Petra F. Mens

Is molecular biology the best alternative for diagnosis of malaria to microscopy? A comparison between microscopy, antigen detection and molecular tests in rural Kenya and urban Tanzania.

Objective  To assess the agreement of different diagnostic methods for the diagnosis and confirmation of the clinical suspicion of Plasmodium infection in children in Tanzania and Kenya.

Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification

BackgroundDecisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (<20 parasites/μl) the technique becomes less sensitive and time consuming. Rapid diagnostic tests based on Plasmodium antigen detection do often not allow for species discrimination as microscopy does, but also become insensitive at <100 parasites/μl.MethodsThis paper reports the development of a sensitive and specific real-time Quantitative Nucleic Acid Sequence Based Amplification (real-time QT-NASBA) assays, based on the small-subunit 18S rRNA gene, to identify the four human Plasmodium species.ResultsThe lower detection limit of the assay is 100 – 1000 molecules in vitro RNA for all species, which corresponds to 0.01 – 0.1 parasite per diagnostic sample (i.e. 50 μl of processed blood). The real-time QT-NASBA was further evaluated using 79 clinical samples from malaria patients: i.e. 11 Plasmodium. falciparum, 37 Plasmodium vivax, seven Plasmodium malariae, four Plasmodium ovale and 20 mixed infections. The initial diagnosis of 69 out of the 79 samples was confirmed with the developed real-time QT-NASBA. Re-analysis of seven available original slides resolved five mismatches. Three of those were initially identified as P. malariae mono-infection, but after re-reading the slides P. falciparum was found, confirming the real-time QT-NASBA result. The other two slides were of poor quality not allowing true species identification. The remaining five discordant results could not be explained by microscopy, but may be due to extreme low numbers of parasites present in the samples. In addition, 12 Plasmodium berghei isolates from mice and 20 blood samples from healthy donors did not show any reaction in the assay.ConclusionReal-time QT-NASBA is a very sensitive and specific technique with a detection limit of 0.1 Plasmodium parasite per diagnostic sample (50 μl of blood) and can be used for the detection, identification and quantitative measurement of low parasitaemia of Plasmodium species, thus making it an effective tool for diagnostic purposes and useful for epidemiological and drug studies.

A randomized trial to monitor the efficacy and effectiveness by QT-NASBA of artemether-lumefantrine versus dihydroartemisinin-piperaquine for treatment and transmission control of uncomplicated Plasmodium falciparum malaria in western Kenya

BackgroundMany countries have implemented artemisinin-based combination therapy (ACT) for the first-line treatment of malaria. Although many studies have been performed on efficacy and tolerability of the combination arthemeter-lumefantrine (AL) or dihydroartemisinin-piperaquine (DP), less is known of the effect of these drugs on gametocyte development, which is an important issue in malaria control.Methods and resultsIn this two-arm randomized controlled trial, 146 children were treated with either AL or DP. Both groups received directly observed therapy and were followed for 28 days after treatment. Blood samples were analysed with microscopy and NASBA. In comparison with microscopy NASBA detected much more gametocyte positive individuals. Moreover, NASBA showed a significant difference in gametocyte clearance in favour of AL compared to DP. The decline of parasitaemia was slower and persistence or development of gametocytes was significantly higher and longer at day 3, 7 and 14 in the DP group but after 28 days no difference could be observed between both treatment arms.ConclusionAlthough practical considerations could favour the use of one drug over another, the effect on gametocytogenesis should also be taken into account and studied further using molecular tools like NASBA. This also applies when a new drug is introduced.Trial registrationCurrent controlled trials ISRCTN36463274

Systematic review and meta-analysis: rapid diagnostic tests versus placental histology, microscopy and PCR for malaria in pregnant women.

BackgroundDuring pregnancy, malaria infection with Plasmodium falciparum or Plasmodium vivax is related to adverse maternal health and poor birth outcomes. Diagnosis of malaria, during pregnancy, is complicated by the absence or low parasite densities in peripheral blood. Diagnostic methods, other than microscopy, are needed for detection of placental malaria. Therefore, the diagnostic accuracy of rapid diagnostic tests (RDTs), detecting antigen, and molecular techniques (PCR), detecting DNA, for the diagnosis of Plasmodium infections in pregnancy was systematically reviewed.MethodsMEDLINE, EMBASE and Web of Science were searched for studies assessing the diagnostic accuracy of RDTs, PCR, microscopy of peripheral and placental blood and placental histology for the detection of malaria infection (all species) in pregnant women.ResultsThe results of 49 studies were analysed in metandi (Stata), of which the majority described P. falciparum infections. Although both placental and peripheral blood microscopy cannot reliably replace histology as a reference standard for placental P. falciparum infection, many studies compared RDTs and PCR to these tests. The proportion of microscopy positives in placental blood (sensitivity) detected by peripheral blood microscopy, RDTs and PCR are respectively 72% [95% CI 62-80], 81% [95% CI 55-93] and 94% [95% CI 86-98]. The proportion of placental blood microscopy negative women that were negative in peripheral blood microscopy, RDTs and PCR (specificity) are 98% [95% CI 95-99], 94% [95% CI 76-99] and 77% [95% CI 71-82]. Based on the current data, it was not possible to determine if the false positives in RDTs and PCR are caused by sequestered parasites in the placenta that are not detected by placental microscopy.ConclusionThe findings suggest that RDTs and PCR may have good performance characteristics to serve as alternatives for the diagnosis of malaria in pregnancy, besides any other limitations and practical considerations concerning the use of these tests. Nevertheless, more studies with placental histology as reference test are urgently required to reliably determine the accuracy of RDTs and PCR for the diagnosis of placental malaria. P. vivax-infections have been neglected in diagnostic test accuracy studies of malaria in pregnancy.

A Magneto-Optic Route toward the In Vivo Diagnosis of Malaria: Preliminary Results and Preclinical Trial Data

We report the development of magneto-optic technology for the rapid quantitative diagnosis of malaria that may also be realizable in a noninvasive format. Hemozoin, the waste product of malarial parasitic action on hemoglobin, is produced in a form that under the action of an applied magnetic field gives rise to an induced optical dichroism characteristic of the hemozoin concentration. Here we show that precise measurement of this induced dichroism may be used to determine the level of malarial infection because this correlates, albeit in a complex manner throughout the infection cycle, with the concentration of hemozoin in the blood and tissues of infected patients. Under conservative assumptions for the production of hemozoin as a function of parasitemia, initial results indicate that the technique can match or exceed other current diagnostic techniques. The validity of the approach is confirmed by a small preliminary clinical trial on 13 patients, and measurements on live parasitized cells obtained from in vitro culture verify the possibility of producing in vivo diagnostic instrumentation.

Molecular diagnosis of malaria in the field: development of a novel 1-step nucleic acid lateral flow immunoassay for the detection of all 4 human Plasmodium spp. and its evaluation in Mbita, Kenya

Microscopy is frequently used for malaria diagnosis, but at low parasitemia, it becomes less sensitive and time consuming. Molecular tools allow for specific/sensitive diagnosis, but current formats, such as polymerase chain reaction (PCR) combined with gel electrophoresis and real-time PCR assays, are difficult to implement in resource-poor settings. Development of a simple, fast, sensitive, and specific detection system, nucleic acid lateral flow immunoassay (NALFIA) for amplified pan-Plasmodium PCR products, is described. The NALFIA lower detection limit is 0.3 to 3 parasites/microL, 10-fold more sensitive than gel electrophoresis analysis. Evaluating 650 clinically suspected malaria cases with the pan-Plasmodium assay under field conditions (rural Kenya) revealed that NALFIA detected more positives than microscopy (agreement, 95%; kappa value = 0.85), and there was an excellent agreement between gel electrophoresis and NALFIA (98.5%; kappa value = 0.96). In conclusion, NALFIA is more sensitive than microscopy and a good alternative to detect PCR products while circumventing using electricity or expensive equipment, making NALFIA the 1st step toward molecular field diagnosis.

Prevalence of Plasmodium spp. in malaria asymptomatic African migrants assessed by nucleic acid sequence based amplification

BackgroundMalaria is one of the most important infectious diseases in the world. Although most cases are found distributed in the tropical regions of Africa, Asia, Central and South Americas, there is in Europe a significant increase in the number of imported cases in non-endemic countries, in particular due to the higher mobility in todays society.MethodsThe prevalence of a possible asymptomatic infection with Plasmodium species was assessed using Nucleic Acid Sequence Based Amplification (NASBA) assays on clinical samples collected from 195 study cases with no clinical signs related to malaria and coming from sub-Saharan African regions to Southern Italy. In addition, base-line demographic, clinical and socio-economic information was collected from study participants who also underwent a full clinical examination.ResultsSixty-two study subjects (31.8%) were found positive for Plasmodium using a pan Plasmodium specific NASBA which can detect all four Plasmodium species causing human disease, based on the small subunit 18S rRNA gene (18S NASBA). Twenty-four samples (38%) of the 62 18S NASBA positive study cases were found positive with a Pfs25 mRNA NASBA, which is specific for the detection of gametocytes of Plasmodium falciparum. A statistically significant association was observed between 18S NASBA positivity and splenomegaly, hepatomegaly and leukopaenia and country of origin.ConclusionThis study showed that a substantial proportion of people originating from malaria endemic countries harbor malaria parasites in their blood. If transmission conditions are available, they could potentially be a reservoir. Thefore, health authorities should pay special attention to the health of this potential risk group and aim to improve their health conditions.

Success or failure of critical steps in community case management of malaria with rapid diagnostic tests: a systematic review

BackgroundMalaria still causes high morbidity and mortality around the world, mainly in sub-Saharan Africa. Community case management of malaria (CCMm) by community health workers (CHWs) is one of the strategies to combat the disease by increasing access to malaria treatment. Currently, the World Health Organization recommends to treat only confirmed malaria cases, rather than to give presumptive treatment.ObjectivesThis systematic review aims to provide a comprehensive overview of the success or failure of critical steps in CCMm with rapid diagnostic tests (RDTs).MethodsThe databases of Medline, Embase, the Cochrane Library, the library of the ‘Malaria in Pregnancy’ consortium, and Web of Science were used to find studies on CCMm with RDTs in SSA. Studies were selected according to inclusion and exclusion criteria, subsequently risk of bias was assessed and data extracted.Results27 articles were included. CHWs were able to correctly perform RDTs, although specificity levels were variable. CHWs showed high adherence to test results, but in some studies a substantial group of RDT negatives received treatment. High risk of bias was found for morbidity and mortality studies, therefore, effects on morbidity and mortality could not be estimated. Uptake and acceptance by the community was high, however negative-tested patients did not always follow up referral advice. Drug or RDT stock-outs and limited information on CHW motivation are bottlenecks for sustainable implementation. RDT-based CCMm was found to be cost effective for the correct treatment of malaria in areas with low to medium malaria prevalence, but study designs were not optimal.DiscussionTrained CHWs can deliver high quality care for malaria using RDTs. However, lower RDT specificity could lead to missed diagnoses of non-malarial causes of fever. Other threats for CCMm are non-adherence to negative test results and low referral completion. Integrated CCM may solve some of these issues. Unfortunately, morbidity and mortality are not adequately investigated. More information is needed about influencing sociocultural aspects, CHW motivation and stock supply.ConclusionCCMm is generally well executed by CHWs, but there are several barriers for its success. Integrated CCM may overcome some of these barriers.

Malaria Prevalence, Spatial Clustering and Risk Factors in a Low Endemic Area of Eastern Rwanda: A Cross Sectional Study

Background Rwanda reported significant reductions in malaria burden following scale up of control intervention from 2005 to 2010. This study sought to; measure malaria prevalence, describe spatial malaria clustering and investigate for malaria risk factors among health-centre-presumed malaria cases and their household members in Eastern Rwanda. Methods A two-stage health centre and household-based survey was conducted in Ruhuha sector, Eastern Rwanda from April to October 2011. At the health centre, data, including malaria diagnosis and individual level malaria risk factors, was collected. At households of these Index cases, a follow-up survey, including malaria screening for all household members and collecting household level malaria risk factor data, was conducted. Results Malaria prevalence among health centre attendees was 22.8%. At the household level, 90 households (out of 520) had at least one malaria-infected member and the overall malaria prevalence for the 2634 household members screened was 5.1%. Among health centre attendees, the age group 5–15 years was significantly associated with an increased malaria risk and a reported ownership of ≥4 bednets was significantly associated with a reduced malaria risk. At the household level, age groups 5–15 and >15 years and being associated with a malaria positive index case were associated with an increased malaria risk, while an observed ownership of ≥4 bednets was associated with a malaria risk-protective effect. Significant spatial malaria clustering among household cases with clusters located close to water- based agro-ecosystems was observed. Conclusions Malaria prevalence was significantly higher among health centre attendees and their household members in an area with significant household spatial malaria clustering. Circle surveillance involving passive case finding at health centres and proactive case detection in households can be a powerful tool for identifying household level malaria burden, risk factors and clustering.

Antigen persistence of rapid diagnostic tests in pregnant women in Nanoro, Burkina Faso, and the implications for the diagnosis of malaria in pregnancy

Objectives  To evaluate persistence of several Plasmodium antigens in pregnant women after treatment and compare diagnostics during treatment follow‐up.