Berit Aydin-Schmidt

Loop-mediated isothermal amplification (LAMP) for point-of-care detection of asymptomatic low-density malaria parasite carriers in Zanzibar

BackgroundAsymptomatic, low parasite density malaria infections are difficult to detect with currently available point-of-care diagnostics. This study piloted a loop-mediated isothermal amplification (LAMP) kit for field-friendly, high-throughput detection of asymptomatic malaria infections during mass screening and treatment (MSAT) in Zanzibar, a malaria pre-elimination setting.MethodsScreening took place in three known hotspot areas prior to the short rains in November. Finger-prick blood was taken for screening by rapid diagnostic test (RDT) and LAMP and collected on filter paper for subsequent polymerase chain reaction (PCR) analyses. LAMP results were compared to RDT and to PCR using McNemar’s test.ResultsApproximately 1,000 people were screened. RDT detected ten infections (1.0% (95% CI 0.3-1.6)) whilst both LAMP and PCR detected 18 (1.8% (95% CI 0.9-2.6)) infections. However, PCR identified three infections that LAMP did not detect and vice versa. LAMP testing was easy to scale-up in field conditions requiring minimal training and equipment, with results ready one to three hours after screening.ConclusionsDespite lower than expected prevalence, LAMP detected a higher number of infections than the currently used diagnostic, RDT. LAMP is a field-friendly, sensitive diagnostic test that could be useful for MSAT malaria campaigns which require quick results to enable prompt treatment.

Loop Mediated Isothermal Amplification (LAMP) Accurately Detects Malaria DNA from Filter Paper Blood Samples of Low Density Parasitaemias

Background 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. Methods and Findings 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. Conclusion 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.

The Usefulness of Rapid Diagnostic Tests in the New Context of Low Malaria Transmission in Zanzibar

Background 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. Methods and Findings 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 <5 years as compared with other age groups. Conclusions The sensitivity of HRP-2 based RDT in the hands of health care workers compared with both PCR and microscopy for P. falciparum case detection was relatively low, whereas adherence to test results with anti-malarial treatment was excellent. Moreover, the results provide evidence that RDT can be reliably integrated in IMCI as a tool for improved childhood fever management. However, the relatively low RDT sensitivity highlights the need for improved quality control of RDT use in primary health care facilities, but also for more sensitive point-of-care malaria diagnostic tools in the new epidemiological context of low malaria transmission in Zanzibar. Trial registration ClinicalTrials.gov NCT01002066

Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability

BackgroundThe need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations.MethodsDNA was extracted from two RDT devices (Paracheck-Pf® and SD Bioline Malaria Pf/Pan®), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman® 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance.ResultsThe P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples.ConclusionsThe results support RDTs as a valuable source of parasite DNA and provide evidence for RDT-DNA extraction for improved malaria case detection, molecular drug resistance surveillance, and RDT quality control.

Prevalence of PCR Detectable Malaria Infection among Febrile Patients with a Negative Plasmodium falciparum Specific Rapid Diagnostic Test in Zanzibar

We screened for malaria in 594 blood samples from febrile patients who tested negative by a Plasmodium falciparum-specific histidine-rich protein-2-based rapid diagnostic test at 12 health facilities in Zanzibar districts North A and Micheweni, from May to August 2010. Screening was with microscopy, polymerase chain reaction (PCR) targeting the cytochrome b gene (cytbPCR) of the four major human malaria species, and quantitative PCR (qPCR). The prevalence of cytbPCR-detectable malaria infection was 2% (12 of 594), including 8 P. falciparum, 3 Plasmodium malariae, and 1 Plasmodium vivax infections. Microscopy identified 4 of 8 P. falciparum infections. Parasite density as estimated by microscopy or qPCR was > 4,000 parasites/μL in 5 of 8 cytbPCR-detectable P. falciparum infections. The infections that were missed by the rapid diagnostic test represent a particular challenge in malaria elimination settings and highlight the need for more sensitive point-of-care diagnostic tools to improve case detection of all human malaria species in febrile patients.

SYBR Green real-time PCR-RFLP assay targeting the plasmodium cytochrome B gene--a highly sensitive molecular tool for malaria parasite detection and species determination.

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.

Carolus Linnaeus, the ash, worm-wood and other anti-malarial plants

Abstract Abstract In 1735 Carolus Linnaeus wrote that quinine was the preferred treatment for malaria but that the bark of the ash (Fraxinus excelsior) and worm-wood (Artemisia absinthium) also had effects on the disease. We here report that lipo- and hydrophilic extracts of the bark of the ash inhibit the in vitro growth of the asexual stages of P. falciparum. The data suggests that the knowledge of the treatment of malaria was already available in Europe some 300 years ago.

Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar

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.

Absence of Plasmodium falciparum K13 Propeller Domain Polymorphisms among Field Isolates Collected from the Brazilian Amazon Basin between 1984 and 2011

Artemisinin resistance, presently confined to Southeast Asia and associated with mutations in the Plasmodium falciparum K13 (PfK13) propeller domain, represents a serious threat to global malaria control. This study aimed to provide baseline information for future artemisinin resistance surveillance, by analyzing the PfK13 propeller domain in P. falciparum field isolates collected from the Brazilian Amazon Basin between 1984 and 2011. A total of 152 P. falciparum mono-infections were assessed, of which 118 (78%) were collected before and 34 (22%) after the introduction of artemisinin-based combination therapy (ACT) in 2006. An 849-base pair fragment encoding the PfK13 propeller was amplified by nested polymerase chain reaction and sequenced in both directions. The sequences were compared with the reference sequence of P. falciparum 3D7. All samples showed wild-type sequences, thus, no mutations were observed. The results are in agreement with other recent reports and do not provide evidence for presence of PfK13 propeller domain polymorphisms associated with artemisinin resistance among P. falciparum field isolates in the Brazilian Amazon Basin neither before nor after the implementation of ACT.