BRIEF REPORT
Molecular Confirmation of Sappinia
pedata as a Causative Agent of Amoebic
Encephalitis
Yvonne Qvarnstrom,1 Alexandre J. da Silva,1 Frederick L. Schuster,2,a
Benjamin B. Gelman,3 and Govinda S. Visvesvara1
1Division of Parasitic Disease, National Center for Zoonotic, Vector-Borne, and
Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia;
2California Department of Public Health, Viral and Rickettsial Disease
Laboratory, Richmond; 3Department of Pathology, Surgery, Internal Medicine,
and Radiology, University of Texas Medical Branch, Galveston, Texas
(See the editorial commentary by Marciano-Cabral, on pages 1104 6.)
Pathogenic free-living amoebae, such as Acanthamoeba spe-
cies, Balamuthia mandrillaris, and Naegleria fowleri, are
known to cause infections of the central nervous system in hu-
man and other animals. In 2001, a case of human encephalitis
was reported that was caused by another amoeba with mor-
phological features suggestive of Sappinia. The amoeba origi-
nally identified as Sappinia diploidea was identified, most
likely as S. pedata, by use of newly developed real-time poly-
merase chain reaction assays. This amoeba had previously
been found only in environmental sources, such as soil and
tree bark. The results illustrate the potential for other free-
living amoebae, which are not normally associated with hu-
man disease, to cause occasional infections.
Free-living amoebae are ubiquitous in the environment world-
wide. A few species are associated with severe infections in hu-
mans. Naegleria fowleri can, under rare circumstances, cause
rapidly progressing primary meningoencephalitis, mainly in
Received 4 September 2008; accepted 22 October 2008; electronically published 24 March
2009.
Potential conflicts of interest: none reported.
Financial support: All funding for this work was provided by the authors respective
institutions.
Use of trade names and commercial sources is for identification only and does not imply
endorsement by the Centers for Disease Control and Prevention or the US Department of
Health and Human Services. The findings and conclusions in this report are those of the
authors and do not necessarily represent the views of the Centers for Disease Control and
Prevention and/or the Agency of Toxic Substances and Disease Registry.
a Deceased.
Reprints or correspondence: Govinda S. Visvesvara, PhD, 4770 Buford Highway, MS-F36,
Atlanta, GA 30341 (gsv1@cdc.gov).
The Journal of Infectious Diseases 2009; 199:1139 42
2009 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2009/19908-0008$15.00
DOI: 10.1086/597473
children. In contrast, Acanthamoeba species and Balamuthia
mandrillaris cause subacute or chronic granulomatous amoebic
encephalitis and infections of the skin and nasal sinuses, pre-
dominantly in immunocompromised individuals [1]. Although
drugs can be effective for treatment of some of these infections,
infections that involve the central nervous system are typically
fatal and often diagnosed late in the disease process.
In 1998, an otherwise healthy male in Texas was admitted to
the hospital with a history of loss of consciousness, emesis, head-
ache, photophobia, and blurry vision [2]. Magnetic resonance
imaging showed a solitary 2-cm mass in the posterior left tem-
poral lobe. A biopsy showed that the mass contained necrotizing
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inflammation with trophozoites of a free-living amoeba, mor-
phologically different from any of the amoeba species previously
found in association with infection in humans. Although the
type of amoeba causing the infection was not identified at the
time, the patient underwent surgical excision of the tumor-like
mass and received antibiotic therapy, as is used to treat Bala-
muthia infection [3], and recovered without any neurologic se-
quelae. The causative agent was later identified as Sappinia dip-
loidea on the basis of distinct morphological features in
hematoxylin-eosin stained brain sections and by transmission
electron microscopy images of sections from the brain lesion [2,
4]. Sappinia is readily recognizable in stained preparations or
when viewed with a phase-contrast microscope because of the
presence of 2 nuclei closely apposed to one another and, in living
amoebae, a cortex that has a rippled appearance as the amoeba
moves [5]. However, the features visible in the images of samples
obtained from the patient were not sufficient to distinguish S.
diploidea from its sister species S. pedata [6]. Both these amoebae
have previously been isolated from herbivore feces and environ-
mental sources but never before or after this case have they been
associated with infectious disease. This report describes the de-
velopment of real-time polymerase chain reaction (PCR) assays
for differential molecular detection of the 2 Sappinia species and
their use to identify S. pedata as the most likely causative agent in
this unique case of granulomatous amoebic encephalitis (GAE)
which was originally described as being caused by S. diploidea.
Materials and methods. Two cultures of Sappinia, infor-
mally identified in our laboratories as cultures I and II, were
provided by Dr. Rolf Michel, who used the terms Sd-plat and
Sd-bio for cultures I and II, respectively (R. Michel, written
communication, 30 June 2008). (Sd-plat is available from the
Culture Collection of Algae and Protozoa [CCAP] as strain
1575/2). Both cultures were presumed to be S. diploidea. The
amoebae were grown on nonnutrient agar (1.5% [wt./vol.] agar
BRIEF REPORT JID 2009:199 (15 April) 1139
prepared in amoeba saline) with Escherichia coli as a food source
[7]. Amoebae were harvested, washed in amoeba saline, counted
and subjected to DNA extraction using proteinase K digestion
[8]. One slide with a paraffin-embedded tissue section obtained
from the brain lesion surgically extracted from the GAE case
patient from Texas was available for analysis. The deparaffinized
section was scraped from the slide, and DNA was extracted from
the tissue using the QIAamp Micro DNA extraction kit (Qia-
gen).
The complete chromosomal 18S rRNA gene was amplified from
the cultured Sappinia strains by using PCR with generic eukaryotic
primers CryptoFL (5'-AAC CTG GTT GAT CCT GCC AGT AGT
CAT-3') and CryptoR (5'-GCT TGA TCC TTC TGC AGG TTC
ACC TAC-3') alone and in combination with the amoeba-
specific primers BNgenR2 (5'-GGA GCT GGA ATT ACC GCG
G-3') and BNgenF1 (5'-GTT CGA TTC CGG AGA GGG AGC-
3'). Amplicons were cloned into pCR2.1-TOPO vectors (In-
vitrogen) and sequenced in both directions with the amplifica-
tion primers and the sequencing primers SappF1428 (5'-TAA
ACG ATG CCG ACC AG-3') and SappR1747 (5'-AAG AAC
GGC CAT GCA C-3') by cycle sequencing with BigDye chemis-
try (version 3.1) and the ABI Prism 3100 sequence analyzer (Ap-
plied Biosystems).
The 18S rRNA sequence of Sappinia strain I was identical to
the GenBank accession number DQ122380 (Sappinia diploidea
[Sd-plat, CCAP 1575/2] 18S rRNA). The sequence from Sap-
pinia strain II was 97%98% identical with the S. pedata entries
in GenBank (entries EU004593-EU004596). The Sappinia strain
II sequence has been deposited in GenBank under accession
number EU980613.
PCR primers and TaqMan probes were designed to detect
both Sappinia species but not other free-living amoebae (i.e., all
known Acanthamoeba genotypes, Balamuthia mandrillaris, and
Naegleria fowleri) or human DNA. The oligonucleotides de-
signed for this purpose were SappF1576 (5'-TCT GGT CGC
AAG GCT GAA AC-3'), SappR1736 (5'-GCA CCA CCA CCC
TTG AAA TC-3'), and SappP1705 (HEX-5'-TGT CAA TCT
GTC AAT CCT CGT CAA GTC T-BHQ-3'). The real-time PCR
was performed on Mx3000P (Stratagene) by using Platinum
qPCR Supermix with ROX and UDG (Invitrogen), 0.2 mol/L
of each primer, 0.1 mol/L probe, and 1 L template DNA in a
20-L reaction. The cycling conditions used were 2 min at 50C
and 2 min at 95C, followed by 45 cycles of 95C for 15 s and 63C for details) also tested negative in this TaqMan assay. After as-
for 1 min. The specificity of this real-time PCR assay was ascer-
tained by testing 34 clinical specimens of cerebrospinal fluid and
brain tissue obtained from patients with suspected cases of in-
fection due to free-living amoebae that were submitted to the
Centers for Disease Control and Prevention during 20052007.
Three of these samples were positive for Acanthamoeba, 2 were
positive for Balamuthia mandrillaris, and 8 were positive for
Naegleria fowleri (no evidence of free-living amoebae could be
found in the remaining 21 samples). Other negative controls
1140 JID 2009:199 (15 April) BRIEF REPORT
included Haemophilus influenzae (1 strain each of serotypes
A-F), Neisseria meningitidis (1 strain each of serogroups A, B, C,
X, Y, Z, W135, and 29E), Streptococcus pneumoniae M4421,
Streptococcus agalactiae M8224, and cerebrospinal fluid samples
from 4 patients with cysticercosis.
By use of available sequence data, 2 sets of primers and Taq-
Man probes were designed to specifically detect S. diploidea and
S. pedata, respectively. The 2 TaqMan probes were labeled with
different fluorescent dyes to facilitate combining the 2 sets into a
duplex assay. The oligonucleotides used were as follows:
SdiplF753, 5'-ATG GGA TGG AGT GGT TAG CTC AA-3';
SdiplR877, 5'-GGA GTA AAG AGA CCT CGG AAG AAA-3';
SpedF756, 5'-AAA GGA TGG GAT GAG AAT TTG G-3';
SpedR903, 5'-CGT TCT CGT CTC TCT CCG AAG A-3';
SpedP784, 5'-Cy5-AGC ACT CGC CTA TCC CTT GAG ATA
TCA CTG-BHQ-3'; and SdiplP777, 5'-FAM-AGG TTA ACG
TAC GCC ACC TAT CTG TCG AGA TT-BHQ-3'. The reaction
conditions were the same as those used for the generic Sappinia
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assay, with the exception of a 60C (instead of 63C) annealing
and extension plateau during thermocycling.
Results and discussion. The aim of this study was to con-
firm Sappinia as the etiologic agent in a case of GAE by use of
molecular techniques. We decided to use a 2-step process in
which we first determined whether a Sappinia species was
present in the clinical material and, if present, determined the
identity of the organism at the species level. The main reason for
using this approach was the inadequate information available
about sequence variability among different Sappinia strains and
species. Given the paucity of sequence data, the causal agent of
this case of GAE could have been an as yet unidentified Sappinia
species, in which case species-specific tools would be inappro-
priate. In addition, the very limited amount and the condition of
the clinical material available for molecular analysis only permit-
ted a few very carefully designed experiments to be performed.
The Sappinia species TaqMan assay produced positive results
for DNA extracted from the 2 cultured Sappinia strains, with a
detection limit of approximately 1.4 cells/mL. The assay did not
detect DNA from cultures of Acanthamoeba, N. fowleri, or B.
mandrillaris. Furthermore, a collection of clinical samples of ce-
rebrospinal fluid and brain tissue obtained from patients with
suspected cases of free-living amoeba infection as well as DNA
from other pathogens that cause similar infections (see Methods
suring the assays sensitivity and specificity, it was used to detect
Sappinia DNA in a sample extracted from the section of brain
tissue obtained from the Sappinia GAE case patient. As illus-
trated in figure 1, this sample was successfully amplified, indi-
cating that the brain lesion removed from the infected patient
did indeed contain Sappinia species. The DNA sample from the
GAE case was then amplified in the species-specific duplex Taq-
Man PCR assay. The GAE sample tested negative for S. diploidea
but had S. pedataspecific priming sites that allowed us to infer
Figure 1. Amplification curves from real-time polymerase chain reaction detection of Sappinia species. Three aliquots of the DNA extracted from

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granulomatous amoebic encephalitis tissue obtained from the Texas case patient (TX case) were analyzed, each with a different DNA concentrations.
dRn, baseline subtracted fluorescence reading normalized to a reference dye.

that the amoeba causing GAE in this case was a different species
of Sappinia and most likely a strain of S. pedata (see figure 2).
The concept of free-living amoebae as causal agents of human
encephalitis was formulated 40 years ago and focused initially
on Naegleria fowleri and Acanthamoeba species [1] . Approxi-
mately 20 years ago, a previously unrecognized amoeba with a
role in encephalitis was described as Balamuthia mandrillaris,
which is now know to be responsible for 150 cases of human
encephalitis. Finding a Sappinia species, most probably S. pe-
data, in samples from a human case of GAE was particularly
unexpected because the organism had previously been isolated
from environmental sources, such as tree bark. This unusual
finding raises the possibility that still other free-living amoebae
have yet to be identified as human pathogens.
Diagnosis of infection due to free-living amoebae is difficult.
For one thing, these infections are so uncommon that clinicians,

Figure 2. Amplification curves from real-time polymerase chain reaction detection of Sappinia pedata. Two aliquots of the DNA extracted from
granulomatous amoebic encephalitis tissue obtained from the Texas case patient (TX case), were analyzed, each with a different DNA concentration.
dRn, baseline subtracted fluorescence reading normalized to a reference dye.

BRIEF REPORT JID 2009:199 (15 April) 1141

pathologists, and laboratory technicians are unfamiliar with symp-
toms and with the appearance of these amoebae in clinical speci-
mens [9, 10]. There are only a few reference laboratories capable of
doing diagnostic detection of the free-living amoeba species that
cause infections. Thus, the development of PCR procedures for di-
agnostic detection is particularly opportune. Both conventional
and real-time PCR techniques for the detection of Acanthamoeba,
Balamuthia mandrillaris, and Naegleria fowleri are now available to
laboratories equipped to perform diagnostic molecular techniques
[8, 1116]. The Sappinia species PCR assay described here can be
used to analyze clinical material from infections for which all other
causative agents have been ruled out or in retrospective analysis of
archived samples from unexplained cases of encephalitis. The assay
design also allows it to be incorporated into the previously pub-
lished triplex TaqMan assay [8] for simultaneous detection of up to
4 genera of free-living amoebae.
Acknowledgments
We thank Dr. Rolf Michel for generously sharing his 2 cultures of Sappinia
with us.
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