Implementation of a novel PCR based method for detecting malaria parasites from naturally infected mosquitoes in Papua New Guinea

<p>Abstract</p> <p>Background</p> <p>Detection of <it>Plasmodium species </it>in mosquitoes is important for designing vector control studies. However, most of the PCR-based detection methods show some potential limitations. The objective of this study was to introduce an effective PCR-based method for detecting <it>Plasmodium vivax </it>and <it>Plasmodium falciparum </it>from the field-caught mosquitoes of Papua New Guinea.</p> <p>Methods</p> <p>A method has been developed to concurrently detect mitochondrial cytochrome b (<it>Cyt b</it>) of four human <it>Plasmodium </it>species using PCR (<it>Cytb</it>-PCR). To particularly discriminate <it>P. falciparum </it>from <it>P. vivax</it>, <it>Plasmodium ovale </it>and <it>Plasmodium malariae</it>, a polymerase chain reaction-repeated fragment length polymorphism (PCR-RFLP) has further been developed to use with this method. However, due to limited samples number of <it>P. ovale </it>and <it>P. malariae</it>; this study was mainly confined to <it>P. vivax </it>and <it>P. falciparum</it>. The efficiency of <it>Cytb</it>-PCR was evaluated by comparing it with two 'gold standards' enzyme linked immunosorbent assay specific for circumsporozoite protein (CS-ELISA) using artificially infected mosquitoes; and nested PCR specific for small subunit ribosomal RNA (<it>SSUrRNA</it>) using field caught mosquitoes collected from three areas (Kaboibus, Wingei, and Jawia) of the East Sepic Province of Papua New Guinea.</p> <p>Results</p> <p>A total of 90 mosquitoes were artificially infected with three strains of <it>Plasmodium</it>: <it>P. vivax-</it>210 (<it>n </it>= 30), <it>P. vivax</it>-247 (<it>n </it>= 30) and <it>P. falciparum </it>(<it>n </it>= 30). These infected mosquitoes along with another 32 unfed mosquitoes were first checked for the presence of <it>Plasmodium </it>infection by CS-ELISA, and later the same samples were compared with the <it>Cytb</it>-PCR. CS-ELISA for <it>P. vivax</it>-210, <it>P. vivax</it>-247 and <it>P. falciparum </it>detected positive infection in 30, 19 and 18 mosquitoes respectively; whereas <it>Cytb</it>-PCR detected 27, 16 and 16 infections, respectively. The comparison revealed a close agreement between the two assays (κ = 0.862, 0.842 and 0.894, respectively for Pv-210, Pv-247 and <it>P. falciparum </it>groups). It was found that the eight CS-ELISA-positive mosquitoes detected negative by <it>Cytb</it>-PCR were false-positive results. The lowest detection limit of this <it>Cytb</it>-PCR was 10 sporozoites. A highly concordance result was also found between nested PCR and <it>Cytb</it>-PCR using 107 field caught mosquitoes, and both tests concordantly detected <it>P. falciparum </it>in an <it>Anopheles punctulatus </it>mosquito collected from Kaboibus. Both tests thus suggested an overall sporozoite rate of 0.9% (1/107) in the study areas. Subsequently, PCR-RFLP efficiently discriminated <it>P. falciparum </it>from <it>P. vivax </it>for all of the <it>Cytb</it>-PCR positive samples.</p> <p>Conclusion</p> <p>A single step PCR based method has been introduced here that is highly sensitive, efficient and reliable for identifying <it>P. vivax </it>and <it>P. falciparum </it>from mosquitoes. The reliability of the technique was confirmed by its ability to detect <it>Plasmodium </it>as efficiently as those of CS-ELISA and nested PCR. Application of the assay offers the opportunity to detect vector species of Papua New Guinea and may contribute for designing further vector control programmes.</p

Agaritine derived from Agaricus blazei Murrill induces apoptosis via mitochondrial membrane depolarization in hematological tumor cell lines

Objectives: Agaritine (AGT) is a hydrazine-containing compound derived from the mushroom Agaricus blazei Murill. We previously reported the antitumor effect of AGT on hematological tumor cell lines and suggested that AGT induces apoptosis in U937 cells via caspase activation. However, the antitumor mechanism of AGT has not been fully understood. Methods: Four hematological tumor cell lines (K562, HL60, THP-1, H929) were used in this study. The cells were incubated in the presence of 50 μM AGT for 24 h and analyzed for cell viability, annexin V positivity, caspase-3/7 activity, mitochondrial membrane depolarization, cell cycle, DNA fragmentation, and the expression of mitochondrial membrane-associated proteins (Bax and cytochrome c). Results: In HL60, K562, and H929 cells, AGT reduced cell viability and increased annexin V- and dead cell-positive rates; however, it did not affect THP-1 cells. In K562 and HL60 cells, caspase-3/7 activity, mitochondrial membrane depolarization, and expression of mitochondrial membrane proteins, Bax and cytochrome c, were all increased by AGT. Cell cycle analysis showed that only K562 exhibited an increase in the proportion of cells in G2/M phase after the addition of AGT. DNA fragmentation was also observed after the addition of AGT. Conclusions: These results indicate that AGT induces apoptosis in K562 and HL60 cells, like U937 reported previously, but showed no effect on THP-1 cells. It was suggested that AGT-induced apoptosis involves the expression of Bax and cytochrome c via mitochondrial membrane depolarization

Evaluation of a new method for the diagnosis of alterations of Lens culinaris agglutinin binding of thyroglobulin molecules in thyroid carcinoma

Background: The measurement of serum thyroglobulin (Tg) is widely used as a marker for recurrence of thyroid carcinoma following total thyroidectomy. However, this method cannot differentiate between benign and malignant disease. We focused on the sugar chain in the Tg molecule and investigated the usefulness of Lens culinaris agglutinin (LCA)-reactive Tg ratios in sera and wash fluids obtained during fine-needle aspiration (FNA) for the detection of thyroid carcinoma. Methods: The study was performed using 203 serum samples (115 from patients with benign thyroid disease and 88 from patients with thyroid carcinomas) and 176 wash fluid samples (143 benign, 21 malignant, and 12 inconclusive). LCA-reactive Tg ratios were determined using an enzyme-linked immunosorbent assay, and a comparison was made between malignant and benign lesions. Results: In serum, the ratio in patients with malignancy was 79.5±6.0 [mean±standard deviation (SD)], significantly lower than in patients with benign lesions (84.9±3.5). The ratios in wash fluid from malignant lesions (75.8±18.9) were also significantly lower than those from benign lesions (85.6±3.9). Conclusions: These results suggest that this method could distinguish between benign and malignant lesions and may be useful for screening serum and wash samples. Clin Chem Lab Med 2009;47:1285–90.Peer Reviewe