Malaria proteases mediate inside-out egress of gametocytes from red blood cells following parasite transmission to the mosquito.

Malaria parasites reside in human erythrocytes within a parasitophorous vacuole. The parasites are transmitted from the human to the mosquito by the uptake of intraerythrocytic gametocytes during a blood meal, which in the midgut become activated by external stimuli and subsequently egress from the enveloping erythrocyte. Gametocyte egress is a crucial step for the parasite to prepare for fertilization, but the molecular mechanisms of egress are not well understood. Via electron microscopy, we show that Plasmodium falciparum gametocytes exit the erythrocyte by an inside-out type of egress. The parasitophorous vacuole membrane (PVM) ruptures at multiple sites within less than a minute following activation, a process that requires a temperature drop and parasite contact with xanthurenic acid. PVM rupture can also be triggered by the ionophore nigericin and is sensitive to the cysteine protease inhibitor E-64d. Following PVM rupture the subpellicular membrane begins to disintegrate. This membrane is specific to malaria gametocytes, and disintegration is impaired by the aspartic protease inhibitor EPNP and the cysteine/serine protease inhibitor TLCK. Approximately 15 min post activation, the erythrocyte membrane ruptures at a single breaking point, which can be inhibited by inhibitors TLCK and TPCK. In all cases inhibitor treatment results in interrupted gametogenesis.

A real-time multiplex-nested PCR system for coprological diagnosis of Echinococcus multilocularis and host species.

A hybridization probe-based real-time multiplex-nested PCR system was developed for the simultaneous detection of Echinococcus multilocularis and host species directly from faecal samples. Species identification was determined by melting curve analysis. Specificity was assessed by using DNA extracted from various cestodes (E. multilocularis, Echinococcus granulosus (G1), Echinococcus ortleppi, Echinococcus canadensis (G6, G7), Taenia crassiceps, Taenia hydatigena, Taenia mustelae, Taenia pisiformis, Taenia serialis, Taenia taeniaeformis, Mesocestoides leptothylacus), carnivores (Vulpes vulpes, Vulpes corsac, Vulpes ferrilata, Canis familiaris, Felis catus, Martes foina), Microtus arvalis and Arvicola terrestris. The analytical sensitivity was 10 fg, evaluated with serially diluted DNA of E. multilocularis to 10 µl total DNA solution from E. multilocularis-negative canid faeces. Based on a comparison of 47 dog samples from China, the proportion of the E. multilocularis-positive-tested samples by the real-time multiplex-nested PCR was moderately higher (38% vs. 30%) as when tested with a previously evaluated nested PCR with a sensitivity of 70-100%, depending on the number and gravidity status of worms present in the intestine (Dinkel et al., J Clin Microbiol 36:1871-1876, 1998). To assess the epidemiological applicability of this method, 227 canid faecal samples collected in the field were analysed. This newly developed real-time multiplex-nested PCR system is a specific, sensitive and reliable method for the detection of E. multilocularis and host species in faecal samples for epidemiological purposes.