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PubMed2001

Nested polymerase chain reaction in detection of Plasmodium vivax sporozoites in mosquitoes.

OBJECTIVE: To detect malaria DNA in mosquitoes. METHODS: A nested polymerase chain reaction (nested PCR) procedure which amplifies a 121 bp DNA of a SSUrRNA gene specific to Plasmodium vivax was used. RESULTS: In laboratory-infected mosquitoes, nested PCR could detect as few as 3 sporozoites or 1 infected mosquito mixed in a group of 99 normal ones. Furthermore, no specific 121 bp band was seen with DNA templates from other malaria parasites or negative mosquitoes. CONCLUSION: Sensitivity and specificity obtained indicated an advantage of the nested PCR over DNA probes or direct PCR for the detection of Plasmodium vivax sporozoites in mosquitoes with low-grade parasitic infections.

Animals↗
PubMed1975

Incorporation of nucleic acid precursors by Plasmodium cynomolgi in Anopheles balabacensis.

Autoradiography was used to study the incorporation of tritium-labelled nucleic acid precursors into the sporogonic stages of Plasmodium cynomolgi in Anopheles b. balabacensis. Infected mosquitoes were fed on either 3H-adenine (5, 25 and 50 muCi/ml or 3H-thymidine (50 muCi/ml) for various periods after the blood meal. 3H-adenine was incorporated into DNA and RNA of the developing oocysts and the resulting sporozoites. Midgut epithelial cells incorporated label from 3H-adenine into nuclear and cytoplasmic regions. In both parasite and host tissue RNA-label was removed after RNase treatment. 3H-thymidine was not taken up by the developing oocysts or sporozoites while incorporation into the cell nuclei of the adjacent midgut epithelium and fat-body of the mosquito was shown. The observed incorporation of 3H-adenine, but not 3H-thymidine, into the sporogonic stages of Plasmodium supports the assumption that the malaria parasite needs exogenous sources of purine but relies on the de novo synthesis of pyrimidine during its development in the mosquito.

Adenine↗
PubMed2001

[Evaluation of in vitro drug sensitivity of antimalarials for Plasmodium falciparum using a colorimetric assay (DELI-microtest)].

The DELI-microtest (Double-Site Enzyme Linked LDH Immunodetection) is a colorimetric method for determination of Plasmodium falciparum in vitro sensitivity to antimalarial drugs. This method is based on the capture of the specific lactate deshydrogenase of Plasmodium falciparum (pLDH) using 2 monoclonal antibodies which recognize 2 different sites of the enzyme. The second monoclonal antibody is biotinylated and may catch a peroxidase-labelled streptavidine and develop a colorimetric reaction in the presence of peroxidase substrate. The level of pLDH released by parasite growth is determined by the measure of optical densities. An excellent correlation has been observed between the IC50's obtained using the DELI-microtest and the classical isotopic microtest, and the high sensitivity of the DELI-microtest can be used to measure the IC50's for isolates with parasitemia as low as 0.005%. It is easier and faster to process than isotopic microtest in field conditions. In addition, due to high sensitivity of the DELI-microtest, isolates with a low parasitemia may be included in the studies.

Animals↗
PubMed1998

[Longitudinal surveillance of sensitivity of Plasmodium falciparum to pyronaridine in south Yunnan].

AIM: To understand the changes in the sensitivity of Plasmodium falciparum to pyronaridine in the south of Yunnan Province. METHODS: WHO standard in vivo and in vitro microtest was used. RESULTS: In 1984-1985 and 1995, 36 and 39 cases of acute falciparum malaria were observed, the average defervescence time being 32.7 +/- 16.0 h and 56.2 +/- 27.4 h, the mean asexual parasite clearance time, 64.2 +/- 22.9 h and 55.3 +/- 11.8 h, and the recrudescence rate of patients followed up for 28 days, 15.2%(5/33) and 37.5%(9/24), respectively. The resistance rates of resistant isolates by in vitro microtest were 0(0/24), 8.8%(3/34), 13.8%(4/29) and 36.4%(4/11) in 1988, 1990, 1992 and 1995, respectively, while the corresponding ID50 were 13.0, 20.0, 32.0 and 40.0 nmol/L, ID95 were 48.0, 136.0, 325.0 and 330.0 nmol/L, the mean concentrations for complete inhibition of schizont formation were 32.6, 99.5, 148.2 and 190.1 nmol/L, respectively. CONCLUSION: The sensitivity of Plasmodium falciparum to pyronaridine was found to have a tendency of continuous dropping in south Yunnan.

Adolescent↗
PubMed1998

[Cloning and sequence analysis of the gene encoding the partial region CS protein of a Plasmodium falciparum isolate from Yunnan].

AIM: Determining nucleotide sequence of the circumsporozoite protein partial gene of the Plasmodium falciparum PFD-3/YN (Yunnan of China) and finding out the differences of the CS gene sequence between Chinese Plasmodium falciparum isolate and other isolates. METHODS: The circumsporozoite protein gene fragment was amplified by polymerase chain reaction and cloned into M13 bacteriophage. M13-CSP single strand DNAs of the three positive clones were extracted respectively. Then, the nucleotide sequence of the CS gene fragment was determined by the dideoxy chain termination method. PCGENE software was used to compare and analyze the CS gene sequence of the six isolates. RESULTS: Different degrees of diversity of the CS gene sequences were found among P. falciparum PFD-3/YN and other isolates(T4, Wellcome, NF54, 3D7 and 7G8). A non-silent substitution at the nucleotide level being found in the P. f Th/Tc antigenic epitope region. CONCLUSION: There were differences in the CS gene sequence among P. falciparum PFD-3/YN and those of other isolates.

Amino Acid Sequence↗
PubMed1998

[Effect of dihydroquinghaosu on the development of Plasmodium yoelii yoelii in Anopheles stephensi].

AIM: To observe the effect of dihydro qinghaosu (DQHS) on the development of Plasmodium yoelii yoelii in Anopheles stephensi and to explore the possibility of whether DQHS has preventive effect against malaria. METHODS: Plasmodium yoelii yoelii-infected mice that had been administered with a single dose of different dosages (60 mg/kg, 120 mg/kg, 180 mg/kg and 240 mg/kg) of DQHS were exposed to mosquitoes to suck blood. The development of malaria parasites in the mosquitoes of the control group and the treated group were observed by light microscopy (LM) and transmission electron microscopy (TEM). RESULTS: DQHS exhibited certain inhibitory effect on the infectivity of gametocyte. The degree of inhibition was related to the developmental stages of gametocyte and the drug dosage. Immature gametocytes were more sensitive to DQHS than the mature gametocytes. With the increases in dosage, the positive rate and the density of oocyst and sporozoite decreased. However, the difference in the density of sporozoite between 180 mg/kg and 240 mg/kg treated groups was insignificant. TEM result showed damage of the membrane system and vacuolation in the cytoplasm of the oocysts (12-13 d) on mosquito midgut of 60 mg/kg 16 h treated group. The oocysts in the mosquitoes continued to develop after treated with 120 mg/kg of DQHS for 16 h on 3-day-instar. There was no significant difference (P > 0.05) in the density of oocyst and sporozoite between the control group and the treated group. CONCLUSION: DQHS can affect the infectivity of gametocyte and decrease the malaria transmission but cannot inhibit the sorogonic stage directly.

Animals↗
PubMed2001

Artemether vs quinine therapy in Plasmodium falciparum malaria in Manipur--a preliminary report.

Qinghaosu and its derivatives are rapidly effective antimalarial drugs derived from a Chinese plant (sweet worm wood). Preliminary studies suggest that these drugs may be more effective than quinine in the treatment of Plasmodium falciparum malaria. A randomised double blind trial was conducted in 52 cases of Plasmodium falciparum malaria cases. In all 26 cases were given artemether and another 26 were given quinine. There were 2 (7.5%) deaths in artemether group and 4 (15%) deaths in quinine group. The parasites were cleared more quickly from the blood in artemether group when compared to quinine group (mean-72 hrs vs 96 hrs). Resolution of fever was comparable in both artemether and quinine group (mean-84 hrs vs 78 hrs) and also the average time of recovery from coma was more earlier in artemether group (mean-60 hrs vs 72 hrs). The only side effect noticed with artemether therapy was gastrointestinal (GI) intolerance while quinine therapy was associated with myocarditis, hypotension, hypoglycemia and GI intolerance.

Adolescent↗
PubMed2001

Reduced levels of chloroquine resistant Plasmodium falciparum in selected foci of the South West Province, Cameroon.

OBJECTIVES: To evaluate the state of chloroquine resistant malaria in the South West Province of Cameroon. To estimate the prevalence of different species of Plasmodium in the area. DESIGN: A cross sectional study. SETTING: South West Province of Cameroon. SUBJECTS: 326 febrile patients. MAIN OUTCOME MEASURES: Chloroquine resistance studies using the WHO seven-day standard in vivo test. Speciation of malaria by polymerase chain reaction. Prevalence of falciparum malaria by light microscopy of thick blood smears. RESULTS: Prevalence of chloroquine resistance ranged from 4.3 to 13.2%. A total of six cases showed RIII resistance, and three cases showed RII resistance. Plasmodium falciparum was the predominant species, 96.8% of those testing positive for P. falciparum, 6.3% of cases showed mixed infections of P. falciparum and P. malariae. P. ovale accounted for 3% of cases. CONCLUSION: P. falciparum resistance to chloroquine may not be on the increase compared to earlier reports from the South West Province. The results have important implications in treatment of malaria in view of the low cost of chloroquine.

Adolescent↗
PubMed2002

[Plasmodium lactate dehydrogenase activity assay and its preliminary application in therapeutic efficacy monitoring of drugs].

OBJECTIVE: To evaluate the activity of Plasmodium lactate dehydrogenase (pLDH) in relation to the degree of parasitemia and laboratory culture time of the parasite, and explore the feasibility of using the activity assessment in monitoring the effect of drug therapies. METHOD: Using enzymatic colorimetric method, pLDH activities were assayed in the blood samples of different degrees of parasitemia and of varied in vitro culture time. Blood samples from a patient with P.falciparum and P.vivax infection were also collected before and after chemotherapy for pLDH activity assay. RESULTS: pLDH activity increased as parasitemia escalated and the culture time prolonged. The strongest activity was observed between 36 and 48 h of incubation when the trophozoites and schizonts were predominant. pLDH activities were reduced in the blood sample of the patient collected after chemotherapy, completely undetectable after 5 d. CONCLUSION: pLDH activity assay would potentially be of value in detecting specific Plasmodium clones and monitoring efficacy of drug therapy.

Animals↗
PubMed2002

Innate immunity to malaria caused by Plasmodium falciparum.

Malaria, a widespread disease caused by protozoa of the genus Plasmodium, contributes to the death of more than 2 million people each year. Resistance to antimalarial drugs is increasing, and an effective vaccine has not yet been designed. In the search for alternative means to control malaria infections, especially those caused by the most lethal species of malaria parasite, Plasmodium falciparum, our attention has turned to elucidating the relationships of the parasite and human host at the molecular level. In this review, we describe possible mechanisms by which naturally occurring genetic mutations might confer resistance to P. falciparum and how our innate immune response mediated by the phagocytic action of monocytes and macrophages acts as a first-line defence in clearing malaria infections. The potential effectiveness of novel therapies to enhance innate phagocytic clearance of malaria parasites, particularly in nonimmune people who are at greatest risk of adverse outcomes, is also discussed.

Animals↗
PubMed1999

[Recombination and cloning of MSP1(19) and PfCMR of Plasmodium falciparum].

AIM: To construct a recombinant plasmid DNA encoding multiantigens of Plasmodium falciparum and to provide the requirements for DNA immunization. METHODS: Two oligonucleotide primers were designed to amplify MSP1(19), the purified PCR products were digested by Sal I + Xba I, and the recombinant plasmid pWR450-1/PfCMR was digested by EcoR I + Sal I to recover PfCMR gene. PfCMR and MSP1(19) gene fragments were linked and recombined with mammalian expression vector pcDNA3. RESULTS: The MSP1(19) gene fragment with about 363 base pairs were specifically amplified by using PCR technique. The positive recombinant pcDNA3-PfCMR-MSP1(19) (named pcDNA3-Pf8) was screened and identified by agarose gel electrophoresis, endonulease digestion and PCR technique, the whole length of Pf8 is 618 bp. CONCLUSION: By specifically amplifying MSP1(19) gene at the C-terminal of MSP1, a recombinant plasmid pcDNA3-Pf8 encoding multiantigens of Plasmodium falciparum was successfully constructed.

Animals↗
PubMed2000

[Relationship between hemolymph phenol oxidase and melanization of oocysts of Plasmodium yoelii in Anopheles stephensi].

OBJECTIVE: To explore the relationship between the hemolymph phenol oxidase and melanization of oocysts. METHODS: Anopheles stephensi-Plasmodium yoelii system was used to determine the activity of monophenol oxidase (MPO) and o-diphenol oxidase (o-DPO) in the hemolymph collected from 4 groups of mosquitoes by polyacrylamide gel electrophoresis (PAGE) followed by density scanning. The 4 groups of mosquitoes were: non-blood-fed (N), normal-blood-fed (B), infected-blood-fed (I) and nitroquine-administrated (D), respectively. RESULTS: No significant difference was found in the activities of MPO and o-DPO between groups N and B. The activities of MPO and o-DPO were not obviously modified in group I, but were significantly increased on day 10 and decreased on day 15 after blood feeding in the group D as compared with those in the groups N and B. CONCLUSION: The alteration in the mosquito hemolymph PO activity coincided at each time point with the melanization of Plasmodium yoelii oocysts.

Animals↗
PubMed2000

[Effect of Nippostrongylus brasiliensis induced alterations in T helper cell subsets on Plasmodium berghei infection in mice].

OBJECTIVE: To observe the anti-P. berghei ability of C57BL/6 mice after infected with Nippostrongylus brasiliensis alterations in T helper cell subsets in the course of Plasmodium infection, and the effect of the alteration on host's prognoses. METHODS: C57BL/6 mice were infected with Nippostrongylus brasiliensis subcutaneously, 3 wk later, the mice were injected with Plasmodium berghei intraperitoneally. The parasitemia was monitored daily. On days 0, 3 and 9, RNA from the spleens of infected mice was prepared for PT-PCR to analyse the changes of IFN-gamma and IL-4 mRNA during the infection course. RESULTS: Compared with control group, the peak-reaching time of parasitemia in the experiment group was delayed, and the bearing capacity of mice to malaria infection and the surviving time increased obviously. The IL-4 level in the experiment group was higher than that in the control group on day 0 of P. berghei infection, but all raised abnormally in both groups at the early stage of the infection; while IFN-gamma level in the experiment group was higher than that in the control group on day 3 after infection, and then began to reduce to some extent on day 9 of the infection in the experiment group. CONCLUSION: T helper cell subsets play an important role in antimalarial immunoregulation in mice.

Animals↗
PubMed2000

[Plasmodium falciparum: antimalarial activities of antisense oligodeoxyribonucleotides targeted to histidine-rich protein genes in vitro].

OBJECTIVE: To study the inhibition of growth of Plasmodium falciparum cultured in vitro with antisense oligodeoxyribonucleotide (AS ODN) against histidine-rich proteins (HRP). METHODS: The ODNs against HRP II and HRP III were synthesized and used to study the antimalarial activities in vitro. Plasmodium falciparum (FCC1/HN strain, China) were exposed to AS DONs for 48 h, and the growth inhibition was determined by microscopic examination. RESULTS: At 1 mumol/L, all ODNs inhibited parasite growth and development in a target-independent manner. However, when the ODN concentrations were between 0.01 and 0.5 mumol/L, the AS ODN significantly inhibited the growth and development of P. falciparum compared with ODN controls (P < 0.01). Inhibition by the sense strand ODN did not differ significantly from the control group (P > 0.05). CONCLUSION: Blockade of the expression of HRP II and HRP III AS ODN could inhibit P. falciparum cultured in vitro.

Animals↗
PubMed2001

[Partial sequence of sporogony stage-specific 18S ribosomal DNA of Plasmodium yoelii and its application for detection of parasites].

OBJECTIVE: To determine sequence of sporogony stage-specific (S type) 18S ribosomal RNA gene of Plasmodium yoelii (P. y) By265 strain, and by using it to detect the malaria parasites within vector mosquito. METHODS: A pair of conserved DNA primers, universe primer (Pu) and reverse transcription one (Pr), was designed and synthesized according to sequence of the 18S rRNA gene of Plasmodium berghei (P. b). The segment of the S type 18S rDNA of P. y was amplified by reverse transcript-polymerase chain reaction (RT-PCR) from dissected midguts of Anopheles stephensi infected with P. y on the 7th day after infective blood-meal, and its sequence was then determined. One P. y sporogony stage-specific primer (Pys) was selected according to the sequence. Using this primer and Pr, the parasites within mosquitoes were semi-quantitatively detected through RT-PCR between 1-7 d post-infection. RESULTS: The length of the amplified segment was 920 bp. Alignment in match region of the 18S rDNA among S type of P. y (PyS), S type of P. b (PbS) and asexual blood stage-specific one of P. y (PyA) revealed that the similarity between the former and the latter two reached 95.3% and 94.0% respectively. The density of amplified band was significantly concordance with the intensity of oocyst in the midgut. Sensitivity of RT-PCR method was higher than that of the traditional dissection and oocyst observation also. The assay could detect the 18S rRNA molecule of the parasites on the third day post-infection while their oocysts were difficult to be recognized under an optical microscope at that time. CONCLUSION: This S type 18S rDNA sequence in P. y species was first reported (AF266261). As a molecular marker, it could be applied to monitoring the parasite development in its vector at an earlier stage semi-quantitatively with an adequate sensitivity and specificity.

Animals↗
PubMed2001

[Gene point mutation in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum].

OBJECTIVE: To investigate the gene point mutation in the dihydrofolate reductase-thymidylate synthase(dhfr) gene of Plasmodium falciparum isolate from Yunnan Province strongly associated with pyrimethamine and cycloguanil resistance. METHODS: Nested PCR and restriction endonuclease digestion were applied to detect the gene mutation using dried blood filter paper collected from the fields in Yunnan Province. RESULTS: Different mutations were found in 4 amino acids at positions 16, 51, 108 and 164 of dhfr gene, particularly, Asn-108 and Ile-51, the mutation frequency being 94.1% and 90.1%, respectively. The frequency of the wild-type genotype (3D7-type) Ser-108 appeared lower (9.1%), while the frequency of the Ala-16 was high(61.8%); the mutation-type was very high, the ratio of HB3-type, 7G8-type/FCR3-type and Cambodian-type was 1:21:7.5. CONCLUSION: The investigation first demonstrated that Plasmodium falciparum Yunnan isolate dihydrofolate reductase-thymidylate synthase gene (dhfr) at positions 16, 51, 108 and 164 exhibited different degrees of point mutation. The frequency of mutation of the 7D8-type involved in pyrimethamine resistance was higher, while that of the FCR3-type involved in cycloquanil resistance was lower.

Animals↗
PubMed2001

Cloning and expressing of the three repeat fragments of Plasmodium falciparum 11.1 gene.

OBJECTIVE: To clone and express the 3R, 6R and 9R repeat fragments of Plasmodium falciparum(Pf11.1) gene. METHODS: Three repeat fragments from the genomic DNA of Plasmodium falciparum 3D7 strain cultivated were amplified by using the designed primers. The PCR products were cloned into the pT7 vector for bi-direction sequencing. The sequencing results were analysised by GENETYX-MAC. And then the amplified fragments were subcloned into pET32a(+) or pET32b(+) in order to express the recombinant proteins under the induction of IPTG in E. coli BL21. RESULTS: 3R,6R and 9R fragments with sizez of 552 bp, 630 bp and 444 bp respectively were successfully amplified by PCR. The sequence analysis showed that there were 4 more 3AA units and one more 6AA unit in Pf11.1 gene of 3D7 strain as compared with Palo Alto strain. The homologies of the nucleotide sequence between the 3R fragment and the 6R fragment of the two strains were 92.8% and 95.1%, respectively. The amplified 9R fragment contains 139AA repeat units. The three recombinant proteins were expressed in BL21 strain with molecular weights of 45, 60 and 42 kDa. CONCLUSION: We got the 3R, 6R and 9R fragments separately by PCR and expressed them in E. coli successfully. The Pf11.1 gene of 3D7 strain is highly homologous to that of the Palo Alto strain.

Animals↗
PubMed2003

Case report: An unusual late relapse of Plasmodium vivax malaria.

We observed an unusual case of Plasmodium vivax malaria who presented with an initial relapse four years after the primary infection. This occurred in Cameroon, where the patient, a 56-year-old priest, acquired a mild form of malaria and was treated with only chloroquine. Since he returned to Italy, he had not experienced any malaria-like symptoms, had not visited any other areas endemic for malaria, and had not received a blood transfusion. Blood smear microscopy confirmed the presence of Plasmodium spp. parasites, but unclear morphologic characteristics did not allow discrimination between P. vivax and P. ovale. A nested polymerase chain reaction-based molecular analysis identified P. vivax as the plasmodial species responsible. This case emphasizes the importance of taking into account the possibility of a very late initial relapse of P. vivax malaria and the relevant issues in terms of infection control.

Animals↗