ISSN 1746-0913part ofFuture MicrobiologyFuture Microbiol. (2009) 4(10), 13631379 10.2217/FMB.09.89 2009 Future Medicine1363General aspects, lifecycle & transmission Toxoplasmagondiiisanapicomplexanpara-site that infects approximately one third of the worldspopulation[1].Itiswidelydistributed and can be foundinmanydifferent species of mammals and birds.ThelifecycleofT.gondiiwasdescribedin 1970, when it was determined that members of the family Felidae, including domestic cats, were the denitive hosts and various warm-blooded animals serve as intermediate hosts. T. gondii is transmitted by three known routes: congenitally, through the consumption of uncooked infected meat and via fecal matter [14,201].Infection of the denitive host occurs follow-ingingestionofmeatcontainingtissuecysts. However, infection can also occur as a result of ingestingtherapidlymultiplyingforms(tach-yzoites)ortheoocystsshedinfeces.Thecyst wall is dissolved by the proteolytic enzymes in thestomachandsmallintestine,releasingthe slow-multiplyingbradyzoitestage.Theforma-tionofnumerousasexualgenerationsbegins afterparasiteinvasionoftheepithelialcellsof thesmallintestine.SexualstagesofT.gondii arehighlyspecic, occurringonlywithingut epithelialcellsoffelinespecies.Oocystsare producedbygametefusionandarethenshed inthefeces.Onceincontactwiththeatmos-phere, the oocysts sporulate to form sporozoites and becomeinfectivetootherdefinitiveor i ntermediate hosts [3,5,201]. Followinginfectionofintestinalepithelial cells of the intermediate host, the infective stages (sporozoites or bradyzoites) transform into tach-yzoites, which multiply rapidly by endodyogeny within an intracellular parasitophorous vacuole. When the cells become packed with tachyzoites, thehost-cellplasmamembrane rupturesand parasitesarereleasedintotheextracellular milieu. Thefreetachyzoitescantheninfect any nucleated cell they encounter and continue intracellularreplication,spreading throughout host tissues. If not controlled by the immune sys-tem, tachyzoites are highly virulent and cause a generalized toxoplasmosis, which is always fatal. HostT-cell-mediatedimmuneresponsesplay an important role in suppression of tachyzoite replication and resistance to T. gondii, resulting in chronic infection or possibly clearance of the parasites [1,2,4,201].Humaninfection isgenerallyinnocuous, asymptomatic and commonly acquired by inges-tion of undercooked or raw meat containing tis-suecysts,orwaterorfoodcontaminatedwith oocystsexcretedinthefeces.Thesebiological characteristics, eating and hygiene habits of pop-ulations are closely related to high prevalence of serum positivity in the different regions, as the oral route is the major source of infection. The infectionriseswithage,doesnotvarygreatly between sexes, and is most common in temperate and hot regions [69].The parasite can also be transmitted by vertical transmissionoftherapidlygrowingtachyzoite form if a mother acquires a new infection dur-ingpregnancy.Theinfectioncancausesevere neonatalmalformationsandocularcomplica-tions in the fetus. Around 1020% of cases of T. gondii infection are symptomatic as ocular and disseminated forms [7,8,10,11,201]. Over the last few decades, the transmission of T. gondii by organ transplantationfromseropositivedonorsto seronegative recipients has become an important issueintransplantpatients[12,13].FIGURES1A&B show the T. gondii lifecycle, as well as the forms Toxoplasma gondii infection and cerebral toxoplasmosis inHIV-infected patientsVera Lucia Pereira-Chioccola, Jos Ernesto Vidal & Chunlei SuAuthor for correspondence: Laboratrio de Parasitologia, Instituto Adolfo Lutz, Av. Dr Arnaldo, 351, 8 andar, CEP 01246902, So Paulo, SP, Brazil n Tel.: +55 11 3068 2991 n Fax: +55 11 3068 2890 n pchioccola@ial.sp.gov.brCerebraltoxoplasmosi si samajorcauseofmorbidityandmortal ityamong HIV-i nfectedpati ents,par ti cul arl yfromdevel opi ngcountri es.Thi sar ti cl e summarizes current literature on cerebral toxoplasmosis. It focuses on: Toxoplasma gondii genetic diversity and its possible relationship with disease presentation; hostresponsestotheparasiteanti gens;hosti mmunosupressi oni nHIVand cerebraltoxoplasmosi saswellasdifferentdiagnosticmethods;clinicaland radiologicalfeatures;treatment;andthedi rectionthatstudiesoncerebral toxoplasmosis will likely take in the future.Keywordsn AIDS n cerebral toxoplasmosis n diagnosis n genotyping n review n Toxoplasma gondiiReviewFuture Microbiol. (2009) 4(10) 1364 future science groupReviewPereira-Chioccola, Vidal & SuDefinitive host Toxoplasmosis transmission Oocysts Fecalcontamination Ingestion of uncooked meatIntermediate hosts infectedAcute infectionChronic infection Cat and otherfeline speciesCongenitalrouteDefinite hosts:cats and other feline speciesSporozoiteoocystsOnly tachyzoites(gropus)Many bradyzoites(cysts)ChronicinfectionCongenitalinfectionAcute infectionImmunityIngestionof oocystOocyst2040 days410 days36 days minimalprepatent periodSporogonyin stool 34 daysCarnivorismIntermediate hosts:rodents, dogs, humanand other mammals and birdsCystsPrimary infectionLatent infection(asymptomatic)During the pregnancy(transplacental transmission)Oral (consumption of infected water or meat) Organ transplantationCongenital infectionReactivation (occurred in immunosuppression)Cerebral toxoplasmosis1020% symptomatic(lymphadenitis, chorioretinitis)8090%asymptomaticFigure 1. Lifecycle of Toxoplasma gondii, transmission and clinical forms of toxoplasmosis. (A) T. gondii lifecycle: felines serve as denitive hosts and are infected by the consumption of meat containing tissue cysts with bradyzoites. The sexual development occurs within the small intestine. Oocysts are formed after fusion of the micro- and macro-gametes, and are shed in the feces. The transmission to the intermediate host (B & C) occurs by ingestion of oocysts, normally in food or water. Infections can also occur via organ transplantation. The acute infection is characterized by fast-growing tachyzoites after invasion within any nucleated cell and subsequent host-cell lysis and reinfection of more cells. Concurrent with the development of immunity, tachyzoites transform into slow-growing bradyzoites, which reside within cysts in the muscles and brain. Around 1020% of the infected individuals develop the systematic form, but the majority of the cases (8090%) are asymptomatic. The chronic infection can persist for the life of the hosts. In immunodecient hosts, bradyzoites reactivate, which causes cerebral toxoplasmosis. When the primary infection occurs during pregnancy, the parasites can also infect the fetus by congenital transmission.Reproduced with permission from [188].www.futuremedicine.com 1365 future science groupRole of Toxoplasma gondii infection in HIV-infected patientsReviewof transmission. The scheme showed in FIGURE 1C demonstrate the transmission routes and clinical forms of toxoplasmosis.Diversity among T. gondii strains GeneticdiversityofT.gondiistrainshas beenaninterestingandimportantsubject ofresearch.Overthelastfewdecades,the developmentofhighlysensitiveandsimple molecularmethodshasfacilitatedthedetec-tion, diagnosis and genotyping of this impor-tant parasitic pathogen. With these molecular methods,itispossibletostudythevariation ofvirulenceamongdifferentparasitestrains, torevealpotentialcorrelationbetweenpara-site genotype and disease patterns in infected patients,andtostudytheepidemiologyand population biology of T. gondii [4].Typing of T. gondii has been achieved based on antigenic variation [14,15], multilocus enzyme electrophoresis[1619],microsatellitesequence polymorphisms[2023],PCRrestrictionfrag-ment length polymorphism (PCR-RFLP) [2428] orDNAsequencetypingofgenesorintrons [2931].StudiesusingPCR-RFLPmarkers revealed that most T. gondii strains from North America,EuropeandAfricacanbedivided into three major groups: type I, II and III line-ages [24,25,31]. Among the three lineages, type I strainsarehighlyvirulentwhiletypeIIand typeIIIareoflowvirulence[24].TypeIand typeI-likestrainsareassociatedwithocular toxoplasmosis,acuteoutbreaksandcerebral toxoplasmosis[3236].Itissuggestedthatthe highvirulence oftypeIstrainsisinpartdue to over stimulation of a Th1 immune response, which leads to pathology [36]. Genotypingstudieswereappliedtoagreat numberofisolatesfromdomesticanimals [27,28,3741],humancongenitalinfection[42,43], human ocular infections [44] and AIDS patients [37,4547].However,earlystudiesusingasingle geneticmarkerwerelimitedtotheidentica-tionofdistinctisolates.Withtheadventof multilocusPCR-RFLPandmicrosatellitetyp-ingmethods,high-resolutiongenotypingwas achieved [22,26]. In North America, Europe and Africa, most isolates belong to the clonal type I, II and III lineages, and there is no host bound-aryfordifferentgenotypes[17,24,25,32,48,49].In theseregions,typeIIstrainsarepredominant andarecommonlyisolatedfromclinicalcases oftoxoplasmosis.Limiteddatasuggestedthat T. gondii population structure in Asia was simi-lartoNorthAmerica,EuropeandAfrica[39]. AttheDNAsequencelevel,thethreelineages are highly similar, differing by less than 1% on average,anditwassuggestedthatthesethree lineageswereexpandedgloballyinthepast 10,000 years [50,51]. However,theideathatT.gondiiwasclonal withverysmallgeneticvariabilitywaschal-lengedbystudiesofT.gondiistrainsfrom Brazil and French Guiana. Genotyping results from these regions showed a higher genetic vari-ability,withdistinctgenotypesnotyetidenti-fiedinNorthAmerica,EuropeandAfrica [21,23,27,28,30,31,47,5256].Analysisof125isolates fromdomesticanimalsinBrazilrevealed48 genotypes, four of which had multiple isolates from different hosts and locations, and they were consideredtobethecommonclonallineages in Brazil [27,55]. In contrast to North America, Europe and Africa, the type II lineage has not beenreportedinBrazilandthetypeIstrains are rare; in this region, most strains are unique [27,28,30,47,53].ItwassuggestedthatT.gondii strainsinSouthAmericahaveahighrateof transmissionandoutcrossing[21,23].Genetic exchange could occur when the denitive host ingestsdifferenttypesofparasitesfromtheir intermediatehostsatnearlythesametime,or the intermediate host has mixed infection of dif-ferentstrains.TheT.gondiiisolatesinSouth Americaseemtohavedifferentphysiological characteristicssinceinsomecasestheyare highlypathogenicinimmunocompetentindi-viduals [47,52,57]. Most of the PCR-RFLP genetic markers were originally developed based on the type I, II and III lineages, which may miss the uniqueallelesthatareonlypresentinSouth Americaand,therefore,mayunderestimate genetic diversity in phylogenetic studies [51,56]. However,forepidemiologicalstudies,these markers are easy to use and can quickly identify T.gondiistrainswithhighresolution,provid-ingcrucialinformationtotracethesourceof infection. Another approach to analyze T. gondii strains with high resolution is multilocus DNA sequence typing [30,31]; however, this method is expensive and time consuming. Ahighprevalenceoftoxoplasmosisexistsin the Brazilian population and there is a consider-able incidence of cerebral toxoplasmosis in AIDS patients. The parasites are easily detected in the bloodandcerebrospinaluid(CSF)ofthese patients[58,59]withanelevatedmorbidityand mortality [60]. However, at present there is lim-ited information regarding genotyping of T. gon-dii samples from human patients, which makes it difcult to determine if parasite genotypes are associated with disease presentations. With the Future Microbiol. (2009) 4(10) 1366 future science groupReviewPereira-Chioccola, Vidal & Suavailable multilocus PCR-RFLP method [26,28,40] itwillbepossibletoaddressthisquestionin future studies.T. gondii antigens & host defensesInfected intermediate hosts, including humans, may die from toxoplasmosis. However, infection is often recovered after the immunity acquisition. The course of infection in humans depends on the doses of inoculated parasites, parasite genetic background, host genetics and immunological sta-tus [61,62]. In the classical infection, orally ingested parasites actively invade intestinal epithelial cells or are phagocytosed by these cells [50,63]. Theinvasionprocessinitiatesthelyticcycle, leadingtocellandtissuedestruction,which results in Toxoplasma pathology. In the host-cell cytoplasm, T. gondii induces the formation of a parasitophorousvacuolethatcontainssecreted parasite proteins and host proteins that normally promote phagosome maturation, thereby prevent-ing lysosome fusion [2,50,64]. This process is rapid, dynamic and relies on the secretion of numerous secretory proteins from micronemes, rhoptries and dense granules [64]. Despite signicant progress in studying these proteins, only a limited number of secretoryproteinshavebeendiscovered[65,202]. The active secretion of these antigens is an essen-tialcomponentof thelow-gradestimulationor boosting of the immune system, as these antigens have been shown to stimulate antibody produc-tion as well as a T-cell response [64,66]. A group of important antigens has been classied as parasite excretory/secretory antigens (ESAs), which rep-resentsthemajorityofthe circulatingantigens in sera from hosts with acute toxoplasmosis. ESA proteinsareexpressedatthetachyzoite,sporo-zoiteandencysted bradyzoitestages[67].Ithas beenproposedthatsecretionbythebradyzoite cystsmaintainslong-lastingimmunityto the parasite[68]. Ontheotherhand,ESAsreleased by tachyzoites are highly immunogenic [64,69] and induce protective immunity, which may be either antibody dependent or cell mediated [7072]. CD4+ T cells specic for ESAs may be involved in the maintenance of long-term immunity in healthy chronically infected individuals. From these cells, theantigensSAG2A[73],SAG1[74],aswellas severaldensegranuleproteins[7477]areabun-dant and highly immunodominant in the stimu-lation of the B-cell response. Immunocompetent infected individuals had low anti-ESA antibody titers, whereas patients with cerebral toxoplasmo-sis and AIDS, and consequently with circulating blood tachyzoites, develop high titers of anti-ESA antibodies [78].TheinfectioncausedbyT.gondiiresults ininammation,usuallyfollowedbynecro-sis.Likemanyobligateintracellularmicrobial pathogens, T. gondii infection induces a strong type1polarizedimmuneresponse, engaging both innate and adaptive immune systems. It is clear that the major mechanism of host resistance totoxoplasmosis ismediatedbyproductionof pro-inammatorycytokines,includingIL-12, IFN-g and TNF-a [79,80]. The major sources of IFN-g, which is an import key in the response to infection, are CD4+ Th1 lymphocytes, CD8+ T cells, natural killer cells and T cells respond-ing to IL-12 [81,82]. These mechanisms prevent rapid replication of tachyzoites and subsequent pathological changes [80,83,84]. After23weeksofinfection,thecombina-tion of the production of cytokines, IgG, IgM, IgA and IgE antibodies against many T. gondii proteins,extracellulartachyzoitesarecleared from host tissues and intracellular parasites dif-ferentiateintolatentbradyzoiteforms,which are surrounded by the parasitophorous vacuole that is enclosed in a cyst wall. The tissue cysts preferentially reside in neural and muscular tis-sues.Thisdifferentiationcanbeincreasedby exposureoftheorganismtostressconditions such as in immune response to the tachyzoites. TlymphocytesareacriticalsourceofIFN-g duringthisstageoftheinfection,asshown severalyearsagoinantibody-mediatedT-cell depletion experiments that resulted in reactiva-tion of infection [83]. In some cases, tachyzoites may persist longer in the spinal cord and brain because immune responses are less effective in these organs. The control of intracerebral para-sites is also dependent on IFN-g-producing CD4 andCD8Tcells,whicharerecruitedtothe brain[79,80,85,86].Theability ofbradyzoitesto escape the host immune response and persist in a quiescent form within the host is another event in the T. gondii lifecycle. In the chronic phase, the tissue cysts can persist indenitely in the brain and muscle, developing lifelong protectiveimmunityagainstre-infec-tion [1,2], although in some cases re-infections are possible, since different T. gondii genotypes were shown in the same patient [47,87]. In this clinical phase, tissue cysts are periodically ruptured, but the bradyzoites released are normally destroyed by the host immune response. Nevertheless, dur-ing the chronic infection around 1020% of the infected immunocompetent individuals are reac-tive, with the rupture of a tissue cyst in the eyes, brain or muscles causing local necrosis accompa-niedbyinammation.Hypersensitivityplaysa www.futuremedicine.com 1367 future science groupRole of Toxoplasma gondii infection in HIV-infected patientsReviewmajor role in such reactions; however, the infec-tionusuallycollapses,withnolocalconverted multiplication of tachyzoites [1]. When asymptomatic individuals present some immunodeciency, reactivation of latent infec-tion may occur, culminating in the conversion of bradyzoites to the active and rapidly replicating tachyzoites,resultinginatissueinjurythatis often fatal. As the cysts have a predilection for neural and muscle tissue as well as the eye, most casesofreactivationleadtochorioretinitisor, more frequently, cerebral toxoplasmosis, which is a life-threatening condition [1,88]. Potentialcorrelationsbetweenthedevelop-ment of cerebral toxoplasmosis and HLA genes (class I and class II) in HIV-patients have been studiedinrecentyears.TheMHCisoneof the most polymorphic genetic systems of many species, including HLA in humans. The MHC controls the adaptive immune response against intra-andextracellularmicroorganismsby class I (HLA-A, HLA-B, HLA-Cw) and class II (HLA-DRB1, HLA-DQB1, HLA-DPB1) and is correlated with infection susceptibility or resist-ance. The association between susceptibility to different diseases and HLA molecules, as well asthedistributionofHLAallelesinlinkage disequilibrium,areimportantfactorsinthe MHC.Linkagedisequilibriumisthesitua-tion in which two specic alleles from separate locicloselylinkedtoeachotheraretransmit-tedtogetheronthesamechromosome[8992]. Class I HLA-B35 antigen was associated with thesusceptibilitytochorioretinitis[93],and classIHLA-B8andclassIIHLA-DRB1*17 antigenswereassociatedwithsusceptibility to cerebral toxoplasmosis [94]. The presence of classIIHLA-DQB1*0402andDRB1*08alle-les [95] and the HLA-DR52 haplotype represent risk factors to the development of cerebral toxo-plasmosis,whereastheHLA-DR53haplotype was associated with infection resistance [96].AIDS & cerebral toxoplasmosisThe introduction of HAART for the treatment of HIV infection has resulted in dramatic reduc-tions in morbidity, mortality and healthcare uti-lization[97].Decreasingratesofopportunistic diseases, including neurological infections, have been reported both in developed and developing countries with access to HAART. However, the impact of HAART seems to be lower in devel-oping countries with access to HAART owing todelayeddiagnosisofHIVinfectionorlack ofopportunitiestostarttreatmentinpatients priortodiagnosisofHIV[98100].Cerebral toxoplasmosis is an HIV-indicative event in 35% of patients and an AIDS-dening event in 75% of cases [60].In addition, neurological infections continue to cause high rates of morbidity and mortality indevelopingcountrieswithoutavailabilityof HAART, where the patients usually present the natural history of most diseases. Cerebraltoxoplasmosisisusuallythemost common cerebral opportunistic disease in both developed and developing countries [101,102]. In some places, particularly in Africa, cases of cer-ebral toxoplasmosis are only exceeded by cases of cryptococcal meningoencephalitis. Globally, T. gondii causes the most common focal brain lesioninHIV-infectedpatients[88,100,103].The incidence of toxoplasmosis varies by country and depends on the prevalence of T. gondii infection in the general population. Differences in geno-types of T. gondii isolates, races and ethnicities and the mode of transmission also seem to inu-encetheoccurrenceoftheinfection[35].Data areavailableregardinginfectionprevalencein differentpartsoftheworld.Thedataindicate thataround25%ofAIDSpatientsfromParis had cerebral toxoplasmosis in the pre-HAART era compared with 10% in some cities from the USA [104,105]. The rate in the USA and UK was found to vary between 16 and 40%, in Spain it was approximately 60%, in Brazil 5080% and in France 7590% [11,106]. Diagnostic approachThe denitive diagnosis of cerebral toxoplasmo-sis requires the presence of the tachyzoite form oftheparasiteincerebraltissuetobedirectly demonstrated. In clinical practice, presumptive cerebral toxoplasmosis diagnosis depends on an association of serological, clinical and radiologi-cal information [107]. Diagnosis is conrmed with aresponsetoempiricanti-Toxoplasmatherapy. A favorable clinical and radiological response is expected within 1014 days of specic treatmentTherearenopathognomonicclinicalor radiological ndings of cerebral toxoplasmosis. Thus,differentialdiagnosisofAIDSpatients with extensive brain lesions is essential and two factorsshouldbealwaysconsidered:thelocal neuroepidemiology and the degree of immuno-suppression in the host [108]. Differential diagno-sis of expansive brain lesions presents geographic particularities. In developed countries, primary lymphoma of the CNS constitutes the main dif-ferential diagnosis of cerebral toxoplasmosis [103]. In developing countries, focal forms of cerebral TB (tuberculomas and, less likely, tuberculous Future Microbiol. (2009) 4(10) 1368 future science groupReviewPereira-Chioccola, Vidal & Subrainabscess)arethemainalternativediag-noses[109].PrimarylymphomaoftheCNS usuallypresentswithaCD4cellcountbelow 50 cells/mm3, cerebral toxoplasmosis frequently below 100 cells/mm3, and cerebral TB usually below 200 cells/mm3 [60]. Of these three etiolo-gies, cerebral TB is more likely to present with CD4 cell count above 200 cells/mm3 [60,110,111]. Inadditiontothesemorefrequentneurologic complications,thedifferentialdiagnosisof expansive brain lesions in HIV-infected patients isbroadandincludesotherinfectionssuchas cryptococcosis,aspergillosisandChagasdis-ease; AIDS- and non-AIDS-associated tumors suchasmetastasesofdisseminatedlympho-mas and glioblastoma multiform, respectively; andvasculardiseases.Forthesereason,more invasive approaches such as stereotactic biopsy should be considered in all HIV-infected patient with expansive brain lesions empirically treated for cerebral toxoplasmosis that do not respond toantiparasitictreatmentwithin1014days. However, at least 10% of cerebral toxoplasmo-siscasesdieddespitewhatwasthoughttobe adequatetreatment[60].Moleculardiagnosis using CSF or peripheral blood samples is a use-ful tool for early, minimally invasive diagnosis of cerebral toxoplasmosis [59,112,113]. However, in clinical practice, results should always be inter-pretedinassociationwithserological,clinical and radiological information.Clinical manifestations& radiological diagnosisCerebral toxoplasmosis causes unifocal or, more commonly,multifocallesionsand,lessfre-quently,diffuseencephalitis.Patientsusually presentsubacutemanifestations,butitcanbe acute in around 10% of cases. The clinical man-ifestations depend on the location and number oflesions.Morefrequentcomplaintsinclude: headache(4963%),fever(4168%),focal decits (2280%), seizures (1929%), mental confusion (1552%), ataxia (1525%), lethargy (1244%), cranial nerve alterations (1219%) andvisualalterations(815%).Othermani-festationsincludedisarthria,cognitivedys-function, intracranial pressure and involuntary movements [60,103,107,114116]. Imagingstudies,eithercomputedtomogra-phy (CT) or MRI, are essential for the presump-tive diagnosisof cerebral toxoplasmosis[97,103]. MRIismoresensitive,particularlyforiden-tifyingsmalllesionsandthoselocatedinthe posteriorfossa.Singlelesionsareobservedin approximately 30% of the patients by CT, but by MRI it is common to identify two or more lesions. Radiological diagnosis can be classied as typical or atypical patterns [60]. Typical pat-terns are observed in around 80% of cases and include hypodense lesions with ring-enhancing and perilesional edema, and hypodense lesions with nodular-enhancing and perilesional edema. Atypical patterns are shown in around 20% of cases and are hypodense lesions without contrast enhancing and with expansive effect, CT with-out focal lesions and MRI demonstrating focal lesions, and diffuse cerebral encephalitis without visible focal lesions.Ahighlysuggestiveimagefortoxoplasmosis, althoughunusual,istheeccentrictargetsign, whichisasmallasymmetricnodulealongthe wall of the enhancing ring. FIGURE 2 shows the main radiological features of cerebral toxoplasmosis in HIV-infected patients.Immunological diagnosis Thehighprevalenceandlifelongpersistence of anti-T. gondii IgG antibodies among healthy individuals in many geographical areas prevent the use of titers in serologic tests to reect recent infection. Another problem is often the lack of reliabilityindiscriminatingrecentfrommore distantinfectionbydetectionofanti-T.gondii IgM, IgA or IgE. In this setting, physicians are often faced with conicting results and disagree-ments about interpretations of results. This often leads to incorrect information being provided by thelaboratoriestothephysiciansaswellasby the physicians to their patients. This situation is very common in congenital toxoplasmosis diag-nosis [117]. In the case of cerebral toxoplasmosis, theidenticationofapositiveserologyisless usefulinsettingswheretheseroprevalencefor T. gondii in the general population is very high. In Brazil, most AIDS patients with focal brain lesionalsoshowpositiveserologicaldiagnosis fortoxoplasmosis.However,theidentication ofanegativeserologicalresultpresentsahigh negative predictive value. [108].Mostpatientshaveserologicalevidence ofinfection,usuallywithhightitresof IgG[59, 60,118]withhighavidity,supporting theideathatthereactivationofthelatent infectionoccursinthesecondaryimmune response[59,119].However,theseantibodies maynotbedetectedinupto5%ofpatients owing to immune suppression. Earlier studies showedthatdifferentlevelsofanti-T.gondii IgG antibodies were inadequate to determine a reactivation or to follow the course of cerebral toxoplasmosis[88,120122].Astheseantibodies www.futuremedicine.com 1369 future science groupRole of Toxoplasma gondii infection in HIV-infected patientsReviewareusuallypresentincerebraltoxoplasmosis, some studies suggested that, statistically, high titers might be indicative of the active disease orahigherriskofdevelopingit[59,123,124]. Thus, a negative serological result or low titers do not exclude a positive diagnosis for cerebral toxoplasmosis and must not delay the start of empiric treatment of cerebral toxoplasmosis in AIDSpatientswithcompatibleclinicaland radiological ndings [60,78,115,120,121,125].In general, anti-T. gondii antibodies are deter-mined in conventional serology, such as ELISA andimmunouorescence,usingtotalextract oftachyzoitesasanantigen,whichconsists ofcytoplasmaticandmembranecomponents. In addition, several studies have demonstrated theusefulnessofrecombinantantigensfor the serological diagnosis of T. gondii infection [126130].TheseantigensusedinELISAfailed to distinguish sera from patients with cerebral toxoplasmosisandasymptomaticinfected immunocompetent individuals, with no or low numbers of circulating tachyzoites, as they have similar reactivity.On the other hand, ESAs constitute an excel-lent serological marker for the diagnosis of cer-ebraltoxoplasmosisinHIV-infectedpatients astheyareproducedbytachyzoites,theform responsiblefordisseminatingtheinfection, which plays an important role in stimulation of the humoral and cellular immune responses to control infection [131133]. Numerous tachyzoites are released from the quiescent cysts and a con-siderableproportionofexcreted/secretedanti-gens are released, eliciting the specic immune responsetoESAs.Unlikeasymptomaticindi-viduals,thesepatientspresentantibodiesfor both ESAs and total crude tachyzoite antigens. WhenESAsareusedasantigensinELISA (ESA-ELISA) and in immunoblot it is possible to distinguish sera from patients with the active disease.Normally,theseseraarethreetimes more reactive than those from seropositive indi-viduals(FIGURE3A,B,E&F)[78].Thus,anti-ESA antibodieswerepresentprincipallyinpatients withactivedisease,suggestingitsimportance, particularly in regions with high prevalence of latent toxoplasmosis in the general population.Previousstudiesanalyzingthepresenceor absence of oligoclonal bands of IgG (OCBs) in serumandCSFdemonstratedthattheintra-thecalhumoralimmuneresponsecanbepro-duced independent of the systemic one. In other situations IgG may be produced in the systemic immune activation but not in intrathecal syn-thesis. In this case the presence of OCBs in the Figure 2. Computed tomography images showing the spectrum of radiological ndings of cerebral toxoplasmosis in HIV-infected patients. Hypodense lesion with ring-enhancing and perilesional edema (A); nodular enhancing and perilesional edema (B); without contrast enhancing and with expansive effect (C). CT scan with contrast enhancement showed no abnormalities (D) and corresponding T2-weighted MRI showed multiple basal ganglia focal lesions, with high-intensity signals (E). T1-weighted MRI showed a ring-enhancing brain lesion with a small, enhancing asymmetric nodule along the wall of the lesion (F) (the eccentric target sign). The arrows show the abnormalities.Future Microbiol. (2009) 4(10) 1370 future science groupReviewPereira-Chioccola, Vidal & SuCSFisexplainedbytheirpassivemovement from the serum into the CSF across the bloodbrain barrier, which does not strongly obstruct serumproteins[134].However,theintrathecal anti-T. gondii IgG antibody production increases inpatientswithcerebraltoxoplasmosisand AIDS [135] and is normally characterized by the presence of T. gondii OCBs [136].For some authors, the immunological diagno-sis value in CSF is limited because the sensitivity and specicity are approximately 6070% [115]. Anti-ESA IgG antibodies are also present in CSF in patients with AIDS and cerebral toxoplasmo-sis and can be determined by ESA-ELISA and immunoblot. CSF samples from these patients clearlydistinguishfromthosewithAIDSand who are seropositive for toxoplasmosis but with other neurological diseases (FIGURE 3C & D). Molecular diagnosisOver recent decades, the development of molec-ular methodologies essentially based upon PCR have allowed the sensitive detection of T. gondii DNAinclinicalspecimenssuchasamniotic uid,aqueoushumor,CSF,bonemarrowand blood [58,59,136146]. Molecular methods are par-ticularlyappropriateforAIDSpatients,since these methods are not affected by immunologi-cal status and have been shown to be rapid, sensi-tive and specic, avoiding the need for invasive and expensive brain biopsy procedures. In CSF samples, the sensitivity of the PCR is extremely variable (11.5100%), but the specicity is high (96100%) [58,138,139,147]. Nevertheless, CSF col-lection can be invasive and is inappropriate in a subset of patients with expansive cerebral lesions. Asanalternativeapproach,PCRinperipheral blood samples has also been used with a range of reported sensitivities (1686%) [59,137,138,148,149]. Thesesensitivityvariationswereshownin reports made over two decades (1990s2000s) when progress in the development of equipment andreagentsimprovedtheassayperformance, as did different parameters, primers and probes. The recent development of real-time quantita-tive PCR (qrtPCR) has revolutionized molecular diagnostics by adding reliability and speed [150]. Its advantages over conventional PCR (cnPCR) includespeed,abroaddynamicrangeoftar-gets, DNA quantitation and reduction of con-tamination. Many reports tend to generalize the ideathatqrtPCRhasanimprovedsensitivity compared with cnPCR, as the rst substantially accelerates the detection of T. gondii DNA in the majorityofpositivespecimens.However,itis difcult to dene the end point of qrtPCR since in some patients the parasitic load is extremely low,principallyinCSFsamples.Assuch,itis necessaryandprudenttoanalyzeresultsfrom both qrtPCR and cnPCR [151153].The sensitivity of PCR for one microorganism in a biological sample primarily depends upon threefactors:thephysicochemicalconditions of the reaction, the concentration and nature of the DNA target, and the selected PCR primers and probes [151,154]. Subsequently, PCR sensitiv-ity and specicity depend on factors including the standardization of reagents and protocols for DNA extraction, the storage of the clinical sam-pleandthetimeelapsingbetweenthestartof specic therapy and collection (blood or CSF), whichoftenaffectsPCRreproducibilityand makes comparison of results difcult. Clinical samplesmustbecollectedbeforeorupuntil therst3daysofthespecictherapyasanti-toxoplasmic therapy decreases diagnostic sensi-tivity, especially if samples were collected after the rst week of treatment [58,139]. The use of a second reaction using in-parallel amplication of a marker that amplies a human sequence, such as the b-globulin gene [155] in human samples, guaranteesthequalityoftheDNAextraction and PCR inhibitors, avoiding false results. PCR (cn or qrt) is a group of assays, each with a vari-able outcome depending on a variety of factors. Comparisonsbetweenlaboratory-developed assays,qrtPCRorotherwise,shouldlikelybe made with even greater caution.Inrecentyears,severalDNAtargetsforcn- and qrtPCR were evaluated and have been used regularly in different laboratories to determine thediagnosisofcongenital,ocular,dissemi-natedorcerebraltoxoplasmosis.Targetssuch asbradyzoitegenesencodingforspecicanti-gens (SAG4, BAG1/hsp30, LDH2, MAG1), B1 gene,P30,ribosomalDNAgenesand529-bp sequencehavefrequentlybeenusedtodetect [112,122,148,151,156164]andquantifytheparasite DNA load at different times during the course ofinfectioninordertomonitorthetreatment effects [161]. Of these targets, two are more frequently used to provide high sensitivity and specicity. One is the 529-bp sequence, which has 200300 copies in the genome of T. gondii. The other is the B1 gene, which has 35 copies in the genome and is conserved in different parasite strains [143,157,164]. The sensitivity and accuracy of the target 529-bp sequenceandB1gene,andthecomparisonof both,werelargelyanalyzedincnPCRand qrtPCR[58,59,137,140,141,144,145,158,160,163,165168]. Themajorityofthesestudies,whichaimedto www.futuremedicine.com 1371 future science groupRole of Toxoplasma gondii infection in HIV-infected patientsReviewcompare sensitivities of both DNA regions, were conducted in European clinical samples. They demonstrated that markers to the 529-bp repeat region were more sensitive than those from the B1 gene. By contrast, recent studies conducted in Brazil [169171] showed that markers directed totheB1geneweremoresensitivethanthose to the 529-bp sequence for diagnosis of cerebral andcongenitaltoxoplasmosisinbothcnPCR and qrtPCR. TreatmentTheantiparasiticdrugcombinationemployed iskeyforeffectivetreatment.However,the recommendeddrugsactprimarilyagainstthe tachyzoites, but do not eradicate the bradyzoites. CerebraltoxoplasmosistherapyinAIDS patientsincludesacutetreatment,second-aryprophylaxis(treatmentmaintenance)and primar y prophylaxis. Acute treatmentThreerandomizeddouble-blindedtrialsof cerebraltoxoplasmosistreatmenthavebeen published comparing pyrimethamine plus sul-fadiazinewithpyrimethamineplusclindamy-cin[172,173],andpyrimethamineplussulfadi-azine with trimethoprim/sulfamethoxazole [174]. In a recent review of these studies The Cochrane Collaboration did not identify any superior regi-menamongthesethreecombinationsforcer-ebral toxoplasmosis treatment [175].Usually,weconsiderthefollowingregimens asrst-choiceinitialtherapy.Therstoption istreatmentfor6weekswithsulfadiazine (1.01.5 g oral route [PO] every 6 h) associated withpyrimethamine(100200mgPOload-ingdose,then50POdaily)andfolinicacid (1020mg PO daily), which reduces the like-lihoodofthehematologictoxicitiesassociated withpyrimethaminetherapy[97].Thesecond associationistrimethroprim/sulfamethoxazol (5/25mg/kgPOorintravenous[IV]every 12hfor46weeks)[176,177].Thislastthera-peutic scheme is uncommon in most developed anddevelopingcountries.However,thereare severalobservationalstudiesthatconrmthe efcacy and safety shown in the single available randomized clinical trial [175181]. The potential advantagesoftrimethroprim/sulfamethoxazol include less adverse events, posology, parenteral formulation, cost and accessibility. These char-acteristicsareparticularlyimportantfortreat-ing severely ill patients. An alternative regimen for patients withouttolerance tosulfa drugs is thecombinationfor6weeksofpyrimetamine 010203040010203040n =94n = 94n = 99n = 99 n = 100n = 100I II IIII II IIICSF groups CSF groupsSerum groups Serum groupsIV V VI010203040CIV V VIELISA-relative valuesELISA-relative values010203040n = 68n = 100n = 103n = 94n = 103n = 68n = 1001 I II III1810960471860472 kDa I II IIIFigure 3. Reactivity of Toxoplasma gondii secreted antigens in sera and cerebrospinal uid from cerebral toxoplasmosis and AIDS patients. Serological reactivity of total crude tachyzoites lysate (A) and T. gondii excretory/secretory antigens (ESA) (B) against sera from cerebral toxoplasmosis and AIDS patients (I), chronic toxoplasmosis individuals (II), and healthy individuals (III) by ELISA. ELISA relative values: ratio of the absorbance of each serum sample at an optical density of 492 nm to the cutoff value. Reactive: values greater than 1.0. (C & D) show the reactivity of total crude tachyzoites lisate and ESA, respectively, against CSF from cerebral toxoplasmosis and AIDS patients (IV), AIDS patients with other neurological diseases, but seropositive (V), or negative for toxoplasmosis (VI). The horizontal lines represent the arithmetic means: 8.7 and 12.7 in group I; 8.2 and 4.3 in group II; 0.5 and 0.4 in group III; 6.9 and 9.1 in group IV; 4.1 and 2.8 in group V; 0.5 and 0.6 in group VI. groups III and groups IVV are statistically similar in (A) and (C), respectively, and different in (B) and (D) at p < 0.05 (Students t-test). (E) and (F) show immunoblot ana lysis of a lysate 1.107 tachyzoites (1) and ESA (2) separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose. A serum from each group (I, II and III) was incubated with the nitrocellulose strips. CSF: Cerebrospinal uid.Future Microbiol. (2009) 4(10) 1372 future science groupReviewPereira-Chioccola, Vidal & Su(100200mgPOloadingdose,then50PO daily), clindamycin (600900 PO or IV every 6 h) and folinic acid (1020 mg PO daily) [97]. Longer treatment courses might be appropriate if the clinical or radiologic diagnoses show that therehasbeenanincompleteresponseorthe degree of infection is still extensive after 6 weeks. Intheexceptionalsettingwherenoneof thepreviousregimenscanbeadministrated, thefollowingoptionsmightbeconsidered. Treatment for 6 weeks with pyrimethamine and folinic acid (as in rst-choice regimen) associ-ated with azithromycin (1.21.5 g PO daily) or atovaquone (750 mg PO every 6 h). However, wearenotawareofanycomparativestudies betweentheefcaciesofthisassociationand the rst-choice therapy.Complications such as expansive brain lesions withamasseffect(e.g.,deviationofthemid-dle line structures or imminent risk of cerebral herniation)andcaseswithdiffuseencephalitis shouldbeadministeredadjunctivecortico-steroids (e.g., dexamethasone). Anticonvulsivant agents should be administrated in the occurrence ofseizures.However,theuseofprophylactics should be discouraged. No consensus has been reached for the timing of HAART when cerebral toxoplasmosis is present in antiretroviral-naive patients. We and others [103] consider that HAART should be started after at least 2 weeks of antiparasitic therapy. Primary prophylaxisPrimary prophylaxis against T. gondii in AIDS patients has been shown to be effective in pre-venting cerebral toxoplasmosis reactivation. For this reason, current guidelines recommend the use of a double-strength tablet daily dose of tri-methroprim/sulfamethoxazolinToxoplasma-seropositivepatientswhohaveaCD4+T-cell countbelow100cells/mm3[180].Inourset-ting,consideringthataround20%ofAIDS-relatedpatientsdevelopcerebraltoxoplasmosis when CD4+ T-cell counts are between 100 and 200cells/mm3[61],werecommendprimary prophylaxis in patients with CD4+ T-cell counts ofbelow200cells/mm3.Primaryprophylaxis shouldbediscontinuedinpatientsshowinga good response to HAART, which can be dened as a CD4 cell count above 200 cells/mm3 after 3 months [180].Secondary prophylaxisThe combination of pyrimethamine (2550 mg/day)plussulfadiazine(500mgevery6h)plus leucovorin(1020mg/day)ishighlyeffective as suppressive therapy for patients with cerebral toxoplasmosis.Whenpatientscannottakethe sulfadiazine four times a day regimen, an alter-native is the use of the same total daily dose in a twice a day regimen [181]. In patients who can-not tolerate sulfa drugs, an alternative option is pyrimethamine plus clindamycin (600 mg clin-damycin every 8 h is recommended) [180]. There is little data available regarding the potential use of trimethroprim/sulfamethoxazol in secondary prophylaxisofcerebraltoxoplasmosis.Asmall uncontrolledstudyinpatientswhohadbeen receivingHAARTforamedianof13months suggestedthattrimethroprim/sulfamethoxa-zol could be used as a suppressive regimen [182]. However, considering its efcacy and safety in the treatment of acute cerebral toxoplasmosis and the reducedpillburden,trimethroprim/sulfameth-oxazol seems to be a reasonable alternative when the conventional maintenance therapy is not pos-sible. In this scenario, we suggest trimethroprim/sulfamethoxazol 2.5/12.5 mg/kg PO every 12 h. Secondaryprophylaxiscanbesafelydiscon-tinuedwhenHIV-infectedpatientsreceiving effective HAART with successfully completed initial therapy for cerebral toxoplasmosis have a sustained increase of CD4+ T-cell count above 200cell/mm3(e.g.,after6months).Onthe otherhand,thesameprophylaxisshouldbe reintroducediftheCD4cellcountdecreases below 200 cells/mm3 [180].Immune reconstitutioninammatory syndromeImmune reconstitution inammatory syndrome (IRIS)hasbeenreportedinassociationwith HAART in patients with AIDS with several neu-rologiccomplications,particularly,tuberculous meningitis, cryptococcal meningitis and progres-sivemultifocalleukoencephalopathy.Despite cerebral toxoplasmosis being the most common opportunistic neurologic disease in HIV-infected patients, there has been doubt regarding the exist-ence of cerebral toxoplasmosis-associated IRIS as a true disease entity. Recently, a neuro pathologic-proven IRIS case in an AIDS patient with cer-ebraltoxoplasmosiswaspublished[183].Thus, cerebral toxoplasmosis-associated IRIS exists but remains uncommon.ConclusionAs cerebral toxoplasmosis persists to cause high morbidity and mortality, particularly in devel-opingcountries,theuseoflaboratorialtools, includingESA-ELISA,immunoblot,cnPCR andqrtPCR,needtobetestedindifferent www.futuremedicine.com 1373 future science groupRole of Toxoplasma gondii infection in HIV-infected patientsReviewclinicalsettings.Thesemethodologiesmaybe associatedwithclinicaldiagnosisandimages (presumptive diagnosis). Identication of T. gon-dii DNA in CSF or peripheral blood samples can contribute not only with the early diagnosis, but alsowiththedifferentialdiagnosisofpatients with expansive brain lesions who also have other opportunisticneurologicaldiseases.However, presumptive diagnosis calls for a prompt start to antiparasitic treatment. For acute cerebral toxo-plasmosistreatmentwerecommendedsulfadi-azinewithpyrimethamineandfolinicacidor trimethroprim/sulfamethoxazol.Maintenance therapycanbesafelydiscontinuedinpatients with consistent immune reconstitution. Future perspective Anumberofthetopicscoveredinthisreview are likely to see continued research. Knowledge oftheT.gondiiinteractionwithitshostcan contributetoimprovementoftheclinicalcare ofpatientsandthiswillrequireaclosercol-laborationbetweenphysiciansandscientists. Understanding the host response and molecular Executive summaryGeneral aspects, lifecycle & transmission n Studies related to Toxoplasma gondii lifecycle and transmission as well as the toxoplasmosis epidemiology allow us to understand: The complexity of the T. gondii lifecycle and its capacity to maintain infection in different host species.How these biological characteristics can compose the epidemiology of toxoplasmosis.Diversity among T. gondii strains n Genotyping studies contribute to our understanding of the epidemiology of toxoplasmosis in different regions of the world thanks to the evolution of molecular methods that facilitated genotyping of diverse T. gondii isolates, including those from domestic animals and human infections.n Results obtained identify two important points:The majority of the T. gondii strains isolated from North America, Europe, Asia and Africa are distributed into three major groups: type I, II and III lineages.Strains isolated in South America showed a higher genetic variability with distinct genotypes, with high rates of transmission and outcrossing, and these strains were not identied in North America, Europe, Asia or Africa.T. gondii antigens & host defensesn Immunological and parasitehost interaction studies contribute to understanding of: How the parasite infection of host cells is correlated with organelles and secretory proteins. The participation of T. gondii excretory/secretory antigens in the stimulation of the host immune system (T- and B-cell responses).The host resistance mediated by production of pro-inammatory cytokines and its interaction with T. gondii infection inducing a type 1 response.The maintenance of the chronic phase in immunocompetent individuals.Infection reactivation in immunodecient patients.The potential correlations between the development of cerebral toxoplasmosis and HLA genes (class I and II).AIDS & cerebral toxoplasmosisn Knowledge concerning AIDS improved knowledge of opportunistic infections, including: The impact of HAART on HIV infection and opportunistic diseases.The epidemiology of cerebral toxoplasmosis in different regions of the world. Diagnostic approach to clinical manifestations & radiological diagnosisn The studies concerning AIDScerebral toxoplasmosis association have established: A denitive and empiric diagnosis as well as the differences between clinical, computed tomography or MRI diagnoses.The different forms of the cerebral infection as dened by location and number of lesions.Immunological & molecular diagnosisn Knowledge of the laboratorial methodologies as well as the relationship between clinical and laboratorial studies improves:The determination, correlation and signicance of IgG antibody titers in active toxoplasmosis. The detection of specic antigens used as serological and intrathecal markers for diagnosis of cerebral toxoplasmosis inHIV-infected patients.Molecular methods such as conventional and real-time quantitative PCR, and T. gondii DNA targets. Treatment n Drug ana lysis, principally since the HIV era, has helped to establish:The antiparasitic drug combination as being the key to cerebral toxoplasmosis treatment. How HAART and antiparasitic therapy must be administrated. The procedures normally used in acute treatment as well as in primary and secondary prophylaxis.The presence of the immune reconstitution inammatory syndrome in cerebral toxoplasmosis.Future Microbiol. (2009) 4(10) 1374 future science groupReviewPereira-Chioccola, Vidal & Supathogenesis of T. gondii infection is critical for thedevelopmentofvaccines,drugsandother infection intervention approaches.Future studies on T. gondii genotyping should focusontwoaspects.First,large-scaletyping studies of T. gondii isolates should be conducted todetermineifhumandiseasepresentationis associatedwithparasitegenotypes.Toaccom-plishthisgoal,itisnecessarytocollectlarge numbersofsamplesfromhumanpatientsand identifyparasitesbyhigh-resolutiontyping methods.Second,high-resolutionserotyping methodsshouldbedevelopedtostudyT.gon-dii isolates in humans and animals. Since most T. gondii infections in human and animals are chronic and without any symptoms, it is difcult to isolate parasites from these hosts. This problem can be alleviated by the recently developed multi-plexnestedPCR-RFLPtypingmethod,which cansuccessfullygenotypesomeDNAsamples extracteddirectlyfromtissues[27,28,47,184,185]. Serotypinghasbeenprovedtobeapromising approachtoovercomethisobstacle[43,186,187]. However,atpresent,theresolutionofsero-typing is limited to differentiating type II from non-type II strains and, therefore, has limited use in typing serum samples from South America. A highresolution,multilocusserotypingmethod ismuchneededtofacilitateepidemiologyand population studies in T. gondii. WithrespecttoT.gondiimoleculardiag-nostics,futureadvanceswillprobablyrelyon the development of methods based on qrtPCR, which provides quantitative results and requires fewerhandlingstepsthancurrentmethods. However,applicationofthistechniqueisstill limited by the low specicity and the relatively high costs of the necessary equipment. Finally,cerebraltoxoplasmosiscontinuesto occur in several settings, such as late HIV diag-nosis (late presenters), patients with prior HIV infection without clinical care or noncompliance with HAART, and HAART failure. For these reasons, clinical and basic research on this chal-lenging disease should be maintained and stimu-lated,particularlyinlow-andmiddle-income countries,wherethetoxoplasmosisepidemic presents a major social and economic cost. Financial & competing interests disclosureThis study was partially supported by grants from the FundaaodeAmparoPesquisadoEstadodeSao Paulo,Brazil(FAPESP).Proc-08/093110.The authors have no other relevant afliations or nancial involvementwithanyorganizationorentitywitha nancial interest in or nancial conict with the sub-jectmatterormaterialsdiscussedinthemanuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.BibliographyPapers of special note have been highlighted as:nof interestnnof considerable interest1.Montoya JG, Liesenfeld O: Toxoplasmosis. Lancet 363(12), 19651976 (2004). 2.Dubey JP: Advances in the life cycle of Toxoplasma gondii. Int. J. 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Dr Arnaldo, 351, 8 andar, CEP 01246-902, So Paulo, SP, Brazil Tel.: +55 11 3068 2991 Fax: +55 11 3068 2890 pchioccola@ial.sp.gov.br.nJose Ernesto Vidal Departamento de Neurologia, Instituto de Infectologia Emlio Ribas, Av. Dr Arnaldo, 165 CEP 05411-000, Sao Paulo, SP, Brazil and Servio de Extenso ao atendimento de Pacientes HIV/AIDS, Diviso de Molstias Infecciosas e Parasitrias, Hospital das Clnicas da Faculdade de Medicina da Universidade de Sao Paulo, Rua Frei Caneca 557, Sao Paulo, SP, Brazil Tel.: +55 11 3120 5290 Fax: +55 11 3120 3472 josevibe@gmail.comnChunlei Su Department of Microbiology F409, Walters Life Sciences Building, The University of Tennessee, 1414 W. Cumberland Ave., Knoxville, TN 37996-0845, USA Tel.: +1 865 974 4015 or +1 865 974 3796 Fax: +1 865 974 4007 csu1@utk.edu