A method for the quantitative assessment of malaria parasite development in organs of the mammalian host

Hulier, Elisabeth; Pétour, Pascal; Snounou, Georges; Nivez, Marie-Paule; Miltgen, F; Mazier, Dominique; Rénia, Laurent

doi:10.1016/0166-6851(96)02584-4

Cited by 30 publications

(30 citation statements)

References 20 publications

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“…The primers rPLU 3 and 4 have been used in single nest PCR assays to amplify DNA specifically from the four human malaria parasites (P. falciparum, P. vivax, P. ovale, and P. malariae), from a simian malaria parasite (P. gonderi), and from all the rodent malaria parasites (P. yoelii, P. berghei, P. chabaudi, and P. v. petteri). 18 These nest 2 genus-specific primers have also been used successfully in nested PCR assays in combination with the nest 1 primers rPLU 1 and rPLU 2 to amplify DNA specifically from rodent, human, and the simian malaria parasites (Jarra W, Snounou G, unpublished data).…”

Section: Methodsmentioning

confidence: 99%

“…The numbers in parentheses indicate the positions of the oligonucleotides on the Plasmodium small subunit ribosomal RNA genes. 3,18 The arrow indicates the approximate position of oligonucleotide rPLU 6, which was used as nest 1 primer with rPLU 5 in previous nested PCR assays. 3,7 The nest 2 species-specific primers rFAL 1 and 2, rVIV 1 and 2, rMAL 1 and 2, and rOVA 1 and 2 are specific for P. falciparum, P. vivax, P. malariae, and P. ovale, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…In previously reported epidemiologic studies, 7,17 the nest 1 genus-specific primers rPLU 5 and rPLU 6 were used in combination with the four pairs of human malaria speciesspecific nest 2 primers. In this study, the genus-specific primer rPLU 1 (Jarra W, Snounou G, unpublished data) was used with rPLU 5 instead of rPLU 6 since this permitted PCR products of nest 1 amplifications to be used as DNA templates with the genus-specific primers rPLU 3 and rPLU 4 18 in nest 2 amplifications. The primers rPLU 3 and 4 have been used in single nest PCR assays to amplify DNA specifically from the four human malaria parasites (P. falciparum, P. vivax, P. ovale, and P. malariae), from a simian malaria parasite (P. gonderi), and from all the rodent malaria parasites (P. yoelii, P. berghei, P. chabaudi, and P. v. petteri).…”

Section: Methodsmentioning

confidence: 99%

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A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies.

Singh¹,

Bobogare²,

Cox-Singh³

et al. 1999

Am J Trop Med Hyg

532

566

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Abstract. A nested polymerase chain reaction (PCR) assay that uses Plasmodium genus-specific primers for the initial PCR (nest 1) amplification and either genus-or species-specific primers for the nest 2 amplifications was tested on laboratory and field samples. With in vitro cultured Plasmodium falciparum-infected blood samples, it was capable of detecting six parasites/l of blood using DNA prepared from 25-l blood spots on filter paper. The assay was evaluated on fingerprick blood samples collected on filter paper from 129 individuals living in a malaria-endemic area in Malaysia. Malaria prevalence by genus-specific nested PCR was 35.6% (46 of 129) compared with 28.7% (37 of 129) by microscopy. The nested PCR detected seven more malaria samples than microscopy in the first round of microscopic examination, malaria in three microscopically negative samples, six double infections identified as single infections by microscopy and one triple infection identified as a double infection by microscopy. The nested PCR assay described is a sensitive technique for collecting accurate malaria epidemiologic data. When coupled with simple blood spot sampling, it is particularly useful for screening communities in remote regions of the world.Microscopy is the method of choice for the diagnosis of malaria in endemic areas because it is an inexpensive and rapid method of detection. Correct identification of the four species of Plasmodium causing human malaria and the level of detection by microscopy depends on a number of factors, including the experience of the microscopist, proper staining of the slides, appropriate maintenance of microscopes, and the time spent examining each slide. At best, the sensitivity of detection by microscopy is approximately 10-30 parasites/l of blood. 1 However, this level of detection is normally not attained in malaria-endemic areas and particularly during epidemiologic studies when many samples need to be screened in a relatively short time. Thus, incorrect speciation is common and mixed infections and low levels of parasitemia may be missed.To overcome some of the limitations of microscopy for detection of malaria, polymerase chain reaction (PCR)-based assays have been developed for the detection and identification of malaria parasites. These methods have proved to be more specific and sensitive than conventional microscopy and some are reported to be able to detect as few as one parasite/l of blood.2-6 However, to attain such a high sensitivity, blood samples collected from individuals have to be processed immediately or stored at low temperatures and the steps involved in DNA template preparation were multistep, often requiring biohazardous chemicals. Since malaria remains a problem of underdeveloped and often remote areas of the world, it is important to couple PCRbased assays with simple sampling and DNA extraction methods to maximize the value of PCR assays. In a previous study, we coupled the nested PCR assay of Snounou and others 3 to blood collection on filter papers and a simple DNA e...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies.

Singh¹,

Bobogare²,

Cox-Singh³

et al. 1999

Am J Trop Med Hyg

532

566

View full text Add to dashboard Cite

show abstract

“…Two g of total RNA were reverse transcribed using a commercial kit as suggested by the manufacturer (Promega). The primers and PCR conditions have been described previously (31,32). The primers used were ␤ 2 -microglobulin (␤ 2 M) forward (5Ј-GGC TCGCTCGGTGACCCTAGTCTTT-3Ј)andreverse(5Ј-TCTGCAGGCGTAT GTATCAGTCTCA-3Ј), iNOS forward (5Ј-GCATGGACCAGTATAAGGC AAGCA-3Ј) and reverse (5Ј-GCTTCTGGTCGATGTCATGAGCAA-3Ј).…”

Section: Rt-pcr and Real Time Pcrmentioning

confidence: 99%

Sporozoite-Mediated Hepatocyte Wounding Limits Plasmodium Parasite Development via MyD88-Mediated NF-κB Activation and Inducible NO Synthase Expression

Torgler

Bongfen

Romero

et al. 2008

The Journal of Immunology

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Plasmodium sporozoites traverse several host cells before infecting hepatocytes. In the process, the plasma membranes of the cells are ruptured, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory/immunogenic and can serve as a danger signal initiating distinct responses in various cells. Thus, our study aimed at characterizing the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-κB, a main regulator of host inflammatory responses, in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-κB occurred shortly after infection and led to a reduction of infection load in a time-dependent manner in vitro and in vivo, an effect that could be reverted by addition of the specific NF-κB inhibitor BAY11-7082. Furthermore, no NF-κB activation was observed when Spect−/− parasites, which are devoid of hepatocyte traversing properties, were used. We provide further evidence that NF-κB activation causes the induction of inducible NO synthase expression in hepatocytes, and this is, in turn, responsible for a decrease in Plasmodium-infected hepatocytes. Furthermore, primary hepatocytes from MyD88−/− mice showed no NF-κB activation and inducible NO synthase expression upon infection, suggesting a role of the Toll/IL-1 receptor family members in sensing cytosolic factors. Indeed, lack of MyD88 significantly increased infection in vitro and in vivo. Thus, host cell wounding due to parasite migration induces inflammation which limits the extent of parasite infection.

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“…Detection of parasites in the blood and quantification of parasite load in the liver of animals were performed according to the method described by Hulier et al (16) with minor modifications. For detection of parasite in the blood, 100 l of blood was mixed with 0.5 ml of digestion buffer (100 mM NaCl, 10 mM Tris-HCl, 25 mM EDTA, and 0.5% SDS).…”

Section: Challenge and Protection Assessmentmentioning

confidence: 99%

Protective T Cell Immunity against Malaria Liver Stage after Vaccination with Live Sporozoites under Chloroquine Treatment

Belnoue

Costa

Frankenberg

et al. 2004

The Journal of Immunology

Self Cite

211

281

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In this study we present the first systematic analysis of the immunity induced by normal Plasmodium yoelii sporozoites in mice. Immunization with sporozoites, which was conducted under chloroquine treatment to minimize the influence of blood stage parasites, induced a strong protection against a subsequent sporozoite and, to a lesser extent, against infected RBC challenges. The protection induced by this immunization protocol proved to be very effective. Induction of this protective immunity depended on the presence of liver stage parasites, as primaquine treatment concurrent with sporozoite immunization abrogated protection. Protection was not found to be mediated by the Abs elicited against pre-erythrocytic and blood stage parasites, as demonstrated by inhibition assays of sporozoite penetration or development in vitro and in vivo assays of sporozoite infectivity or blood stage parasite development. CD4+ and CD8+ T cells were, however, responsible for the protection through the induction of IFN-γ and NO.

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A method for the quantitative assessment of malaria parasite development in organs of the mammalian host

Cited by 30 publications

References 20 publications

A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies.

A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies.

Sporozoite-Mediated Hepatocyte Wounding Limits Plasmodium Parasite Development via MyD88-Mediated NF-κB Activation and Inducible NO Synthase Expression

Protective T Cell Immunity against Malaria Liver Stage after Vaccination with Live Sporozoites under Chloroquine Treatment

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