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Molecular biology for detection and characterization of protozoan infections in humans

Posted on: Sunday, 14 March 2004, 06:00 CST

Molecular biology (and particularly PCR) has been increasingly used for the diagnosis of parasitic protozoa of medical interest. Technical improvements in the past decade include: simplification of DNA extraction, development of automated procedures and of real- time PCR permitting precise quantification of parasitic load. In clinical practice, PCR is routinely used for the diagnosis of toxoplasmosis in the amniotic fluid of pregnant women and in immuno- compromised patients. It can be useful for the diagnosis of cutaneous and visceral leishmaniasis and to differentiate the pathogenic amoeba Entamoeba histolytica from the morphologically identical but non-pathogenic Entamoeba dispar. Its use for malaria remains limited due to high cost. Molecular biology methods facilitate the study of parasitic populations and could be useful to identify possible relationships between a particular genotype and virulence.

Keywords: Parasite; PCR; Diagnosis; Human disease; Protozoa; Molecular biology.

Molecular biology has now been used to study or diagnose all parasitic protozoa usually found in humans and this can only be a partial review. The excellent review by J. Weiss (1995) already described the main aspects of molecular probes and PCR for the diagnosis of parasitic infections. However, we will here select some representative examples routinely used in clinical practice. The development of molecular biology (and particularly of PCR) for diagnostic purposes is certainly a major breakthrough, comparable with the use of microscopy and serological methods. Molecular biology has several advantages over these methods. Microscopy is labour-intensive and requires well-trained microscopists; its specificity is, in theory, perfect (but depends on the skills of the microscopist) and its sensitivity is sometimes disappointing for some parasitic diseases. Immunoassays have the benefits of technical simplicity and rapidity but are often unable to differentiate an active from an ancient infection and are subject to possible crossreactions with other pathological conditions. Cultures or animal inoculation require sophisticated equipment and results are obtained after a delay ranging from days to weeks. Molecular biology does not require large biological samples; it has the ability to amplify the target DNA by 10^sup 5^-10^sup 6^ fold and is now quick to perform. Molecular biology has proved its efficiency, not only for the diagnosis of many parasitic protozoa of medical interest but also for differentiating isolates of the same species with consequences for our knowledge about parasite transmission. Some examples of a possible link between genotypes and virulence will be given.

Recent technical improvements

The routine use of molecular biology in research labs has resulted in many technical improvements and simplification allowing the easy transfer of these techniques to clinical laboratories. The long, toxic and awkward phenol/chloroform or chloroform/isoamylic alcohol separations and ethanol or isopropanol precipitations which were used in the past, have been now replaced by simple and commercially available kits using gel filtration (Qiagen SA) or magnetic separation (Roche Diagnostic) methods. Methods to reveal amplified DNA are also key factors in PCR specificity and sensitivity. Some detection systems are particularly interesting because of their low cost and universal applicability to all DNA targets. In classical PCR, amplified DNA and a molecular weight marker are separated in agarose gels and visualized on a UV ramp after staining with ethidium bromide. The use of restriction fragment length polymorphism (RFLP), sequencing of the amplified DNA and use of specific probes, increase PCR specificity. Hybridization of the amplified DNA also increases the sensitivity of the detection and is classically performed by Southern blot or ELISA hybridization.

The use of fluorescent labels in the PCR reaction and its continuous detection by a fluorimetric method throughout the reaction allowed the development of real-time PCR (Cockerill et al. 2002). Real-time PCR has been developed recently for parasites and is a technology of choice allowing easy quantification of the number of copies of a gene in a sample (Bell et al. 2002). It is a fast and automated method which achieves amplification and post- amplification analysis in the same step, limiting the risk of contamination. The possibility of distinguishing a range of optical frequencies permits the use of different probes in the same assay. The concentration of the target nucleic acid is determined by either an "absolute" quantification performed with a known set of controls from a reference external standard or a "relative" quantification made with a reference co-amplified or no gene. "Absolute" quantification was used to quantify different types of pathogens and also to determine plasma viral load. "Relative" quantification was applied to the analysis of gene expression in different conditions. Real-time detection systems may be divided into two groups, both of which are applicable for quantification: detection with non- specific double stranded DNA intercalators (SYBR Green) or hybridizations with different types of fluorescent probes (Table 1). The fluorescent probes are coupled to both a fluorophore (reporter) and a quencher of fluorescence. The two molecules are often bound at the 5' and 3' ends of the probe. The energy emitted by the reporter is transferred by resonance towards the quencher which absorbs its fluorescence (fluorescence resonance energy transfer or FRET). The fluorescent signal can also be generated after the physical separation of the reporter from the quencher by a mechanism related to the presence of a target sequence. Many real-time PCR machines (e.g. ABI PRISM, LightCycler) are available on the market, but all are quite expensive.

Table 1. Fluorescent detection systems used in real-time PCR

Diagnosis of parasitic infections

Two approaches are used to identify parasites in clinical samples: either a set of specific primers of the pathogen is chosen to amplify a DNA fragment identified by its size and by hybridization, or a set of consensus primers is used to amplify a region shared by different pathogens and species diagnosis is made by hybridization with specific probes or sequencing. PCR analysis for human disease diagnosis requires some commitments to prevent false positive or false negative results. The use of dUTP, instead of dTTP, is compulsory to prevent post PCR contamination by amplicons (amplified DNA). Uracyl N-glycosylase added in the assay will destroy possible contaminating dUTP amplicons. An internal control is required to eliminate false negative results due to possible Taq polymerase inhibitors in biological samples. In addition, extraction, preparation of the reactive mixture, amplification and revelation should be carried out in different locations and with specific lab wares devoted to each step of the reaction. In real-time PCR, tubes or glass capillaries are not opened after amplification thus minimising the risk of contamination. The highly sensitive PCR has been used for detection in clinical samples of many parasites and we will review now some of the most demonstrative examples.

PCR has revolutionized the diagnosis of congenital toxoplasmosis (CT), enabling early detection and thereby avoiding the use of more invasive procedures on the foetus. We have been routinely using this technique during the last decade (Dupouy-Camet et al. 1992). We recently reviewed the files of 110 women with Toxoplasma seroconversion during pregnancy that had been followed in our lab (Robert-Gangneux et al. 1999). Prenatal diagnosis was performed for 94 women by amniotic fluid sampling and neonatal diagnosis by placental examination. Toxoplasma gondii was detected by PCR, with or without tissue culture and mouse inoculation. The sensitivity and specificity of prenatal diagnosis were 81 and 100%, respectively. Placental examination was positive for 66.7% of individuals with CT and was always negative for neonates without CT. PCR was also used to detect T. gondii DNA in brain tissue, CSF, vitreous and aqueous fluids, broncho-alveolar lavage fluid and blood in patients with AIDS or other immuno-compromising diseases (Dupouy-Camet et al. 1993). In all these cases identification of the parasite by microscopy had a very low sensitivity and cell culture or mouse inoculation required long delay (a few days for culture; a few weeks for inoculation). Numerous papers have reported on the use of PCR for T. gondii diagnosis in these different occurrences and have been reviewed by Weiss (1995), Morgan (2000), Bastien (2002) and Montoya (2002).

Real-time PCR has also been used for T. gondii detection (Lin et al. 2000; Kupferschmidt et al. 2001; Dworkin et al. 2002; Reischl et al. 2003). Quantitative accuracy was achieved over six logs of DNA concentration with low inter-assay variation. A multiplex FRET- based LightCycler assay incorporating an internal control to avoid false negative results was able to detect less than 10 T, gondii parasites per ml of amniotic fluid (Costa et al. 2001), and was also used to diagnose and follow-up Toxoplasma reactivation after bone marrow transplantation (Costa et al. 2000). Homan et al. (2000) evaluated two DNA targetsfor the diagnosis of toxoplasmosis: the 35- fold repeated Bl gene and a newly described 300-fold repeated 529 bp genomic DNA fragment. Comparison of LightCycler analyses showed a 10 to 100-fold higher sensitivity of the PCR assay targeting the newly described repeated fragment (Reischl et al. 2003). However, the clinical value of real-time PCR remains to be determined and particularly it would be important to find whether there is a relationship between amniotic fluid parasitic burden and severity of foetal lesions.

The main drawback of T. gondii PCR diagnosis is that there are no commercially available kits and, consequently, there are risks of discrepancies between laboratories as all of them use procedures designed "in-house". This was particularly well illustrated by a collaborative study involving 15 European laboratories (Pelloux et al. 1998). Each team received 12 aliquots (four negative, eight positive) of 'artificial samples' made of amniotic fluid spiked with tachyzoites of the RH strain of T. gondii. Each team performed its own PCR protocol (all were different!). Nine of the 15 laboratories were able to detect a single parasite, but two of the 15 found all samples negative. Four of the 15 laboratories found one or more control samples to be falsely positive. This study highlights the lack of homogeneity between PCR protocols and performance and underlines the need for an external quality assurance scheme which could provide 'reference' samples that could be used by any laboratory wanting to establish and maintain an accurate diagnostic test based on PCR.

PCR has been used to diagnose both visceral and cutaneous leishmaniasis and to identify asymptomatic carriers in endemic diseases areas (Schallig and Oskam 2002). PCR in most cases was shown to be more sensitive than microscopy or serology. Gangneux et al. (2003) assessed the prospective value of PCR amplification of a repetitive sequence from Leishmania nuclear DNA and sequencing for the diagnosis and typing of Old World Leishmania infection in a non- endemic area. This molecular approach showed excellent sensitivity (97%) and specificity (100%) compared to direct examination (86 and 100%, respectively) and in vitro culture (72 and 100%, respectively). The parasite loads of mouse tissues experimentally infected with Leishmania were precisely determined using T^Man multiplex, SYBR Green or FRET real-time quantitative PCR (Bretagne et al. 2001; Nicolas et al. 2002; Schulz et al. 2003).

Similar approaches have been used by South American scientists for detection of Trypanosoma cruzi (Weiss 1995; Gomes 1997). In the acute phase of Chagas' disease, when the parasitaemia is high, diagnosis can be easily made using conventional parasitological methods. During the chronic phase, due to the low parasitaemia, diagnosis is generally performed by immunological methods (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay ELISA), which are limited by cross-reactivity with other parasitic diseases (leishmaniasis), non-standardization of reagents, and the diversity of technical procedures. PCR is therefore very useful during this phase.

Molecular techniques have provided useful tools to differentiate the pathogenic species Entamoeba histolytica from the non- pathogenic and microscopically identical Entamoeba dispar; this is essential both for treatment decisions and public health knowledge. Formerly, this differentiation required difficult Entamoeba cultures followed by isoenzyme analysis. PCR has greatly simplified this step. As early as 1993, Acuna-Soto et al. showed that both E. dispar and E. bistolytica coexisted in a community of rural Mexico. Gonin and Trudel (2003) tested 95 stool samples by microscopy, ELISA and in-house PCR. The target for the PCR amplification was a small region (135 bp) of the multicopy small-subunit (SSU) rRNA. Sixty- eight specimens were tested positive by PCR: 2 for E. histolytica and 66 for E. dispar. For detection of E. dispar, ELISA performance was lower than that of microscopy in this reference context, while PCR was much more sensitive than microscopy.

A real-time PCR assay has been developed for detection and differentiation of these two related species, directly from human faeces (Blessmann et al. 2002). This assay was performed with the LightCycler system using fluorescence-labelled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, nbosomal DNA gene. This assay was able to detect as little as 0.1 parasites per g of faeces. The two pairs of primers used were specific for the respective amoeba species, and results were not influenced by the presence of other Entamoeba species even when present in excess amounts. This assay was evaluated in several hundred stool samples from areas of amoebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and amoeba culture. PCR was found to be significantly more sensitive than microscopy or culture and revealed a considerable number of additional E. histolytica- or E. dispar-pos'iuve samples. A multiplex PCR assay has been performed for accurate detection and differentiation of the two species from stool samples, by a single amplification reaction (Nunez et al. 2001). The protocol showed a specificity of 100% and a sensitivity of 94% and was able to detect less than 100 parasites per 0.5 g of faeces.

Finally, a recent study in Bangladesh has shown that a third species, Entamoeba moshkovskii, which was non-pathogenic, could be found in human faeces (AIi et al. 2003). The cysts of this species are morphologically indistinguishable from those of E. histolytica and E. dispar. Amongst 109 stool specimens from preschool children in Bangladesh tested by PCR; 17 were positive for E. histolytica (15.6%) and 39 were positive for E. dispar (35.8%). In addition, 23 (21.1%) were positive for E. moshkovskii, and 17 (73.9%) of these also carried E. histolytica or E. dispar. The high association of E. moshkovskii with E. histolytica and E. dispar may have obscured its identification in previous studies. The high prevalence found in this study suggests that humans may be a true host for this amoeba.

Free-living amoebae of the genus Acanthamoeba are of medical interest as causative agents of keratitis among contact lens wearers. We and other authors used PCR assays targeting Acanthamoeha ribosomal DNA (Lehmann et al. 1998; Mathers et al. 2000; Yera et al. 2001) to detect the presence of Acanthamoeba in epithelial specimens from patients presenting with keratitis or corneal ulcer. PCR is useful to differentiate infections caused by Enterocytozoon bieneusi and Encephalitozoon intestinalis in stool specimens (Liguory et al. 1997) and to have, when real-time PCR is used, a quantification allowing assessment of treatment efficiency (Menotti et al. 2003). The microscopic differentiation of these 2 species is only possible by transmission electron microscopy (Garcia 2002) and is important because E. intestinalis is sensitive to the anthelminthic albendazole (Franzen and Muller 1999).

PCR has also been extensively used for the diagnosis of malaria, though cheaper methods that are easy to perform are still available. Hanscheid and Grobusch (2002) reviewing its use considered that "PCR assays were the most sensitive and specific method to detect malaria parasites, and have acknowledged value in research settings. However, the time lag between sample collection, transportation and processing, and dissemination of results back to the physician limits the usefulness of PCR in routine clinical practice. Furthermore, in most areas with malaria transmission, factors such as limited financial resources, persistent subclinical parasitaemia, and inadequate laboratory infrastructures in the poorer, remote rural areas preclude PCR as a diagnostic method. Even in affluent, non-endemic countries, PCR is not a suitable method for routine use

Many other parasites (e.g. Trypanosoma brucei, Giardia, Cyclospora, Babesia) have been detected by PCR in clinical samples but in most of these cases PCR was an expensive alternative to sensitive classical methods that are easier to perform (Weiss 1995; Morgan 2000). PCR has been used to diagnose Trichomonas vaginitis or urethritis from urine specimens and had a good sensitivity, particularly in male patients (Kaydos et al. 2002; KaydosDaniels et al. 2003).

Genetic polymorphism of parasites: epidemiology and virulence

Molecular tools are increasingly being used to address questions about parasite epidemiology and are similar to those used for other pathogens (Arens 1999). Constantine (2003) rightly considers that testing hypotheses on the polymorphism of parasitic populations depends on "correct sampling, appropriate marker choice, appropriate analysis and careful interpretation". Formerly studying parasite populations was based on the analysis of patterns obtained after digestion of substantial amounts of DNA by restriction enzymes and probe hybridization. Now, in most cases, PCR is a valuable tool to increase this amount before analysis of polymorphism. Different approaches are used to type parasitic isolates: amplification of a conserved region and DNA hybridization with specific probes, sequencing of specific regions of amplified DNA (Gangneux et al. 2003), restriction fragment length polymorphism (RFLP) of amplified DNA (Pelandakis and Pernin 2002; Kaneda et al. 2001), single strand conformation polymorphism (SSCP) of amplified DNA (in denaturing medium, DNA strands with single nucleotide differences can be separated by polyacrylamide gel electrophoresis) (Fedorko et al. 2001), analysis of patterns obtained after amplification with low stringency conditions by short randomly chosen primers (RAPD) (Sedinova et al. 2003).

Amongst protozoa of medical interest, the polymorphism of T. gondn populations has particularly been studied. Mole\cular analysis has revealed that the population structure of T. gondii was clonal and consisted of three predominant lineages: types I, II and III (Boothroyd and Grigg 2002). Acute virulence in mice was associated with the type I genotype. Mordue et al. (2001) showed different patterns in mice infected either by the type I or type II strains. The virulent type I strain reached high tissue burdens accompanied by extremely elevated levels of ThI cytokines in the serum, including IFN-gamma, TNF-alpha and IL-12. Non-lethal infections with a low dose of type II strain parasites were characterized by a modest induction of ThI cytokines that led to control of infection and minimal damage to host tissues. The type II (chronic type) was found to be the most prevalent in human isolates (Howe et al. 1997; Morgan 2000) though geographical variations could occur. Ajzenberg et al. (2002a) studied eight microsatellite markers to type 84 independent isolates from humans and animals. Two microsatellite markers were present in the introns of two genes, one coding for beta-tubulin and the other for myosin A, and six were found in expressed sequence tags. With 3-16 alleles detected, these markers can be considered as the most discriminating multi-locus single- copy markers available for typing T. gondii isolates. This high discriminatory power of microsatellites made it possible to detect mixed infections and epidemiologicalIy related isolates. Evolutionary genetic analyses of diversity show that the T. gondii population structure consists of only two clonal lineages. To study the influence of T. gondii genotypes on the severity of human congenital toxoplasmosis, these microsatellite markers were used to analyze 86 T. gondii isolates collected from patients with congenital toxoplasmosis (Ajzenberg et al. 2002b). Seventy-four different genotypes were detected, some identical genotypes originating probably from the same source of contamination. The 3 less-polymorphic microsatellite markers associated with 6 isoenzymatic markers allowed a classification of isolates into the 3 classical types and detected atypical genotypes. Type II isolates were largely predominant (85% of the whole collection) and seemed to be independent from the clinical findings. Grigg et al. (2001) genotyped T. gondii isolates from vitreous fluid of 12 patients with severe or atypical ocular toxoplasmosis and found that most strains were atypical. Particularly virulent Toxoplasma strains, acquired after wild game consumption, were observed in patients from French Guiana. In immuno-competent adults these strains resulted in severe infections consisting of a marked, non-specific infectious syndrome accompanied by an altered general status with visceral localization and particularly pulmonary involvement. These strains showed atypical multi-locus genotypes, with one allele found only for isolates of this region (Carme et al. 2002).

Molecular biology techniques are now currently used to study the association of molecular markers with antimalarial drug resistance. The association between pyrimethamine resistance and point mutations on the dihydrofolate reductase (dhfr) gene as well as sulfadoxine resistance and point mutations on the dihydropteroate synthetase (dhps) gene are the most well documented genomic alterations observed in Plasmodium falciparum. Substitution of threonme for lysine at position 76 on the chloroquine resistance transporter (pfcrt) gene is known to be a key determinant for chloroquine resistance. Available evidence suggests the roles of the multidrug- resistance gene 1 (pfmdrl) on the resistance of P. falciparum to several drugs. Molecular beacons were developed to detect the chloroquine resistance-associated pfcrt K76T point mutation (Durand et al. 2002) and the antifolate resistance-associated S108N point mutation (Durand et al. 2000). This rapid and inexpensive genomic assay appears to be a promising tool in comparison with the PCR- restriction fragment length polymorphism method. The precise relationship between particular polymorphism and specific drugs still needs further research and analysis (Noedl et al. 2003). Snounou et al. (1998) reported on the use of PCR genotyping in the assessment of recrudescence or re-infection after antimalarial drug treatment. Finally, the possibility that specific parasite characteristics contribute to severity has been investigated in French Guiana, a hypo-endemic area, where parasite diversity is low and all patients with severe cases are referred to a single intensive care unit (Ariey et al. 2001). Parasite genotyping in geographically and temporally matched patients with mild and severe disease showed that the association of a specific msp-1 allele (B- K1) with a specific var gene (var-D) was over-represented among patients with severe versus mild disease (47% vs. 3%, respectively; p < 0.001). Similarly, isolates of P. falciparum from Senegal were typed using amplification of polymorphic genetic loci such as MSP- I, MSP-2, HRP1, GLURP ... (Robert at al. 1996). A high prevalence, up to 60% of MSP-2 alleles, was specifically observed in the severe malaria isolates. In addition, the presence of MSP-I/RO33 alleles was significantly associated with a higher plasma level of TNF- alpha receptor 1 (p < 0.05), a reported indicator of severity in human malaria. Additional data are needed to identify with certainty genetic factors of parasitic virulence and micro-arrays will provide tools to approach this problem (Rathod et al. 2002).

Conclusions and prospects

The use of molecular biology for diagnosis of human protozoan infections has two major drawbacks: a high cost limiting its use in low income countries (Wilson 1998) and the absence of standardized and commercialized diagnostic kits resulting in discrepant results between labs. However, there are still fascinating perspectives for these techniques. Nowadays, real-time PCR is an important improvement because it is sensitive, quantitative, quick to perform and does not require post-amplification manipulations. The real usefulness of quantification in the field of parasitic protozoa remains to be determined. Following the precise parasite burden during the treatment of immuno-compromised persons could be of great help to assess drug efficacy. A relationship between a high parasitic burden and a particular clinical expression remains still quite speculative. Real time RT-PCR assays could be also useful to monitor expression of parasite genes, such as genes of resistance. The development of multiplex PCR assays would enable the simultaneous detection, in one assay, of several pathogens (such as T. gondii and Leishmania in immuno-compromised patients) and would be a cost-effective procedure. Detection of DNA or RNA sequences within tissue sections by in situ PCR or in situ RT-PCR is a valuable tool in other fields of microbiology (Alzahrani et al. 2002; Comar et al. 2001). T. cruzi has been successfully detected via in situ PCR within paraffin-embedded murine cardiac tissue sections (Lane et al. 2003). In situ PCR use remains to be developed and evaluated in human parasitic diseases. The development of automated nucleic acid extraction would enable the analysis of a high number of samples in a couple of hours. Several types of machines associating automated extraction, amplification and revelation are already available though very expensive (BioRobot, Qiagen SA or Gobas Amplicor, Roche Diagnostic).

Molecular biology is increasingly relevant to the diagnosis and control of infectious diseases. Information on DNA sequences has been extensively exploited for the development of polymerase chain reaction-based assays for the diagnosis of different protozoa and the identification of parasite species. It has also led to the use of cloned antigen for serodiagnosis. It is expected that the sequencing of the genome of the different parasitic protozoa will enable important progress in further improving diagnosis and control. Techniques such as micro-arrays (Boothroyd et al. 2003) and nucleic acid sequence-based amplification (NASBA) will eventually allow rapid screening for specific parasite genotypes and assist in diagnostic and epidemiological studies. Curiously, the NASBA method based on RNA amplification, which is thus able to confirm the viability of the detected pathogens, has not been properly evaluated in the field of medical parasitology, except in Plasmodium falciparum (Schoone et al. 2000).

One of the greatest threats to human mankind is the development of drug resistance in protozoa. Knowing the molecular basis of drug resistance and the ability to monitor its development with sensitive and specific DNA-based assays for 'resistance alleles' may aid in maintaining the effectiveness of available anti-protozoan drugs. A better knowledge of the genotype of an infecting protozoan strain could result in better management of the resulting human disease.

References

Acuna-Soto R., Samuelson J., De Girolami P., Zarate L., MiIlan- Velasco F, Schoolnick G. and Wirth D. (1993): Application of the polymerase chain reaction to the epidemiology of pathogenic and nonpathogenic Entamoeba bistolytica. Am. J. Trop. Med. Hyg. 48, 58- 70.

Ajzenberg D., Banuls A. L., Tibayrenc M. and Darde M. L. (2002a): Microsatellite analysis of Toxoplasma gondii shows considerable polymorphism structured into two main clonal groups. Int. J. Parasitol. 32, 27-38.

Ajzenberg D., Cogne N., Paris L., Bessieres M. H., Thulliez P., Filisetti D., Pelloux H., Marty P. and Darde M. L. (2002b): Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings. J. Infect. Dis. 186, 684-689.

AIi I. K., Hossain M. B., Roy S., Ayeh-Kumi P. R, Petri W. A. Jr., Haque R. and Clark C. G. (2003): Entamoeba, moshkovskii infections in children, Bangladesh. Emerg. Infect. Dis. 9, 580-584.

Alzahrani A. J., Valley P. J. and McM\ahon R. F. (2002): Developpement of a novel nested in situ PCR-ISH method for detection of hepatitis C virus RNA in liver tissue. J. Virol. Methods 99, 53- 61.

Arens M. (1999): Methods for subtyping and molecular comparison of human viral genomes. Clin. Microbiol. Rev. 12, 612-626.

Ariey F., Hommel D., Le Scanf C., Duchemin J. B., Peneau C., HuIm A., Sarthou J. L., Reynes J. M., Fandeur T. and Mercereau-Puijalon O. (2001): Association of severe malaria with a specific Plasmodium faldparum genotype in French Guiana. J. Infect. Dis. 184, 237-241.

Bastien P. (2002): Molecular diagnosis of toxoplasmosis. Trans. R. Soc. Trop. Med. Hyg. 96, S205-215.

Bell A. S. and Ranford-Cartwright L. C. (2002): Real-time quantitative PCR in parasitology. Trends Parasitol. 18, 337-342.

Blessmann J., Buss H., Nu P. A., Dinh B. T., Ngo Q. T, Van A. L., Alla M. D., Jackson T. E, Ravdin J. I. and Tannich E. (2002): Real- time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples. J. Clin. Microbiol. 40, 4413- 4417.

Boothroyd J. C., Blader I., Cleary M. and Singh U. (2003): DNA microarrays in parasitology: strengths and limitations. Trends Parasitol. 19, 470-476.

Boothroyd J. C. and Grigg M. E. (2002): Population biology of Toxoplasma gondii and its relevance to human infection: do different strains cause different disease? Curr. Opin. Microbiol. 5,438-442.

Bretagne S., Durand R., Olivi M., Garin J. E, Sulahian A., Rivollet D., Vidaud M. and Deniau M. (2001): Real-time PCR as a new tool for quantifying Leishmania infantum in liver m infected mice. Clin. Diagn. Lab. Immunol. 8, 828-831.

Carme B., Bissuel E, Ajzenberg D., Bouyne R., Aznar C., Demar M., Bichat S., Louvel D., Bourbigot A. M., Peneau C., Neron P. and Darde M. L. (2002): Severe acquired toxoplasmosis in immunocompetent adult patients in French Guiana. J. Clin. Microbiol. 40, 4037-4044.

Cockerill E R. and Smith T. E (2002): Rapid-Cycle real-time PCR: a revolution for clinical microbiology. ASM News, 68, 77-83

Comar M., Spano A., Canova S., Bogoni S., Marziliano N., Cernigoi E., Boniotto M., Amoroso A., Parodi S., Campello C. and Crovella S. (2001): Direct in situ PCR allows rapid and sensitive detection of high risk human papillomavirus in cytologie specimens and formalin- fixed paraffin tissues by fluorescent labelling. Int. J. Oncol. 18, 181-185.

Constantine C. C. (2003): Importance and pitfalls of molecular analysis to parasite epidemiology. Trends Parasitol. 19, 346-348.

Costa J. M., Pautas C., Ernault P., Foulet E, Cordonnier C. and Bretagne S. (2000): Real-time PCR for diagnosis and follow-up of Toxoplasma reactivation after allogeneic stem cell transplantation using fluorescence resonance energy transfer hybridization probes. J. Clin. Microbiol. 38, 2929-2932.

Costa J. M., Ernault P., Gautier E. and Bretagne S. ( 2001): Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes. Prenat. Diagn. 21, 85-88.

Dupouy-Camet J., Bougnoux M. E., Lavareda de Souza S., Thulliez P., Dommergues M., Mandelbrot L., Ancelle T., Tourte-Schaefer C. and Benarous R. (1992): Comparative value of polymerase chain reaction and conventional biological tests for the prenatal diagnosis of congenital toxoplasmosis. Ann. Biol. Clin. (Paris) 50, 315-319.

Dupouy-Camet J., de Souza S. L., Maslo C., Paugam A., Saimot A. G., Benarous R., Tourte-Schaefer C. and Derouin E (1993): Detection of Toxoplasma gondii in venous blood from AIDS patients by polymerase chain reaction. J. Clin. Microbiol. 31,1866-1869.

Durand R., Eslahpazire J., Jafari S., Delabre J. E, MarmoratKhuong A, di Piazza J. P. and Le Bras J. (2000): Use of molecular beacons to detect an antifolate resistance-associated mutation in Plasmodwm falciparum. Antimicrob. Agents Chemother. 44, 3461-3464.

Durand R., Huart V., Jafari S. and Le Bras J. (2002): Rapid detection of a molecular marker for chloroquine-resistant falciparum malaria. Antimicrob. Agents Chemother. 46, 2684-2686.

Dworkin L. L., Gibier T. M. and Van Gelder RN. (2002): Real-time quantitative polymerase chain reaction diagnosis of infectious posterior uveitis. Arch. Ophthalmol. 120, 1534-1539.

Fedorko D. P., Nelson N. A., Didier E. S., Bertucci D., Delgado R. M. and Hruszkewycz A. M. (2001): Speciation of human Microsporidia by polymerase chain reaction single-strand conformation polymorphism. Am. J. Trop. Med. Hyg. 65, 397-401.

Franzen C. and Muller A. (1999): Molecular techniques for detection, species differentiation, and phylogenetic analysis of Microsporidia. Clin. Microbiol. Rev. 12, 243-285.

Gangneux J. P., Menotti J., Lorenzo E, Sarfati C., Blanche H., Bui H., Pratlong F., Garin Y. J. and Derouin F. (2003): Prospective value of PCR amplification and sequencing for diagnosis and typing of old world Leiskmania infections in an area of nonendemicity. J. Clin. Microbiol. 41, 1419-1422.

Garcia L. S. (2002): Laboratory identification of the Microspondia. J. Clin. Microbiol. 40,1892-1901.

Gomes Y. M. (1997): PCR and sero-diagnosis of chronic Chagas' disease. Biotechnological advances. Appl. Biochem. Biotechnol. 66, 107-119.

Gonin P. and Trudel L. (2003): Detection and differentiation of Entamoeba histolytica and Entamoeba dispar isolates in clinical samples by PCR and enzyme-linked immunosorbent assay. J. Clin. Microbiol. 41, 237-241.

Grigg M. E., Ganatra J., Boothroyd J. C. and Margolis T. P. (2001): Unusual abundance of atypical strains associated with human ocular toxoplasmosis. J. Infect. Dis. 184, 633-639.

Hanscheid T. H. and Grobusch M. (2002): How useful is PCR in the diagnosis of malaria? Trends Parasitol. 18, 395-398.

Homan W. L., Vercammen M., De Braekeleer J. and Verschueren H. (2000): Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnosic and quantitative PCR. Int. J. Parasitol. 30, 69-75.

Howe D. K., Honore S., Derouin F. and Sibley L. D. (1997): Determination of genotypes of Toxoplasma gondii strains isolated from patients with toxoplasmosis. J Clin Microbiol. 35,1411-1414.

Kaneda Y, Horiki N., Cheng X. J., Fujita Y, Maruyama M. and Tachibana H. (2001): Ribodemes of Blastocystis hominis isolated in Japan. Am. J. Trop. Med. Hyg. 65, 393-396.

Kaydos S. C., Swygard H, Wise S. E., Sena A. C., Eeone P. A., Miller W. C., Cohen M. S. and Hobbs M. M. (2002): Development and validation of a PCR-based enzyme-linked immunosorbent assay with urine for use in clinical research settings to detect Trichomonas vaginalis in women. J. Clin. Microbiol. 40, 89-95.

Kaydos-Daniels S. C., Miller W. C., Hoffman L, Banda T, Dzinyemba W., Martinson F, Cohen M. S. and Hobbs M. M. (2003): Validation of a urine-based PCR-enzyme-linked immunosorbent assay for use in clinical research settings to detect Trichomonas vaginalis in men. J. Clin. Microbiol. 41,318-323.

Kupferschmidt O., Kruger D., held T. K, Ellerbrok H., Siegert W. and Janitschke K. (2001): Quantitative detection of Toxoplasma gondii DNA in human body fluids by TaqMan polymerase chain reaction. Clin. Microbiol. Infect. 7, 120-124.

Eane J. E., Ribeiro-Rodrigues R., Olivares-Villaomez D., Vnencak- Jones C. E., McCurley T. E. and Carter C. E. (2003): Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction. Mem. Inst. Oswaldo Cruz 98, 373- 376.

Lehmann O. J., Gren S. M., Morlet N., Kilvington S., Keys M. R, Matheson M. M, Dart J. K. G., McGiIlJ. I. and Watt P. J. (1998): Polymerase chain reaction analysis of corneal epithelial and tear samples in the diagnosis of Acanthamoeba keratitis. Invest. Ophthalmol. Vis. Sei. 39,1261-1265.

Liguory O., David E, Sarfati C., Schuitema A. R., Hartskeerl R. A., Dcrouin E, Modai J. and Molina J. M. (1997): Diagnosis of infections caused by Enterocytozoon bieneusi and Encephalitozoon intestinalis using polymerase chain reaction in stool specimens. AIDS. 11, 723-726.

Lin M. H., Chen T. C., Kuo T. T, Tseng C. C. and Tseng C. P. (2000): Real-time PCR for quantitative detection of Toxoplasma gondii. J. Clin. Microbiol. 38, 4121-4125.

Mathers W. D, Nelson S. E, Lane J. L, Wilson M. E., Alien R. C. and Folberg R. (2000): Confirmation of confocal microscopic diagnosis of Acanthamoeba keratitis using Polymerase Chain Reaction analysis, Arch. Ophthal. 118, 178-183.

Menotti J., Cassinat B., Porcher R., Sarfati C., Derouin F. and Molina J. M. (2003): Development of a real-time polymerase-chain- reaction assay for quantitative detection of Enterocytozoon bieneusi DNA in stool specimens from immunocompromiscd patients with intestinal microsporidiosis. J. Infect. Dis.187, 1469-1474.

Montoya J. G. (2002): Laboratory diagnosis of Toxoplasma gondii infection and toxoplasmosis. J. Infect. Dis. 185, S73-82.

Mordue D. G., Monroy E, La Regina M., Dinarello C. A. and Sibley L.D. (2001): Acute toxoplasmosis leads to lethal overproduction of ThI cytokincs. J. Immunol. 167, 4574-4584.

Morgan U. M. (2000): Detection and characterisation of parasites causing emerging zoonoses. Int. J. Parasitol. 30, 1407-1421.

Nicolas L., Prina E., Lang T. and Milon G. (2002): Real-time PCR for detection and quantitation of Leishmania in mouse tissues. J. Clin. Microbiol. 40, 1666-1669.

Noedl H., Wongsrichanalai C. and Wernsdorfer W. H. (2003): Malaria drug-sensitivity testing: new assays, new perspectives. Trends Parasitol. 19, 175-181.

Nunez Y. O., Fernandez M. A., Torres-Nunez D., Suva J. A., Montano I., Maestre J. L. and Fonte L. (2001): Multiplex polymerase chain reaction amplification and differentiation of Entamoeba histolytica and Entamoeba dispar DNA from stool samples. Am. J. Trop. Med. Hyg. 64, 293-297.

Pelandakis M. and Pernin P. (2002): Use of multiplex PCR and PCR restriction enzyme analysis for detection and exploration of the variability m the free-living amoeba Naegleria in the environment. Appl. Environ. Microbiol. 68, 2061-2065.

Pelloux H., Guy E., AngeliciM. C., Aspock H., Bessieres M. H., Blatz R., Del Pezzo M, Girault V., Gratzl R., Holberg-Petersen M., Johnson J., Kruger D., Lappalainen M., Nacssens A. and Olsson M. (1998): A second European collaborative study on polymerase chain reaction for Toxoflasma gondii, involving 15 teams. FEMS Microbiol. Lett. 165,231-237.

Rathod P. K., Ganesan K., Hayward R. E., Bozdech Z., DeRisi J. E. (2002): DNA microarrays for malaria. Trends Parasitol. 18, 39-45.

Reischl U., Bretagne S., Kruger D., Ernault P. and Costa J. M. (2003): Comparison of two DNA targets for the diagnosis of toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. BMC Infect. Dis. 3, 7.

Robert E, Ntoumi E, Angel G., Candito D., Rogier C., Fandeur T., Sarthou J. E. and Mercereau-Puijalon O. (1996): Extensive genetic diversity of Plasmodium falciparum isolates collected from patients with severe malaria in Dakar, Senegal. Trans. R. Soc. Trop. Med. Hyg. 90, 704-711.

Robert-Gangneux F., Gavinet M. F., Ancelle T., Raymond J., Tourte- Schaefer C. and Dupouy-Camet J. (1999): Value of prenatal diagnosis and early postnatal diagnosis of congenital toxoplasmosis: retrospective study of 110 cases. J. Clin. Microbiol. 37, 2893- 2898.

Schallig H. D. and Oskam E. (2002): Molecular biological applications in the diagnosis and control of Lcishmaniasis and parasite identification.Trop. Med. Int. Health. 7, 641-651.

Schoone G. J., Oskam E., Kroon N. C., Schallig H. D. and Omar S. A. (2000): Detection and quantification of Plasmodium falciparum in blood samples using quantitative nucleic acid sequence-based amplification. J. Clin. Microbiol. 38, 4072-4075.

Schulz A., Mellenthin K., Schonian G., Fleischer B. and Drosten C. (2003): Detection, differentiation, and quantitation of pathogenic Leishmania organisms by a fluorescence resonance energy transfer-based real-time PCR assay. J. Clin. Microbiol. 41,1529- 1535.

Sedinova J., Flegr J., Ey P. E. and Kulda J. (2003): Use of random amplified polymorphic DNA (RAPD) analysis for the identification of Giardia intestinalis subtypes and phylogenetic tree construction. J. Eukaryot. Microbiol. 50, 198-203.

Snounou G. and Beck H. P. (1998): The use of PCR genotyping in the assessment of recrudescence or reinfection after antimalarial drug treatment. Parasitol. Today 14, 462-467.

Weiss J. B. (1995): DNA probes and PCR for diagnosis of parasitic infections. Clin. Microbiol. Rev. 8, 113-130.

Wilson S. M. (1998): Application of molecular methods to the study of diseases prevalent in low income countries. Trans. R. Soc. Trop. Med. Hyg. 92, 241-244.

Yera H., Zamfir O., Chaumeil C., Dussard P., Batellier L., Dupouy- Camet J., Tourte-Schaefer C. and Scat Y. (2001): PCR Analysis for the diagnosis ofAcanthamoeba Isolated from corneal ulcer. Preliminary results. In: Proc. IXth Internat. Meeting Biol. Pathogenicity free-living amoebae, pp. 39-44. John Eibbey Eurotext, Paris.

Helene Yera, MonZenTzen and Jean Dupouy-Camet*

Parasitology, Hopital Cochin, Universite R. Descartes, 27 rue du Faubourg St Jacques, 75014, Paris, France;

E-mail: Jean.dupouy-camet@cch.ap-hop-paris.fr

Received: 4 September 2003. Accepted: 13 October 2003

*corresponding author

Copyright Urban & Fischer Verlag Dec 2003

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