“Role of routine TORCH testing with emphasis on Toxoplasmosis in pregnancy” 1
2
Kulvinder Kochar Kaur ,Gautam Nand Allahbadia ,Mandeep Singh
3
1.Deparment of reproductive neuroendocrinology,Dr Kulvinder Kaur Centre for Human Reproduction,Jalandhar,Punjab,India
2Department of reproductive neuroendocrinology,Rotunda Centre for Human Reproduction,Mumbai,India
3.C onsultan Neurologist SWAMI Satyanand Hospital Jalandhar,Punjab,India
Corresponding author-DR kulvinder Kochar
[email protected]
ABSTRACT There is a common misconception regarding TORCH group of infections as being the aetiological factors for recurrent abortions/bad obstetric history.The significance of serological investigations for TORCH infections is illunderstood by many.While reviewing TORCH infections in relation to infertility I soon became aware of the global extent of toxoplasmosis and the significance of understanding the aetiology ,pathogenesis,and treatment which can go a long way to prevent the potential hazards ,such as congenital deafness,blindness and mental retardation in unborn children.It is important to take preventive measures at the initiation as toxoplasmosis can occur in pregnant mothers without history of any contact with infected cat or eating infected meat with subclinical infection and lymphadenopathy going unnoticed , and early measures taken like start of spiramycin/pyrimethamine and folinic acid according to the investigations can go a long way in preventing these as has been shown by long term studies of Mcleod et al and other studies where early treatment has been implemented.
1.Introduction Being a reproductive endocrinologist and having been invited to write on “TORCH in infertility” for an infertility journal ,although reluctant ,I decided to review the role of these antibody titres in reproduction itself as a lot of unnecessary antibody titres and investigations are carried out in India without the treating physicians actually understanding the significance of the same. The most unfortunate part of which I find is that none of the infections included in the “TORCH” group is responsible for a repeat abortion and unfortunately I find an article included in an international journal for investigations for bad obstetrics history(1). Since on reviewing I realized with the era of HIV the opportunistic infection Toxoplasma gondii has become a big global threat I decided to review it in detail from an infertility specialists perspective and management both in developed countries like USA as well as developing countries like India.Funnily thoughtoxoplasma infection does not cause recurrent fetal losses this has been the commonest indication for investigating for toxoplasmosis in india .There is considerable confusion and uncertainty concerning T.gondii as a cause of multiple abortions,sterility and other reproductive failures in india since most data is based on serology .There are several shortcomings in reports linking habitual abortions to T.gondii infection egi)lownumber of patients, ii)uncontrolled studies andiii)absence of serological data before pregnancy(2,3,4). Although one recognizes there are technical difficulties in conducting a well controlled prospective study in India, that is necessary to establish a causal relationship between toxoplasmosis and abortion.Even isolation of T.gondii several weeks after abortion does not prove congenital toxoplasmosis because
T.gondii has been found in the uteri of nonpregnant women as well(5).Also even when placenta is infected the , fetus may escape the infection(2,3). The original “TORCH complex” described clinically similar congenital infections caused by toxoplasma gondii,rubella virus,cytomegalus virus and herpes simplex virus types 1and 2.Further Epps et al described a “TORCH syndrome”(6),where they described the commonality of the symptoms eg chorioretinopathy,and intracerebral calcification in congenital toxoplasmosis,CMV,and involvement of central nervous system in herpes simplex as well.The implementation of widescreening “TORCH” screening programmes has been questioned due to several factors including1)potential overuse 2)lack of consistent and reliable serological methods 3)cost 4)misinterpretetion of results(7-10).For e.g the presence of TORCH IgG class antibodies in the mother does not differentiate between past exposure(ie low risk of congenital infection)and recent acute infection(increased risk of congenital infection).Furthermore detection of TORCH IgM antibodies is often misinterpreted as a recent infection,even though IgM antibodies may persist for months to years following exposure.Also prenatal IgM screening may lead to needless worry due to numerous false positives (11).It is estimated that a third of world’s population has been infected with the Toxoplasma.gondii which is an unicellular coccidian parasite with worldwide distribution.Although the seroprevalence is unevenly distributed across countries and different socioeconomic strata,congenital toxoplasmosis can be prevented and treated across gestation and less severe disease is reported in countries where prenatal screening and treatment has been systematically implemented(12).By contrast ,severe disease appears to be observed primarily in infants born to untreated mothers .Hence I decided to review what exactly is toxoplasmosis, and how should it be approached and investigated during pregnancy and treatment since the advent of HIV and the importance of cooking meat properly and mode of transmission and freezing techniques . Initially I will just touch upon TORCH in infertility briefly. 1.1Role of TORCH in Infertility: There is yet no direct evidence showing the association between toxoplasmosis and sterility in women.Nevertheless some studies have demonstrated that T.gondii infection could cause reproductive failure in mice which was due to an acquired hypogodadotropic hypogonadism,secondaryto hypothalamic dysfunction ,exhibiting histopathological changes with estrus cycle cessation,impaired folliculogenesis and few corpora lutea(13,14 ).Asurvey of T.gondii in oviductal sterility also revealed prevalence of 44.1%,which was significantly different from that in normal pregnant women(3.3%)(15).Similarly in a survey of T.gondii infection in female infertility in china 15.9%(140/882) for anti T.gondii igG antibodies(16).In an antenatal surveillance program of IVF patients from Palestine ,gaza strip anti toxoplasma,anti rubella,anti CMV and anti Chlamydia IgM antibodies prevalence was 7.9%,6%,7%,and 12.8%respectively(17).Also in male infertility recently zooperty studies reported that reproductive parameters including sperm parameters including sperm motility and sperm concentration were significantly decreased in T.gondii infected rats and a marked increase in sperm abnormalities was also found in the infected male rats(18).Similar results were also observed in male mice experimentally infected with T.gondii(19).Zhou (2002) found that toxoplasma infection was higher in infertile couples ,possibly related to the antisperm antibodies which were higher in Toxoplasma infected couples.A recent investigation of T.gondii infection in 100 men with infertility revealed that 16% of them were IgM positive and13%were CAg positive ,significantly higher than in healthy men. 2.Toxoplasmosis(Ts) 2.1Life cycle: The obligate intracellular parasite Toxoplasma gondii,is a protozoan of the phylum Apicomplexa,which is highly prevalent in humans and animals worldwide(20).The parasite was discovered in 1908 but its mode of transmission remained a mystery until 1970 ,when the full lifecycle was discovered(2).Like othermembers of phylum Apicomplexa the parasite posseses an elaborate apical apparatus dedicated to host cell invasion .The successive exocytosis by secretory compartment including rhoptries and micronemes ,play a key role
in the invasion process .Micronemal proteins are apparently used for host cell recognition ,binding and motility ,whereas the contents of rhoptries likely contribute to the formation of a functional parasitophorous vacuole(PV). Its clinical relevance isdue topotential congenital transmission to the fetus by seronegative women,causing intrauterine death or severe sequelae in the newborn(21).Following infection the parasite can persist for the life of the organism,thus approximately 50%of the world’s population is currently carrying T.gondii.In developing countries seroprevalence can be as high as 80%(22).Despite the worldwide distribution and capacity to infect virtually all mammals and birds species(23),the genetic diversity of T.gondii wasfound to be remarkably low(23,24)and geographic variation was virtually absent(24).Despite the essential sexual cycle T.gondii population structure was found to consist of three clonal lineages namely I,II,and IIIand recombinants among them were rare(24,25).Lehmann et al revealed that while studying the contribution of genetic variations among parasites to patterns of disease transmission ,that south American and Eurasian population have evolved separately until recently when ships populated by rats,mice and cats provided T.gondii with unprecedentedmigration opportunities ,probably during the transatlantic slave trade(26)(fig1).
Legend for figure 1 Courtesy reference 26 showing that the SA1 and SA2 predominates in south America ,RW is common in all continents ,but is rare in South America .WW is cosmopolitan out of a sample size of 275-Aschematic showing the origin of the samples (n=275),vertical bars over sites wheren>=5depict sample composition with respect to the four populations identified by the population structure.Also this diagram illustrates the geographical distribution of T.gondii.
Most of the drugs used for toxoplasmosis treatment are toxic (27)and T.gondii can readily develop resistance (28).Not only are these drugs being administered to infected pregnant women,but also to infected newborns,who are more weak and susceptible to toxicity(21).Recently endochin like quinolones ELQ 271 and ELQ 316 ,two orally
bioactive drugs have been found to be effective against acute and latent toxoplasmosis,likely acting as inhibitors of the Q1 site of the T.gondii cytochrome bc1 complex(29). The complex lifecycle of T.gondii includes three infectious stages,the tachyzoite,the bradyzoite and the sporozoite(30).Only asexual cycle occurs in intermediate hosts.T.gondii is capable of multiplying in virtually any nucleated cell of the body .It multiplies by a special form of binary fission(called endyogeny) until the host cell ruptures.The rapidly dividing tachyzoites(tachy=fast,zoite=organism)is responsible for the clinical signs of toxoplasmosisas. It disseminates the infection to virtually all organs and tissues of the host,and can reach the fetus transplacentally.After repeated divisions ,tachyzoites become encysted in the body ,mostly in the muscle and neural tissues.The encysted stage (tissue cyst contains relatively slowly multiplying organisms (called bradyzoites,brady=slow).Tissue cyst is a dead end phase of the parasite ,in the intermediate host ,waiting to be eaten by the definitive host ,namely cat.Upon ingestion of tissue cysts by the cat ,bradyzoites are liberated in the stomach and intestines .In feline intestines epithelial cells ,bradyzoites first undergo a special form of asexual development ,and merozoites liberated from the schizont produce male and female gametes .After fertilization ,awall is formed around the zygote ,and the oocyst is produced.After rupture of epithelial cells ,oocysts are liberated in the intestinal lumen and are expelled in the faeces of infected cats.Oocysts are unsporulated (noninfective)when passed in faeces .They can sporulate within one day within feline faeces(2).However the major role in disease transmission is played by the oral ingestion of either bradyzoites encysted in the tissues of the chronically infected host(through undercooked infected meat) or sporozoites contained in the oocyst .This highly resistant environmental stage is the result of sexual reproduction ,which occurs in the intestinal epithelium of the cats and virtually all species of the felids and culminates in the fecal shedding of unsporulated oocysts(31).In the external environment oocysts undergo sporogamy(32) and develop two ellipsoidal sporocysts containing four infectious sporozoites each of,which can survive in moist soil or water for months or years (20 ).After initial infection T,gondii establishes latent infection.Reactivation of latent infection in immunocomprised persons causes encephalitis,myocarditis and pneumonitis .Most immunocomprised individuals with AIDS live in the developing world and do not have access to the firstline anti toxoplasma therapy.Moreover ,the impact of toxoplasmosis is expected increase as immunosuppression for solid organ and stem cell transplant patients becomes more frequent in the developing world where latent T.gondii infection is common(33).However even in immunocompetent people toxoplasmosis has been known to present as pyrexia of unknown origin(PUO)(34) or as a case of schizophrenia(35). 2.2Metabolism: Numerous metabolic routes were identified underlying the metabolism of a widerange of molecules including lipids,carbohydrates,aminoacids,nucleotides,and vitamins in the metabolic organization of the oocyst/sporozoite unveiling stage specific differences with respect to the extensively studied tachyzoites.Possenti et al found that T.gondii oocysts posssess the full complement of both glycolytic and TCA cycle enzymes differing from tachyzoites for the alternative expression of isoenzymes enolase(ENOI),citratesynthaseand phosphoenolpyruvate carboxykinase which connects the two pathways by fuelling gluconeogenesis(36).The relative contribution of glycolysis and TCA cycle to energy production is still an open issue .Previous works compared the invitroactivity of select enzymes involved in either of the two pathways highlighting stage specific differences between tachyzoites and bradyzoites .Biochemical data revealed that in bradyzoites glycolysis largely predominates over the TCA cycle,suggesting that the latent parasite stage lacks a functional transport chain(37).Conversely tachyzoites rely on both pathways with glycolysis playing a major role in extracellular parasitesby supplying most of the energy required for the gliding motility and invasion(38,39),whereas oxidative phosphorylation is important for ATP homeostasis in intracellular tacyzoites(40).Thus Possenti et al identified a subset of 154 putative oocyst/sporozoite specific protein(POSP),whose analysis as compared to tachyzoites ,showed that tachyzoites oocysts have a greater capacity of denovo amino acid biosynthesis and are well equipped
to fuel the krebs cycle with the acetyl-coA generated through the fatty acid β oxidation and the degradation of branched amino acids .Hence T.gondii oocysts have adaptation to the nutrient poor and stressing extracellular compartment( 36 ). T.gondii has the remarkable ability to invade a broad range of host cell types.This parasite is believed to attach to host cell via ubiquitously expressed surface molecules of the host ,or each host cell type may carry a unique receptor ,that is bound by a particular parasite molecule(41).Fourteen PAN/apple domain proteins have been detected in T.gondii although only two TgAMA I and TgMIC I have been described(42,43).TgAMAI was shown to form a complex called a moving junction(MJ)with the neck of the rhoptries for RON2/RON1/RON5proteins during the invasion process(44).The depletion of TgAMA1 prevented MJ formation and the parasite was consequently unable to invade the host cell(45).TgMIC4 has been shown to bind with and serve as a bridge between the parasite and the host cell(45).Gaji et al found the parasite invasion occurs by modifying host cell actin dynamicsresulting in increased deposition of F-actin at the periphery of the cell suggesting that host cell contributes rather than being a passive victim (46). 3.Mode of Transmission:Principle source of transmission remains uncertain-mainly 1)oral-Transmission usually takes place by oral route and can be attributable to ingestion of sporulated oocysts from contaminated soil eg in mexico or usuallyby bradyzoites from undercooked meat.During acute feline infection,a cat may excrete as many as 100 million parasites/day which are very stable and may remain viable for many years in the soil.Humans infected develop stage specific antibodies.Undercooking/insufficient freezing,of meat is a important source in children and adults in developed world. 2)blood/transplantation-In addition viable parasites can be cultured from refrigerated anticoagulated blood, which maybe a source of infection in individuals receiving blood transfusions where transmission occurs at a low rate.ii)After kidney/heart transplant patients reported to become T.gondii positive who were negative earlier. rd
3)transplacental Transmission-About 1/3 women who acquire infection with T.gondii during pregnancy transmit the parasite to their fetus:the remainder give birth to normal uninfected babies.Women who are sero positive before pregnancy usually are protected against acute infection and do not give birth to congenitally infected neonates.(1-3,47-51) 4.Clinical Manifestations during Pregnancy Transmission in the fetus occurs predominantly in women who acquire their primary infection during gestation.In rare cases congenital transmission occured in chronically infected women whose infection was reactivated because of their immunocompromised state(AIDS/glucocorticoid treatment).But importantly most pregnant women with acute infection do not experience obvious signs/symtoms(21,52).A minority may experience malaise ,low grade fever ,lymphadenopathy.Very rarely pregnant women present with visual changes due to toxoplasmic chorioretinitis(53) as a result of recent/reactivation of chronic infection. 4.1Effects on fetus and newborn-The frequency of vertical transmission increases with gestation age (21,54).In contrast severe clinical signs in the infected infant are more commonly observed with in offspring of women whose infection was acquired early in gestation. In the neonate,manifestations of congenitaltoxoplasmosis might include hydrocephalaus, microcephaly,intracranial calcifications, retinochoroiditis, strabismus, blindness, epilepsy, psychomotor and mental retardation,petechiae due to thrombocytopenia,and anemia(55,56).Although infected children may initially be asymptomatic the persistence of T.gondii can result in reactivation and clinical disease – most frequently chorioretinitis –decades later.Factors associated with relatively severe disabilities include delays in
diagnosis and an initiation of therapy ,neonatal hypoxia and hypoglycemia,profound visual impairments,uncorrected hydrocephalaus and increases in intracranial pressure.If treated appropriately upwards of 70%of children have normal developmental,neurologic and ophthalmologic findings at followup evaluation.Treatment for one year with pyrimethamine a sulfonamide and folinic acid is tolerated with minimal toxicity(57). Finally for congenital toxoplasmosis it has been shown that hearing loss likely results as a result of postnatal infection response of tachyzoites form and hence early identification can prevent hearing loss(58).Also majority of newborns with congenital toxoplasmosis do not have any clinical signs of disease at birth;however 3070%of those with clinical abnormalities were not detected initially and are found to develop chorioretinitis later in life(12). 4.2Diagnosis during Pregnancy -Serological tests and PCR are used to diagnose toxoplasmosis in pregnant women.Transmission of parasite to fetus frequently occurs in pregnant women with no history of illness or exposure to undercooked meat or cat(59).Therefore the decision to perform T.gondii serological tests during pregnancy should not be based solely on clinical or epidemiological grounds. Systemic serological screening for T.gondii IgG and IgM antibodies in all pregnant women as early in gestation st as feasible(ideally during 1 trimester)and for seronegative women each month /trimester would be optimal .Although usually such screening tests are not performed in most countries in France and Austria it has been mandated by law to facilitate early detection of recently acquired infection.Although important ,the cost, demographic characteristics, availability of appropriate tests,and low incidence of acute infection dictate the above. 4.2.1 Laboratory Diagnosis: The detection of Toxoplasma specific antibodies(Ab’s)is the primary diagnostic method to determine infection with T.gondii ‘s Ab’s,which are detected by numerous serologic kits and most of the test kits are commercially available to detect T.gondiispecific IgG ,IgM,IgA,or IgE Ab’s.The Sabin -Feldman dye test(DT),Indirect haemagglutination Test(IHAT),latex agglutination test(LAT),Direct agglutination test(DAT),and enzyme linked immunosorbent assay(ELISA)are some of these tests used to detect T.gondii Ab’s.Although the DT’is the most specific test ,it is rarely used now because it uses live virulent T.gondii.The IFAT is nearly as sensitive as the DT but it requires fluoresecent microscope .The IFAT ,LAT,DATand ELISAare used more commonly .The IHAT lacks sensitivity specially during acute infection.In addition it lacks specificity ,specially in titres of100,000) of T.gondii Ab titres in DAT or IFATand titres may remain elevated for several years or even whole life if repeated exposures are encountered .Although an 8fold rise in Ab titres taken 2 weeks apart is indicative of a recent infection,this is seldom achieved in practice because by the time the patients are seen in the clinic ,Ab titres have usually peaked.As compared to IgG Ab’s IgM Ab’s are shortlived,and they appear before IgGAb’s(60-63).If more precise knowledge at the time of infection is necessary then an IgG Positive person should have an IgM test performed by a procedure with minimal nonspecific reactions ,such as IgG capture enzyme immune assay(EIA).Anegative IgM test essentially excludes recent infection,but a positive IgM test is difficult to interpret because Toxoplasma –specific IgM Ab’s maybe detected by EIA for6-12 monthsbut rarely as long as 18 months after acute acquired infection(64).Amajor problem with Toxoplasma specific IgM Ab’stesting is lack of specificity.Two situations occur frequently i)positive IgM but negative IgGii)positive IgGand IgM.In the first situation ,the patients blood should be redrawn two weeks after the first and tested together with the first specimen.If the first specimen was drawn in early infection ,the patient should have highly positive IgGand IgMAb’s in the second sample.If the IgG is negative and the IgM is positive ,in both specimens ,the IgM result should be considered to be a false positive and the patient should be considered to be not infected.In the second situation ,a second specimen should be drawn and both specimens submitted together to a reference laboratorywhich
employs a different IgM testing system for confirmation.In a pregnant woman whose sample is taken in second or third trimester rather than ideally in first trimester ,and she is found IgG positive but IgM negative ,it is more advisable to perform IgG avidity tests.High avidity IgG tests indicates that she acquired the infection more than 4 months ago .But the low avidity is not a confirmatory test for recent infection(60,61).Prior to initiation of patient management for acute toxoplasmosis ,all IgM positives should be verified by a reference laboratorywith experience with experimental toxoplasmosis,like in India,Toxoplasma reference laboratory(AIIMS-TSL),Department of Laboratory Medicine,All India Institute of Medical Sciences,New Delhi or similarly the nearest lab for the patient say in PGIMER,Chandigarh or whatever is closest In a vast country like india with cost factor and waiting lists to be taken into account (for review read details Singh S 2003)(5). Yang et al assessed the efficiency of 3 methods of detecting Toxoplasma IgG,Goldmarked method IHA,ELISA and sensitivity, specificity And Youden index were compared .They found the sensititivity and specificity of the ELISA method for Toxoplasma IgG Ab’s are higher and in addition it can be automated.Therefore it is suitable for a large scale Toxoplasma IgG Ab screening(65). Hashoosh et al investigated IgG avidity ELISA test for detecting recent T.gondii infection among pregnant women in eastern Iraq and compared immunological methods and PCR as molecular assays in the diagnosis of T.gondii .Serum was taken from 130 pregnant women at risk of toxoplasmosis and a control group of 25 women with normal pregnancy.Of 50 IgM and /or IgG positive samples ,only 15 showed low IgG avidity Ab’s.PCR was performed on 25 selected samples.Toxoplasma DNA was detected in 15/15IgM-positive with low IgG avidity and 1/3 IgM positive with high IgG avidity.None of the IgM negative with high IgG avidity showed any Toxoplasma DNA .ELISA IgG-avidity when used in combination with ELISA-IgG/IgM is a valuable assay for the exclusion of ongoing i.e recently acquired T.gondii infection in pregnant women was their final conclusions(66). Laboratory diagnosis of Toxoplasma infection is usually based on the detection of specific Ab’s.The specificity and sensitivity of these methods depend primarily on the diagnostic antigens(67). Many serologic tests used in the detection of T.gondii immunoglobulin are commercially available,the majority of which use native parasite antigens prepared from tachyzoites grown in mice and/or in tissue culture that control various non-parasite materials from the culture media and the eukaryote host cells( 68-70).The ELISA is one of the easiest test to perform.Due to the lack of a purified standardized antigen or a standard method for preparing the antigen ,it is not surprising that some interassay variability exists(69). The major advantages of using recombinant antigens of T.gondii infection are as followsi)the antigen composition of the test is precisely known.ii)more than one defined antigen can be used and iii)the method can be easily standardized.Therefore ,the use of recombinant antigens would allow better standardization of the tests and would reduce the costs of production.These considerations are very important when, as often happens ,only one serum sample is available for testing(71).To develop a standardized antigen ,recombinant SAG1(previously namedp30)was produced in bacterial cells and purified.This antigen is one of the principle proteins in tachyzoites and because of its immunological structureSAG1 is considered an important candidate for the development of effective diagnostic reagents or subunit vaccines that induce an immunodominant response(72).This Ag is suitable for use in diagnostic systems for detecting anti-SAG1-specific IgG and IgM Ab’s.The recombinant SAG1has no crossreactivity with proteins from other microorganisms(73).Jallallou et al subcloned SAG1 into an expression vector and this was subsequently transformed into BL2(DE3)pLvsS competent bacterial cells.After inducing the expression of the recombinant Ag ,the protein product was purified using Ni-affinitychromatography.The immunoreactivity of rSAG1was analyzed by SDS-Page blotting.The reactivity of the rec-SAG1 protein was examined using ELISA.They found sensitivity and specificity of the generated rec-ELISA compared to a commercially available ELISA(com-ELISA) and were 88.4% and 88%respectively.Thus they concluded that rec-SAG1 produced in E.coli is a
promising Ag that can be used in diagnostic sensitivity for the detection of specific Ab’s against T.gondii in their laboratory in Iran(74). Besides SAG2,the surface antigen2(SAG2)also known as P22,is another protein that has been shown to be an attachment ligand and also to have good immunogenicity(69,75).Monoclonal Ab’s raised against these2major tachyzoite Ag’s(SAG1,SAG2)are 100 fold more reactive with the tachyzoite stage than with oocyst(sporozoite) and bradyzoite stages(76).Recombinant surface antigens like SAG1or SAG2 are useful in serodiagnosis in cats(77,78)and humans(71,79).The surface Ag 3 SAG3 (P43),is found both in tachyzoites and bradyzoites.and like SAG1,it is anchored in the membrane via glycosylphosphatidylinositol groups.It is very similar in structure to SAG1 in structure an d function ,and SAG1and SAG3 proteins participate in cellular invasion and attachment(80-82).In this study Khanaliha et al in shiraz university iran,used the 3 recombinant Ag’s of T.gondii,(SAG1,SAG2 and SAG3)to evaluate their diagnostic usefulness and efficacy compared with the commercially available ELISA for diagnosis of Toxoplasmosis and improvement of diagnostic methods.The best outcome in terms of sensitivity ,specificity ,positive predictive value ,negative predictive value ,and agreement was found with recombinant SAG2(100%and 89.4% in diagnosing IgM Ab’s compared with recombinant SAG1(93.6%and 92.9%)and SAG3(95.4%and 91.2%).This showed the usefulness of this Ag in the diagnosis of acute infection as shown in another study(79).Thus they concluded that although recombinant SAG1,SAG2 and SAG3 produced in E.coli are suitable Ag’s which can be used in the diagnosis of IgG anti-toxoplasma-Ab’s ,recombinant SAG3 is more sensitive and specific,SAG2 showed the best performance in the diagnosis of IgM –specific Ab against T.gondii(83). Drapala et al have evaluated the preliminary diagnostic utility of two mixtures of T.gondii recombinant antigens(rROP1+rSAG2and rROP1+rGRA6)in IgG ELISA avidity test.Atotal of 173 serum samples from patients with toxoplasmosis and seronegative people were examined.The sensitivity of IgG ELISAfor rROP1+rSAG2and rROP1+rGRA6 was 91.6%and76.7%respectivelywhile the reactivity for sera from patients where acute toxoplasmosis was suspected was higher at 100%and 95.4%respectively ,than for people with chronic infection,at 88.2%and 70.6%.In this study a different trend in avidity maturation of IgG Ab’s for 2 mixturesof proteins in comparison with native antigen was observed.This result suggests that the new IgG avidity test using the mixtures of recombinant antigens maybe useful for the diagnosis of difficult to identify phases of toxoplasmosis.For this reason,selected mixtures after the additional tests on groups of sera with well defined dates of infection could be used as a better alternative to the native antigens of the parasite in the serodiagnosis of human T.gondii infection(84). rd
Serological Tests(inUSA):Mostly only 1/3 of samples submitted are from 1st trimester in USA-the best lab(read review for details by Montoya et al 2008(85).Serological test results of serum samples from later gestations more difficult to interpret,and testing after second trimester most often will not exclude that an infection was acquired during pregnancy.Further detailed serological diagnosis igA,IgG,IgM,IgE,antibodies,IgG avidity and the differential agglutination(AC/HS) tests have been successfully employed to distinguish the acute vs chronic stage of infection(86).Except for IgG/IgM most tests available in reference laboratories like Palo Alto Medical Foundation Toxoplasma Serology Laboratory(PAMF-TSL). For details read review Montaya et al 2008(85). Serological testing for IgG/IgM in nonreference laboratories should be done initially-If done early in gestation in vast majority by the absence of both one can determine that infection has not occurred at all,or occurred at a distant past,by positive IgG,and negative IgM antibody test result.Additional assistance with confirmatory testing in reference laboratories such as PAMF-TSL/AIIMS-TSL often can determine whether a patient positive for IgM antibody test result acquired infection recently/in distant past .(See review for further details on amniocentesis and PCR) (85).Recently multiplex flow immunoassay (MFI)technologies emerged as a novel approach to assess the serologic response to various infectious diseases(87-89).This technology is similar to traditional EIA but allows for
the simultaneous detection and identification of multiple analysis in a single reaction.Binnicker et al found that with the use of multiplex detection of IgG/IgM to T.gondii,Rubella virus and CMV that Bioplex ToRC IgG immunoassays showed a comparative performance to the routine EIA/ELFA.However the IgM for all three showed lower specificities but with the advantage of lower cost and lower number of running time and multiple samples could be run at a time and suggest improving the sensitivity of the test may be important in the future(90).Further lesser invasive tests using urine for DNA extraction are being targeted(91).The Vidas system fits the need for laboratories working on small routine series first line testing as well as expert laboratories due to a high specificity and a powerful avidity test( 92). 4.2.2Ultrasound:--USG is recommended in women with suspected or diagnosed acute infection acquired during or shortly before gestation.USG may reveal the presence of fetal abnormalities including,hydrocephalaus,brain/hepatic calcification,splenomagaly and ascites(24).The clinical outcome of congenitally infected infants whose mothers acquired infection during the first trimester of pregnancy ,whose fetal ultrasound was normal and mothers had received spiramycin during gestation revealed no difference in two year follow up children born to mothers who had acquired infection during the second and third trimester(93).Therefore termination of pregnancy was not indicated however treatment was essential,and prenatal USG findings should be free of any abnormality(93).Also besides USG, MRI has been used to search for other abnormalities in the fetus. Histological analysis and parasite isolation-Occasionally placental/fetal tissues from pregnant women suspected of having acquired infection –available to determine vertical transmissionof parasite has occurred.i)T.gondii cysts may be isolated by Wright –giemsa stain ii)immunoperoxidase staining using T.gondii specific antibodies are more sensitive(94). 2)Isolation of parasite can be attempted by inoculation of tissues into tissue culture or mice(24). 4.2.3 PCR: Amplification of T.gondii DNA in amniotic fluid has been used successfully for prenatal diagnosis of congenital toxoplasmosis at 18weeks of gestation age(GA)(95-97).Its sensitivity and specificity for amniotic fluid obtained before 18 weeks of GA have not been studied.The procedure done early in gestation is associated with higher risk to the fetus,besides being less likely to be useful.A defining study of routine use of PCR of amniotic fluid obtained at 18weeks GA or later was reported to have an overall sensitivity of 64% for the diagnosis of congenital infection in the fetus ,a negative predictive value of 88% and a specificity and positive predictive value of 100%(ie a positive result signifies infection of the fetus(95).Sensitivity was statistically significantly higher when maternal infection occurred 17-21 weeks of GA as compared with when infection occurred before 17weeks or after 21 weeks of GA(95). However the negative predictive value of PCR of amniotic fluid from women who acquired the infection early in gestation eg before 7wks of GAwas 100%because of the very low transmission rate during that time of gestation(95).Maternal infections acquired before 20wks of GA with a parasite load 1100 parasites/ml of amniotic fluid was associated with the highest risk of severe outcome in the fetus ,in clinical practice ,amniocentesis has essentially replaced fetal blood sampling for diagnosis of congenital toxoplasmosis because of the inherent lower risk and higher sensitivity(21,97). However Paquet et al recommends amniocentesis only if maternal primary infection is confirmed ,if maternal serologic test results do not confirm or exclude acute infection,or if ultrasonographic feautures are consistent with congenital toxoplasmosisin countries like canada with low prevalence of the disease(98).It should be offered only after 18weeks GA and atleast 4weeks after the suspected acute maternal infection to decrease the false negative results(98). 5.Management of patients with suspected/diagnosedT.gondiiinfection acqired during gestation
Once it has been established that serological tests are consistent with a recently acquired infection during the first 18wks of gestation shortly before conception can’t be excluded,an attempt to prevent vertical transmissionof parasite through treatment with spiramycin is recommended for the mother.If fetal infection is confirmed by a positive result of positive PCR of amniotic fluid at 18 weeks of gestation or later treatment with pyrimethamine,sulphadiazine and folinic acid is recommended.(if patient is already receivingspiramycin,the recommendation is to switch to this combination .In some centres this switch takes place as early as 14 -16 weeks(99). Because of the high transmission rates observed after 18 weks gestation ,treatment with pyrimethamine ,sulfadiazine and folinic acid is also used for patients who have acquired the infection after 18 weeks of gestation in an attempt to prevent fetal infection from occurring and,if transmission has occurred to provide ,treatment for the fetus (fig2).
Pregnant woman is suspected or confirmed to have acquired toxoplasmosis during gestation
< 18 wks of gestation
>18 wks of gestation
Pyrimethamine + sulfadiazine + folinic acid
Spiramycin Fetal ultrasound(s) should be performed
Fetal ultrasound(s) should be performed
Amniotic fluid PCR at > 18 wks or as soon thereafter as feasible
PCR negative and ultrasound negative
Continue spiramycin
Amniotic fluid PCR at >18 wks or as soon thereafter as feasible
PCR positive and ultrasound positive
Pyrimethamine +sulfadiazine + folinic acid
PCR negative and ultrasound negative
Consider switch to spiramycin continue pyrimethamine + sulfadiazine +folinic acid
Delivery .Pyrimethamine is not used earlier because it is potentially teratogenic.
5.1Spiramycin-Althogh use of macrolide antibiotic spiramycin has been reported to decrease the frequency of vertical transmission(100,101),carefully designed prospective studies demonstrating this effect have not been performed.The protection has been more distinct in women infected during their first trimester.Spiramycin does not cross the placenta and thus is not reliable for treatment of infection in the fetus.There is no evidence that spiramycin is teratogenic(table1).
Treatment
Dosage
Comments
Spiramycin
1g(3 million U)every 8 h(for a total of 3g or 9 million U per day )
Not teratogenic:does not treat infection in the fetus:indicated for pregnant women suspected of having acquired the infection at 18 weeks of gestation.Spiramycin treatment should be continued until delivery in women with low suspicion of fetal infection or those with documented negative results of amniotic fluid PCR and negative findings on ultrasounds at follow up.Available in United states only through Investigational New Drug process at FDA.Prior consultation with medical consultants is required.
Pyrimethamine, sulfadiazine and folinic acid
Pyrimethamine:50 mg every 12 h for 2 days followed by 50mg daily: sulfadiazine:initial dose of 75mg/kg followed by 50mg/kg every 12 h (maximum 4g/day):folinic acid (leucovorin):10-20 mg daily (during and 1 week after completion of pyrimethamine therapy)
Pyrimethamine is teratogenic: therefore this combination should no be used before week 18 of gestation(in some centers in europe.it is used as early as week 14-16).Indicated for women suspected of having acquired infection at >18weeks of gestation and those with documented fetal infection (positive result of amniotic fluid PCR) or abnormal ultrasound findings suggestive of congenital toxoplasmosis, given when patient is at >18weeks of gestation.
The drug is administered until deliveryeven in patients with negative results of amniotic fluid PCR because of the theoretical risk that fetal infection can occur later in pregnancy from a placenta that was infected earlier in gestation(102).Where probability of fetal infection is high or fetal infection has been established ,treatment with th spiramycin should be switched after the 18 week of gestation to treatment with pyrimethamine,sulphadiazine,and folinic acid.Dosage-daily dose of 1.0 (or 3million) every 8h(total dosage of 3g/9million units/day). Although in recent years use of spiramycin has become controversial . Recent EMSCOT(European muticentric study on congenital toxoplasmosis)studies suggest that spiramycin maybe more effective when administered after early seroconversion(103).Still large randomized trials are recommended before spiramycin can be given up .
5.2Pyrimethamine,Sulfadiazine and folinic acid Pyrimethamine and trimethoprim inhibit the enzyme dihydrofolate reductase.Inhibitors of protein synthesis including clindamycin,chlortetracycline, azithromycin affect the growth of the parasite.Inhibition of purine synthesis ,such as arprinocid may prove to be important.Atovaquone ,which blocks pyrimidine salvage ,has demonstrated activity against both T.gondii and P.carnii.But until further information is available Montaya et al recommend the combination of pyrimethamine ,sulfadiazine.,and folinic acid as treatment for pregnant women who acquire the infection after 18weeks gestation and those in whom fetal infection has been confirmed eg after positive PCR on amniotic fluid .This combination is used with hope of treating infection in the fetus and in some instances preventing transmission especially if amniocentesis can’t be performed and infection is acquired after 18 week of gestation. Pyrimethamine is potentially teratogenic ,and should not be used in the first trimesterof pregnancy.The drug produces reversible,usually ,dose related depression of bone marrow.All patients receiving pyrimethamine should have complete blood count, frequently monitored,folinic acid and not folic acid is used for
reduction and prevention of haematological toxicities of the drug. Recently Li et al carried out a meta analysis on risks of adverse outcomes in T.gondii infection and although studied 53 of the 2632 studies in literature most of their studies included work in china as they could find detailed analysis trimesterwise only in their files.They concluded that pooled risk of vertical transmission was 20%in maternal infection and the incidence of vertical transmission increased in first ,second or third trimester of pregnancy .The pooled rates of transmission in groups treated with spiramycin only ,PSF(pyrimethamine+sulfadiazine+folinic acid) or PScombined with spiramycinor other atypical treatments was not statistically different.This appears slightly in contrast to studies of Montaya who quote infection acquired in latter half of pregnancy usually have lower chance of vertical transmission(104). 6. Guidelines for management-i)Generally no risk if mother is infected >=6mths before conception. 2)If the infection is acquired6months before conception Since the incidence of congenital toxoplasmosis is very low treatment is not indicated with either of the above mentioned drugs if serological tests reveal infection was acquired in the distant past. 4)In case of immunocompromised patients who are coinfected wth HIV and T.gondii and who have developed AIDS and are at risk of reactivating T.gondii infection,developing severe toxoplasmosis(encephalitis,pneumonia etc)and/or transmitting parasite to their offspring.Fortunately ,such transmission is surprisingly rare(21,110).At present data are insufficient to define effectiveness of Rx intended to prevent vertical transmissionof T.gondii in a HIV infected woman Until more data is available Montaya et al suggested that Toxoplasma seropositive pregnant women whose CD4cell count is 1200 cells/mm receive trimethprim/sulphmethoxazole(80mg trimethoprimand 400mg sulfmethoxazole in a single strength tablet), 1tablet/day;this treatment is commonly used to prevent pneumocystis pneumonia in such patients in an attempt to prevent reactivation of their toxoplasma infection and transmission of the parasite to their offspring.Trimethoprim is usually avoided in the first trimester because it is a 3 folic acid antagonist .For women whose CD4 cell count is 1200cells/mm and for non HIV infected infected immunocomprised women spiramycin treatment is suggested for the duration of the pregnancy.Unfortunately no studies are available regarding effectiveness of these strategies .Performance of amniotic fluid PCR is not advisable because of risk of facilitating transmission of HIV to the fetus during the procedure. 5)For a patient who recently acquired toxoplasma infection it is advisable to wait for 6 months before undertaking pregnancy. 6) Patients who develop Toxoplasmic chorioretinitis need a serological evaluation regarding whether infection was acquired recently or in distant past.Patients with reactivation of chorioretinitis as a result of latent infection(acquired before gestation) don’t have a higher risk of transmission of parasite to their offspring than pregnant women who were infected before gestation and do not have ocular toxoplasmosis(111).But those with a
toxoplasmic chorioretinitis are considered to be a manifestation of recently acquired infection (112).They should be given treatment for the infection for both the eye disease and the risk of transmission to their fetus. 7.Prevention: The most promising measure for the protection of humans and animals against T.gondii infection is vaccination .Vaccination with SAG1 affinity purified from the RH strain produced high survival rates and significantly decreased brain cyst load in mice (113,114).Also the use of a combination of antigen delivered as plasmids coding for regions of micronemal proteins ,including MIC2,MIC3,MIC4,M2AP and AMA1 resulted in a significant reduction(84%)in the number of cysts(106).Almost all protection molecules seem to be involved in the parasite –host interaction. Due to so much increasing prevalence of toxoplasmosis worldwide attempts are being made to control it actively by devicing vaccines which are safe eg MIC 3 gene and aspartic protease 1 have been identified as potential candidate genes respectively(115,116). 8.Conclusions Thus it is important to understand that the term “TORCH” used loosely is considered outmoded and needs to be used judiciously.It should be replaced by targeted testing for specific pathogens in well defined circumstances.The patient history ,risk factors and local regulations should guide the screening procedure for the specific component of “TORCH”with no demonstrable role in infertility of this“TORCH”group of pathogens.Considering the impact of worldwide spread of toxoplasmosis it is important to include in the armametatrium and further understand the role of timing of reports and their significance . Reports from pregnant women from Croatia,Brazil,Tunisia further confirm the importance of early screening in pregnancy and prevention of congenital toxoplasmosis respectively(117-119).For e.g.in Brazil although prenatal care was given only 13%were treated for congenital toxoplasmosis resulting in 31 children having toxoplasmosis in a study over 10years.While in Tunis prenatal diagnosis allowed to decrease severity of congenital toxoplasmosis.Still a lot of ignorance along with cost factors prevent the early diagnosis and treatment specially in developing countries like India where patients have to face a lot of hurdles including lack of standardization of several test kits available with differing results from different laboratories due to lack of standardization,need to go to a reference laboratory for confirmation by IgG avidity tests before treatment can be instituted and some of them can not afford either to travel the distance to reference laboratory forconfirmation or may not afford the cost of medicines.Alot of efforts are made to develop cost effective kits from recombinant antigens with reproducibility. Still a lot of mass awareness and education is needed to reduce the incidence of congenital toxoplasmosis. Similarly Andiappan et al studied in southern Thailand 760 patients for Toxoplasma IgG,IgM by ELISA along with IgG avidity tests.He found 25%were positive Ab titres(22%IgG,3%IgM).All were high avidity indicating infection had occurred 4-5 months beforeand multivariate regression analysis indicated that significant factors associated with serpositivity are age>=26yrs,those working as labourers and drinking unclean water(piped tap water )and advocated health education and the awareness of risk exposures regarding the parasitic diseases was required to minimize the effects of this parasitic infection in pregnant women as well as general population(120). Further Andiappan et al studied 2598 pregnant women from Malaysia,Phillipines,and Thailand in an attempt to know the knowledge and practices in pregnant womenand found demographic profile such as age,level of education ,pregnancy term and number of children of the pregnant women showed significant association with their response towards prevention knowledge and preventive behaviour related questions.Thus they suggested that health education on Toxoplasmosis and primary behavioral practices should be consistently offered to reproductive age women in general and pregnant women in particular.Thus information could help to reduce vertical transmission of Toxoplasma infection during pregnancy(121).
REFERENCES 1.
2. 3.
4.
5. 6. 7.
8. 9. 10.
11.
12. 13. 14.
15.
16.
Sen MR,Shukla BN,Banerjee T.Prevalence of Serum Antibodies to TORCH Infection in and around Varanasi,Northern India.J Clin Diagn Res 2012 Nov;6(9):1483-5. Dubey JP Toxoplasmosis in India.In:Perspect Prasitol(CBS Publishers and Distributors ,Delhi)1988;2:131-152. RemingtonJS,Mcleod R,Desmonts G.Toxoplasmosis .In;JS Remington and JO th Klein(ed)Infectious disease of the fetus and newborninfant 4 edition(WB Saunders Co,Philadelphia) 1995:140-226. Singh S,Singh N,Maniar JK.AIDS Associated Toxoplasmosis in India and its correlation with serum tumor necrosis factor –alpha.J Parasitol Dis 1996;20:4952. Singh S.Mother –to-child Transmission and diagnosis of toxoplasma gondii infection during pregnancy.Indian J Med Microbiol 2003;21(2):69-76. Epps RE,Pittlekow MR,Su PW.TORCH syndrome.Semin Dermatol 1995;14:179186. Abdel-Fattah SA,Bhat A,Illanes S,Bartha JL,Carrington D.TORCH test for fetal medicine are indicationsonlyCMV is necessary in the United Kingdom.Prenat Diagn 2005;25:1028-1031. Khan NA,Kazzi SA.Yield and costs of screening growth retarded infants for torch infections.Am J Perinarol 2000;17:131-135. Leland D,French ML,Kleiman MB,Schreiner RL.The use of TORCH titers.Pediatrics 1983;72:41-43. Garland SM,Gilbert GL.Investogation of congenital infection-theTORCH screen is not a legitimate test..Pediatric Infectious Disease Group of the Australian Society for Infectious diseases.Med J Aust 1993;159:346-348. Averhoff F,Zucker J,Vellozi C,Redd S,Wooddfill S,Waterman S,Baggs J,Weinberg M,Rodriquez-Lainz A,Carrison V,Goto C,Reef SE.Adequacy of surveillance to detect endemic rubella transmission in the united states.Clin Infect Dis 2006;43(Suppl 3):S151-S157. Moncada PA,Montaya JG.Toxoplasmosis in the fetus and newborn :an update on prevalence ,diagnosis and treatment.Expert Rev Infect Ther 2012;10(7):815-823. Stahl W,KanedaY,Noguchi I.Reproductive failure in mice chronically infected with Toxoplasma gondii.Physiol Res 1994;80:22-27. Antonios SN,Small HI,Essa T.Hypothalamic origin of reproductive failure in mice chronically infected with experimental toxoplasmosis .J Egypt Soc Parasitol 2000;30:593-599. Wei M,Jing DX,Luo RY,Zhang W,Wu XZ,Yang LD,Chen CY,Lu M.Study on the relationship between toxoplasmosis and oviductal sterility.J Pub Health Prev Med 2005;16:31in Chinese. Li S,Cui L,Zhao J,Dai P,Zong S,Zuo W,Chen C,Jin H,Gao H,Liu Q.Seroprevalence of Toxoplasma gondii infection in female sterility patients in china.J Parasitol 2011;97(3):529-530.
17. Al-Hindi A,,Al-h=Helou T,Al-Helou Y.Seroprevalence of Toxoplasma gondii,cytomegalovirus,rubella virus and Chlamydia trachomatis among infertile women attending in vitro fertilization center,Gaza strip ,Palestine.J Egypt Soc Parasitol 2010;40(2):451-458. 18. Terpsidis KI,Papazahariadou MG,Taitzoglou IA,Papaioannou NG,Georgiadis MP,Theodoridis IT.Toxoplasma gondii:reproductive parameters in experimentally infected male rats.Exp Parasitol 2009;121:238-241. 19. Yang YD,Chen CY,LuM,.Wu XZ,Gang F.Infertility experiment on male mice infected with toxoplasmosis.Chin J Zoonosis 2005;21:592-594.(In Chinese) 20. Dubey JP Toxoplasmosis of animals and humans.Boca Ratan,Canto CRG Press 2009. 21. Remington IS,Mcleod R,Thuilliez P,Desmonts G.Toxoplasmosis,In:Remington IS,Klein IO,Wilson CB,Baker C, eds .Infectious diseases of the fetusand newborn infant6th ed.Philadelphia:Elsevier Saunders 2006;947-1091. 22. Arevalo JF,Belfort RJr,Muccioli C,Espinoza JV.Ocular toxoplasmosis in the developing world.Int Ophthalmol Clin 2010;50:57-69. 23. Dubey JP,&Beatie CP Toxoplasmosis of animal and man (CRC,Boca Raton(L) 1992. 24. Darde ML,Bouteille B,&Pestre-Alexandere MI Isoenzyme analysis of 35 toxoplasmagondiiisolates aqnd the biological and epidemiological implications. JParasitol 1992;78(5):786-94. 25. Howe DK,Sibley LD.Toxoplasma .gondii comprises three clonal lineages:correlation of parasite genotype with human disease.J Infect Dis 1995;172(6):1561-1566. 26. Lehmann T,Marcet PL,Graham DH,Dahl ER,Dubey JP Globalization and the population structure of Toxoplasma gondii PNAS 2006;103(30):11423-11428. 27. Kaufman HF,Geisler PH .The haematoligic toxicity of Pyrimethamine Daraprim in man,Arch Opthalmol 1960;64:140-146. 28. Fohl LM,Rous DS,.Fitness effects of DHFR-LS mutations associated with pyrimethamine resistancein apicomplexan parasites.Mol Microbiol 2003;50:1319-1327. 29. Doggert JS,Nilsen A,Forquer I,Wegmann KW,Jones-Brando L,Yolken RH,Bordon CH,Charman SA,Karneni K,Carruthers VB et al.Endochin –like quinolones are highly efficacious against acute and latent experimental toxoplasmosis. PNAS 2012;109(39):15396-15941. 30. Dubey JP,Lindsay DS,Speer CA Structure of Toxoplasma gondii Tachyzoites,Bradyzoites,and Sporozoitesand biology and development of tissue cysts.Clin Microbiol Rev 1998;11(2):267-299. 31. Belli SI,Smith NC,Ferguson DJ.The coccidian oocyst:atough nut to crack!Trends Parasitol 2006;22(9):416-423. 32. Ferguson DJ,Birsch-Anderson A,Hutchinson WM,Siim JC.Ultrastructural studies on the endogenous development of Eimeria brunette II,Microgametogony and the Microgamete.Acta Pathol Microbiol Scand B1977;858(1):67-77. 33. Mulanovich VE,Ahmed SI,Oztur ET,Khokhar FA,Kontoyiannis DF,deLima M. Toxoplasmosis in allo-SCT patients ;Risk factors and outcomes at a
34.
35. 36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
transplantation centre with a low incidence.Bone Marrow Transplant 2011;46:273-277. Abhilash KP,Roshine MK,Vandana K,Varghese GM.Aprobable case of acquired toxoplasmosis presenting as pyrexia of unknown origin in an immunocompetent individual.Int J Inf Dis 2013;May 28,pii:s1201-9712(13)00168-9. Torrey EF,Yolken RH.Editors’introduction:Schizophrenia and toxoplasmosis.Schizophrenia Bull 2007:37-58 Possenti A,Fratini F,Fantozzi L,Pozio E,Dubey JP,Ponzi M,Pizzi E,Spano F.Global proteomic analysis of the oocyst/sporozoite of Toxoplasma gondii reveals commitment to a host –independent lifestyle.BMC Genomics 2013;14:183(1-18) Denton H,Roberts cw,Alexander J,Thong KW,Coombs GH.Enzymes of energy metabolism in the bradyzoite and tachyzoite of Toxoplasma gondii.FEMS Microbiol Lett 1990;137(1):103-108. Lin SS,Blume H,vonAhsen N,Gross U,Bohne W.Extracellular Toxoplasmosis gondii tachyzoites do not require carbon source uptake for ATP maintainance ,gliding motility and invasionin the first hour of their extracellular life.Int J Parasitol 2011;41(8):835-841. Pomel S,Luk FC,Beckers CJ.Host cell egress and invasion induce marked relocation of glycolytic enzymes in Toxoplasma gondii tachyzoites.PLoS Pathog 2008;4(10)e1000188. Lin SS,Gross U,Bohne W.TypeII NADH dehydrogenase inhibitor 1hydroxy 2diodecyl 4(1H)quinolone leads to collapse of mitochondrial innermembrane potential and ATP depletion in Toxoplasma gondii Eukaryot Cell 2009;8(6):877887. Oerega –Barria F,Boorthroyd JC,A Toxoplasma lectin like activity specific for sulfated polysaccharides is involved in host cell infection .JBiol Chem 1999;274:1267-1276. Brecht S,Carrithers VB,Ferguson DJ,Gaddings OK,Wang G et al The Toxoplasma microneml ptotein MIC4is an adhesin composed of six conserved apple domains J Biol Chem 2001;276:4119-4227. Crawford J,Toukin ML,Grujic O,Boulanger MJ.Structural characterization of apical membrane antigen1AMA1 from Toxoplasma gondii J Biol Chem 2010;285:15644-15652. Bestejro S,Michelin A,Poncet J,Dubremetz JF,Lebrun M.Export of aToxoplasma gondii shptrynack protein couples at the host call membrane in form thr moving junction during invasion PLoS Pathog 2009;5:e1000309 doi :10.13717journal.ppai.1000309. Maal J,Mejssur M,Soldati D,Ward GE.Conditioned expression of Taoxoplasma gondii apical membrane antigen TgAMA1 demonstrates that TgAMA1 plays a critical role on onvasion.Mol Biol Cell 2005;16:4341-4349. Gaji RY,Huynh MH,Carruthers VB.Anovel high throughput invasion screen identifieshost actin regulators required for efficient cell entry by Toxoplasma gondii.PLoS One.2013;8(5):e64693. Choi WY,Nam HW,Kwak NH,Huh W,Kim YR,Kang MW,Cho SY,Dubey JP.Foodborne outbreaks of human txoplasmosis.J Infect Dis 1997;175(5):1280-2.
48. Slavin MA,Meyers JD,Remington JS et al.Toxoplasma gondii infection in bonemarrow recipient:A20 year experience .Bone Marrow Transplant 1994;54:64-6. 49. Wreghitt TG,Hakim M,Gray JJ et al .Toxoplasmosis in heart and lung transplant recipients.J Clin Pathol 1989;42:194-199. 50. Singh S,Ahlawat S,Singh N,Chaudhary VP.High Torch seroprevalence in multiply transfused b-thallasemic children in India.Eur J Haematol 1994;54:64-6. 51. Roberts T,Frenkel JK.Estimating income lossesand other preventable costs caused by congenital toxoplasmosisin people in the united states.J Am Vet MedAssoc 1990;196:249-256. 52. Boyer KM,Holfels E,Roizen N,et al.Risk factors for Toxoplasma.gondii infection in mothers of infants with congenital toxoplasmosis:implications forprenatal managementand screening.Am JObstet Gynecol 2005;192:564-571. 53. Garvey IG,Scherrer J,Wallon M,Kudkikian I,Peyron F.Reactivation of ocular toxoplasmosis during pregnancy.BIOG 2005;112:241-2. 54. Dunn D,Wallon M,Peyron F,Peterson E,Peckham C,Gilbert R.Mother-to child transmission of toxoplasmosis:risk estimates forclinical counseling.1999;353:1829-1833. 55. Mcauley J,Boyer KM,Patel D,Mets M,Swisher C,Roizen N et al.Early and longitudinal evaluations of treated infants and children and untreated historical patients with congenital toxoplasmosis:the Chicago Collaborative Treatment Trial .Clin Infect Dis 1994;18(1):38-72. 56. Swisher CN,Boyer K,Mcleod R.Congenital toxoplasmosis.The Toxoplasmosis Study group .Semin Pediatr Neurol 1994;1(1):4-25. 57. Mcleods R,Boyer K,Karrison T,KaszaK,Swisher C,Roizen N,Jalbrzikowski J,Remington Jet al.Outcome of Treatment for Congenital Toxoplasmosis,18812004,:The National Collaborative Chicago-Based,Congenital Toxoplasmosis Study.Clin Infect Dis 2006; 42(10):1083-94. 58. Salviz M,Montoya JB,Nadol JB,Santos F.Otopathology in congenital toxoplasmosis.Otol Neurol 2013 Apr 17Epub ahead of print. 59. Dubey JP,Hill DE,Jones JL,et al.Prevalence of viable Toxoplasma gondiiin beef ,chicken and porkfrom retail meat storesin the united states:risk assessment to consumers.J Parasitol 2005;91:1082-1093 60. Pinon JM,Dumon H,Chemla et al .Strategy for diagnosis of congenital toxoplasmosis:evaluationof methods comparing mothers and newborns and standard methods for postnatal detection of innunoglobulin G,M and IgA antibodies.J Clin Microbiol 2001;39:2267-2271. 61. Peleoux H,Brun E,Vernet G et al.Determination of anti-Toxoplasma gondii immunoglobulin G avidity :adaptation to the Vidas system(biomereux).Diagnostic Microbiolo Infect Dis 1998;32:69-72. 62. Wilson M,Remington JS,Clavet C,Varney G,Press C,Ware W,The FDA Toxoplasmosis Ad hoc Working Group.Evaluation of six commercial kits for detection of human IgM antibodies to Toxoplasma gondii.J Clin Microbiol 1997;35:3112-3115.
63. Singh S,Singh N,Dwivedi SN.Evaluation of seven commercially available kits for the serodiagnosis of Toxoplasmosis.Indian J Med Res 1997;105:103-7. 64. Remington JS,McLeod R,Desmonts G.Toxoplasmosis .In:JSremington and JO Klein(ed).Infectious diseases of the fetus and nreborn infant,4thed.,W.B.Saunders Co..Philadelphia)1995:140-226. 65. Yang XX,Chen YK,Wei SJ,Song RH.Efficiency of three methods for detecting Toxoplasma IgG antibody.Zhonguo Xue Xi Chong Bing Fang Zhi Za Zhi 2014;26(1):109-10. 66. Hashoosh DA,Majeed IA.Comparison of two Assays in the diagnosis of Toxoplasmosis :Immunological and Molecular.East Mesiterr Health J 2014;20(1):46-50. 67. Joyson DHM,Guy EC.Laboratory diagnosis of Toxoplasma infection .In Joynson DHM,Wreghitt TG.editors.Toxoplasmosis,Acomprehensive clinical guide.Cambridge:Cambridge University Press;2001:pp296-318. 68. Johnson AM,,Roberts H,Tenter AM.Evaluation of a recombinant antigen ELISA for the diagnosis of acute toxoplasmosisand comparison with traditional antigen ELISA’s.J Med Microbiol 1992;37:404-9. 69. Aubert D,Maine GT,Villena I,Hunt JC,Howard L,Sheu M et al.Recombinant antigens to to detect Toxoplasma gondii –specific immunoglobulin Gand immunoglobulin M,in human seraby enzyme immunoassays.J Clin Microbiol 2000;38:1144-50. 70. LiS,Maine G,Suzuki YAraujo FG,Galvan G,Remington JS et al.Serodiagnosis of recently acquired Toxoplasma gondii infection with arecominant antigen.JcLIN Microbiol 2000;38:179-84. 71. Pietkiewicz H,Hisczynska-Sawicka E,Kur J,Petersen E,Nielsen HV,Paul M,et al.Usefulness of Toxoplasma gondii recombinant antigens(GRA1,GRA7 andSAG1)in an immunoglobulin G avidity test for the serodiagnosis of toxoplasmosis.Parasitology Res 2007;100:333-7. 72. Burg JL,Perelman D,Kasper LH,Ware PL,Boothrovd JC.Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii.J Immunol 1988;141:3584-91. 73. Harning D,Spenter J,Metsis A,VuustJ,Petersen E.Recombinant Toxoplasma gondii surface antigen 1(p30)expressed in Escherichia coli is recognized by human Toxoplasma specific immunoglobulin M(IgM)and IgG antibodies.Clin Diagn Lab Immunol 1996;3:355-7. 74. Jallallou N,Bandapour M,Khazam H,Haghghi A,Abdollah S,Kazami S.Recombinant SAG1 Antigen to detect Toxoplasma gondii specific immmuunoglobulin G in human sera by ELISA test.Iran J Parasitol 2010;5(2):1-9. 75. Grimwood J,SmithJE.Toxoplasma gondii:the role of parasite surface and secreted proteins in host cell invasion.Int J Parasitol 1996;26:169-173. 76. Kasper LH.Identification of stage specific antigens of Toxoplasma gondii.Infect Immun 1989;57:668-672. 77. Huang X,Xuan X,Kimbita EN,Battur B,Miyazawa T,Fukumoto S,Mishima LH,Suzuki Het al.Development and evaluation of anenzyme linked immunosorbent assay with recombinant SAG2 for the diagnosis of Toxoplasma gondii infection in cats.J Parasitol 2002;88:804-807.
78. Kimbita EN,Xuan X,Huang X,MiyazawaT,Fukumoto S,Mishima M,Suzuki H et al.Serodiagnosis of Toxoplasma gondii infectio in cats by enzyme linked immunosorbent assay using recombinant SAG1.Vet Parasitol 2001;102:35-44. 79. Parmley SF,Sgarlato GD,Mark J,Prince JB,Remington JS.Expression,characterization,and serologic reactivity of recombinant surface antigenP22 of Toxoplasma gondii.J Clin Microbiol 1992;30:1127-1133. 80. Burg JL,Perelman D,Kasper LH,Ware PL,Boothroyd JC.Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii.J Immunol 1988;141:3584-3591. 81. Prince JB,Auer KL,Huskinson J,Parmley SF,Araujo FG,Remington JS.Cloning ,expression,and Cdna sequence of surface antigen P22 from Toxoplasma gondii.Mol Biochem Parasitol 1990;43:97-106. 82. Jacquet A,Coulon L,DeNeve J,Daminet V,Haumont M,Garcia L,Bollen A,Jurado M,Biemans R.The surface antigen SAG3 mediates the attachment of Toxoplasma gondiitocell-surface proteoglycans.Mol Biochem Parasitol 2001;116:35-44. 83. Khanaliha K,Motazedian MH,Kazemi B,Shahriari B,Bandehpour M,Sharifriya Z.Evaluation of Recombinant SAG1,SAG2,andSAG3Antigens for serodiagnosis of Toxoplasmosis.Korean J Parasitol 2014;52(2):137-142. 84. Drapala D,Holec-Gasior L,Kur J,Hiszczyriska-SawickaE,Lautenbach D.Anew human IgG avidity test ,using mixtures of recombinant antigens(rROP1,rSAG1,rGRA6)for the diagnosis of difficult to identify phases of toxoplasmosis.Diagn Microbiol Infect Dis 2014;79(3):342-6. 85. Montoya JG,Remington JS.Management of Toxoplasma gondii infection during pregnancy.Clin Infect Dis 2008;47554-566. 86. Montoya JG.Laboratory diagnosis of Toxoplasma gondii infectionand toxoplasmosis.J Infect Dis 2002;185:Suppl1:s73-82. 87. Berth M,Bosmans E.Comparisons of three automated immunoassay methods for the determination of Epstein Barr virus specific immunoglonin M.Clin Vaccine Immunol 2010;17:559-563. 88. Binnicker MJ,Jesperson JD,Harring JA.Evaluation of three multip[lex flow immunoassay s compared to the an enzyme immunoassay for the detection and differentiation of IgG class antibodies to herpes simplex virus type 1 and 2.Clin Vaccine Immunil 2010;17:253-257. 89. Klutts JS,Liao RS,Dunne Jr WM,Gronowski AM.Evaluation of a multiplex bead assay for assessment of Epstein –Barr virusimmunologic status.J Clin Microbiol 2004;42:4996-5000. 90. Binnicker MJ,Jespersen DJ,Harring JA.Multiplex detection of IgM and IgG class antibodies to Toxoplasma gondii,Rubella virus and cytomegalo virus using a multiplex flow immunoassay Clin Vaccine Immunol 2010;17(11):1734-1738. 91. Eskandarian AA.Improving sample efficiency as a convenient alternative for invasive samples in molecular diagnosis of toxoplasmosis.Iranian J Parasitol 2013;8(1):177-181. 92. Murat JB,Dard C,Fricker Hidalgo H,Darde ML,Brenier-Pinchart MP,Pelloux H The VIDAS system for the diagnosis in pregnant women and newborns :comparison with two recently fully automated assays.Clin Vaccine Immunol 2013 Jun 5 epub ahead of print.
93. Berrebi A,Bardon M,Bessieres MH et al.Outcome of children infected with congenital toxoplasmosisin the first trimesterand with normal ultrasonography findings:a study of 56 cases.Eur J Obstet Gynecol Reprod Biol 2007;135:53-57. 94. Canley FK,Jenkins KA,Remington JS.Toxoplasma gondii infection of the central nervous system :use of the peroxidase-antiperoxidase method to demonstrate Toxoplasma in formalin fixed,paraffin embeddedtossue sections.Hum Pathol 1981;12:690-698. 95. Romand S,Wallon M,Franck J,Thulliez P,Peyron F,Dumon H.Prenatal diagnosis using polymerase chain reaction on amniotic fluid for congenital toxoplasmosis.Obstet Gynecol 2001;97(2):296-300.
96. Grover CM,Thulliez P,Remington JS,Boothroyd JD.Rapid prenatal diagnosis of congenital Toxoplasma infection by using polymerase chain reactionand amniotic fluid.J Clin Microbiol 1990;28:2297-301. 97. Hohlfeld P,Daflos F,Costa JM,Thulliez P,Forestier F,Vidaud M.Prenmatal diagnosis of congenital toxoplasmosis with polymerase chain reaction test on amniotic fluid.NEngl J Med 1994;331:695-9.
98. Paquet C,Yudin MH.Toxoplasmosis in pregnancy:prevention,screeningand treatment.J Obstet Gynaecol Can 2013;35(1):78-9. 99. Gilbert R,Gras L.European multicentre study of Congenital toxoplasmosis :Effect of timing and type of treatment on the risk of mother to child transmission of Toxoplasma gondii.BJOG 2993;110:112-120. 100. Desmons G,Couvieur J.Congenital toxoplasmosis:a prospective study of the offspring of 542 women who acquired toxoplasmosisduring pregnancy.In:Thalhammer O,Pollack A,Baumgarten K ed.Perinatol Proceedings th of the 6 European Congress in ,Vienna ,Stuttgart ,Germany;Georg Theme Publishers 1979;51-60. 101. Forrestier F.Les foetopathiesinfectieuses:prevention,diagnostic,prenatal,attitudepratique.Presse Med 1991;20:1448—1454. 102. Couvreur I.Desmonts G,Thulliez P.Prophylaxis of congenital toxoplasmosis:effects of spiramycin on placental infection.J Antimicrob Chemother 1988;22:193-200 103. Thiebaut R,Leproust S,Chene G,Gilbert R.Effectiveness of prenatal treatment for congenital toxoplasmosis:a metaanalysis of individual patient data.Lancet 2007;369:113-122. 104. Li XL,Wei HX,Zhang H,Peng HJ,Lindsay DS.Ameta analysis on risks of adverse pregnancy outcomes in Toxoplasma gondii infection.PLoS One 2014;9(5):e97775.
105. Vogel N,Karists M,Michael F,Bach M,Hostetter M,Boyer K,et al.Congenital toxoplasmosis transmitted from an immunologically competent mother infected before conception..Clin Infect Dis 1996;23(5):1055-60. 106. Gavinet MF,Robert F,Firtion G,Delouvrier E,Heenequin C,Maurin JR et al.Congenital toxoplasmosis due to maternal infection during pregnancy.J Clin Microbiol 1997;33(5):1276-7. 107. Berrebi A,Assaouline C,Bessierees MH,Lethiere M,Cassaing SMinvilleV et al.Longterm outcome of children with congenital toxoplasmosis.Am J Obstet Gynecol 2010;203(6):552e1-6 108. Wilson CB,Remington JS,StagooS,Reynolds DW.Development of adverse sequelae in chi;ldren born with subcliunical congenital toxoplasma infection.Pediatrics 1980;66(5):767-74. 109. Mitchell CD,Edich SS,Mastrucci MT,Hutto DC,Parks WP,Scott GB.Congenital toxoplasmosis occurring in infants perinatally infected with human immunodeficiency virus1.Pediatr Infect Dis J 1999;9:512-518. 110. Garweg R,Scherrer I,Wallon M,Kodjikian I,Peyron F.Reactivation of ocular toxoplasmosis during pregnancy.BJOG 2003;112:241-2. 111. Montoya JG,Remington JS.Toxoplasma chorioretinitisin the setting of acquired toxoplasmosis during pregnancy.Clin Infect Dis 1996;23:277-282. 112. Bulow R,Boothroyd JC.Protection of mice from failed Toxoplasma gondii infection by immunization with p30 antigen in liposomes.J Immunol 1991;147:3501-3506. 113. Velge-Roussel I,Marcelo P,Lepange AC,Buzoni-Ganel D,Danier-Poisson J,Ferer M et al.Intranasal immunization with Toxoplasma gondii SAG1 induces protective cells into both NAL1 and GAL 1compartments.Infect Immun 2000;68:969-972. 114. Beghetto F,Nielsen HV,Del Porto P,Buffolano W,Guglietta S et al.Acombination of antigenic regions of Toxoplasma gondii microneme proteins induces protective immunity against oral infection with parasite cysts.J Inf Dis 2005;191:637-645. 115. Ismael AB,Sekkai D,Collin C,Bout D,Mvelrc MN.The MIC3 Gene of Toxoplasma gondii is a novel potent vaccine candidate against toxoplasmosis.Inf and Immunity 2003;71(11)6222-6228. 116. Zhao G,Zhou A,Lu G,Meng M,Sun M,Bai Y,Han Y,Wang L,Zhou H,Cong H,Zhao Q,He S.Identificationand characterizationof Toxoplasma gondii aspartic protease 1 as a novel vaccine candidate against toxoplasmosis.Pasrasites and Vectors.2013;6175-188. 117. Jerant PV,Milosevic V,Hrnjakovic-Cvjetkovic I,Patic A,Stefan-Mikic S,Ristic M.Toxoplasma gondiiinfection in pregnant women.Med Pregl 2013;66(1112):459-63. 118. Capobiango JD,Mitsuka Bregano R,Navarro IT,Rezende Neto CP,Barbante CasellaAM,Ruiz Lopes Mon FM,Paglian S,Inoue IT,Reiche em.Congenital Toxoplasmosis in a reference center of Parana,Southern Brazil.Braz J Infect Dis 2014;(epub ahead of print)doi:10.1016/jbjid.2013.11.009. 119. Siala E,Ben Abdallah R,Laouti F,Maatoung R,Souissi O,Aoun K,Bouratbine A.Toxoplasmic infections in pregnancy :about94 bcases diagnosed at the Pasteur Institite of Tunis.Gynecol Obstet Fertil 2014;42(5):312-6.
120. Andappian H,Nissapatom V,Sawangiaroen N,ChemonW,Lau YL,Kumar T,Onichandran S,Suwanrath C,Chandeying V.Toxoplasma infection in pregnant women :a current status in Songklanagarind hospital ,southern Thailand.Prasit Vectors 2014;7:239. 121. Andiappan H,Nissapatom V,Sawangjaroen N,Khaing SL,Salibay CC,Cheung MM,Dunca JZ,ChemohW,Xiao Teng C,Lau YLMat Adenan NA.Knowledge and practices on Toxoplasma infection in pregnant women from Malaysia,Philippines,andThailand.Front Microbiol 2014;jun 11;5:291. .
