Thomas Hänscheid

How useful is PCR in the diagnosis of malaria

Polymerase chain reaction (PCR) assays are the most sensitive and specific method to detect malaria parasites, and have acknowledged value in research settings. However, the time lag between sample collection, transportation and processing, and dissemination of results back to the physician limits the usefulness of PCR in routine clinical practice. Furthermore, in most areas with malaria transmission, factors such as limited financial resources, persistent subclinical parasitaemia, inadequate laboratory infrastructures in the poorer, remote rural areas preclude PCR as a diagnostic method. Even in affluent, non-endemic countries, PCR is not a suitable method for routine use. Nonetheless, PCR could be clinically useful in selected situations.

Haemozoin: from melatonin pigment to drug target, diagnostic tool, and immune modulator

Plasmodium spp produce a pigment (haemozoin) to detoxify the free haem that is generated by haemoglobin degradation. Haemozoin was originally thought to be an inert waste byproduct of the parasite. However, recent research has led to the recognition that haemozoin is possibly of great importance in various aspects of malaria. Haemozoin is the target of many antimalarial drugs, and the unravelling of the exact modes of action may allow the design of novel antimalarial compounds. The detection of haemozoin in erythrocytes or leucocytes facilitates the diagnosis of malaria. The number of haemozoin-containing monocytes and granulocytes has been shown to correlate well with disease severity and may hold the potential for becoming a novel, automated laboratory marker in the assessment of patients. Finally, haemozoin has a substantial effect on the immune system. Further research is needed to clarify these aspects, many of which are important in clinical practice.

Cerebral malaria and the hemolysis/methemoglobin/heme hypothesis: Shedding new light on an old disease

Malaria causes more than 1 million deaths every year with cerebral malaria (CM) being a major cause of death in Sub-Saharan African children. The nature of the malaria-associated pathogenesis is complex and multi-factorial. A unified hypothesis involving sequestration of infected red blood cells, systemic host inflammatory response and hemostasis dysfunction has been proposed to explain the genesis of CM. In this review, we discuss the role of hemolysis, methemoglobin and free heme in CM, brought to light by our recent studies in mice as well as by other studies in humans.

Comparison of three antigen detection tests for diagnosis and follow-up of falciparum malaria in travellers returning to Berlin, Germany

Abstract. We determined the sensitivity and specificity of three rapid immunochromatographic malarial antigen detection test systems (RDTs) for the detection of Plasmodium falciparum and assessed the quality of follow-up results. ParaSight-F and ICT Malaria detect histidine-rich protein-2 (HRP-2), whereas OptiMal detects plasmodial lactate dehydrogenase (pLDH). ParaSight-F performed with 95.1% sensitivity and 97.1% specificity (554 patients tested of whom 144 had falciparum malaria). ICT Malaria performed with 95.7% sensitivity and 99.2% specificity (718 patients tested of whom 184 had falciparum malaria). OptiMal performed with 76.2% sensitivity and 99.7% specificity (539 patients tested of whom 130 had falciparum malaria). In follow-up investigations, HRP-2 did not appear to be a useful antigen due to its long half-life, whereas pLDH offers a reasonable correlation with the presence of viable parasites in those cases initially detected. We therefore conclude that a combination of both antigens might be the best option for creating a reliable RDT for the diagnosis of falciparum malaria.

Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins

Significance Plasmodium parasites have two distinct intracellular growth stages inside the mammalian host—the first stage, which is clinically silent, in liver hepatocytes, and the second, which causes the symptoms of malaria, in red blood cells. This study reports the discovery of a class of antimalarial compounds called torins, which are extremely potent inhibitors of both intracellular stages of Plasmodium. We show that torins block trafficking of liver stage parasite proteins to the physical host–parasite interface, called the parasitophorous vacuole membrane (PVM), and that without continuous trafficking of PVM-resident proteins, the parasite is subject to elimination by its host hepatocyte. Residence within a customized vacuole is a highly successful strategy used by diverse intracellular microorganisms. The parasitophorous vacuole membrane (PVM) is the critical interface between Plasmodium parasites and their possibly hostile, yet ultimately sustaining, host cell environment. We show that torins, developed as ATP-competitive mammalian target of rapamycin (mTOR) kinase inhibitors, are fast-acting antiplasmodial compounds that unexpectedly target the parasite directly, blocking the dynamic trafficking of the Plasmodium proteins exported protein 1 (EXP1) and upregulated in sporozoites 4 (UIS4) to the liver stage PVM and leading to efficient parasite elimination by the hepatocyte. Torin2 has single-digit, or lower, nanomolar potency in both liver and blood stages of infection in vitro and is likewise effective against both stages in vivo, with a single oral dose sufficient to clear liver stage infection. Parasite elimination and perturbed trafficking of liver stage PVM-resident proteins are both specific aspects of torin-mediated Plasmodium liver stage inhibition, indicating that torins have a distinct mode of action compared with currently used antimalarials.

Full blood count and haemozoin-containing leukocytes in children with malaria: diagnostic value and association with disease severity

BackgroundDiligent and correct laboratory diagnosis and up-front identification of risk factors for progression to severe disease are the basis for optimal management of malaria.MethodsFebrile children presenting to the Medical Research Unit at the Albert Schweitzer Hospital (HAS) in Lambaréné, Gabon, were assessed for malaria. Giemsa-stained thick films for qualitative and quantitative diagnosis and enumeration of malaria pigment, or haemozoin (Hz)-containing leukocytes (PCL) were performed, and full blood counts (FBC) were generated with a Cell Dyn 3000® instrument.ResultsCompared to standard light microscopy of Giemsa-stained thick films, diagnosis by platelet count only, by malaria pigment-containing monocytes (PCM) only, or by pigment-containing granulocytes (PCN) only yielded sensitivities/specificities of 92%/93%; 96%/96%; and 85%/96%, respectively. The platelet count was significantly lower in children with malaria compared to those without (p < 0.001), and values showed little overlap between groups. Compared to microscopy, scatter flow cytometry as applied in the Cell-Dyn 3000® instrument detected significantly more patients with PCL (p < 0.01). Both PCM and PCN numbers were higher in severe versus non-severe malaria yet reached statistical significance only for PCN (p < 0.0001; PCM: p = 0.14). Of note was the presence of another, so far ill-defined pigment-containing group of phagocytic cells, identified by laser-flow cytometry as lymphocyte-like gated events, and predominantly found in children with malaria-associated anaemia.ConclusionIn the age group examined in the Lambaréné area, platelets are an excellent adjuvant tool to diagnose malaria. Pigment-containing leukocytes (PCL) are more readily detected by automated scatter flow cytometry than by microscopy. Automated Hz detection by an instrument as used here is a reliable diagnostic tool and correlates with disease severity. However, clinical usefulness as a prognostic tool is limited due to an overlap of PCL numbers recorded in severe versus non-severe malaria. However, this is possibly because of the instrument detection algorithm was not geared towards this task, and data lost during processing; and thus adjusting the instruments algorithm may allow to establish a meaningful cut-off value.

Use of flow cytometry (Sysmex) UF-100) to screen for positive urine cultures: in search for the ideal cut-off.

Abstract Background: Cultures for urinary tract infections (UTI) constitute a large workload in the clinical microbiology laboratory, although up to 80% are usually negative. Several automated methods are available to screen urines for UTI, one being the flow cytometry-based Sysmex® UF-100. Methods: The performance of the UF-100 was evaluated over a 16-month period using urine culture as the reference method. Results: During this period, a total of 5356 urine samples were studied (469 children; 3229 women and 1658 men), of which 706 were culture positive (593 grew Gram negative bacilli). Receiver operating characteristics (ROC) curve analysis showed an area under the curve (AUC) of 0.83 for leukocytes and 0.85 for bacterial count. Applying cut-off values reported in the literature gave sensitivities ranging from 75% to 90%, resulting in 73–174 false negatives (FN). Using a logical combination (leukocytes ≥15×106/L OR bacteria ≥500×106/L) gave a sensitivity of 98%. However, the specificity dropped to 25%, resulting in 15 FN. Conclusions: Screening urine samples for UTI detects a large number of culture positive samples. However, the rather large number of FN observed precludes the use of the UF-100 as a routine screening method to exclude urine samples from culture. Clin Chem Lab Med 2010;48:289–92.

Simple flow cytometric detection of haemozoin containing leukocytes and erythrocytes for research on diagnosis, immunology and drug sensitivity testing

BackgroundMalaria pigment (haemozoin, Hz) has been the focus of diverse research efforts. However, identification of Hz-containing leukocytes or parasitized erythrocytes is usually based on microscopy, with inherent limitations. Flow cytometric detection of depolarized Side-Scatter is more accurate and its adaptation to common bench top flow cytometers might allow several applications. These can range from the ex-vivo and in-vitro detection and functional analysis of Hz-containing leukocytes to the detection of parasitized Red-Blood-Cells (pRBCs) to assess antimalarial activity.MethodsA standard benchtop flow cytometer was adapted to detect depolarized Side-Scatter. Synthetic and Plasmodium falciparum Hz were incubated with whole blood and PBMCs to detect Hz-containing leukocytes and CD16 expression on monocytes. C5BL/6 mice were infected with Plasmodium berghei ANKA or P. berghei NK65 and Hz-containing leukocytes were analysed using CD11b and Gr1 expression. Parasitized RBC from infected mice were identified using anti-Ter119 and SYBR green I and were analysed for depolarized Side Scatter. A highly depolarizing RBC population was monitored in an in-vitro culture incubated with chloroquine or quinine.ResultsA flow cytometer can be easily adapted to detect depolarized Side-Scatter and thus, intracellular Hz. The detection and counting of Hz containing leukocytes in fresh human or mouse blood, as well as in leukocytes from in-vitro experiments was rapid and easy. Analysis of CD14/CD16 and CD11b/Gr1 monocyte expression in human or mouse blood, in a mixed populations of Hz-containing and non-containing monocytes, appears to show distinct patterns in both types of cells. Hz-containing pRBC and different maturation stages could be detected in blood from infected mice. The analysis of a highly depolarizing population that contained mature pRBC allowed to assess the effect of chloroquine and quinine after only 2 and 4 hours, respectively.ConclusionsA simple modification of a flow cytometer allows for rapid and reliable detection and quantification of Hz-containing leukocytes and the analysis of differential surface marker expression in the same sample of Hz-containing versus non-Hz-containing leukocytes. Importantly, it distinguishes different maturation stages of parasitized RBC and may be the basis of a rapid no-added-reagent drug sensitivity assay.

A flow cytometric protocol for detection of Cryptosporidium spp.

Cryptosporidium parvum is transmitted through water and can cause severe diarrhea. The diagnosis is usually based upon observer‐dependent microscopic detection of oocysts, with rather low sensitivity and specificity. Our objective was to optimize a flow cytometric (FC) protocol for the detection of C. parvum. A specific monoclonal antibody conjugated with R‐phycoerythrin was incubated with dead oocysts to determine the optimal antibody concentration. Serial concentrations of oocysts were stained with the optimized concentration and analyzed by FC. The lower detection limit was determined, and the possibility of cross‐reaction was investigated using prokaryotic and eukaryotic microorganisms. A FC protocol was optimized to detect oocysts in spiked human stools. The optimal antibody concentration was found to be 3.0 μg/ml. The lowest number detectable was 2 × 103 oocysts/ml. Staining procedure was specific, as no cross‐reactions were observed. This reliable and easy FC protocol allow the specific detection of Cryptosporidium oocysts, even at very low concentrations, which is important for public health and further studies of treatment efficacy.

Probing the aurone scaffold against Plasmodium falciparum: design, synthesis and antimalarial activity.

A library comprising 44 diversely substituted aurones derivatives was synthesized by straightforward aldol condensation reactions of benzofuranones and the appropriately substituted benzaldehydes. Microwave enhanced synthesis using palladium catalyzed protocols was introduced as a powerful strategy for extending the chemical space around the aurone scaffold. Additionally, Mannich-base derivatives, containing a 7-aminomethyl-6-hydroxy substitution pattern at ring A, were also prepared. Screening against the chloroquine resistant Plasmodium falciparum W2 strain identified novel aurones with IC50 values in the low micromolar range. The most potent compounds contained a basic moiety, with the ability to accumulate in acidic digestive vacuole of the malaria parasite. However, none of those aurones revealed significant activity against hemozoin formation and falcipain-2, two validated targets expressed during the blood stage of P. falciparum infection and functional in digestive vacuole of the parasite. Overall, this study highlight (i) the usefulness of aurones as platforms for synthetic procedures using palladium catalyzed protocols to rapidly deliver lead compounds for further optimization and (ii) the potential of novel aurone derivatives as promising antimalarial compounds.