myanmar and Nagongera, Uganda. RESULTS: The HS ELISA has an analytical sensitivity of less than 25 pg/mL and shows strong specificity for P. falciparum HRP2 when tested against P. falciparum native culture strains with pfhrp2 and pfhrp3 gene deletions. Additionally, the Z'-factor statistic of 0.862 indicates the HS ELISA as an excellent, reproducible assay, and the coefficients of variation for inter- and intra-plate testing, 11.76% and 2.51%, were acceptable. Against clinical whole blood specimens with concordant microscopic and PCR results, the HS ELISA showed 100% (95% CI 96.4-100) diagnostic sensitivity and 97.9% (95% CI 94.8-99.4) diagnostic specificity. For P. falciparum positive specimens with HRP2 concentrations below 400 pg/mL, the sensitivity and specificity were 100% (95% CI 88.4-100) and 88.9% (95% CI 70.8-97.6), respectively. The overall sensitivity and specificity for all 352 samples were 100% (CI 95% 96-100%) and 97.3% (CI 95% 94-99%). CONCLUSIONS: The HS ELISA is a robust and reproducible assay. The findings suggest that the HS ELISA may be a useful tool as an affordable reference assay for new ultra-sensitive HRP2-based RDTs.

pfk13-Independent Treatment Failure in Four Imported Cases of Plasmodium falciparum Malaria Treated with Artemether-Lumefantrine in the United Kingdom.

We present case histories of four patients treated with artemether-lumefantrine for falciparum malaria in UK hospitals in 2015 to 2016. Each subsequently presented with recurrent symptoms and Plasmodium falciparum parasitemia within 6 weeks of treatment with no intervening travel to countries where malaria is endemic. Parasite isolates, all of African origin, harbored variants at some candidate resistance loci. No evidence of pfk13-mediated artemisinin resistance was found. Vigilance for signs of unsatisfactory antimalarial efficacy among imported cases of malaria is recommended.

British Society for Haematology guidelines for the laboratory diagnosis of malaria.

The laboratory diagnosis of malaria depends on skilled examination of well-stained thick and thin blood films. Rapid diagnostic tests are a useful supplement and the use of nucleic acid-based testing in diagnostic laboratories should also be considered. These British Society for Haematology guidelines update the 2003 guidelines for malaria diagnosis. Training, quality control, incidental diagnosis, differential diagnosis and reference laboratory referral are considered.

Analytical sensitivity of current best-in-class malaria rapid diagnostic tests.

BACKGROUND: Rapid diagnostic tests (RDTs) are today the most widely used method for malaria diagnosis and are recommended, alongside microscopy, for the confirmation of suspected cases before the administration of anti-malarial treatment. The diagnostic performance of RDTs, as compared to microscopy or PCR is well described but the actual analytical sensitivity of current best-in-class tests is poorly documented. This value is however a key performance indicator and a benchmark value needed to developed new RDTs of improved sensitivity. METHODS: Thirteen RDTs detecting either the Plasmodium falciparum histidine rich protein 2 (HRP2) or the plasmodial lactate dehydrogenase (pLDH) antigens were selected from the best performing RDTs according to the WHO-FIND product testing programme. The analytical sensitivity of these products was evaluated using a range of reference materials including P. falciparum and Plasmodium vivax whole parasite samples as well as recombinant proteins. RESULTS: The best performing HRP2-based RDTs could detect all P. falciparum cultured samples at concentrations as low as 0.8 ng/mL of HRP2. The limit of detection of the best performing pLDH-based RDT specifically detecting P. vivax was 25 ng/mL of pLDH. CONCLUSION: The analytical sensitivity of P. vivax and Pan pLDH-based RDTs appears to vary considerably from product to product, and improvement of the limit-of-detection for P. vivax detecting RDTs is needed to match the performance of HRP2 and Pf pLDH-based RDTs for P. falciparum. Different assays using different reference materials produce different values for antigen concentration in a given specimen, highlighting the need to establish universal reference assays.