Toxo in HIV Patients - FutureMicrobiology2009

Author for correspondence: Laboratrio de Parasitologia, Instituto Adolfo Lutz, Av.
Dr Arnaldo, 351,
8andar, CEP 01246902, So Paulo, SP, Brazil n Tel.: +55 11 3068 2991 n Fax: +55 11 3068 2890
n pchioccola@ial.sp.gov.br
Cerebral toxoplasmosis is a major cause of morbidity and mortality among

HIVinfected patients, particularly from developing countries. This article
summarizes current literature on cerebral toxoplasmosis. It focuses on: Toxoplasma
gondii genetic diversity and its possible relationship with disease presentation;
host responses to the parasite antigens; host immunosupression in HIV and
cerebral toxoplasmosis as well as different diagnostic methods; clinical and
radiological features; treatment; and the direction that studies on cerebral
toxoplasmosis will likely take in the future.
General aspects, lifecycle & transmission
Toxoplasma gondii is an apicomplexan parasite that infects approximately one third of the
worlds population [1] . It is widely distributed
and can be found in many different species of
mammals and birds.
The lifecycle of T. gondii was described in
1970, when it was determined that members of
the family Felidae, including domestic cats, were
the definitive 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 definitive host occurs following ingestion of meat containing tissue cysts.
However, infection can also occur as a result of
ingesting the rapidly multiplying forms (tachyzoites) or the oocysts shed in feces. The cyst
wall is dissolved by the proteolytic enzymes in
the stomach and small intestine, releasing the
slow-multiplying bradyzoite stage. The formation of numerous asexual generations begins
after parasite invasion of the epithelial cells of
the small intestine. Sexual stages of T.gondii
are highly specific, occurring only within gut
epithelial cells of feline species. Oocysts are
produced by gamete fusion and are then shed
in the feces. Once in contact with the atmosphere, the oocysts sporulate to form sporozoites
and become infective to other definitive or
intermediate hosts [3,5,201] .
Following infection of intestinal epithelial
cells of the intermediate host, the infective stages
(sporozoites or bradyzoites) transform into tachyzoites, which multiply rapidly by endodyogeny
within an intracellular parasitophorous vacuole.
When the cells become packed with tachyzoites,
the host-cell plasma membrane ruptures and

parasites are released into the extracellular
milieu. The free tachyzoites can then infect
any nucleated cell they encounter and continue
intracellular replication, spreading throughout
host tissues. If not controlled by the immune system, tachyzoites are highly virulent and cause a
generalized toxoplasmosis, which is always fatal.
Host Tcell-mediated immune responses play
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] .
Human infection is generally innocuous,
asymptomatic and commonly acquired by ingestion of undercooked or raw meat containing tissue cysts, or water or food contaminated with
oocysts excreted in the feces. These biological
characteristics, eating and hygiene habits of populations are closely related to high prevalence of
serum positivity in the different regions, as the
oral route is the major source of infection. The
infection rises with age, does not vary greatly
between sexes, and is most common in temperate
and hot regions [69] .
The parasite can also be transmitted by vertical
transmission of the rapidly growing tachyzoite
form if a mother acquires a new infection during pregnancy. The infection can cause severe
neonatal malformations and ocular complications 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
transplantation from seropositive donors to
seronegative recipients has become an important
issue in transplant patients [12,13] . Figures1A&B
show the T.gondii lifecycle, as well as the forms
10.2217/FMB.09.89 2009 Future Medicine
Future Microbiol. (2009) 4(10), 13631379
Review
Vera Lucia Pereira-Chioccola, Jos Ernesto Vidal & Chunlei Su
Future Microbiology
Toxoplasma gondii infection and

cerebral toxoplasmosis in
HIV-infected patients
Keywords
n AIDS n cerebral
toxoplasmosis n diagnosis
n genotyping n review
n Toxoplasma gondii
part of
ISSN 1746-0913
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Review
Pereira-Chioccola, Vidal & Su
Definite hosts:
cats and other feline species
Sporozoite
oocysts
2040 days
Only tachyzoites
(gropus)
Cysts
410 days
Many bradyzoites
(cysts)
Carnivorism
36 days minimal
prepatent period
Chronic
infection
Immunity
Congenital
infection
Sporogony
in stool 34 days
Acute infection
Intermediate hosts:
rodents, dogs, human
and other mammals and birds
Ingestion
of oocyst
Oocyst
Toxoplasmosis transmission
Definitive host
Oocysts
Ingestion
of uncooked
meat
Cat and other

feline species
Fecal
contamination
Intermediate
hosts infected
Congenital
route
Chronic infection
Acute infection
Oral (consumption of
infected water or meat)
Organ transplantation
Primary infection
1020% symptomatic
(lymphadenitis, chorioretinitis)
8090%
asymptomatic
Latent infection
(asymptomatic)
During the pregnancy

(transplacental transmission)
Congenital
infection
Reactivation (occurred
in immunosuppression)
Cerebral toxoplasmosis
Figure1. Lifecycle of Toxoplasma gondii, transmission and clinical forms of toxoplasmosis.

(A) T.gondii lifecycle: felines serve as definitive 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 immunodeficient 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].
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Future Microbiol. (2009) 4(10)
future science group
Role of Toxoplasma gondii infection in HIV-infected patients
of transmission. The scheme showed in Figure1C

demonstrate the transmission routes and clinical
forms of toxoplasmosis.
Diversity among T.gondii strains
Genetic diversity of T. gondii strains has

been an interesting and important subject
of research. Over the last few decades, the
development of highly sensitive and simple
molecular methods has facilitated the detection, diagnosis and genotyping of this important parasitic pathogen. With these molecular
methods, it is possible to study the variation
of virulence among different parasite strains,
to reveal potential correlation between parasite genotype and disease patterns in infected
patients, and to study the epidemiology and
population biology of T.gondii [4] .
Typing of T.gondii has been achieved based
on antigenic variation [14,15] , multilocus enzyme
electrophoresis [1619] , microsatellite sequence
polymorphisms [2023] , PCR restriction fragment length polymorphism (PCRRFLP) [2428]
or DNA sequence typing of genes or introns
[2931] . Studies using PCRRFLP markers
revealed that most T.gondii strains from North
America, Europe and Africa can be divided
into three major groups: typeI, II and III lineages [24,25,31] . Among the three lineages, typeI
strains are highly virulent while type II and
typeIII are of low virulence [24] . TypeI and
typeI-like strains are associated with ocular
toxoplasmosis, acute outbreaks and cerebral
toxoplasmosis [3236] . It is suggested that the
high virulence of typeI strains is in part due
to over stimulation of a Th1 immune response,
which leads to pathology [36] .
Genotyping studies were applied to a great
number of isolates from domestic animals
[27,28,3741] , human congenital infection [42,43] ,
human ocular infections [44] and AIDS patients
[37,4547] . However, early studies using a single
genetic marker were limited to the identification of distinct isolates. With the advent of
multilocus PCR-RFLP and microsatellite typing methods, high-resolution genotyping was
achieved[22,26] . In North America, Europe and
Africa, most isolates belong to the clonal typeI,
II and III lineages, and there is no host boundary for different genotypes [17,24,25,32,48,49] . In
these regions, typeII strains are predominant
and are commonly isolated from clinical cases
of toxoplasmosis. Limited data suggested that
T.gondii population structure in Asia was similar to North America, Europe and Africa [39] .
At the DNA sequence level, the three lineages
Review
are highly similar, differing by less than 1% on

average, and it was suggested that these three
lineages were expanded globally in the past
10,000years [50,51] .
However, the idea that T.gondii was clonal
with very small genetic variability was challenged by studies of T. gondii strains from
Brazil and French Guiana. Genotyping results
from these regions showed a higher genetic variability, with distinct genotypes not yet identified in North America, Europe and Africa
[21,23,27,28,30,31,47,5256] . Analysis of 125 isolates
from domestic animals in Brazil revealed 48
genotypes, four of which had multiple isolates
from different hosts and locations, and they were
considered to be the common clonal lineages
in Brazil [27,55] . In contrast to North America,
Europe and Africa, the typeII lineage has not
been reported in Brazil and the typeI strains
are rare; in this region, most strains are unique
[27,28,30,47,53] . It was suggested that T. gondii
strains in South America have a high rate of
transmission and outcrossing [21,23] . Genetic
exchange could occur when the definitive host
ingests different types of parasites from their
intermediate hosts at nearly the same time, or
the intermediate host has mixed infection of different strains. The T.gondii isolates in South
America seem to have different physiological
characteristics since in some cases they are
highly pathogenic in immunocompetent individuals [47,52,57] . Most of the PCRRFLP genetic
markers were originally developed based on the
typeI, II and III lineages, which may miss the
unique alleles that are only present in South
America and, therefore, may underestimate
genetic diversity in phylogenetic studies [51,56] .
However, for epidemiological studies, these
markers are easy to use and can quickly identify
T.gondii strains with high resolution, providing crucial information to trace the source of
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.
A high prevalence of toxoplasmosis exists in
the Brazilian population and there is a considerable incidence of cerebral toxoplasmosis in AIDS
patients. The parasites are easily detected in the
blood and cerebrospinal fluid (CSF) of these
patients [58,59] with an elevated morbidity and
mortality [60] . However, at present there is limited information regarding genotyping of T.gon
dii samples from human patients, which makes
it difficult to determine if parasite genotypes are
associated with disease presentations. With the
www.futuremedicine.com
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available multilocus PCRRFLP method [26,28,40]

it will be possible to address this question in
future studies.
T.gondii antigens & host defenses
Infected 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 status [61,62] . In the classical infection, orally ingested
parasites actively invade intestinal epithelial cells
or are phagocytosed by these cells [50,63] .
The invasion process initiates the lytic cycle,
leading to cell and tissue destruction, which
results in Toxoplasma pathology. In the host-cell
cytoplasm, T.gondii induces the formation of a
parasitophorous vacuole that contains secreted
parasite proteins and host proteins that normally
promote phagosome maturation, thereby preventing 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 significant progress in
studying these proteins, only a limited number of
secretory proteins have been discovered [65,202] .
The active secretion of these antigens is an essential component of the low-grade stimulation or
boosting of the immune system, as these antigens
have been shown to stimulate antibody production as well as a Tcell response [64,66] . A group of
important antigens has been classified as parasite
excretory/secretory antigens (ESAs), which represents the majority of the circulating antigens
in sera from hosts with acute toxoplasmosis. ESA
proteins are expressed at the tachyzoite, sporozoite and encysted bradyzoite stages [67] . It has
been proposed that secretion by the bradyzoite
cysts maintains long-lasting immunity to the
parasite [68] . On the other hand, ESAs released
by tachyzoites are highly immunogenic [64,69] and
induce protective immunity, which may be either
antibody dependent or cell mediated [7072] . CD4+
Tcells specific for ESAs may be involved in the
maintenance of long-term immunity in healthy
chronically infected individuals. From these cells,
the antigens SAG2A [73] , SAG1 [74] , as well as
several dense granule proteins [7477] are abundant and highly immunodominant in the stimulation of the B-cell response. Immunocompetent
infected individuals had low anti-ESA antibody
titers, whereas patients with cerebral toxoplasmosis and AIDS, and consequently with circulating
blood tachyzoites, develop high titers of anti-ESA
antibodies [78] .
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The infection caused by T. gondii results

in inflammation, usually followed by necrosis. Like many obligate intracellular microbial
pathogens, T.gondii infection induces a strong
type 1 polarized immune response, engaging
both innate and adaptive immune systems. It is
clear that the major mechanism of host resistance
to toxoplasmosis is mediated by production of
pro-inflammatory cytokines, including IL12,
IFNg and TNFa [79,80] . The major sources of
IFNg, which is an import key in the response
to infection, are CD4 + Th1 lymphocytes, CD8 +
Tcells, natural killer cells and Tcells responding to IL12 [81,82] . These mechanisms prevent
rapid replication of tachyzoites and subsequent
pathological changes [80,83,84] .
After 23weeks of infection, the combination of the production of cytokines, IgG, IgM,
IgA and IgE antibodies against many T.gondii
proteins, extracellular tachyzoites are cleared
from host tissues and intracellular parasites differentiate into latent bradyzoite forms, which
are surrounded by the parasitophorous vacuole
that is enclosed in a cyst wall. The tissue cysts
preferentially reside in neural and muscular tissues. This differentiation can be increased by
exposure of the organism to stress conditions
such as in immune response to the tachyzoites.
Tlymphocytes are a critical source of IFNg
during this stage of the infection, as shown
several years ago in antibody-mediated Tcell
depletion experiments that resulted in reactivation 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 parasites is also dependent on IFNg-producing CD4
and CD8 T cells, which are recruited to the
brain [79,80,85,86] . The ability of bradyzoites to
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
indefinitely in the brain and muscle, developing
lifelong protective immunity against re-infection[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, during the chronic infection around 1020% of the
infected immunocompetent individuals are reactive, with the rupture of a tissue cyst in the eyes,
brain or muscles causing local necrosis accompanied by inflammation. Hypersensitivity plays a
major role in such reactions; however, the infection usually collapses, with no local converted
multiplication of tachyzoites [1] .
When asymptomatic individuals present some
immunodeficiency, reactivation of latent infection may occur, culminating in the conversion of
bradyzoites to the active and rapidly replicating
tachyzoites, resulting in a tissue injury that is
often fatal. As the cysts have a predilection for
neural and muscle tissue as well as the eye, most
cases of reactivation lead to chorioretinitis or,
more frequently, cerebral toxoplasmosis, which
is a life-threatening condition [1,88] .
Potential correlations between the development of cerebral toxoplasmosis and HLA genes
(classI and classII) in HIV-patients have been
studied in recent years. The MHC is one of
the most polymorphic genetic systems of many
species, including HLA in humans. The MHC
controls the adaptive immune response against
intra- and extracellular microorganisms by
classI (HLAA, HLAB, HLACw) and classII
(HLADRB1, HLADQB1, HLADPB1) and is
correlated with infection susceptibility or resistance. The association between susceptibility to
different diseases and HLA molecules, as well
as the distribution of HLA alleles in linkage
disequilibrium, are important factors in the
MHC. Linkage disequilibrium is the situation in which two specific alleles from separate
loci closely linked to each other are transmitted together on the same chromosome [8992] .
ClassI HLAB35 antigen was associated with
the susceptibility to chorioretinitis [93] , and
class I HLAB8 and class II HLADRB1*17
antigens were associated with susceptibility
to cerebral toxoplasmosis [94] . The presence of
classII HLADQB1*0402 and DRB1*08 alleles[95] and the HLADR52 haplotype represent
risk factors to the development of cerebral toxoplasmosis, whereas the HLADR53 haplotype
was associated with infection resistance [96] .
AIDS & cerebral toxoplasmosis
The introduction of HAART for the treatment

of HIV infection has resulted in dramatic reductions in morbidity, mortality and healthcare utilization [97] . Decreasing rates of opportunistic
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 developing countries with access to HAART owing
to delayed diagnosis of HIV infection or lack
of opportunities to start treatment in patients
prior to diagnosis of HIV [98100] . Cerebral
Review
toxoplasmosis is an HIV-indicative event in 35%

of patients and an AIDS-defining event in 75%
of cases [60] .
In addition, neurological infections continue
to cause high rates of morbidity and mortality
in developing countries without availability of
HAART, where the patients usually present the
natural history of most diseases.
Cerebral toxoplasmosis is usually the most
common cerebral opportunistic disease in both
developed and developing countries [101,102] . In
some places, particularly in Africa, cases of cerebral toxoplasmosis are only exceeded by cases
of cryptococcal meningoencephalitis. Globally,
T.gondii causes the most common focal brain
lesion in HIV-infected patients [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 genotypes of T.gondii isolates, races and ethnicities
and the mode of transmission also seem to influence the occurrence of the infection [35] . Data
are available regarding infection prevalence in
different parts of the world. The data indicate
that around 25% of AIDS patients from Paris
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 approach
The definitive diagnosis of cerebral toxoplasmosis requires the presence of the tachyzoite form
of the parasite in cerebral tissue to be directly
demonstrated. In clinical practice, presumptive
cerebral toxoplasmosis diagnosis depends on an
association of serological, clinical and radiological information [107] . Diagnosis is confirmed with
a response to empiric anti-Toxoplasma therapy.
A favorable clinical and radiological response is
expected within 1014days of specific treatment
There are no pathognomonic clinical or
radiological findings of cerebral toxoplasmosis.
Thus, differential diagnosis of AIDS patients
with extensive brain lesions is essential and two
factors should be always considered: the local
neuroepidemiology and the degree of immuno
suppression in the host [108] . Differential diagnosis of expansive brain lesions presents geographic
particularities. In developed countries, primary
lymphoma of the CNS constitutes the main differential diagnosis of cerebral toxoplasmosis [103] .
In developing countries, focal forms of cerebral
TB (tuberculomas and, less likely, tuberculous
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brain abscess) are the main alternative diagnoses [109] . Primary lymphoma of the CNS
usually presents with a CD4 cell count below
50cells/mm3, cerebral toxoplasmosis frequently
below 100cells/mm3, and cerebral TB usually
below 200cells/mm3 [60] . Of these three etiologies, cerebral TB is more likely to present with
CD4 cell count above 200cells/mm3 [60,110,111] .
In addition to these more frequent neurologic
complications, the differential diagnosis of
expansive brain lesions in HIV-infected patients
is broad and includes other infections such as
cryptococcosis, aspergillosis and Chagas disease; AIDS- and non-AIDS-associated tumors
such as metastases of disseminated lymphomas and glioblastoma multiform, respectively;
and vascular diseases. For these reason, 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
to antiparasitic treatment within 1014days.
However, at least 10% of cerebral toxoplasmosis cases died despite what was thought to be
adequate treatment [60] . Molecular diagnosis
using CSF or peripheral blood samples is a useful tool for early, minimally invasive diagnosis
of cerebral toxoplasmosis [59,112,113] . However, in
clinical practice, results should always be interpreted in association with serological, clinical
and radiological information.
Clinical manifestations
& radiological diagnosis
Cerebral toxoplasmosis causes unifocal or, more

commonly, multifocal lesions and, less frequently, diffuse encephalitis. Patients usually
present subacute manifestations, but it can be
acute in around 10% of cases. The clinical manifestations depend on the location and number
of lesions. More frequent complaints include:
headache (4963%), fever (4168%), focal
deficits (2280%), seizures (1929%), mental
confusion (1552%), ataxia (1525%), lethargy
(1244%), cranial nerve alterations (1219%)
and visual alterations (815%). Other manifestations include disarthria, cognitive dysfunction, intracranial pressure and involuntary
movements [60,103,107,114116] .
Imaging studies, either computed tomography (CT) or MRI, are essential for the presumptive diagnosis of cerebral toxoplasmosis [97,103] .
MRI is more sensitive, particularly for identifying small lesions and those located in the
posterior fossa. Single lesions are observed in
approximately 30% of the patients by CT, but
1368
by MRI it is common to identify two or more

lesions. Radiological diagnosis can be classified
as typical or atypical patterns [60] . Typical patterns 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 without focal lesions and MRI demonstrating focal
lesions, and diffuse cerebral encephalitis without
visible focal lesions.
A highly suggestive image for toxoplasmosis,
although unusual, is the eccentric target sign,
which is a small asymmetric nodule along the
wall of the enhancing ring. Figure2 shows the main
radiological features of cerebral toxoplasmosis in
HIV-infected patients.
Immunological diagnosis
The high prevalence and lifelong persistence

of anti-T.gondii IgG antibodies among healthy
individuals in many geographical areas prevent
the use of titers in serologic tests to reflect recent
infection. Another problem is often the lack of
reliability in discriminating recent from more
distant infection by detection of anti-T.gondii
IgM, IgA or IgE. In this setting, physicians are
often faced with conflicting results and disagreements about interpretations of results. This often
leads to incorrect information being provided by
the laboratories to the physicians as well as by
the physicians to their patients. This situation is
very common in congenital toxoplasmosis diagnosis [117] . In the case of cerebral toxoplasmosis,
the identification of a positive serology is less
useful in settings where the seroprevalence for
T.gondii in the general population is very high.
In Brazil, most AIDS patients with focal brain
lesion also show positive serological diagnosis
for toxoplasmosis. However, the identification
of a negative serological result presents a high
negative predictive value. [108] .
Most patients have serological evidence
of infection, usually with high titres of
IgG [59,60,118] with high avidity, supporting
the idea that the reactivation of the latent
infection occurs in the secondary immune
response [59,119] . However, these antibodies
may not be detected in up to 5% of patients
owing to immune suppression. Earlier studies
showed that different levels of anti-T. gondii
IgG antibodies were inadequate to determine a
reactivation or to follow the course of cerebral
toxoplasmosis [88,120122] . As these antibodies
Review
Figure2. Computed tomography images showing the spectrum of radiological findings 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.
are usually present in cerebral toxoplasmosis,

some studies suggested that, statistically, high
titers might be indicative of the active disease
or a higher risk of developing it [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
AIDS patients with compatible clinical and
radiological findings [60,78,115,120,121,125] .
In general, anti-T.gondii antibodies are determined in conventional serology, such as ELISA
and immunofluorescence, using total extract
of tachyzoites as an antigen, which consists
of cytoplasmatic and membrane components.
In addition, several studies have demonstrated
the usefulness of recombinant antigens for
the serological diagnosis of T.gondii infection
[126130] . These antigens used in ELISA failed
to distinguish sera from patients with cerebral
toxoplasmosis and asymptomatic infected
immunocompetent individuals, with no or low
numbers of circulating tachyzoites, as they have
similar reactivity.
On the other hand, ESAs constitute an excellent serological marker for the diagnosis of cerebral toxoplasmosis in HIV-infected patients
as they are produced by tachyzoites, the form
responsible for disseminating the infection,

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 considerable proportion of excreted/secreted antigens are released, eliciting the specific immune
response to ESAs. Unlike asymptomatic individuals, these patients present antibodies for
both ESAs and total crude tachyzoite antigens.
When ESAs are used as antigens in ELISA
(ESA-ELISA) and in immunoblot it is possible
to distinguish sera from patients with the active
disease. Normally, these sera are three times
more reactive than those from seropositive individuals (Figure3A,B,E&F) [78] . Thus, anti-ESA
antibodies were present principally in patients
with active disease, suggesting its importance,
particularly in regions with high prevalence of
latent toxoplasmosis in the general population.
Previous studies analyzing the presence or
absence of oligoclonal bands of IgG (OCBs) in
serum and CSF demonstrated that the intra
thecal humoral immune response can be produced independent of the systemic one. In other
situations IgG may be produced in the systemic
immune activation but not in intrathecal synthesis. In this case the presence of OCBs in the
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CSF is explained by their passive movement

from the serum into the CSF across the blood
brain barrier, which does not strongly obstruct
serum proteins [134] . However, the intrathecal
anti-T.gondii IgG antibody production increases
in patients with cerebral toxoplasmosis and
AIDS [135] and is normally characterized by the
presence of T.gondii OCBs [136] .
For some authors, the immunological diagnosis value in CSF is limited because the sensitivity
and specificity are approximately 6070% [115] .
Anti-ESA IgG antibodies are also present in CSF
in patients with AIDS and cerebral toxoplasmosis and can be determined by ESA-ELISA and
immunoblot. CSF samples from these patients
clearly distinguish from those with AIDS and
who are seropositive for toxoplasmosis but with
other neurological diseases (Figure3C&D) .
Molecular diagnosis
Over recent decades, the development of molecular methodologies essentially based upon PCR
have allowed the sensitive detection of T.gondii
DNA in clinical specimens such as amniotic
fluid, aqueous humor, CSF, bone marrow and
blood [58,59,136146] . Molecular methods are particularly appropriate for AIDS patients, since
these methods are not affected by immunological status and have been shown to be rapid, sensitive and specific, 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 specificity is high
(96100%) [58,138,139,147] . Nevertheless, CSF collection can be invasive and is inappropriate in a
subset of patients with expansive cerebral lesions.
As an alternative approach, PCR in peripheral
blood samples has also been used with a range of
reported sensitivities (1686%) [59,137,138,148,149] .
These sensitivity variations were shown in
reports made over two decades (1990s2000s)
when progress in the development of equipment
and reagents improved the assay performance,
as did different parameters, primers and probes.
The recent development of real-time quantitative PCR (qrtPCR) has revolutionized molecular
diagnostics by adding reliability and speed [150] .
Its advantages over conventional PCR (cnPCR)
include speed, a broad dynamic range of targets, DNA quantitation and reduction of contamination. Many reports tend to generalize the
idea that qrtPCR has an improved sensitivity
compared with cnPCR, as the first substantially
accelerates the detection of T.gondii DNA in the
majority of positive specimens. However, it is
difficult to define the end point of qrtPCR since
1370
in some patients the parasitic load is extremely

low, principally in CSF samples. As such, it is
necessary and prudent to analyze results from
both qrtPCR and cnPCR [151153] .
The sensitivity of PCR for one microorganism
in a biological sample primarily depends upon
three factors: the physicochemical conditions
of the reaction, the concentration and nature of
the DNA target, and the selected PCR primers
and probes [151,154] . Subsequently, PCR sensitivity and specificity depend on factors including
the standardization of reagents and protocols for
DNA extraction, the storage of the clinical sample and the time elapsing between the start of
specific therapy and collection (blood or CSF),
which often affects PCR reproducibility and
makes comparison of results difficult. Clinical
samples must be collected before or up until
the first 3days of the specific therapy as anti
toxoplasmic therapy decreases diagnostic sensitivity, especially if samples were collected after
the first week of treatment [58,139] . The use of a
second reaction using in-parallel amplification of
a marker that amplifies a human sequence, such
as the b-globulin gene [155] in human samples,
guarantees the quality of the DNA extraction
and PCR inhibitors, avoiding false results. PCR
(cn or qrt) is a group of assays, each with a variable outcome depending on a variety of factors.
Comparisons between laboratory-developed
assays, qrtPCR or otherwise, should likely be
made with even greater caution.
In recent years, several DNA targets for cnand qrtPCR were evaluated and have been used
regularly in different laboratories to determine
the diagnosis of congenital, ocular, disseminated or cerebral toxoplasmosis. Targets such
as bradyzoite genes encoding for specific antigens (SAG4, BAG1/hsp30, LDH2, MAG1), B1
gene, P30, ribosomal DNA genes and 529bp
sequence have frequently been used to detect
[112,122,148,151,156164] and quantify the parasite
DNA load at different times during the course
of infection in order to monitor the treatment
effects [161] .
Of these targets, two are more frequently used
to provide high sensitivity and specificity. One is
the 529bp sequence, which has 200300copies
in the genome of T.gondii. The other is the B1
gene, which has 35copies in the genome and is
conserved in different parasite strains [143,157,164] .
The sensitivity and accuracy of the target 529bp
sequence and B1 gene, and the comparison of
both, were largely analyzed in cnPCR and
qrtPCR [58,59,137,140,141,144,145,158,160,163,165168] .
The majority of these studies, which aimed to
compare sensitivities of both DNA regions, were

conducted in European clinical samples. They
demonstrated that markers to the 529bp repeat
region were more sensitive than those from the
B1 gene. By contrast, recent studies conducted
in Brazil [169171] showed that markers directed
to the B1 gene were more sensitive than those
to the 529bp sequence for diagnosis of cerebral
and congenital toxoplasmosis in both cnPCR
and qrtPCR.
ELISA-relative values
Three randomized double-blinded trials of

cerebral toxoplasmosis treatment have been
published comparing pyrimethamine plus sulfadiazine with pyrimethamine plus clindamycin [172,173] , and pyrimethamine plus sulfadiazine with trimethoprim/sulfamethoxazole [174] .
In a recent review of these studies The Cochrane
Collaboration did not identify any superior regimen among these three combinations for cerebral toxoplasmosis treatment [175] .
Usually, we consider the following regimens
as first-choice initial therapy. The first option
is treatment for 6 weeks with sulfadiazine
(1.01.5g oral route [PO] every 6h) associated
with pyrimethamine (100200 mg PO loading dose, then 50 PO daily) and folinic acid
(1020mg PO daily), which reduces the likelihood of the hematologic toxicities associated
with pyrimethamine therapy [97] . The second
association is trimethroprim/sulfamethoxazol
(5/25 mg/kg PO or intravenous [IV] every
12 h for 46 weeks) [176,177] . This last therapeutic scheme is uncommon in most developed
and developing countries. However, there are
several observational studies that confirm the
efficacy and safety shown in the single available
randomized clinical trial [175181] . The potential
advantages of trimethroprim/sulfamethoxazol
include less adverse events, posology, parenteral
formulation, cost and accessibility. These characteristics are particularly important for treating severely ill patients. An alternative regimen
for patients without tolerance to sulfa drugs is
the combination for 6weeks of pyrimetamine
30
30
20
n = 100
n = 100
20
n = 99
10
n = 99
10
n = 94
n = 94
0
II
Serum groups
III
II
III
Serum groups
40
40
C
ELISA-relative values
Acute treatment
40
Treatment
The antiparasitic drug combination employed

is key for effective treatment. However, the
recommended drugs act primarily against the
tachyzoites, but do not eradicate the bradyzoites.
Cerebral toxoplasmosis therapy in AIDS
patients includes acute treatment, secondary prophylaxis (treatment maintenance) and
primary prophylaxis.
40
Review
30
30
n = 100
n = 103
20
20
10
0
n = 100
n = 103
10
nn =94
= 68
n = 68
0
IV
V
CSF groups
VI
II
III
kDa
IV
V
CSF groups
II
VI
III
109
60
47
18
Figure3. Reactivity of Toxoplasma gondii secreted antigens in sera and

cerebrospinal fluid 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 492nm 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 groupI; 8.2 and 4.3 in groupII; 0.5 and 0.4 in groupIII; 6.9 and 9.1 in
groupIV; 4.1 and 2.8 in groupV; 0.5 and 0.6 in groupVI. groupsIII and
groupsIVV 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 analysis
of a lysate 1.107tachyzoites (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 fluid.
1371
Review
(100200 mg PO loading dose, then 50 PO

daily), clindamycin (600900 PO or IV every
6h) and folinic acid (1020mg PO daily) [97] .
Longer treatment courses might be appropriate
if the clinical or radiologic diagnoses show that
there has been an incomplete response or the
degree of infection is still extensive after 6weeks.
In the exceptional setting where none of
the previous regimens can be administrated,
the following options might be considered.
Treatment for 6weeks with pyrimethamine and
folinic acid (as in first-choice regimen) associated with azithromycin (1.21.5g PO daily) or
atovaquone (750mg PO every 6h). However,
we are not aware of any comparative studies
between the efficacies of this association and
the first-choice therapy.
Complications such as expansive brain lesions
with a mass effect (e.g., deviation of the middle line structures or imminent risk of cerebral
herniation) and cases with diffuse encephalitis
should be administered adjunctive cortico
steroids (e.g., dexamethasone). Anticonvulsivant
agents should be administrated in the occurrence
of seizures. However, the use of prophylactics
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 2weeks of antiparasitic therapy.
Primary prophylaxis
Primary prophylaxis against T.gondii in AIDS

patients has been shown to be effective in preventing cerebral toxoplasmosis reactivation. For
this reason, current guidelines recommend the
use of a double-strength tablet daily dose of trimethroprim/sulfamethoxazol in Toxoplasmaseropositive patients who have a CD4 + Tcell
count below 100 cells/mm 3 [180] . In our setting, considering that around 20% of AIDSrelated patients develop cerebral toxoplasmosis
when CD4 + Tcell counts are between 100 and
200 cells/mm 3 [61] , we recommend primary
prophylaxis in patients with CD4 + Tcell counts
of below 200cells/mm3. Primary prophylaxis
should be discontinued in patients showing a
good response to HAART, which can be defined
as a CD4 cell count above 200cells/mm3 after
3months [180] .
Secondary prophylaxis
The combination of pyrimethamine (2550mg/

day) plus sulfadiazine (500mg every 6h) plus
leucovorin (1020 mg/day) is highly effective
1372
as suppressive therapy for patients with cerebral

toxoplasmosis. When patients cannot take the
sulfadiazine four times a day regimen, an alternative is the use of the same total daily dose in
a twice a day regimen [181] . In patients who cannot tolerate sulfa drugs, an alternative option is
pyrimethamine plus clindamycin (600mg clindamycin every 8h is recommended) [180] . There
is little data available regarding the potential use
of trimethroprim/sulfamethoxazol in secondary
prophylaxis of cerebral toxoplasmosis. A small
uncontrolled study in patients who had been
receiving HAART for a median of 13months
suggested that trimethroprim/sulfamethoxazol could be used as a suppressive regimen [182] .
However, considering its efficacy and safety in the
treatment of acute cerebral toxoplasmosis and the
reduced pill burden, trimethroprim/sulfamethoxazol seems to be a reasonable alternative when
the conventional maintenance therapy is not possible. In this scenario, we suggest trimethroprim/
sulfamethoxazol 2.5/12.5mg/kg PO every 12h.
Secondary prophylaxis can be safely discontinued when HIV-infected patients receiving
effective HAART with successfully completed
initial therapy for cerebral toxoplasmosis have a
sustained increase of CD4 + Tcell count above
200 cell/mm 3 (e.g., after 6 months). On the
other hand, the same prophylaxis should be
reintroduced if the CD4 cell count decreases
below 200cells/mm3 [180] .
Immune reconstitution
inflammatory syndrome
Immune reconstitution inflammatory syndrome

(IRIS) has been reported in association with
HAART in patients with AIDS with several neurologic complications, particularly, tuberculous
meningitis, cryptococcal meningitis and progressive multifocal leukoencephalopathy. Despite
cerebral toxoplasmosis being the most common
opportunistic neurologic disease in HIV-infected
patients, there has been doubt regarding the existence of cerebral toxoplasmosis-associated IRIS as
a true disease entity. Recently, a neuropathologicproven IRIS case in an AIDS patient with cerebral toxoplasmosis was published [183] . Thus,
cerebral toxoplasmosis-associated IRIS exists but
remains uncommon.
Conclusion
As cerebral toxoplasmosis persists to cause high

morbidity and mortality, particularly in developing countries, the use of laboratorial tools,
including ESA-ELISA, immunoblot, cnPCR
and qrtPCR, need to be tested in different
clinical settings. These methodologies may be

associated with clinical diagnosis and images
(presumptive diagnosis). Identification of T.gon
dii DNA in CSF or peripheral blood samples can
contribute not only with the early diagnosis, but
also with the differential diagnosis of patients
with expansive brain lesions who also have other
opportunistic neurological diseases. However,
presumptive diagnosis calls for a prompt start to
antiparasitic treatment. For acute cerebral toxoplasmosis treatment we recommended sulfadiazine with pyrimethamine and folinic acid or
Review
trimethroprim/sulfamethoxazol. Maintenance
therapy can be safely discontinued in patients
with consistent immune reconstitution.
Future perspective
A number of the topics covered in this review

are likely to see continued research. Knowledge
of the T. gondii interaction with its host can
contribute to improvement of the clinical care
of patients and this will require a closer collaboration between physicians and scientists.
Understanding the host response and molecular
Executive summary
General aspects, lifecycle & transmission
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

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:
typeI, 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 identified in North America, Europe, Asia or Africa.
n
T.gondii antigens & host defenses

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 Bcell responses).
The host resistance mediated by production of pro-inflammatory cytokines and its interaction with T.gondii infection inducing a
type1 response.
The maintenance of the chronic phase in immunocompetent individuals.
Infection reactivation in immunodeficient patients.
The potential correlations between the development of cerebral toxoplasmosis and HLA genes (classI and II).
AIDS & cerebral toxoplasmosis

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 diagnosis

The studies concerning AIDScerebral toxoplasmosis association have established:
A definitive and empiric diagnosis as well as the differences between clinical, computed tomography or MRI diagnoses.
The different forms of the cerebral infection as defined by location and number of lesions.
Immunological & molecular diagnosis

Knowledge of the laboratorial methodologies as well as the relationship between clinical and laboratorial studies improves:
The determination, correlation and significance of IgG antibody titers in active toxoplasmosis.
The detection of specific antigens used as serological and intrathecal markers for diagnosis of cerebral toxoplasmosis in
HIV-infected patients.
Molecular methods such as conventional and real-time quantitative PCR, and T.gondii DNA targets.
Treatment
Drug analysis, 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 inflammatory syndrome in cerebral toxoplasmosis.
1373
Review
pathogenesis of T.gondii infection is critical for

the development of vaccines, drugs and other
infection intervention approaches.
Future studies on T.gondii genotyping should
focus on two aspects. First, large-scale typing
studies of T.gondii isolates should be conducted
to determine if human disease presentation is
associated with parasite genotypes. To accomplish this goal, it is necessary to collect large
numbers of samples from human patients and
identify parasites by high-resolution typing
methods. Second, high-resolution serotyping
methods should be developed to study T.gon
dii isolates in humans and animals. Since most
T.gondii infections in human and animals are
chronic and without any symptoms, it is difficult
to isolate parasites from these hosts. This problem
can be alleviated by the recently developed multi
plex nested PCRRFLP typing method, which
can successfully genotype some DNA samples
extracted directly from tissues [27,28,47,184,185] .
Serotyping has been proved to be a promising
approach to overcome this obstacle [43,186,187] .
However, at present, the resolution of sero
typing is limited to differentiating typeII from
nontypeII strains and, therefore, has limited use
in typing serum samples from South America. A
high resolution, multilocus serotyping method
is much needed to facilitate epidemiology and
population studies in T.gondii.
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Review
Vera Lucia Pereira Chioccola

Laboratrio de Parasitologia, Instituto
Adolfo Lutz, Av. 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.
Jose 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.com
Chunlei 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
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Toxo in HIV Patients - FutureMicrobiology2009

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Author for correspondence: Laboratrio de Parasitologia, Instituto Adolfo Lutz, Av.

Cerebral toxoplasmosis is a major cause of morbidity and mortality among

the host-cell plasma membrane ruptures and

10.2217/FMB.09.89 2009 Future Medicine

Future Microbiol. (2009) 4(10), 13631379

Vera Lucia Pereira-Chioccola, Jos Ernesto Vidal & Chunlei Su

Toxoplasma gondii infection and

Pereira-Chioccola, Vidal & Su

Cat and other

During the pregnancy

Figure1. Lifecycle of Toxoplasma gondii, transmission and clinical forms of toxoplasmosis.

Future Microbiol. (2009) 4(10)

future science group

Role of Toxoplasma gondii infection in HIV-infected patients

of transmission. The scheme showed in Figure1C

Genetic diversity of T. gondii strains has

are highly similar, differing by less than 1% on

Pereira-Chioccola, Vidal & Su

available multilocus PCRRFLP method [26,28,40]

Infected intermediate hosts, including humans,

Future Microbiol. (2009) 4(10)

The infection caused by T. gondii results

Role of Toxoplasma gondii infection in HIV-infected patients

The introduction of HAART for the treatment

toxoplasmosis is an HIV-indicative event in 35%

Pereira-Chioccola, Vidal & Su

Cerebral toxoplasmosis causes unifocal or, more

Future Microbiol. (2009) 4(10)

by MRI it is common to identify two or more

The high prevalence and lifelong persistence

Role of Toxoplasma gondii infection in HIV-infected patients

Figure2. Computed tomography images showing the spectrum of radiological findings of

are usually present in cerebral toxoplasmosis,

responsible for disseminating the infection,

Pereira-Chioccola, Vidal & Su

CSF is explained by their passive movement

Future Microbiol. (2009) 4(10)

in some patients the parasitic load is extremely

compare sensitivities of both DNA regions, were

Role of Toxoplasma gondii infection in HIV-infected patients

Three randomized double-blinded trials of

The antiparasitic drug combination employed

Figure3. Reactivity of Toxoplasma gondii secreted antigens in sera and

Pereira-Chioccola, Vidal & Su

(100200 mg PO loading dose, then 50 PO

Primary prophylaxis against T.gondii in AIDS

The combination of pyrimethamine (2550mg/

Future Microbiol. (2009) 4(10)

as suppressive therapy for patients with cerebral

Immune reconstitution inflammatory syndrome

As cerebral toxoplasmosis persists to cause high

Role of Toxoplasma gondii infection in HIV-infected patients

clinical settings. These methodologies may be

A number of the topics covered in this review

Diversity among T.gondii strains

T.gondii antigens & host defenses

AIDS & cerebral toxoplasmosis

Diagnostic approach to clinical manifestations & radiological diagnosis

Immunological & molecular diagnosis

future science group

Pereira-Chioccola, Vidal & Su

pathogenesis of T.gondii infection is critical for

Papers of special note have been highlighted as: