A Rapid FRET Real-Time PCR Protocol for Simultaneous Quantitative Detection and Discrimination of Human Plasmodium Parasites.

Malaria is the most important parasitic disease worldwide, and accurate diagnosis and treatment without delay are essential for reducing morbidity and mortality, especially in P. falciparum malaria. Real-time PCR is highly sensitive and highly specific, therefore an excellent diagnostic tool for laboratory detection and species-specific diagnosis of malaria, especially in non-endemic regions where experience in microscopic malaria diagnostics is limited. In contrast to many other real-time PCR protocols, our new fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) allows the quantitative and species-specific detection of all human Plasmodium spp. in one run. Species identification is based on single nucleotide polymorphisms (SNPs) within the MalFL probe, detectable by melting curve analysis. A significant advantage of our FRET-qPCR is the short turn-around time of less than two hours, including DNA extraction, which qualifies it for routine diagnostics. Rapid and reliable species-specific malaria diagnosis is important, because treatment is species-dependent. Apart from first-line diagnosis, the quantitative results of our new FRET-qPCR can be helpful in therapy control, to detect early treatment failure. Graphic abstract.

Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites.

Increasing numbers of travelers returning from endemic areas, migrants, and refugees have led to a significant rise in the number of imported malaria cases in non-endemic countries. Real- time PCR serves as an excellent diagnostic tool, especially in regions where experience in microscopy is limited. A novel fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) was developed and evaluated using 56 reference samples of the United Kingdom National External Quality Assessment Service (UK NEQAS) for molecular detection of malaria, including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Species identification is based on single nucleotide polymorphisms (SNPs) within the genome where the MalLC640 probe binds, lowering the melting temperature in the melting curve analysis. The novel FRET-qPCR achieved 100% (n = 56) correct results, compared to 96.43% performing nested PCR. The high sensitivity, with a calculated limit of detection of 199.97 parasites/mL blood for P. falciparum, is a significant advantage, especially if low-level parasitemia has to be ruled out. Even mixed infections of P. falciparum with P. vivax or P. ovale, respectively, were detected. In contrast to many other real-time PCR protocols, this novel FRET-qPCR allows the quantitative and species-specific detection of Plasmodium spp. in one single run. Solely, P. knowlesi was detected but could not be differentiated from P. vivax. The turnaround time of this novel FRET-qPCR including DNA extraction is less than two hours, qualifying it for routine clinical applications, including treatment monitoring.

Development of a pharmacodynamic screening model with Crithidia fasciculata.

The genus Crithidia is a member of the family Trypanosomatidae and is related to the genera Leishmania and Trypanosoma with which it shares a variety of biochemical mechanisms, such as polyamine synthesis and methionin salvage. In consequence, a screening system for antiparasitic candidate material has been developed with Crithidia fasciculata, a parasite naturally occurring in insects and amphibians, but devoid of pathogenicity for humans. Initially a variety of culture media were evaluated of which TPS was best suited for the maintenance of stock cultures, and E-medium - a newly developed formula - for sensitivity testing. Optimal growth of C. fasciculata was observed under microaerophilic conditions. A system for sensitivity testing was developed and applied to the investigation of extracts from higher tropical plants of the genera Stemona and Aglaia for anticrithidial activity. Extracts with significant anti-crithidial activity were scheduled for chromatographic fractionation and the subsequent isolation, purification and structural identification of individual compounds for further sensitivity testing. Encouraging results were obtained with extracts from Aglaia odorata leaves, A. elaeagnoidea stem bark and A. edulis leaves, with EC(90) values of 1213 ng/ml, 1606 ng/ml, and 1462 ng/ml, respectively.

[Adjuvants and additives in vaccines--medical relevance]. / Hilfs- und Zusatzstoffe von Impfstoffen -- Medizinische Relevanz.

Side effects of vaccinations can have different causes. Substances admixed to vaccines may produce allergic or toxic reactions. The significance and importance of causal associations is discussed in this paper. A table is added listing the most important substances in vaccines, such as inactivating substances, preservatives, stabilizers, adjuvants, and residual substances derived from production processes. Among possible allergic reactions, type I reactions, as the most undesirable ones, should be avoided. In this respect, vaccines against yellow fever are the most important ones. With respect to antibiotics, it should be stressed that penicillin and cephalosporins are not contained in any of the vaccines. The significance of side effects caused by ethylmercury as a preservative (thiomersal) is extensively discussed in the literature. Allergy against this substance is common among the population, manifested as type IV reactions following superficial antigen administration. It has been shown that deep intramuscular injection of thiomersal-containing vaccines may be administered even to persons who are allergic to this substance without risk of side effects. Regarding toxic side effects after application of thiomersal, several studies have disproved a causal relation between thiomersal exposure and developmental disorders. Nevertheless, the general recommendation is to use thiomersal-free vaccines, unless no other preparations are available. In these cases risk of morbidity and mortality from the vaccine-preventable diseases outweigh by far any theoretical risk from ethylmercury.