Annals of Tropical Medicine & Parasitology, Vol. 102, No.
3, 271–274 (2008)
SHORT COMMUNICATION
Symptomatic infection with Blastocystis sp. subtype 8
successfully treated with trimethoprim–sulfamethoxazole
After returning from a 3-week trip to
Indonesia (Java, Borneo and Sulawesi) in
March–April 2006, a 40-year-old Danish
female office assistant was hospitalized for 3
days because of diarrhoea and fever. The three
faecal samples that were submitted for para-
sitological, bacteriological and viral examina-
tion at this time were only found positive for
Blastocystis (Table). Four bloodsmears were
all negative for haemoparasites and no bacter-
aemia or fungaemia could be demonstrated.
The woman was seronegative for Chlamydia,
Legionella, Mycoplasma, Leptospira and
Brucella, and there was no evidence of acute
cytomegalovirus infection. Sigmoidoscopy
revealed nothing unusual. The woman was
treated with metronidazole (400 mg three
times/day for 10 days) in May 2006. Two
stool specimens collected immediately after
this treatment ended were negative for patho-
genic parasites, viruses and bacteria (Table)
and the patient’s symptoms subsided a little
for a while. Intermittent gastro-intestinal
symptoms, including diarrhoea, bloating,
flatulence and abdominal pain, rapidly reap-
peared, however, and persisted throughout
the summer and autumn of 2006. In
November 2006, the woman was again
treated with metronidazole (this time at
800 mg three times/day for 10 days) but
this resulted in very little clinical improve-
ment, and faecal specimens collected after
this round of treatment were positive only
for Blastocystis (Table). In December 2006,
the woman received 10 days of treatment
with trimethoprim–sulfamethoxazole (TMP/
SMX), in thrice-daily doses of 80 mg
trimethoprim/400 mg sulfamethoxazole, and
subsequently reported that most of her
symptoms had been markedly alleviated.
# 2008 The Liverpool School of Tropical Medicine
DOI: 10.1179/136485908X278847
Her abdominal pain and bloating had
subsided, although episodes of multiple
loose stools persisted. Three faecal speci-
mens taken up to 4 weeks after completion
of the TMP/SMX treatment were negative
for all parasites (Table). Even 5 months
after completing the TMP/SMX treatment,
the patient reported no abdominal pain or
bloating. The data presented in this article
are published with the informed consent of
the patient.
To evaluate the efficacies of the second
round of metronidazole treatment and the
TMP/SMX treatment, faecal specimens
collected post-treatment were cultured in
Jones’ medium to check for the presence of
Blastocystis (Stensvold et al., 2006, 2007a).
In order to rule out Dientamoeba fragilis
infection, smears of stools fixed in sodium-
acetate–acetic-acid–formalin (SAF) imme-
diately after voiding were stained with Gomori’s
trichrome and examined for trophozoites.
A rough susceptibility test was performed
by inoculating 750,000 Blastocystis organisms
from one of the cultures into 3-ml volumes of
Jones’ medium containing no metronidazole
or 0.01 mg or 0.1 mg metronidazole/ml
(Haresh et al., 1999). When, after 48 h, each
of these cultures was evaluated for the
presence of Blastocystis, multiplication of the
parasite was found to have been markedly
and equally inhibited by the two concentra-
tions of metronidazole, with .100-fold more
parasites in the cultures without the drug
than in those with the drug.
From a faecal sample obtained in
November 2006 (Table), DNA was extracted
using the QIAGEN Stool Mini Kit
(QIAGEN, Hilden, Germany) according to
the manufacturer’s recommendations. The
TABLE. Results of the various analyses of faecal specimens collected from the patient between May 2006 and January 2007
272

Date of: Results of:

ZiehlBNeelsen
staining
Type of Bacterial of FECT Blastocystis Gomori’s
Treatment Stool collection* specimen{ examination ELISA for viruses FECT{ concentrates culture Trichrome staining{

7 May 2006 Unpreserved No enteropatho- Negative for Few Blastocystis No sporozoa ND ND

genic bacteria1 rotavirus
7 May 2006 Unpreserved No enteropatho- Negative for Some Blastocystis No sporozoa ND ND
STENSVOLD ET AL.

genic bacteria rotavirus

8 May 2006 Unpreserved ND ND Few Blastocystis No sporozoa ND ND
10–19 May
2006
18 May 2006 Unpreserved No enteropatho- Negative for No parasites No sporozoa ND ND
genic bacteria rotavirus
18 May 2006 Unpreserved No enteropatho- Negative for No parasites No sporozoa ND ND
genic bacteria rotavirus
21 October 2006 Preserved and ND ND Some Blastocystis ND ND Many Blastocystis
unpreserved
23 October 2006 Preserved and ND ND Few Blastocystis ND ND Many Blastocystis
unpreserved
3 November 2006 Unpreserved ND ND No parasites No sporozoa Positive ND
5–14 November
2006
14 November 2006 Unpreserved ND ND Some Blastocystis ND Positive ND
21 November 2006 Unpreserved ND ND Some Blastocystis ND Positive ND
22 November 2006 Unpreserved ND ND No parasites ND Positive ND
22–31 December
2006
3 January 2007 Unpreserved ND ND No parasites ND Negative ND
4 January 2007 Unpreserved ND ND No parasites ND Negative ND
23 January 2007 Unpreserved ND ND No parasites No sporozoa Negative ND

*
On each of two dates (7 May 2006 and 18 May 2006), two, independent, consecutive samples were collected.
{
Preserved specimens were fixed in sodium-acetate–acetic-acid–formalin.
{
In these results, ‘few’, ‘some’ and ‘many’ indicate, respectively, one to five, six to 10 and .10 organisms/field, at a magnification of 6400.
1
Tested for Salmonella, Shigella, Yersenia, Campylobacter, Aeromonas, Plesiomonas, Vibrio, and verotoxin-producing (VTEC), enterotoxigenic (ETEC) and entero-invasive
(EIEC) Escherichia coli.
FECT, FormolBethyl-acetate concentration; ND, not determined.

primers and protocol described by Stensvold
et al. (2006) were successfully used for the
PCR-based amplification of a 310-bp pro-
duct, which was purified, using a QIAquick
PCR Purification kit (QIAGEN), and
then dideoxy-sequenced (MWG-Biotech,
Ebersberg, Germany). The sequence so
obtained was deposited in the nucleotide
sequence database of the European
Molecular Biology Laboratory (EMBL), as
accession AM495096. When submitted to
phylogenetic analysis, using the MEGA 3.1
software package (Center for Evolutionary
Functional Genomics, Tempe, AZ), this
nucleotide sequence clustered within
Blastocystis sp. subtype 8 (data not shown).
Blastocystis is a common, enteric, unicel-
lular parasite, the clinical importance of
which is unclear; the parasite can be isolated
from both symptomatic and asymptomatic
individuals (Stenzel and Boreham, 1996).
Blastocystis exhibits extensive genetic diver-
sity (Clark, 1997) and nine subtypes are
currently recognised (Stensvold et al.,
2007b). It is possible that the parasites’
pathogenic properties might be subtype-
dependent. Traditionally, symptomatic
individuals found infected with Blastocystis
but no other potential pathogen have been
treated with metronidazole. This approach
is supported by a number of reports,
although only one placebo-controlled trial
of the treatment of Blastocystis with metro-
nidazole has been performed (Nigro et al.,
2003). A few studies have addressed the
efficacy of TMP/SMX, with varied results
(Ok et al., 1999; Moghaddam et al., 2005).
In this report, a woman who suffered
from intermittent, moderately severe gastro-
intestinal symptoms for about 8 months is
described. In spite of multiple, thorough viral,
bacterial, parasitological and endoscopic
examinations, the only potentially disease-
causing organism isolated was Blastocystis sp.
subtype 8. This subtype, which has been
isolated from birds, humans and other pri-
mates, has not previously been detected in
Denmark, where subtypes 1, 2, 3 and 4 seem
to predominate (Stensvold et al., 2006).
INFECTION WITH Blastocystis SUBTYPE 8 273
Although the two stool specimens sub-
mitted after the first period of metronidazole
treatment in May 2006 were found negative
for Blastocystis when checked by the micro-
scopical examination of formol–ethyl-
acetate concentrates, this technique only
has limited sensitivity, compared with, for
example, in-vitro culture (Suresh and Smith,
2004) or Blastocystis-specific PCR (Stensvold
et al., 2007a). The irregular shedding of
Blastocystis by infected humans (Vennila
et al., 1999) and the lysis of the vacuolar
forms of Blastocystis (Pinel et al., 1991;
Leelayoova et al., 2002) probably limit the
sensitivity of formol–ethyl-acetate concentra-
tion (FECT). It is possible that the metroni-
dazole given in May reduced the intensity of
the patient’s Blastocystis infection below the
detection limit of microscopy but did not
fully eradicate the infection. Haresh et al.
(1999) found that Blastocystis of different
geographical origins had different levels of
in-vitro resistance to metronidazole, an
Indonesian isolate still being viable in med-
ium containing 1.0 mg metronidazole/ml.
Although the isolate tested in the present
study did not show this level of resistance,
being inhibited in vitro by metronidazole
concentrations of >0.01 mg/ml, treatment of
the patient with high doses of metronidazole
(800 mg given three times/day) for 10 days
did not lead to eradication of her Blastocystis
infection. This treatment failure may have
been the result of the metronidazole being
extensively absorbed in the proximal gut.
Low-level resistance of the parasites cannot
be excluded as an alternative cause of this
treatment failure, however, as in-vitro sus-
ceptibility to metronidazole at concentra-
tions of ,0.01 mg/ml was not investigated.
Unfortunately, it was not possible to inves-
tigate the in-vitro susceptibility of the isolate
towards TMP/SMX.
The possibility that the patients’ main
symptoms resolved spontaneously and/or
independently of the eradication of her
Blastocystis infection cannot be excluded
and it is also possible that the TMP/SMX
treatment was effective in eradicating an
274 STENSVOLD ET AL.
unidentified pathogen that was causing the
patient’s intestinal symptoms. The fact that
the symptom resolution occurred simulta-
neously with the Blastocystis eradication is,
however, strongly indicative of the success-
ful treatment of a symptomatic Blastocystis
infection with TMP/SMX.
In conclusion, TMP/SMX seems to be a
relevant alternative to metronidazole in the
treatment of symptomatic Blastocystis infec-
tions. It is highly likely that the gastro-
intestinal symptoms reported in this case
were attributable to Blastocystis sp. subtype
8, since those symptoms were remarkably
alleviated after eradication of this parasite.
C. R. STENSVOLD
M. C. ARENDRUP
H. V. NIELSEN
Department of Bacteriology, Mycology
and Parasitology, Statens Serum
Institut, Artillerivej 5, DK-2300
Copenhagen S, Denmark
A. BADA
Department of Internal Medicine,
Sygehus Øst Køge, Lykkebækvej 1,
DK-4600 Køge, Denmark
S. THORSEN
Department of Internal Medicine,
Roskilde University Hospital, Køgevej
7–13, DK-4000 Roskilde, Denmark
Received 30 August 2007,
Revised 5 October 2007,
Accepted 9 October 2007
Reprint requests to: C. R. Stensvold.
E-mail: run@ssi.dk; fax: z45 32 68 30 33/
REFERENCES
Clark, C. G. (1997). Molecular and Biochemical
Parasitology, 87, 79–83.
Haresh, K., Suresh, K., Khairul Anus, A. &
Saminathan, S. (1999). Isolate resistance of
Blastocystis hominis to metronidazole. Tropical
Medicine and International Health, 4, 274–
277.
Leelayoova, S., Taamasri, P., Ragnsin, R., Nagglor, T.,
Thathaisong, U. & Mungthin, M. (2002). Annals of
Tropical Medicine and Parasitology, 96, 803–807.
Moghaddam, D. D., Ghadirian, E. & Azami, M.
(2005). Parasitology Research, 96, 273–275.
Nigro, L., Larocca, L., Massarelli, L., Patamia, I.,
Minniti, S., Palermo, F. & Cacopardo, B. (2003).
Journal of Travel Medicine, 10, 128–130.
Ok, U. Z., Girginkardesler, N., Balcioglu, C., Ertan, P.,
Pirildar, T. & Kilimcioglu, A. A. (1999). American
Journal of Gastroenterology, 94, 3245–3247.
Pinel, C., Rejasse, C., Picot, S., Brenier-Pinchart, M.
P., Grillot, R. & Ambroise-Thomas, P. (1991).
Annales de Biologie Clinique, 57, 601–604.
Stensvold, R., Brillowska-Dabrowska, A., Nielsen, H.
V. & Arendrup, M. C. (2006). Journal of Parasitology,
92, 1081–1087.
Stensvold, C. R., Arendrup, M. C., Jespersgaard, C.,
Mølbak, K & Nielsen, H. V. (2007a). Diagnostic
Microbiology and Infectious Disease, 59, 000–000.
Stensvold, C. R., Suresh, G. K., Tan, K. S. W.,
Thompson, R. C. A., Traub, R. J., Viscogliosi, E.,
Yoshikawa, H. & Clark, C. G. (2007b). Trends in
Parasitology, 23, 93–96.
Stenzel, D. J. & Boreham, P. F. (1996). Clinical
Microbiology Reviews, 9, 563–584.
Suresh, K. & Smith, H. (2004). European Journal of
Clinical Microbiology and Infectious Diseases, 23, 509–
511.
Vennila, G. D., Suresh Kumar, G., Khairul Anuar, A.,
Rajah, S., Saminathan, R., Sivanandan, S. &
Ramakrishnan, K. (1999). Parasitology Research, 85,
162–164.