Detection and characterisation of parasites causing emerging zoonoses

U.M. Morgan
*
World Health Organization Collaborating Centre for the Molecular Epidemiology of Parasitic Infections and State Agricultural Biotechnology Centre,
Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia
Received 22 June 2000; received in revised form 24 August 2000; accepted 1 September 2000
Abstract
Understanding the epidemiology of zoonotic parasitic infections is dependent upon the availability of accurate and sensitive diagnostic
techniques. The development of molecular diagnostic methods, particularly those utilising PCR for the detection of zoonoses will contribute
greatly to the identication and control of these pathogens, by increasing the speed of diagnosis, specicity and sensitivity, reproducibility
and ease of interpretation. Molecular characterisation studies allow us to distinguish between closely related infectious agents and to
document the patterns of transmission of `strains' and species within populations. This will allow precise determinations to be made
about the aetiological agent, its characteristics and the source of infection. This review focuses on recent detection and characterisation
techniques for both emerging and re-emerging parasite zoonoses. q 2000 Australian Society for Parasitology Inc. Published by Elsevier
Science Ltd. All rights reserved.
Keywords: Detection; Characterisation; Molecular; Zoonoses
1. Introduction
Zoonoses involving parasites are both common and
important, some causing serious diseases [1]. Of the 374
species of parasite recorded as naturally infecting Homo
sapiens, all but some 40 are zoonotic [2]. Understanding
the epidemiology of any infectious agent is dependent
upon the availability of accurate and sensitive diagnostic
techniques. Traditionally, parasitic infections have been
diagnosed using microscopy. However, microscopy is
labour-intensive and requires well-trained microscopists
for accurate identication and interpretation [3]. In addition,
the diagnostic skills of microscopists can vary greatly from
laboratory to laboratory, resulting in some infections being
mis-diagnosed or missed completely. Immunoassays have
the benets of technical simplicity, rapidity, specicity and
cost-effectiveness, but often have poor sensitivity and a low
negative predictive value. Molecular biology can now
provide epidemiologists with information that goes much
further than traditional microscopy, culture and immunolo-
gical procedures. This review will focus on the improved
detection and characterisation of re-emerging and emerging
parasite zoonoses.
2. Toxoplasma
Toxoplasmosis, caused by the protozoan parasite, Toxo-
plasma gondii, can be acquired by the ingestion of infected
undercooked meat or from oocysts shed in cat faeces [4].
Generally, toxoplasmosis has mild symptoms, or is asymp-
tomatic, in patients with intact immune systems [5]. The
infection, however, may have serious consequences in
immunodecient or immunosuppressed patients, as well as
in the offspring of pregnant women. In AIDS patients, it
may preferentially infect the CNS, resulting in a wide
range of clinical presentations [6]. Pregnant mothers
infected during early pregnancy can infect their foetus trans-
placentally, often during the second or third trimester of
foetal development. Acute toxoplasmosis in children is
often fatal, although the mothers of infected newborns are
characteristically without symptoms [7].
Early detection of toxoplasmosis is essential for success-
ful treatment. Occasionally Toxoplasma tachyzoites can be
detected and identied in smears of the lymph nodes, bone
marrow, spleen, brain, or other material. The organisms are
readily identied in impression smears, but in histological
preparations of xed tissues, they do not exhibit the typical
morphology [7]. A variety of new clinical techniques,
including neuroimaging and thallium-201 brain single-
photon emission CT have been applied to the diagnosis of
International Journal for Parasitology 30 (2000) 14071421
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PII: S0020-7519(00)00129-6
www.parasitology-online.com
* Tel.: 161-8-9360-2457; fax: 161-8-9310-4144.
E-mail address: morgan@numbat.murdoch.edu.au (U.M. Morgan).
toxoplasmosis [4,8]. However, these are expensive and not
always conclusive [4].
Serological tests for the detection of Toxoplasma-specic
antibodies, such as the SabinFeldman dye test, rst
described by Sabin and Feldman 50 years ago, have been
widely used in the past [4]. The test is highly specic and
sensitive, and considerable information is available on the
development and persistence of dye test antibodies after
primary Toxoplasma infection. However, the test uses live
T. gondii and is now only employed in a few laboratories,
but it is still the reference method for the serodiagnosis of
toxoplasmosis [9]. A recent multicentre evaluation of the
test involving 19 laboratories in eight countries, revealed
overall satisfactory standardisation among the laboratories,
but there were differences in the test protocols, the use of
reference/standard preparations and the interpretation of
results. There is still no agreement on the level of dye test
values which reect infection with the parasite, and the
conversion from titres to international units (IUs) did not
improve standardisation. However, the results indicated that
a value of .4 IU or a titre of 1:16 met the denition of
positivity for most participants [9].
A number of PCR-based assays have been developed for
the detection of Toxoplasma in a variety of tissues which
offer increased sensitivity, many based on the B1 repeat and
the P30 antigen gene [1015]. The results to date have indi-
cated that PCR has not proven to be sufciently robust to
serve as a diagnostic test alone, but that a PCR-based on the
detection of a sequence with a much higher copy number
than the B1 would warrant further investigation [11]. As
PCR-based diagnosis of Toxoplasma has been recently
reviewed [11], only those most recent publications will be
discussed here.
A one tube hemi-nested PCR has been developed using
primers to the B1 gene with a reported sensitivity of 0.1
parasite [14]. In a comparison of different DNA purication
methods, the High Pure PCR Template Preparation kit
(Boehringer, Mannheim) gave the best results. With this
purication method and the one-tube hemi-nested PCR, T.
gondii DNA was detected in 14 of 16 (87.5%) clinical speci-
mens (amniotic uid, broncho-alveolar lavage, bone
marrow, blood, liver biopsy) for which the parasite was
demonstrated by cell culture [14]. Another study modied
a commercially available Toxoplasma PCR probe capture
assay by including uracil N-glycosylase (UNG) to prevent
carryover amplicon contamination [15]. The results
revealed that the probe capture assay showed a log increase
in detection sensitivity compared with standard agarose gel
electrophoresis. To assess the sensitivity and possible inhi-
bition of amplication, different sample types were spiked
with Toxoplasma organisms. After DNA extraction and
PCR amplication, a sensitivity of two tachyzoites for the
assay was determined in buffered saline, CSF serum and
amniotic uid; 20 tachyzoites for whole blood; and 200
tachyzoites for brain tissue [15]. An additional 20 human
serum and CSF samples submitted for Toxoplasma serolo-
gical testing were assayed by the PCR method. Of these,
only an IgM-positive CSF sample was PCR-positive. The
Toxoplasma PCR probe capture assay showed good sensi-
tivity and was not substantially inhibited by several different
clinically relevant samples [15].
The biological diagnosis of congenital toxoplasmosis at
birth is important to determine the infant's treatment. A
recent study analysed a total of 94 placentas, of which
33 came from foetuses suspected of or with proven conge-
nital toxoplasmosis (CT 1) and 61 from denitely or prob-
ably non-infected foetuses (CT 2), by in vitro culture,
mouse inoculation and PCR [12]. The results showed that
the PCR sensitivity (60.9%) was higher than that of cell
culture (29.6%) and mouse inoculation (51.5%), but the
number of PCR-positive results in CT-patients was also
higher (9.5%). The presence of T. gondii in the placenta
tissues was the only indication at birth (IgM and
neosynthesised Ig were negative) in three out of the 33
CT 1 cases. Overall, the detection of IgM by ELISA
and immunosorbent agglutination assay (ISAGA) and the
detection of neosynthesised Ig by immunoblotting were
more satisfactory to diagnose congenital toxoplasmosis,
but the placental analysis was important to improve the
sensitivity of the diagnosis at birth, especially when the
prenatal diagnosis was negative or not performed [12].
Toxoplasma gondii infection can also cause chorioretini-
tis resulting from congenital infection [13]. Patients are
often asymptomatic during life, with a peak incidence of
symptomatic illness in the second and third decades of
life. Diagnosis is mainly supported by ophthalmological
examination and a good response to installed therapy.
However, establishment of a diagnosis by ophthalmological
examination alone can be difcult in some cases. A recent
study evaluated the PCR detection of T. gondii in 56 blood
and 56 aqueous humor samples from 56 immunocompetent
patients [13]. Fifteen patients with a diagnosis of ocular
toxoplasmosis had increased serum anti-T. gondii IgG
levels, but were negative for anti-T. gondii IgM (group 1),
and 41 patients were used as controls (group 2). The
samples were taken before antiparasitic therapy was
initiated, and only one blood sample and one aqueous
humor sample were obtained for each patient. Single nested
PCRs and Southern-blot hybridisation were performed with
DNA extracted from these samples. The results obtained
showed sensitivity and specicity values of 53.3 and 83%,
respectively. Interestingly, among all patients with ocular
toxoplasmosis, a positive PCR result with the aqueous
humor sample was accompanied by a positive PCR result
with the blood sample. This result suggests that ocular toxo-
plasmosis should not be considered a local event, as PCR
testing of blood samples from patients with ocular toxoplas-
mosis yielded the same result as PCR testing of aqueous
humor samples. PCR testing may be useful for discriminat-
ing between ocular toxoplasmosis and other ocular diseases,
and also can avoid the problems associated with ocular
puncture [13].
U.M. Morgan / International Journal for Parasitology 30 (2000) 14071421 1408
Another study compared the PCR amplication of three
T. gondii genes (B1, P30 and 18S rDNA) in aqueous humor
[16]. Nested PCRs carried out using published methods
were optimised for maximum sensitivity and specicity.
Five pairs of oligonucleotide primers, directed against the
BI, P30 and ribosomal genes, were compared to determine
which sequences were most effective in detecting T. gondii
DNA. The methods were developed with DNA templates in
water and were subsequently applied to both normal and
inamed aqueous. After one round of PCR amplication,
P30 and ribosomal primers were able to detect 1 pg genomic
T. gondii DNA. However, those directed against the B1
gene were able to detect 50 fg (approximately a single
tachyzoite). This level of sensitivity was also achieved
using the P30 primers after a second round of PCR.
However, only primers based on the B1 gene maintained
this level of sensitivity in both normal and inamed
aqueous. B1-specic primers did not amplify sequences
from fungal, bacterial or human lymphocyte DNA. The
authors reported that `the B1 PCR protocol appears to be
the most sensitive protocol in the detection of T. gondii
DNA and has been successful in identication of T. gondii
DNA in ocular uids and retinal sections. This provides
direct evidence of the presence of T. gondii within the eye
and may therefore help in the management of toxoplasma
retinochoroiditis' [16].
To facilitate studies of vaccines and antimicrobial agents
effective against T. gondii infection, an assay system was
developed to semi-quantitate parasitaemia using PCR
amplication of T. gondii DNA obtained from the blood
of mice infected with the parasite. A competitive internal
standard DNA fragment of the B1 gene of T. gondii was
generated and used in PCR so that the amplied product
could be semi-quantitated and false negative results
avoided. The levels of parasitaemia in immunised and anti-
microbial treated mice were assayed by this semi-quantita-
tive PCR at various times after infection with T. gondii. The
results of these studies indicate that this highly sensitive
detection method is a rapid and reliable procedure that
can be employed to assess the abilities of vaccines or anti-
microbial agents to provide early protection following T.
gondii infection [17].
Understanding the population structure and epidemiol-
ogy of Toxoplasma is essential to control the spread of
infection. Molecular analysis has revealed that the popula-
tion structure of T. gondii is remarkably clonal, consisting
of just three predominant lineages (strain types I, II and III)
that are geographically widespread and found in a variety
of hosts, including humans [18]. Acute virulence is
strongly associated with the type I genotype, which exhi-
bits an enhanced replication rate in vitro and higher tissue
burdens in vivo relative to non-virulent lineages [18].
Molecular tools have been used to determine the preva-
lence of these three primary clonal lineages of T. gondii
in a potential food source of infection for humans [19]. A
total of 43 isolates of T. gondii that had been collected
from pigs at an abattoir in Iowa and their genotypes were
determined at the SAG1 and SAG2 loci. The results
revealed that type II strains were by far the most prevalent,
accounting for 83.7% of the isolates. The type III genotype
was identied in only 16.3% of the isolates. These preva-
lences differ signicantly from a previous sampling of
isolates from animals, but are similar to the frequencies
with which they occur in human disease cases. The type
I genotype was not identied in the isolates from pigs,
although these strains have previously been shown to
account for approximately 1025% of toxoplasmosis
cases in humans [19].
Another study used restriction fragment length poly-
morphism (RFLP) analysis of chromosomal DNA from 22
strains of T. gondii from the Czech Republic. Two virulent
strains, RH and P-CZ, had previously been isolated from
humans, the remaining 20 strains were isolated from
animals. Among the 20 recently isolated strains, nine
belonged to an avirulent lineage and only one strain, from
the wild cat Felis silvestris, belonged to a virulent lineage
[20].
To examine the correlation between T. gondii genotype
and congenital human toxoplasmosis, the polymorphism of
the microsatellite, consisting of a dinucleotide (TG) repeat
in the intron of the beta-tubulin gene, was investigated by
PCR [21]. Thirty-four reference strains were studied,
including seven strains virulent in mice and 27 strains
avirulent in mice. The seven virulent strains had a (TG)
8
microsatellite, and the avirulent strains had a (TG)
7
micro-
satellite. This conrms the dichotomy already observed for
virulent and avirulent strains. Additionally, 37 samples of
amniotic uid from infected foetuses were tested. All of
them had the (TG)
7
microsatellite marker. This result
conrms that most of the human cases of congenital toxo-
plasmosis are due to strains avirulent in mice. Neverthe-
less, their virulence in human foetuses was obvious, as
numerous abnormalities were observed on ultrasonic exam-
ination. More polymorphic microsatellites are required to
obtain new genetic markers for the direct screening of
biological samples [21].
In recent years, genomics has increased the understanding
of many diseases. Proteomics is a rapidly growing research
area that encompasses both genetic and environmental
factors [22]. A eld where proteomics can be a valuable
tool in identifying proteins of importance for diagnosis is
the proteome analysis of T. gondii and also other pathogenic
micro-organisms, such as Borrelia burgdorferi (Lyme
disease). Results have shown that antibody reactivity to
seven different marker antigens of T. gondii allowed differ-
entiation between acute and latent toxoplasmosis, an impor-
tant diagnostic tool in both pregnancy and
immunosuppressed patients. Similarly, sera from patients
with early or late symptoms of Lyme borreliosis contained
antibodies of various classes against about 80 antigens each,
containing the already described antigens OspA, B and C,
agellin, p83/100, and p39 [22].
U.M. Morgan / International Journal for Parasitology 30 (2000) 14071421 1409
3. Giardia
The intestinal protozoan Giardia duodenalis is a wide-
spread opportunistic parasite of humans and animals [23].
This parasite inhabits the upper part of the small intestine
and has a direct life-cycle. After ingestion of cysts, which
are the infective stage, the trophozoites emerge from the
cysts in the duodenum and attach to the small intestinal
mucosa of the host, which can result in malabsorption and
diarrhoea.
Traditionally, diagnosis of giardial infection has been
based on the detection of cysts or trophozoites in the faeces.
Due to the intermittent nature of cyst excretion by animals
and humans [24], three faecal specimens taken on non-
consecutive days are required for reliable assessment of
the presence of Giardia, since a single sample identies
only 5075% of positive patients [2426]. Faecal examina-
tion is tedious and relies on the visual identication of cysts
by light microscopy, which may be facilitated by concen-
tration using zinc sulphate or formal-ether and the use of
trichrome or iron haematoxylin stains [27]. In addition, low
numbers of cysts in stools and the inexperience of labora-
tory technicians can contribute to low sensitivity of conven-
tional detection of giardial infections [24].
A number of immunological assays have been developed,
including an improved immunouorescence assay [28]. The
detection limit was determined as 500 G. duodenalis cysts,
and the specicity was conrmed using a species-specic
PCR following immunomagnetic capture (IC) of cysts [28].
A recent study compared nine ELISAs using 222 individual
faecal samples submitted for the detection of G. duodenalis
[29]. The assays evaluated were manufactured by Alexon,
Inc., Cambridge Biotech Corp., Meridian, Inc., and Trend
Scientic, Inc. All assays used polyclonal antibodies, except
the `new and improved' Microplate (direct and diluted
methods) by Alexon, which is a mAb assay. Sensitivities
and specicities ranged from 88.6 to 100% and 99.3 to
100%, respectively [29]. The total hands on time needed
to run one specimen ranged from 1 to 2 min 17 s/specimen.
All except one commercially available ELISA were found
to be rapid, sensitive and specic for the detection of G.
duodenalis in faecal specimens [29]. However, Giardia
antigens have been shown to exhibit considerable variation
among isolates [30,31], and Moss et al. [32] warn that, as a
result of this antigenic variation, immunodiagnostic assays
based on mAbs may miss some Giardia infections.
Recently, Becton Dickinson has developed the ColorPAC
Giardia/Cryptosporidium solid-phase qualitative immuno-
chromatographic assay, which detects and distinguishes
between G. duodenalis and Cryptosporidium parvum in
human stools [33]. The agreement between the Alexon-
Trend ProSpecT Giardia Rapid EIA and the ColorPAC
assay was 166 of 172 (96.5%). The agreement between
the Alexon-Trend ProSpecT Cryptosporidium Rapid EIA
and the ColorPAC assay was 169 of 171 (98.8%). No
cross-reactions were seen with other parasites or human
cells [33]. A combination cassette format non-enzymatic
rapid immunoassay for the detection of Giardia and Cryp-
tosporidium antigens has also been developed and was eval-
uated using 556 patient stool specimens from three clinical
laboratories [34]. This assay (Genzyme Diagnostics
Contrast Giardia/Cryptosporidium), which can be used
with fresh or formalin-xed specimens, had unadjusted
sensitivities and specicities of 96.1 and 98.5% for Giardia
and 100 and 98.7% for Cryptosporidium, respectively in this
study [34].
In the last 30 years, endemic and epidemic water-borne
and food-borne outbreaks of giardiasis and also cryptospor-
idiosis in developed countries have led to a reappraisal of
conventional isolation and detection methods [35]. Robust,
efcient detection, viability and typing methods are required
to assess risks and for further epidemiological understand-
ing. A greater awareness of parasite contamination of our
environment and its impact on health has precipitated the
development of better detection methods [35,36]. The
upsurge in molecular techniques, particularly PCR, for
determining occurrence and viability have brought with
them the added benets of increased sensitivity and speci-
city, yet many methods still have to be shown to address
these issues consistently in the eld [35].
Filter-feeding molluscan shellsh can concentrate zoono-
tic and anthroponotic water-borne pathogens. Recently,
molecular tools were used to identify G. duodenalis geno-
type A from tissues of the clams, Macoma balthica and
Macoma mitchelli, from Rhode River, a Chesapeake Bay
(MD) subestuary [37]. Genotype A is the most common
genotype recovered from humans. Macoma clams are
burrowers in mud or sandy-mud substrata, and preferen-
tially feed on the surface sediment layer. Water-borne Giar-
dia cysts settle rapidly to the bottom in slow-moving waters
and contaminate the sediment. Macoma clams do not have
economic value, but can serve as biological indicators of
sediment contamination with Giardia sp. cysts, which are of
importance for public health. These clams can be used for
the sanitary assessment of water quality [37].
Giardia is capable of infecting a wide range of vertebrate
hosts, including species of mammals, birds and amphibians
[24,38]. The zoonotic potential of isolates of Giardia from
mammalian hosts has been difcult to determine due to the
lack of morphological differences. A recent study used
sequence analysis of the 18S rDNA gene to genotype Giar-
dia isolates from humans and dogs living in isolated Abori-
ginal communities in Western Australia where Giardia
infections are highly endemic and were presumed to be
zoonotic [38]. Interestingly, comparison of the 185-rRNA
sequences from 13 human and nine dog isolates suggested
that zoonotic transmission of Giardia infections between
humans and dogs does not occur frequently in these commu-
nities.
Other studies have used PCR-RFLP analysis, the triose-
phosphate isomerase (tpi) gene locus, as well as pulsed eld
gel electrophoresis (PFGE) and isoenzyme electrophoresis,
U.M. Morgan / International Journal for Parasitology 30 (2000) 14071421 1410
to genotype Giardia isolates from outbreaks [39]. This study
has highlighted the need for more sensitive molecular meth-
ods to detect and characterise Giardia in original host and
environmental samples [39]. The development of nger-
printing analysis of the more variable, non-coding inter-
genic rDNA spacer (IGS) will greatly assist in our
understanding of the transmission dynamics of Giardia in
the future [40].
4. Leishmania
Leishmaniasis is a spectrum of diseases, ranging in sever-
ity from cutaneous (CL), post-kala-azar dermal (PKDL) and
diffuse cutaneous (DCL) to mucocutaneous (MCL) and
visceral (VL) infections that are endemic in 86 tropical
and subtropical countries around the world, accounting for
75 000 deaths/year [41]. Different forms of leishmaniases
are generally caused by different distinct species of Leish-
mania having a digenetic life-cycle, alternating between an
aagellated amastigote form replicative within the macro-
phages of the host and a agellated promastigote form that
multiplies within the gut of the sandy [41,42].
The serodiagnosis of leishmaniasis is an alternative to
parasite detection in biopsy samples, either by the staining
of amastigotes or by culturing the amastigotes which trans-
form to a promastigote form and replicate. A battery of
immunological procedures have been developed or adapted
to demonstrate either humoral or cell-mediated immune
responses against Leishmania for diagnosis and epidemio-
logical survey. The sensitivity and specicity of such diag-
nostic methods depend on the type, source and purity of
antigen employed, as some of the leishmanial antigens
have common cross-reactive epitopes shared with other
micro-organisms, particularly Trypanosoma, mycobacteria,
Plasmodia and Schistosoma. Serodiagnostic techniques for
the detection of anti-leishmanial antibodies have been
employed with about 72100%, 2390%, 83 and 33
100% success in VL, CL, MCL and PKDL patients, respec-
tively [41].
The Leishmanin skin test (LST) is useful to detect MCL
and CL, with about 100 and 84% success, respectively [41].
In PKDL, the gradual fall of anti-leishmanial antibody titre
to some extent and the rise of delayed hypersensitivity to the
parasite antigen are the characteristic features associated
with the chronicity of the disease. The use of whole promas-
tigote as the source of antigens in the direct agglutination
test (DAT) and IFAT gave cross-reactions with the sera
from leprosy, tuberculosis and African trypanosomiasis
patients. Again, the use of cell-free extracts of promasti-
gotes generally gave false positive results with the sera
from normal human and individuals with Chagas' disease,
leprosy, tuberculosis and malaria patients in ELISA, dot
ELISA, immunodiffusion, immunoelectrophoresis and
counter-current immunoelectrophoresis tests. Leishmanial
proteinase, gp63, is not species-specic, but appears to be
Leishmania-specic, so it can be used to detect a case of
leishmaniasis. Acid phosphatase and the dp72-kDa protein
of Leishmania donovani cross-reacts with the sera of
leprosy patients. Active VL cases are conrmed by the posi-
tive results obtained for anti-leishmanial antibody detection
and negative results for LST. The titres of antibodies against
excreted factor (EF) or lipophosphoglycan (LPG) or lipo-
phosphopolysaccharide (LPPS) of Leishmania were found
to be insignicant for serodiagnostic purposes. The use of
ConA-positive glycoproteins released or secreted by
promastigotes of L. donovani showed 100% positive reac-
tions with VL in immunoelectrophoresis [41].
PCR is used increasingly widely for the diagnosis of both
CL and VL and the identication of asymptomatic carriers
in the population in endemic disease areas. A recent study
compared PCR diagnosis and the typing of Leishmania
parasites with conventional diagnostic techniques in clinical
specimens from patients suspected of CL [43]. Using
cultures as the reference standard, the PCR detection
method was shown to be more sensitive (92%) than classical
diagnostic techniques, including microscopy (42% sensitiv-
ity), histological staining (33%) and IgG ELISA (20%). The
PCR assay was also 100% specic. Parasites in both lesion
biopsies and isolates cultured from lesion aspirates were
identied as Leishmania braziliensis by PCR [43].
Another study compared the effectiveness of PCR with
microscopy for the diagnosis of VL [44]. PCR was
performed with bone marrow, lymph node and blood
samples from 492 patients, 60 positive controls and 90 nega-
tive controls. The results were compared with microscopy
results for Giemsa-stained smears. PCR and microscopy of
lymph node and bone marrow aspirates from patients with
microscopically conrmed VL were equally sensitive.
However, in patients clinically suspected of having VL
and in whom parasites could not be demonstrated by micro-
scopy, PCR was positive for 12 of 23 (52.2%) lymph node
aspirates and eight of 12 (66.7%) bone marrow aspirates,
thus conrming the clinical diagnosis of VL. With PCR on
lter paper, Leishmania DNA was detected in the blood of
33 of 47 (70%) patients with conrmed VL and in two of 11
(19%) patients suspected of having VL. Positive PCR
results were more frequently found for blood samples on
lter paper than for samples stored in EDTA. The authors
concluded that `PCR is a more sensitive method than micro-
scopy for the detection of Leishmania in lymph node and
bone marrow aspirates, being especially useful for the
conrmation of cases of suspected VL'. Blood from a nger
prick may be used for the initial PCR screening of people
suspected of having VL. If the PCR of blood is negative, one
should perform PCR with lymph node and/or bone marrow
material, because PCR with these materials is more often
positive [44].
The same group used PCR to detect leishmanial DNA
from 14 patients from lymph-node aspirates from 35
patients from eastern Sudan, who had had VL but were
believed to have been cured [45]. There were no signicant
U.M. Morgan / International Journal for Parasitology 30 (2000) 14071421 1411
differences between the PCR-positives and -negatives in
terms of age, sex, spleen size, malaria status or presence
of anti-Leishmania antibodies. However, PCR was more
often positive in the patients who tested negative by the
LST than in those who gave positive skin tests. Moreover,
patients with a positive PCR and a negative LST converted
more often to LST positivity than those with a negative PCR
and a negative LST. The most important nding was that,
during follow-up, eight (57%) of the PCR-positives, but
none of the 21 negatives, developed PKDL leishmaniasis.
Therefore, PCR-based testing of lymph-node aspirates after
treatment may be used as a prognostic marker for the future
development of PKDL and may be useful in the follow-up
of patients [45].
The use of complex-specic hybridisation probes in
conjunction with PCR increases the specicity as well as
the sensitivity of the diagnostic procedure as it discriminates
between different infecting Leishmania species. A minicir-
cle kinetoplast DNA probe, B4 Rsa, which hybridises to all
members of the Leishmania (L.) donovani complex has been
identied and characterised. It is a segment of a minicircle
highly conserved in Bangladeshi and Indian L. (L.) dono-
vani isolates [46].
A semi-nested PCR assay has been developed in order to
amplify the kinetoplast minicircle of Leishmania species
from individual sand ies [47]. The kinetoplast minicircle
is an ideal target because it is present in 10 000 copies/cell
and its sequence is known for most Leishmania species. The
two-step PCR is carried out in a single tube using three
primers, which were designed within the conserved area
of the minicircle and contain conserved sequence blocks.
The assay was able to detect as few as three parasites/indi-
vidual sand y and to amplify minicircle DNA from at least
eight Leishmania species. This technique permits the
processing of a large number of samples synchronously,
as required for epidemiological studies in order to study
infection rates in sand y populations and to identify poten-
tial insect vectors. Comparison of the sequences obtained
from sand ies and mammalian hosts will be crucial for
developing hypotheses about the transmission cycles of
Leishmania spp. in areas of endemicity [47].
A sensitive and specic PCR-ELISA technique has been
developed for use as a diagnostic test in cases of MCL [48].
DNA was extracted from cultures of L. braziliensis, Leish-
mania infantum, Leishmania tropica, Leishmania mexicana,
Trypanosoma cruzi, and blood samples from individuals
who presented a clinical diagnosis of leishmaniasis as
well as from healthy individuals. The DNA was PCR ampli-
ed and the product obtained was hybridised with a biotin-
labelled probe. The result of the hybridisation was visua-
lised by means of an ELISA technique, using antiuorescein
antibody labelled with alkaline phosphatase and p-nitrophe-
nylphosphate (pNFF) as the chromogen. The optical density
of the products of the pNFF hydrolysis was quantied in a
spectrophotometer at a wavelength of 405 nm. Using this
technique, the percentage of detection was 83.3% in blood
samples from patients clinically diagnosed as having MCL.
No false positive results were obtained [48].
Leishmania species of the subgenus Viannia are respon-
sible for a large proportion of New World leishmaniasis.
The mode of reproduction in and the population structure
of Leishmania (Viannia) parasites were deduced from the
nature and extent of genetic variation in the parasites [49].
Forty-seven zymodemes belonging to the Viannia subgenus
were analysed by enzyme electrophoresis and the proles
compared with those of two zymodemes belonging to the
Leishmania subgenus. The results revealed extensive
genetic variation in Viannia. The widespread occurrence
of certain zymodemes, the level of heterozygosity and the
great similarity observed among the samples are indicative
of a clonal population structure. However, the large number
of recurrent mutations needed to explain the variation
observed and the presence of hybrid parasites indicate that
occasional bouts of genetic exchange may occur [49]. Addi-
tional studies are required to better estimate the long-term
stability of clonal genotypes and the possible interference of
gene exchange at an evolutionary scale [49,50].
Recently, a set of microsatellite markers have been devel-
oped which can discriminate among all species within the
subgenus Viannia, including the closely related species
pairs: L. braziliensis and Leishmania peruviana; Leishma-
nia panamensis and Leishmania guyanensis [51]. Potential
species hybrids were uncovered in the analysis. These
markers are sufciently polymorphic such that within-
species epidemiological, population and genetic studies
are theoretically possible for all species analysed [51].
5. Echinococcus
Echinococcosis is an infectious disease of humans caused
by the larval (metacestode) stage of the cestode species,
Echinococcus granulosus (cystic echinococcosis or hydatid
disease) [52]. Alveolar echinococcosis (AE) in humans is
caused by Echinococcus multilocularis, which exhibits a
tumour-like growth, initially in the liver, with the potential
to induce serious disease [53]. At the end of the 1980s, E.
multilocularis was known to occur in four countries of
central Europe, but has now been identied in 10 countries.
Red foxes are the principal denitive hosts of E. multilocu-
laris and sources of human infection, but dogs and cats can
also be infected. Growing populations of foxes and their
increasing immigration to urban areas are new risk factors
[53]. The nal/intermediate cycles of E. granulosus include:
(a), dog/sheep; (b), dog/horse; (c), dog/cattle; (d), dog/pig;
(e), dog/reindeer; (f), wallaby/dingo [54]. Infection in
humans is accidental and occurs via the ingestion of eggs
shed by the denite host.
Medically, hydatid disease is signicant because surgery
remains the only effective cure for patients with active,
viable hydatid cysts [55,56], although advanced technolo-
gies (intraoperative ultrasonography, CT scan, argon coagu-
U.M. Morgan / International Journal for Parasitology 30 (2000) 14071421 1412
lator, etc) have changed the surgical approach of liver hyda-
tid disease, allowing even multiple or deeply located cysts
to be detected and treated successfully [57]. Diagnosis relies
predominantly on imaging procedures and serology [53,58].
Immunodiagnosis must be performed early, especially when
a preclinical diagnosis is desired upon exposure to infection
[52,56].
In view of the public health signicance of Echinococcus,
there is an urgent need for reliable and simple techniques for
the diagnosis of the infection in populations of nal hosts
[59]. Recently, extensions of the range of E. multilocularis
in Europe and North America and drastic increases in fox
populations in Europe have put an increasing proportion of
the human population at risk of AE [60]. To obtain data on
the local infection pressure, studies of the prevalence of the
parasite in the animals that transmit the parasite, foxes, dogs
and cats, are urgently required. Such investigations,
however, have been hampered by the need for necropsy of
the host animal to specically diagnose infection with the
parasite [60]. An ELISA has been evaluated using rabbit and
chicken polyclonal antibodies against E. multilocularis anti-
gens (afnity-puried coproantigens and somatic adult
worm antigens). The specicity of this test (evaluated in
20 foxes and 661 dogs with helmintic infections other
than E. multilocularis) was very high (9599.5%). The aver-
age sensitivity in 35 foxes infected with E. multilocularis
was 80%, but reached 93% in foxes with individual worm
burdens over 55 [59]. A PCR assay was used for detecting
the DNA of E. multilocularis in faecal samples of foxes after
the parasite eggs had been isolated by a sieving procedure.
In a total of 55 foxes, the specicity was 100% and the
sensitivity was 94%. For eld applications, the coproantigen
ELISA has the potential of replacing parasite detection at
necropsy, and PCR is a valuable method for the conrma-
tion of positive coproantigen results and for diagnosis in
individual animals [59]. The detection of circulating anti-
Em2 antibodies by ELISA may be useful for the primary
screening of fox populations, but antibody prevalence rates
do not correlate with the prevalence rates of the intestinal
infection with E. multilocularis [59].
More recently, a sandwich-ELISA for the detection of E.
multilocularis coproantigens (EM-ELISA) was developed
with polyclonal rabbit (solid phase) and chicken egg (catch-
ing) antibodies that were directed against E. multilocularis
coproantigens and somatic worm antigens, respectively
[61]. The EM-ELISA was used in surveys of `normal' dog
and cat populations in Switzerland. Among 660 dogs and
263 cats, ve dogs and two cats exhibited a positive reac-
tion. In two of these dogs (0.30%) and one cat (0.38%),
intestinal E. multilocularis infections were conrmed by
necropsy, PCR, or both. The specicites of the ELISA in
these groups were found to be 99.5 and 99.6%, respectively,
if positive ELISA results that could not be conrmed by
other methods were classied as `false positive' reactions
[61].
Another study developed a nested PCR and an improved
method for DNA extraction to allow the sensitive and speci-
c diagnosis of E. multilocularis infections directly from
diluted faecal samples from foxes with echinococcosis
[60]. The target sequence for amplication is part of the
E. multilocularis mitochondrial 12S rRNA gene. The speci-
city of the method was 100% when it was tested against 18
isolates (metacestodes and adult worms) of 11 cestode
species, including E. granulosus. The sensitivity of the
method was evaluated by adding egg suspensions and indi-
vidual eggs to samples of diluted faeces from uninfected
foxes. The presence of one egg was sufcient to give a
specic signal. PCR results were conrmed with an internal
probe. Necropsy and PCR of the rectal contents of 250 wild
foxes from an area in southern Germany where echinococ-
cosis is highly endemic revealed that the sensitivity of the
traditional and widely used necropsy method was only
approximately 76% [60].
In a recent study, E. multilocularis was demonstrated in
ve out of 272 foxes in the Netherlands close to the border
with Germany and Belgium [62]. Besides microscopic
examination of mucosal scrapings, two different PCR assays
were used based on the detection of E. multilocularis DNA
in colon content. The results revealed that two distinct areas
in The Netherlands were positive for E. multilocularis. Two
positive foxes were found in the northern province of
Groningen and three positive foxes were found in the south-
ern province of Limburg. Both PCR assays detected more
positive foxes compared with microscopic examination of
the intestinal content. This is the rst report of E. multi-
locularis occurring in foxes in the Netherlands [62].
The diagnosis of E. granulosus infections in canids has
traditionally involved unreliable coproparasitological and
purging methods [63]. The detection of coproantigens in
faeces of infected dogs by ELISA is suitable for detecting
patent and prepatent infections with a high degree of sensi-
tivity and specicity. In a recent study, natural and experi-
mental infections in domestic and wild Australian canids
were investigated using a coproantigen capture ELISA.
The results suggest that the coproantigen ELISA could be
successfully used to monitor E. granulosus prevalence rates
in Australian domestic dogs, foxes and wild dogs [63].
Another study which compared a coproantigen enzyme-
immunoassay (EIA) test and arecoline purging for the diag-
nosis of E. granulosus in canines revealed the frequency of
canine echinococcosis to be 46% by EIA and 32% by
purging, indicating that immunological methods are more
sensitive [64]. Similarly, the frequency of sheep echinococ-
cosis was shown to be 65% by an enzyme-linked immunoe-
lectrotransfer blot (EITB) assay and 38% by necropsy [64].
Evidence for strain diversity within E. granulosus has
been conrmed based on morphological, biological and
biochemical features and genetic studies [64]. Several mole-
cular techniques which allow the identication of E. gran-
ulosus strains are now available [65,66]. Recently, dideoxy
ngerprinting (a hybrid between single-strand conformation
polymorphism (SSCP) and dideoxy sequencing, employing
U.M. Morgan / International Journal for Parasitology 30 (2000) 14071421 1413
only one dideoxynucleotide in the sequencing reaction) has
been used to genetically type E. granulosus parasites, utilis-
ing the mitochondrial cytochrome c oxidase subunit I (COI)
as the gene sequence for analysis [67]. All of the seven
genotypes (G1, G4, G6, G8, O, V and M2) examined
could be readily differentiated from one another by their
characteristic and reproducible dideoxy ngerprinting
proles. Only a subtle variation in proles was detected
among some of the eight isolates representing genotype
G1, and no variation was detected between two samples
of genotype G4 and of genotype M2 [67]. Another study
analysed 16 human and animal isolates of E. granulosus
using PCR-RFLP analysis of the COI and NADH dehydro-
genase subunit I (NDI) genes [68]. The analysis clearly
indicated that the camel/dog strain (G6 genotype) of E.
granulosus, as well as the cosmopolitan, common sheep
strain (G1 genotype), occur in Iran. The G1 genotype was
found to be present in all four human isolates examined and
it was more prevalent in domestic animals than the camel-
restricted G6 genotype. These results indicate that in E.
granulosus-endemic areas of Iran, the majority of E. gran-
ulosus-infected livestock animals can potentially act as
reservoirs for human infection, and this has important impli-
cations for hydatid control and public health [68].
Epidemiological evidence and molecular studies indicate
that the so-called sheep, cattle and cervid strains of E. gran-
ulosus are infective to humans, while the horse, camel and
pig strains may be less or not infective, but this question
warrants further studies. A recent study indicates that E.
granulosus infecting patients in Poland shares a close mole-
cular afnity with a genotype of pig origin (G7), but exhibits
some clear differences [66]. Therefore, it may represent a
previously undescribed genotype of E. granulosus, desig-
nated as G9 [66]. Phylogenetic analysis of molecular data
has demonstrated the need to reappraise the taxonomic
status of currently recognised strains. Clear evidence for
strain variation in the other species of Echinococcus does
not exist at present [69].
6. Angiostrongylus
Abdominal angiostrongyliasis is a nematode disease
produced by Angiostrongylus costaricensis [70]. The de-
nitive hosts of A. costaricensis are forest rodents, while
snails and slugs are its intermediate hosts. Infection in
humans is accidental and occurs via the ingestion of food
or water contaminated with third-stage larvae present in the
mucous secretion of the intermediate hosts, or by direct
contact with the mucus [71]. Abdominal angiostrongyliasis
is clinically characterised by prolonged fever, anorexia,
abdominal pain in the right-lower quadrant and peripheral
blood eosinophilia. The disease is endemic in Southern
Brazil and a number of cases are diagnosed every year.
Larvae are not discharged in the faeces of humans as they
are in the rat, although larvae in eggs and free larvae may be
seen in sections of human tissue [7]. In the past, the diag-
nosis has been based on anatomo-pathological examination
of biopsies or surgical specimens [7]. Recently, an ELISA
test was standardised for the detection of IgG antibodies,
recognising a surface antigen prepared from female worms
of A. costaricensis [70]. Competitive absorption of sera with
Ascaris suum crude antigen resulted in a test with 86%
sensitivity and 83% specicity. According to seroepidemio-
logical studies, the prevalences in two transmission foci are
23.8 and 66%, attesting to the widespread occurrence of the
infection in those endemic areas [70].
Another nematode parasite, Angiostrongylus cantonensis,
is the most common cause of eosinophilic meningitis in
humans. In the past 50 years, it has spread from Southeast
Asia to the South Pacic, Africa, India, the Caribbean, and
recently, to Australia and North America, mainly carried by
cargo ship rats [72]. Humans are accidental, `dead-end'
hosts infected by eating larvae from snails, slugs, or
contaminated, uncooked vegetables. These larvae migrate
to the brain, spinal cord and nerve roots, causing eosinophi-
lia in both spinal uid and peripheral blood. Infected
patients present with severe headache, vomiting, paresthe-
sias, weakness, and occasionally, visual disturbances and
extraocular muscular paralysis. Most patients have a full
recovery; however, heavy infections can lead to chronic,
disabling disease and even death [7,72].
Historically, the diagnosis of A. cantonensis has been
based on clinical symptoms and sometimes by the identi-
cation of worms in the CSF [7]. A double-antibody, sand-
wich-ELISA has been developed to detect circulating
antigens of A. cantonensis in patients with eosinophilic
meningitis or meningoencephalitis (EME) and in mice
experimentally infected with A. cantonensis [73].The
ELISA values in the detection of circulating antigens in
CSF from patients were markedly higher than those in
serum. Immunodiagnosis of patients with angiostrongyliasis
by this technique proved to be highly specic for circulating
antigens in serum and CSF specimens; however, the sensi-
tivity in CSF was signicantly higher than in serum [73].
More recently, an antigen from A. cantonensis L5 was
puried by immune-afnity chromatography with a specic
mAb [74]. The puried antigen showed only a single band
with a Mr of 204 kDa in SDS-PAGE, and no cross-reactivity
to antibodies induced by several other species of helminths
was observed in ELISA. When the puried antigen was used
to examine serum and CSF specimens by ELISA, the anti-
body levels in patients with eosinophilic meningitis or EME
were signicantly higher than those of control subjects. The
antibody levels in serum were slightly higher than those in
CSF, and the levels in serum were positively correlated with
the levels in CSF. The reliability of the detection of anti-
bodies in serum was slightly higher than that for the detec-
tion of antibodies in CSF specimens. The purication of a
specic A. cantonensis antigen and its subsequent use in the
development of an ELISA for detection of A. cantonensis-
specic antibodies in serum specimens constitutes an
U.M. Morgan / International Journal for Parasitology 30 (2000) 14071421 1414
important step towards an improvement in the accuracy of
diagnosis for A. cantonensis infections [74].
7. Babesia
Babesiosis is a world-wide distributed protozoal zoono-
sis, and Babesia spp. are the most ubiquitous of the blood
parasites of mammals, except the trypanosomes [75]. These
tick-transmitted protozoa infect various vertebrate reservoir
hosts, like rodents, cattle and horses. Approximately 100
million cattle are exposed to the disease. In tropical and
subtropical regions, the infection causes considerable losses
in the livestock industry, but clinical cases of human babe-
siosis in these areas have not certainly been identied. The
rst demonstrated case of human babesiosis in the world
was reported in Europe, in 1957. Since then, more than 28
babesial infections in man have been reported in Europe.
Most (83%) of the infections were in asplenic individuals
and most (76%) were with Babesia divergens, a cattle para-
site [76]. Hundreds of cases of human infection with Babe-
sia spp. have been reported in the USA. Most cases were
infected by ticks carrying the rodent parasite Babesia
microti, but other emerging Babesia spp. (currently known
as WA1, CA1 and MO1) are increasingly involved. Several
cases have been the result of blood transfusions. In terms of
clinical manifestations, human infections with B. microti
can vary widely, from asymptomatic infection to a severe,
rapidly fatal disease [75,76]. The splenectomised, the
elderly, the immunocompromised and HIV-infected
patients are predisposed to severe infection [76,77]. Infec-
tion with B. microti can remain subclinical or asymptomatic
and only be detected through serological surveys. A few
other cases of human babesial infection have been described
in China, Egypt, Mexico, South Africa and Taiwan [76].
Improved testing methods for the detection of infection
with B. microti are needed, since asymptomatic B. microti
infection represents a potential threat to the blood supply in
areas where B. microti is endemic [78]. The specic diag-
nosis of babesiosis is made by microscopic identication of
the organism in Giemsa-stained thin blood smears, detection
of the babesial antibody in acute- and convalescent-phase
sera, or identication of the organism following the injec-
tion of patient blood into laboratory animals [79]. Although
rapid diagnosis can be made with thin blood smears, para-
sites are often not visualised early in the course of infection.
Usually less than 1% of erythrocytes are parasitised early in
the course of the illness [80]. Antibody testing is useful for
the conrmation of babesosis, but it may also yield a nega-
tive result in the early phase of illness [81]. In cases in which
the presence of Babesia is suspected, but not demonstrated
by blood or antibody studies, blood from the patient can be
injected by the i.v. or i.p. route into small laboratory
animals, such as hamsters or gerbils. If present in the
patient, B. microti usually does not appear in the blood of
the test animal until 24 weeks after inoculation [82].
The use of PCR for the detection of Babesia spp. has not
been extensively compared with standard methods for the
detection of babesiosis. A recent study conducted a blinded
study of the sensitivity, specicity and reproducibility of a
PCR-based test with patients with babesiosis and a group of
asymptomatic subjects residing in a region in southern New
England where babesiosis is enzootic [79]. Among 19
patients with recent babesial illness, PCR was as sensitive
and specic as the use of Giemsa-stained blood smears and
inoculation of hamsters. Among asymptomatic subjects, the
PCR result was positive for three persons with recent babe-
sial infection and negative for 41 persons without previous
babesial infection. The researchers concluded that `the B.
microti PCR procedure is sufciently sensitive, specic and
reproducible for use in the diagnosis of acute babesiosis'
[79].
Babesiosis was recently detected in three residents of
New Jersey who were suspected of local acquisition of B.
microti infection [83]. Serial blood samples from these resi-
dents were positive for B. microti by immuno uorescent
assay (IFA) and PCR. All three residents experienced symp-
toms suggestive of acute babesiosis. The sera of each of the
patients reacted against a babesial antigen at a titer four-fold
or higher in sequentially collected blood samples. PCR-
ampliable DNA, characteristic of B. microti, was detected
in their blood. These data suggest that human B. microti
infections were acquired recently in New Jersey, extending
the range of this piroplasmosis in the north-eastern United
States [83].
The presence of non-pathogenic piroplasms may confound
eld estimates of the risk of B. microti infection. A recent
study demonstrated the utility of PCR in assessing risk
factors for Babesia infection in the USA [84]. Sporozoites
infecting the salivary glands of deer ticks (Ixodes dammini)
were identied by parallel microscopy and PCR assays [84].
Piroplasms were evident in 14.4% of adult ticks from sites in
the north-central and north-eastern United States. Of these,
83.3% contained DNA characteristic of Babesia odocoilei.
This cervid piroplasm was detected in all of the sites exam-
ined and was generally more prevalent than B. microti. As
deer ticks transmit both B. odocoilei and B. microti, estimates
of pathogen prevalence based solely on microscopy may
overestimate the risk of human babesiosis [84].
There have been few studies on the molecular character-
isation of B. microti isolates. However, a recent study has
shown that that considerable genetic and antigenic diversity
exists among isolates of B. microti from the United States
and that geographic clustering of subtypes may exist [78].
Further studies are required in order to understand the popu-
lation structure and extent of genetic variability between
isolates of B. microti.
8. Trichinella
Trichinosis is a world-wide zoonotic disease closely
U.M. Morgan / International Journal for Parasitology 30 (2000) 14071421 1415
related to cultural and dietary habits caused by nematodes
belonging to the genus Trichinella [85]. Trichinella spp.
have the widest geographical distribution and the largest
range of host species of all parasitic nematodes [86].
There are more than eight distinct types of species: Trichi-
nella spiralis, which infects swine, wild boars, bears, horses
and foxes; Trichinella nativa, which infects bears and
horses; Trichinella britovi, which infects wild boars and
horses; Trichinella pseudospiralis, which infects birds and
omnivorous mammals; and Trichinella nelsoni, which
infects warthogs, as well as some undened species desig-
nated T5, T6 and T8 [87]. More recently, Trichinella T5,
collected from sylvatic carnivores in North America has
been proposed as a distinct species, Trichinella murrelli,
on the basis of distinct biological and genetic differences
[88]. Another new species, Trichinella papuae has been
proposed from domestic and sylvatic swine of Papua New
Guinea [89]. The majority of human infections are caused
by T. spiralis, and it remains a signicant human pathogen
[87,90]. Human infection is acquired through the ingestion
of undercooked meat containing infective encysted larvae.
The clinical presentations of signs and symptoms are
remarkably constant for most of the species of Trichinella,
but in infections with T. nativa and T. britovi, classical
symptoms of trichinellosis may be absent. It is important
to be able to correlate the clinical presentation of trichinel-
losis with the life-cycle of these helminths in order to make
an accurate diagnosis [90].
The diagnosis generally involves examining a muscle
biopsy for the presence of larvae which can be seen in
heavy infections or by histopathological examination of
the biopsy sample as infected muscle cells undergo baso-
philic changes once they are penetrated by larvae [87].
Immunouorescent and ELISA-based assays have been
developed [9193]. One study compared testing for circu-
lating parasite antigens versus antiparasite antibodies in sera
collected from patients with suspected or conrmed expo-
sure to T. spiralis [92]. The results indicated that antibody
detection is a more sensitive diagnostic method for human
trichinellosis, but that antigen detection might be a useful
conrmatory test because it is a direct demonstration of
parasite products in the circulation [92].
Several investigators have successfully applied PCR to
the amplication of DNA from T. spiralis muscle-stage
larvae [94,95]. It has also been shown that specic DNA
can be amplied from T. spiralis migratory larvae in the
blood of experimentally infected mice [96] and human
patients [97]. Knowledge of the epidemiology of the disease
is essential for identifying other potential cases, since most
epidemics can be traced back to a common source of raw or
undercooked meat [87]. Therefore, rapid and sensitive
differentiation between different genotypes and species of
Trichinella is important. Several groups have developed
genotyping tools for Trichinella [98103].
A PCR-based SSCP analysis of the expansion segment 5
(domain IV) of the LSU of rDNA was employed to char-
acterise seven isolates of Trichinella from China (AG),
including six of pig origin from regions in Dengxian (A),
Tianjin (B), Harbin (D), Baoshan (E), Xinye (F) and Xian
(G), and one of canine origin from Changchun (C) [99].
Isolates A, D, E, F and G were classied as T. spiralis
based on SSCP patterns, while the patterns for isolates B
and C were consistent with those of T. nativa or Trichinella
T6. The results were supported by random amplied poly-
morphic DNA (RAPD) analysis using ve decamer primers
and were in accordance with ecological information for the
isolates. SSCP results also allowed the direct display of
mutational sequence variation in the expansion segment
among the ve isolates of T. spiralis from China, indicating
its usefulness for studying population variation within that
species [99].
A single PCR test for the simple and unequivocal differ-
entiation of all currently recognised genotypes of Trichi-
nella has been developed recently [103]. Partial DNA
sequence data were generated from internal transcribed
spacers ITS1 and ITS2, and from the expansion segment
V region of the rDNA repeat from ve species of Trichinella
and two additional genotypes, designated T5 and T6. Five
different PCR primer sets were identied which, when used
simultaneously in a multiplex PCR, produce a unique elec-
trophoretic DNA banding pattern for each species and geno-
type including three distinct genotypes of T. pseudospiralis.
The technique was developed further to distinguish geno-
types at the level of single muscle larvae using a nested,
multiplex PCR, whereby the entire internal transcribed
spacer region, as well as the gap region of the expansion
segment V of the large subunit ribosomal DNA were ampli-
ed concurrently in a rst-round PCR using primer sets
specic for each region, followed by the multiplex PCR
for nal diagnosis [103].
Wu et al. [102] have used PCR-RFLP analysis to identify
ve species (T. spiralis, T. britovi, T. nativa, T. nelsoni and
T. pseudospiralis) and three phenotypes of uncertain taxo-
nomic status (Trichinella T5, T6 and T8). PCR-RFLP analy-
sis of the mitochondrial cytochrome c-oxidase subunit I has
been used to identify nine species/genotypes (T. spiralis, T.
britovi-European strains, T. britovi-Japanese strains, T.
nativa, T. nelsoni, Trichinella T5, Trichinella T6, Trichi-
nella T8 and T. pseudospiralis) [101]. The results obtained
with this PCR-RFLP assay conrmed those previously
reported by others and support the separation of the Japa-
nese isolates as a new genotype, namely Trichinella T9
[101].
There has been a decline in the number of human trichi-
nellosis cases associated with consumption of commercial
pork in the United States, while the relative importance of
trichinellosis from game meats has increased. An investiga-
tion of an outbreak of trichinellosis in Idaho occurring after
the consumption of improperly prepared cougar jerky
revealed that 10 cases of trichinellosis were identied
among 15 persons who ate the implicated meat [98]. Viable
Trichinella larvae were recovered from frozen cougar
U.M. Morgan / International Journal for Parasitology 30 (2000) 14071421 1416
tissue. PCR on parasite DNA yielded results consistent with
genotypes T. nativa and Trichinella type T6. This report of
cougar meat as a source of human trichinellosis and the
nding of freeze-resistant Trichinella organisms in wildlife
in Idaho extends the range of this genotype. Consumers of
game need to cook the meat thoroughly, since even frozen
meat may harbour viable Trichinella that can cause illness
[98].
Future molecular epidemiological studies to identify
Trichinella genotypes will greatly assist in distinguishing
between sylvatic and synanthropic life-cycles. Such infor-
mation will be critical in tracing sources of trichinellosis by
easily and unambiguously identifying likely host reservoirs
and will provide valuable information for instituting meth-
ods of control [100].
9. Helminth zoonoses
Although much remains to be done, many improved
detection and characterisation procedures have been devel-
oped for the clinical diagnosis and epidemiological studies
and control programs directed against the helminth
zoonoses, a few of which are discussed below.
Anisakiasis, or anisakidosis, is a parasitic zoonosis due to
the infestation by nematodes of the Anisakidae family,
mainly by Anisakis simplex [104], which parasitises several
sea-sh and cephalopods [105]. Human infection occurs
when raw or undercooked sh is consumed [106]. The clin-
ical features of anisakiasis may simulate acute abdominal
pain, such as that found in patients with gastric ulcers,
appendicitis and Crohn's disease [106].
Anisakis simplex induces not only anisakiasis, but also
anaphylactic reactions [105]. In one study, 28 patients
developed immediate hypersensitivity to A. simplex, after
ingestion of parasitised sh. Each case was diagnosed by
suggestive anamnesis, skin prick tests with an A. simplex
extract, specic IgE detection in serum (CAP System) and
histamine release test. The clinical manifestations were urti-
caria/angioedema in all 28 patients and respiratory arrest in
one. It was also proven that the allergen(s) involved may be
resistant to cooking and deep freezing. Hence, anaphylactic
reactions may result either from infection or, more
frequently, from mere exposure to the allergen. Therefore,
physicians must take into account that the consumption of
parasitised sh may cause severe reactions, even if sh of
the same kind is subsequently tolerated [105].
Anisakiasis has a world-wide distribution, but in Spain,
its appearance is quite recent (1991) with only 19 cases
previously reported. A recent report details 13 cases diag-
nosed in different hospitals in the province of Cordoba,
Spain, from September 1994 to July 1998 which represents
the biggest series described in Spain to date [104]. Only in
one of the cases were parasitic fragments detected in the
intestinal mucosa, and in the 12 remaining cases, the diag-
nosis was immunological by IgE specic for A. simplex
determination and antigen detection of the nematode with
mAb [107].
PCR-based studies have recently been developed for
three species of anisakid nematode from sh, A. simplex,
Hysterothylacium aduncum and Contracaecum osculatum
[107]. The nuclear rDNA region spanning the ITS1, the
5.8S gene and the ITS2 was amplied and sequenced.
Based on the sequence differences, PCR-RFLP and SSCP
polymorphism methods were established for the unequivo-
cal delineation of the three species. These methods should
provide valuable tools for studying the life-cycle, transmis-
sion pattern(s) and population structure of each of the three
anisakid nematodes examined herein, and for the diagnosis
of anisakiasis in humans and animals [107].
Toxocarosis is the clinical disease in humans caused by
infection of zoonotic roundworms of dogs and cats, Toxo-
cara canis and Toxocara cati [108,109]. The disease
comprises three syndromes: visceral larva migrans, ocular
toxocarasis and mild hypereosinophilia. The diagnosis
relies upon microscopy and also immunological methods
(ELISA and Western-blot) that use Toxocara larval ES anti-
gens [109]. Morphologically, it is very similar to other
nematode species, such as T. canis, T. cati and Toxascaris
leonina. Recently, PCR-RFLP analysis of the rDNA ITS-2
spacer region have been developed to differentiate these
three species [110].
Capillaria hepatica is a helminth that can cause parasitic
hepatitis in humans and is transmitted by deer mice (Pero-
myscus maniculatus). In the past, the only way of detecting
C. hepatica was to perform a liver biopsy. Recently, an
indirect immunouorescence (IIF) assay, based on liver
sections of naturally infected mice and human serum
samples, has been developed for detecting early stages of
human infections and for screening purposes. No cross-
reactivity with other parasitic infections was detected [111].
Cerebral sparganosis is a rare parasitic disease caused by
infestation by the plerocercoid larva of Spirometra mansoni
[112]. Humans become infected with spargana in one of
three ways: ingestion of water containing copepod crusta-
ceans (Cyclops spp.) infected with procercoids, penetration
of wounds or mucous membranes by spargana as a result of
direct contact with the esh of intermediate hosts, or inges-
tion of spargana in intermediate hosts [113]. The diagnosis
of sparganosis can be made on the basis of brain MRI and
ELISA. Comparison of a colorimetric ELISA and chemilu-
minescence ELISA for the detection of specic IgG in the
serum of nine patients with S. mansoni and nine healthy
controls revealed that the chemiluminescence ELISA was
able to measure serum levels of specic IgG over a far wider
range than the colorimetric assay, and its detection limit was
at least 10-fold lower [114].
Oesophagostomiasis in humans due to infection with
Oesophagostomum bifurcum (nodular worm) is of major
human health signicance in northern Togo and Ghana
where Necator americanus (the human hookworm) also
exists at high prevalence. However, very little is known
U.M. Morgan / International Journal for Parasitology 30 (2000) 14071421 1417
about the transmission patterns of O. bifurcum, partly due to
the difculty in differentiating O. bifurcum from N. amer-
icanus at some life-cycle stages using morphological
features [115]. Accurate diagnosis of O. bifurcum infection
in humans is central to studying the epidemiology and
controlling the parasite. However, the diagnosis of oesopha-
gostomiasis on clinical grounds alone is difcult. Ultra-
sound has been used to aid diagnosis [116], but a
denitive diagnosis is still difcult. To overcome some of
these limitations, a molecular approach utilising genetic
markers in ITS-2 of rDNA was developed [115]. The ITS-
2 sequence of each species was determined, and specic
oligonucleotide primers were designed to the regions of
greatest sequence difference between the species. Utilising
these primers, rapid PCR assays were developed for the
specic amplication of DNA of O. bifurcum or N. amer-
icanus, which have the potential to conrm the identity of
eggs from faeces and larvae from the intestine or environ-
ment [115]. More recently, a two-step, semi-nested PCR
method for the specic amplication of minute amounts
(fg) of O. bifurcum DNA from human faecal samples was
used [117]. Using a panel of 155 well dened faecal and
DNA samples, the assay achieved a sensitivity of 94.6% and
a specicity of 100%. This PCR assay will be useful for the
diagnosis of O. bifurcum infection and as a molecular tool
for elucidating the epidemiology of human oesophagosto-
miasis [117].
10. Future needs and opportunities for molecular
diagnosis and characterisation
Molecular diagnostics are fast, efcient and more amen-
able to large through-put processing. Since conventional
microscopic parasitological examinations are amongst the
most time-consuming procedures in a clinical laboratory,
the use of molecular-based tests may lead to a more cost-
effective and clinically efcient use of the diagnostic para-
sitology laboratory. However, many PCR methods will need
improvement before reliable diagnostic methods become
available. The clinical applicability will depend on the
availability of highly standardised detection systems,
including methods and ingredients for sample collection,
sample preparation and detection, and identication of
PCR products [118]. In the future, the development of
multiplex PCR assays which would enable the simultaneous
detection of several pathogens in one assay, such as the tick-
borne pathogens B. microti, Ehrlichia spp. and B. burgdor-
feri, would be an extremely useful public health tool, and an
attractive, cost-effective procedure for clinical diagnostic
laboratories.
Molecular characterisation allows us to distinguish
between closely related infectious agents or to document
the patterns of transmission of `strains' and species within
populations. This will allow precise determinations to be
made about the aetiological agent, its characteristics and
the source of infection. Phenotypic markers will hopefully
soon be available so that more accurate predictions can be
made about the clinical course of any resultant disease and
the most appropriate therapy. Techniques that utilise viru-
lence markers for parasites and susceptibility markers of
human populations are also needed to provide information
on why infections occur and if they are likely to occur in the
future; for example, ligands and receptors that mediate the
attachment of infectious agents to the surface of host cells
are being described at the molecular level [119]. Similarly,
processes associated with the invasion of tissues by infec-
tious agents and dissemination within the host are also being
investigated. Characterisation of the molecules and genes
involved may provide the basis for the development of
newer techniques.
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