1. Introduction
Over the past two decades, molecular diagnosis of toxoplasmosis, which is based on the detection of
Molecular methods based on polymerase chain reaction (PCR) are simple, sensitive, reproducible and can be applied to all clinical samples (Bell and Ranford-Cartwright, 2002; Contini et al., 2005; Calderaro et al., 2006; Bastien et al., 2007). These methods are divided into two groups. The first group consists of techniques focused on detection of
However, it is important to emphasize that molecular diagnostics, being a constantly improving modern methodology, is not standardized even among the world's leading laboratories. The differences are substantial and numerous, and they extend to all segments of the methodology such as target genes for parasite detection and markers for genotyping, equipment manufacturers and different protocols (various sets of primers and probes and their concentration, different internal controls, etc...).
2. Molecular diagnostics
2.1. Methodology
Conventional PCR was, in the beginning, the molecular detection method of choice for the majority of laboratories dealing with the diagnosis of toxoplasmosis and it was based on both in-house protocols and commercial kits (Lavrard et al., 1995). To increase the sensitivity of molecular diagnostics of toxoplasmosis nested PCR was introduced, although in recent years real-time PCR has shown a significantly higher sensitivity as well as specificity (Jauregui et al., 2001; Reischl et al., 2003; Contini et al., 2005; Calderaro et al., 2006; Edvinsson et al., 2006). Real-time PCR detection also has the capability of quantification of
Molecular diagnostics of toxoplasmosis is generally based on the detection of a specific DNA sequence, using different assays and protocols, mostly from highly conserved regions such as the B1 gene repeated 35 times in the genome, 529 bp repetitive element with about 200-300 copies in the genome, ITS-1 (internal transcribed spacer ) that exists in 110 copies and 18S rDNA gene sequences (Table 1). Qualitative PCR protocols for the detection of single copy genes such as the P30 gene appeared less sensitive and they are rarely used for diagnostic purposes (Jones et al., 2000).
≈ 35 | Wahab et al., 2010 Correia et al., 2010 Okay et al., 2009 | |
200-300 | da Silva RC et al., 2011 Yera et al., 2009 Vujanić et al., 2011 | |
≈110 | Truppel et al., 2010 Miller et al., 2004 | |
Single copy gene | Buchbinder et al., 2003 Eida et al., 2009 Cardona et al., 2009 |
The first protocol for molecular detection of
However, it can be of great methodological significance to further clarify the specificity of using a multicopy target of unknown function before the introduction of such protocol into the laboratory diagnostics (Edvinsson at al., 2006)
2.2. Clinical significance in various biological samples
Molecular detection of
91 | 28 (30.8) | |
28 | 10 (35.7) | |
9 | 3 (33.3) | |
1 | 1 (100) | |
10 | 6 (60) | |
7 | 4 (57.1) | |
146 | 52 (35.6) |
2.2.1. Peripheral blood
In our laboratory, the presence of
We have analyzed real-time PCR results from peripheral blood samples originating from patients suspected of acute toxoplasmosis according to the serological criteria for acute infection, i.e. avidity of specific IgG antibodies and the finding of specific IgM antibodies. The results showed that positive real-time PCR correlates better with the finding of specific IgM antibodies, than with low avidity of specific IgG antibodies (Vujanić, 2012).
Comparison of molecular detection and bioassay findings on peripheral blood samples of the patients with specific IgM antibodies and specific IgG antibodies of low avidity, suggesting acute toxoplasmosis, has been done as well. It was shown that in nearly one-third (29%) of the analyzed cases
Although the detection of parasite DNA in peripheral blood of adults may not always be direct evidence of active parasitemia,
2.2.2. Amniotic fluid
In the last two decades, the detection of
Given that in many published studies real-time PCR and bioassay results from the amniotic fluid did not match, which is the case in our research as well, and as congenital infection cannot be excluded by negative PCR (Romand et al., 2001; Golab et al., 2002), for prediction of congenital toxoplasmosis it is optimal to combine both molecular detection and bioassay. In one study of prenatal diagnosis of congenital toxoplasmosis in patients from 6 European centers of reference it was shown that PCR from amniotic fluid has a higher sensitivity (81%) in regard to both bioassay (58%) and cell culture (15%) (Foulon et al., 1999). The combination of PCR and bioassay increases the sensitivity to 91%, and represents the best diagnostic approach (Foulon et al., 1999).
In European countries such as France and Austria regular serological monitoring of pregnant women for
2.2.3. Cord blood
Cord blood is not considered the ideal sample for prenatal diagnosis of congenital toxoplasmosis. For example, the results of a survey carried out in France did not show any positive PCR result among 19 tested cord blood samples from children with proven congenital toxoplasmosis (Filisetti et al., 2003). Nevertheless, cord blood samples that are occasionally provided to our laboratory, have shown a rate of positivity in real-time PCR of 33%. All cord blood samples in our study were inoculated into mice and the rate of positivity of bioassay was 55.5%. A higher rate of isolation of viable parasites by bioassay compared to the detection of parasitic DNA by real-time PCR may be explained by a larger sample volume used for mouse inoculation in comparison to the amount used for DNA extraction, as well as by probable presence of PCR inhibitors. In one study performed on a representative sample of pregnant women in China a similar rate of real-time PCR positive results was obtained from the amniotic fluid and fetal blood samples (Ma et al., 2003).
It can be concluded that the diagnosis of congenital toxoplasmosis from fetal blood samples should be based on the results of both bioassay and molecular detection.
2.2.4. Aqueous humor
Prior to the introduction of molecular methods, the laboratory diagnosis of ocular toxoplasmosis has been based primarily on a comparison of the level of antibodies detected in the humor aqueous and serum in order to detect intraocular synthesis of specific antibodies (Witmer-Goldman's coefficient). Lately, molecular methods are becoming a standard diagnostic approach in the diagnosis of ocular toxoplasmosis as well. A number of studies has already shown that a positive PCR result is not always accompanied by positive serology indicating local synthesis of IgG antibodies (Villard et al., 2003; Talabani et al., 2009) and thus can be the only confirmation of the diagnosis (Okhravi et al., 2005).
We have so far studied 10 humor aqueous samples from patients clinically suspected of ocular toxoplasmosis of which 60% (6/10) were real-Time PCR positive. A similar result was obtained in a French study when 55% (22/40) of humor aqueous samples were positive by real-Time PCR using AF146527 as a marker (Talabani et al., 2009). Also, the detection of the same AF146527 marker by real-Time PCR in another French study, revealed somewhat lower rate of positive samples, 38.2% (13/34) (Fekkar et al., 2008). It is interesting that in the latter study the sample volume of 10 μL used for DNA extraction was unusually small, which certainly could affect the success of PCR reactions. However, in another study performed in Strasbourg, the amplification of 18S rRNA and B1 gene by conventional PCR resulted in the 28% (5/18) of the humor aqueous samples positive for the presence of
2.2.5. Cerebrospinal fluid
Cerebral toxoplasmosis usually affects immunosuppressed patients and is mostly the result of reactivation of chronic infection which may be fatal if left untreated. Definitive diagnosis of toxoplasmosis can be made by the detection of tachyzoites in brain tissue samples obtained by biopsy, but this method, because of its invasiveness, is seldom applied, and certainly not since the PCR, giving consistent and quick result, has been introduced in the diagnostics (Vidal et al., 2004). A study of cerebral toxoplasmosis in HIV-infected patients infected in Brazil, showed that 27.4% (14/51) of cerebrospinal fluid samples were positive for
2.3. Comment
In summary, all above-mentioned results confirm the value of the use of molecular methods, due to their high sensitivity and specificity, in the diagnosis of toxoplasmosis. Coupled with conventional parasitological diagnostic methods, PCR-based methods allow for the timely diagnosis especially of congenital toxoplasmosis and of reactivated toxoplasmosis in immunosuppressed patients. Further advances of the technology itself along with its wide, (universal) use may be expected to markedly improve diagnostics and monitoring of the course of infection as well as of the therapeutic effect.
3. Genotyping
In the early days of strain designation, isolates of
Nevertheless, most recent phylogenetic studies indicate that the population structure of
A realistic picture of the distribution of genotypes in Europe is also difficult to obtain because research on
Further work on the genotyping of
blood | toxoplasmosis in pregnancy | II | |
amniotic fluid | congenital toxoplasmosis | II | |
blood | congenital toxoplasmosis | I | |
blood | bone marrow transplantation | II | |
bronchoalveolar lavage fluid | bone marrow transplantation | II |
We have also genotyped isolates from both a blood and BAL sample from an immunosuppressed patient after bone marrow transplantation, which were found to belong to type II. In another study, genotyping of strains isolated from immunosuppressed patients, HIV infected or patients who had undergone organ transplantation, has shown predominance of type II in patients who were infected in Europe (Ajzenberg et al., 2009). On the other hand, isolates that do not belong to this type usually come from people who are infected with
Furthermore, results of a study performed in the USA, based on genotyping of strains isolated from cerebrospinal fluid originating from eight HIV-positive patients showed that most of them were infected with type I strain or strains that have type I alleles (Khan et al., 2005). Although the possible association between clinical entities induced by
Numerous studies of the
But even the use of multiple markers does not always provide satisfactory results, mainly due to insufficient amounts of extracted parasite DNA. Therefore, there are cases when amplification of all markers in each sample is not successful, as it can be observed in studies performed in the United States and Poland, where PCR-RFLP analysis was carried out also using four genetic markers SAG2, SAG3, BTUB and GRA6 (Khan et al., 2005; Nowakowska et al., 2006). Using these genetic markers, it was possible to discriminate types I, II and III, but also strains that have a genotype with two allele types at the same locus. Such was the case with one sample in our study which, after the digestion of the product of the amplified GRA7 gene, turned out to possess alleles of both types I and II (Fig. 1,
Although PCR-RFLP has a limited ability to distinguish between closely related isolates within a clonal line as compared to microsatellite analysis, analysis of up to 9 or 10 genetic markers by this method has been successfully performed in world-class laboratories (Su et al., 2006; Dubey & Su, 2009). On the other hand, the microsatellite analysis is presumed to be more informative to distinguish recent mutations in closely related isolates of the same line, while the RFLP markers are better for detection of time period when the separation of distinct strains in different clonal group has occurred (Su et al., 2006). Multilocus PCR-RFLP genotyping is still the first method of choice in clinical research, mainly for its simplicity and favorable reagent prices, but the best approach for successful genotyping is the use of both methods.
Along with the phylogenetic study of
Even the generally accepted concept of major clinical importance that immunized mothers are resistant to reinfection thereby preventing infection of the offspring, have been recently challenged by insight into the strain variation at the genotype level. Six cases of reinfection among chronically infected pregnant women resulting in a vertical transmission and congenital infection either with a distinct typical or atypical strain have already been reported (Lindsay & Dubey, 2011).
Despite this significant new knowledge, the clinical relevance of the infecting genotypes is an issue that will continue to intrigue researchers in the coming years. Insight into the global population structure of
4. Conclusion
The introduction of highly sensitive molecular methods into the diagnosis of toxoplasmosis is of great importance and this paper emphasizes its practical importance and potential as a part of the standard laboratory protocols. Nevertheless, it can be concluded that, at the moment, the best diagnostic approach is a combination of both conventional and molecular methods.
We also present the very first and original phylogenetic data on the
Acknowledgement
The work was supported by a grant (project No. III41019) from the Ministry of Education and Science of Serbia.
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