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1101
BRIEF REPORTS
Outbreak of Amebiasis in a Family
in The Netherlands
Human-to-human transmission of Entamoeba histolytica is rare in industrialized countries. We describe an outbreak of amebiasis in a family in The Netherlands, demonstrating that even with Western standards of hygiene,
persistent cyst passage may result in the transmission of
E. histolytica to household contacts. If E. histolytica is
isolated from a person living in an area of nonendemicity,
it may be worthwhile to test all family members for cyst
passage.
Isoenzyme analysis [1] and, more recently, immunologic and
molecular techniques [2] showed conclusively that there are 2
genetically distinct but morphologically (at the cyst and small
trophozoite stage) identical Entamoeba species: 1 noninvasive
species, Entamoeba dispar, and 1 species clearly associated with
disease in humans, Entamoeba histolytica [3].
The noninfectious, motile trophozoite stage of E. histolytica
can invade the colonic mucosa, ingest erythrocytes, and cause
ulcerative colitis resulting in diarrhea, which often contains
blood. In some cases, E. histolytica trophozoites break through
the mucosal barrier, enter the portal circulation, and then are
carried to the liver, where they can cause amebic liver abscess.
Within the colon, trophozoites encyst, and the infectious, nonmotile cyst stage is passed in the stool, where it can infect new
hosts.
Although rare, human-to-human transmission of E. histolytica can occur in industrialized countries. We describe an outbreak of amebiasis in a family in The Netherlands. The use of
molecular tools proved to be an important contribution to the
diagnosis and subsequent clinical management of amebiasis.
Finally, we call attention to the problems that can arise if treatment of E. histolytica cyst passage fails.
The index case was a 5-year-old girl who presented in November 1994 with a 6-week history of abdominal pain and
bloody diarrheal stools without fever. Stool cultures for bacterial pathogens remained negative. Because the girl had never
been abroad, the possibility of amebiasis was not considered
by the physician until her mother gave a history of amebic
dysentery and persistent cyst passage after return from India
13 years before. The diagnosis of amebic dysentery was made
after demonstration of many large trophozoites with ingested
RBCs in the stools.
In 1981, the mother of the index case had been admitted to
a hospital in India under the clinical suspicion of appendicitis.
Reprints or correspondence: Dr. L. G. Visser, Dept. of Infectious Diseases,
Leiden University Medical Centre, C5-P, P.O. Box 9600, 2300 RC Leiden,
The Netherlands ([email protected]).
Clinical Infectious Diseases 2000; 31:1101–4
q 2000 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2000/3104-0037$03.00
She developed bloody stools, and amebic dysentery was diagnosed after demonstration of amebae in the stools. Despite
treatment with tissue (metronidazole) and luminal (clioquinol)
amebicides, complaints of abdominal pain and diarrhea, sometimes containing blood, recurred after her return to The Netherlands. Subsequent treatment with metronidazole, clioquinol,
and later diloxanide furoate failed to end cyst excretion. Because bowel symptoms had disappeared, no further treatment
was advised.
In 1987, the mother returned to the outpatient clinic of gastroenterology because of complaints of recurrent abdominal
pain and diarrhea, sometimes containing blood, at irregular
intervals. Despite high serum titers of antibody to Entamoeba,
the presumptive diagnosis of inflammatory bowel disease was
made because none of the microscopic examinations of stool
samples and colon biopsies revealed any amebae. The mother
was treated with 5-aminosalicylic acid enemas. During the last
few years, she had no complaints.
When it became clear that the mother was the most likely
source of infection for her daughter, we decided to investigate
whether the remaining household contacts were infected as well.
An individual was regarded to be infected if excretion of cysts
or trophozoites, identified by PCR analysis as belonging to E.
histolytica, was demonstrated. An individual was believed not
to be infected if at least 3 consecutive parasitological examinations remained negative. An infection was considered asymptomatic if there were no signs or symptoms of abdominal pain
and diarrhea, dysentery, or amebic liver abscess.
Blood samples were obtained from all household members
for serological testing. IgG antibodies to unpurified E. histolytica–derived antigens were assessed by ELISA and immunodiffusion assay [4, 5].
Stool samples were examined for protozoal cysts and trophozoites by means of a variety of methods. Both preserved
and fresh stool samples were examined. The preservative fluid
contained sodium acetate, acetic acid, and formaldehyde
(SAF). Direct smears of SAF-preserved stool specimens were
examined for trophozoites and cysts of Entamoeba species.
Fresh stool samples were examined after formol-ether concentration. Identification of E. histolytica or E. dispar was based
on the presence of cysts with a diameter between 10–15 mm.
Fresh stool samples were also cultured for amebae [6].
PCR analysis of both DNA extracted from amebae cultured
from the stools and DNA extracted directly from stool specimens by using phenol-chloroform isolation and ethanol precipitation was performed. Repetitive regions from extrachromosomal DNA specific to E. histolytica (125-bp region) and to
E. dispar (133-bp region) were amplified by PCR analysis in
which 1 primer of each pair was labeled with digoxigenin. The
PCR product was hybridized with a specific biotin-labeled
probe and visualized by ELISA (PCR-soluble hybridization
enzyme-linked assay) [7, 8].
1102
Brief Reports
Lysates of E. histolytica cultured from stool samples were
subjected to isoenzyme electrophoresis on starch gels, and isoenzyme patterns of phosphoglucomutase, glucose-phosphate
isomerase, malate dehydrogenase, and hexokinase were determined [1].
All infected individuals were treated with metronidazole for
10 days (adult dosage, 750 mg t.i.d.; pediatric dosage, 20 mg/kg
t.i.d.) and diloxanide furoate for 10 days (adult dosage, 500 mg
t.i.d.; pediatric dosage, 6 mg/kg t.i.d.).
Stool examinations were repeated in all cases 6, 10, 18, 30,
and 41 weeks and 4 years after therapy: at each occasion, 1
fresh and 2 SAF-preserved stool samples were collected on
separate days.
In addition to the index case, the household contacts of the
mother were her husband, a 4-year-old son, a 2-year-old son,
and a nanny. None of the household contacts had traveled
outside western Europe or had developed signs or symptoms
that were consistent with dysentery or extraintestinal invasive
disease. However, the oldest son had an episode of abdominal
cramps and nonbloody diarrhea a few weeks after his sister
had developed amebic dysentery.
Serum antibodies to amebae were detected by ELISA in all
family members, except for the nanny. Results of immunodiffusion assay were negative for the father and the nanny (table
1). Trophozoites and/or cysts of E. histolytica/E. dispar were
found in stool specimens from the index case, her mother, and
all other household contacts (table 1). Hematophagous trophozoites were found only in stool samples from the index case.
With use of PCR-soluble hybridization enzyme-linked assay,
the cysts were later identified as belonging to E. histolytica in
5 of 6 cases. No E. dispar DNA was detected in any stool
sample (table 1). Cultures of stool specimens from 4 (67%) of
6 individuals who excreted cysts became positive for amebae.
With use of isoenzyme electrophoresis, all cultured amebae were
shown to belong to the IIa2 zymodeme of E. histolytica [9].
All infected individuals were treated orally with metronidazole, followed by diloxanide furoate. The father interrupted
treatment with metronidazole because of gastrointestinal intolerance. Because of persistent cyst passage in the children,
treatment had to be repeated up to 3 times. Treatment consisted
of tinidazole at week 6 (all children); metronidazole, diloxanide
Table 1.
CID 2000;31 (October)
furoate, and paromomycin at week 12 (all children); and diloxanide furoate and paromomycin at week 18 (2-year-old boy).
All children remained asymptomatic during that time.
This outbreak of amebiasis demonstrates that, even with
Western standards of hygiene, persistent cyst passage may result
in transmission of E. histolytica to household contacts many
years later.
Without any doubt, the outbreak originated from the mother,
who had persistent cyst passage and recurrent complaints of
abdominal pain and bloody diarrhea at irregular intervals after
treatment for amebic dysentery 13 years earlier. She was the
only person who had traveled outside western Europe. Furthermore, all cultured isolates of E. histolytica belonged to the
comparatively rare IIa2 zymodeme (John Williams, unpublished data), supporting the hypothesis of a single source of
infection. Unfortunately, the cultures remained negative for
amebae for the index case and the nanny. However, it is extremely unlikely that they were infected with a different strain
of E. histolytica.
Normally, transmission of E. histolytica occurs by the fecaloral route under conditions of poor personal hygiene or poor
domestic sanitation. However, an inquiry made by the municipal health officer with use of a standardized questionnaire
did not reveal any of these circumstances. Therefore, the mode
of transmission in this family cluster remains unknown. There
were no secondary cases of dysentery observed in the day care
center and school that were attended by the children.
PCR analysis of DNA specific to E. histolytica that was extracted directly from stool samples proved to be an important
contribution to the laboratory diagnosis and subsequent clinical
management of amebiasis. The extraction of DNA from amebae isolated directly from stool samples obviates the need for
stool cultures. Furthermore, PCR analysis allows for the distinction between morphologically identical cysts of E. histolytica and E. dispar. This distinction is important because most
amebae found in areas of nonendemicity as well as areas of
endemicity belong to the noninvasive species E. dispar. In a
survey of 174 stool samples containing cysts of Entamoeba species that were obtained at various laboratories of parasitology
in The Netherlands, cysts were identified by PCR analysis as
E. dispar in 153 samples (88%) and as E. histolytica in 18 sam-
Clinical and parasitological characteristics of family members in an outbreak of amebiasis in November 1994.
Serological finding
Family
member
5-Year-old girl
Mother
Father
4-Year-old boy
2-Year-old boy
Nanny
PCR-SHELA for Entamoeba histolytica
Symptomatic
disease
ELISA (AU)
ID assay
Trophozoites
and/or cysts
DNA extracted
directly from stool
DNA extracted
from stool culture
Zymodeme
IIa2
Yes
No
No
Yes
No
No
1:320
>1:2560
1:80
1:1280
1:160
!1:40
1
1
2
1
1
2
1
1
1
1
1
1
1
1
1
2
1
2
NG
1
1
1
1
NG
ND
1
1
1
1
ND
NOTE. The girl was the index case. AU, arbitrary units; ID, immunodiffusion; ND, not done; NG, no growth; PCR-SHELA, PCRsoluble hybridization enzyme-linked assay; 1, positive; 2, negative.
CID 2000;31 (October)
Brief Reports
ples (10%). It was impossible to make a differentiation in only
3 samples (in 2 cases, heating or dilution of the extracted DNA
neutralized inhibitory factors in the stools) [10].
It is common practice to treat asymptomatic persons with
Entamoeba cysts in their stools who are returning from an area
of endemicity with a luminal amebicide to prevent symptomatic
disease in the future [11]. As a result, 9 of 10 persons will be
unnecessarily treated. Therefore, the Expert Consultation on
Amoebiasis recommended that “for the treatment of cyst carriers, ideally, E. histolytica should be specifically identified and,
if present, treated” [3]. PCR analysis is a sensitive and specific
diagnostic tool to reliably differentiate infection with pathogenic E. histolytica and infection with noninvasive E. dispar. In
addition, follow-up stool examinations after treatment can be
reserved for those patients excreting E. histolytica cysts.
The presence of serum antibodies to E. histolytica in asymptomatic persons excreting E. histolytica cysts may be suggestive
of subclinical invasive intestinal disease. The history of the
mother of the index case is a clear example of this concept.
After an initial episode of asymptomatic cyst excretion, she
developed recurrent complaints of abdominal pain and bloody
diarrheal stools, which were mistaken for chronic inflammatory
bowel disease. The inadvertent administration of glucocorticosteroids could have resulted in potentially life-threatening invasive disease [12].
Serum antibodies to amebae were detected in all asymptomatic infected family members. Similar observations were made
in a longitudinal study of asymptomatic carriers of E. histolytica in a semirural area south of Durban, South Africa; all
subjects had a strongly positive serological reaction [13]. Together, these findings strongly support the hypothesis that
asymptomatic excretion of E. histolytica cysts is accompanied
by tissue invasion by E. histolytica.
Persistent excretion of E. histolytica cysts despite treatment
with luminal amebicides should not be accepted even in the
absence of complaints, because it is associated with the risk for
transmission of E. histolytica to close contacts (in this study,
up to 13 years after infection). An earlier report described the
transmission of E. histolytica within an Italian family that most
likely originated from a Philippine housemaid [14]. If treatment
with contact amebicides alone fails, a course of tissue and luminal amebicides should be given, since treatment failure most
likely indicates subclinical tissue invasion.
The children required several courses of tissue and luminal
amebicidal drugs before repeated stool examinations became
negative for amebic cysts. There are several possible explanations for treatment failure. The taste of metronidazole or diloxanide furoate can be prohibitive to guarantee adequate compliance in children. On the other hand, the failure of the
mother’s treatment and the difficulty in clearing the infection
in the children may suggest that the strain of E. histolytica was
less susceptible to amebicidal drugs. Although drug-resistant
E. histolytica mutants have emerged in the laboratory by means
1103
of the selection effect [15, 16], their role in treatment failures
remains to be demonstrated.
The index case demonstrates the problems in an isolated case
of amebiasis in an area of nonendemicity. The main obstacle
in the proper diagnosis of amebiasis is that clinicians fail to
consider this possibility. Generally, as with so many exotic diseases, a stay in the Tropics is the key to the diagnosis. However,
in this case, the origin of infection was the mother who had
been in the Tropics many years before. It illustrates the fact
that in an isolated case of amebiasis in an area of nonendemicity
one should thoroughly investigate all close contacts as the possible source of infection. Finally, if E. histolytica cysts are found
in a member of a family, all close contacts should be tested for
cyst passage.
Acknowledgment
We are indebted to John Williams for his advice in the zymodeme
characterization of cultures.
S. G. S. Vreden, 1 , a L. G. Visser, 1 J. J. Verweij, 2 J. Blotkamp, 2
P. C. Stuiver, 1 A. Aguirre, 3 and A. M. Polderman 2
Departments of 1Infectious Diseases and 2Parasitology, Leiden
University Medical Centre, Leiden, The Netherlands; and 3Department
of Parasitology, London School
of Hygiene and Tropical Medicine, London, United Kingdom
a
Current affiliation: Diakonessenhuis, Paramaribo, Suriname.
References
1. Sargeaunt PG, Williams JE. The differentiation of invasive and non-invasive
Entamoeba histolytica by isoenzyme electrophoresis. Trans R Soc Trop
Med Hyg 1978; 72:519–21.
2. Clark CG, Diamond LS. Ribosomal RNA genes of “pathogenic” and “nonpathogenic” Entamoeba histolytica are distinct. Mol Biochem Parasitol
1991; 49:297–302.
3. González-Ruiz A, Wright SG. Disparate amoebae. Lancet 1998; 351:
1672–3.
4. Bos HJ, Van den Eijk AA, Steerenberg PA. Enzyme-linked immunosorbent
assay (ELISA) in the serodiagnosis of amoebiasis. Trans R Soc Trop Med
Hyg 1975; 69:440.
5. Bos HJ, Van der Kaay HJ, Van den Eijk AA. De waarde van serodiagnostiek
bij amoebiasis. Ned Tijdschr Geneeskd 1977; 121:287–90.
6. Robinson GL. The laboratory diagnosis of human parasitic amoebae. Trans
R Soc Trop Med Hyg 1968; 62:285–94.
7. Aguirre A, Warhurst DC, Guhl F, Frame IA. Polymerase chain reactionsolution hybridization enzyme-linked immunoassay (ELISA) for the differential diagnosis of pathogenic and non-pathogenic Entamoeba histolytica. Trans R Soc Trop Med Hyg 1995; 89:187–8.
8. Aguirre A, Molina S, Blotkamp C, et al. Diagnosis of Entamoeba histolytica
and Entamoeba dispar in clinical specimens by PCR-SHELA. Arch Med
Res 1997; 28(Suppl):S282–4.
9. Blanc D, Nicholls R, Sargeaunt PG. Experimental production of new zymodemes of Entamoeba histolytica supports the hypothesis of genetic
exchange. Trans R Soc Trop Med Hyg 1989; 83:787–90.
10. Polderman AM, Verweij JJ. Diagnosis of E. histolytica infections. In: Visser
LG, Polderman AM, ed. Advances in diagnosis and understanding of
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protozoal enteric infections. Leiden, The Netherlands: Boerhaave Commissie voor Post Academisch Onderwijs in de Geneeskunde, Rijksuniversiteit Leiden, 1997:17–21.
11. Bell DR. Lecture notes on tropical medicine. 3d ed. Oxford, United Kingdom:
Blackwell Scientific Publications, 1990.
12. Stuiver PC, Goud TJLM. Corticosteroids and liver amoebiasis. BMJ 1978;
2:394–5.
13. Gathiram V, Jackson TF. A longitudinal study of asymptomatic carriers of
pathogenic zymodemes of Entamoeba histolytica. S Afr Med J 1987; 72:
669–72.
14. Gatti S, Cevini C, Bruno A, Novati S, Scaglia M. Transmission of Entamoeba
histolytica within a family complex. Trans R Soc Trop Med Hyg 1995;
89:403–5.
15. Orozco E, de la Cruz Hernandez F, Rodriguez MA. Isolation and characterization of Entamoeba histolytica mutants resistant to emetine. Mol
Biochem Parasitol 1985; 15:49–59.
16. Samarawickrema NA, Brown DM, Upcroft JA, Thammapalerd N, Upcroft
P. Involvement of superoxide dismutase and pyruvate:ferredoxin oxidoreductase in mechanisms of metronidazole resistance in Entamoeba histolytica. J Antimicrob Chemother 1997; 40:833–40.
Failure of Pentavalent Antimony in Visceral
Leishmaniasis in India: Report from the Center
of the Indian Epidemic
Sb was expressed in India as early as the 1970s [4], incremental
increases in both the recommended dosage (to 20 mg/kg/day)
and the duration of treatment (to >28 days) satisfactorily compensated for the growing resistance to Sb in India until approximately 1990 [3–5]. Since that time, however, there has been
a steady erosion in the capacity of Sb to induce long-term cure
in patients with kala-azar who live in Bihar [4–7].
In 1994, we set out to formally document the level of efficacy
of Sb treatment in patients with kalaz-azar who live in Bihar
and to determine whether unresponsiveness to Sb had spread
to an adjacent region, eastern Uttar Pradash. From May 1994
through June 1997, we enrolled a total of 320 subjects in a 2site study in which conventional Sb therapy, 20 mg/kg/day for
30 days, was given to previously untreated patients in a similar
and closely supervised fashion.
The Kala-Azar Medical Research Center consists of 2 treatment units, one of which is located in Muzaffarpur in north
Bihar and the other of which is located 200 miles to the northwest at Banaras Hindu University Hospital in Varanasi, Uttar
Pradesh [6]. From May 1994 through June 1997, consecutive
patients were enrolled at both sites to receive conventional Sb
therapy.
Male and female patients of any age were eligible for the
study if they had typical symptoms and signs of kala-azar (e.g.,
fever, weight loss, hepatosplenomegaly, and pancytopenia), if
they had characteristic amastigotes observed microscopically
on splenic or bone marrow aspirate smears, and if they had
not received prior antileishmanial treatment [6]. Patients were
excluded from this study if they had a serious underlying illness
(e.g., congestive heart failure, renal dysfunction, or diabetes)
or an associated infection (e.g., tuberculosis or malaria), if they
were receiving immunosuppressive therapy, or if one of the
following laboratory abnormalities was present: a serum glutamic oxaloacetic transaminase value 15 times the normal
value, a serum creatinine level 12.0 mg/dL, or a prothrombin
time 15 seconds above the control value. Eleven eligible patients
(9 at the Bihar site and 2 at the Uttar Pradesh site) either were
excluded because of the preceding criteria (9 patients) or chose
not to participate (2).
In addition to routine biochemical and hematologic tests,
evaluation of history, complete physical examination, and assessment of the Karnofsky performance status, other standard
In India, 320 patients with visceral leishmaniasis (209
in the state of Bihar and 11 in the neighboring state of
Uttar Pradesh) received identical pentavalent antimony
(Sb) treatment. Sb induced long-term cure in 35% (95%
confidence interval [CI], 28%–42%) of those in Bihar versus
86% (95% CI, 79%–93%) of those in Uttar Pradesh. In
Bihar, the center of the Indian epidemic, traditional Sb
treatment should be abandoned.
Of the estimated 500,000 new cases of visceral leishmaniasis
(kala-azar) that occur annually throughout the world, as many
as one-half are thought to occur in India, and up to 90% of
these cases are found in the northern region of the state of
Bihar [1, 2]. In India, kala-azar is also endemic in neighboring
states, including Uttar Pradesh and West Bengal; sporadic cases
are found elsewhere as well [1, 2]. In addition to representing
the center of what is now the decades-old kala-azar epidemic
in India, north Bihar has also been watched closely, since it
appears to be the first region in the world where large-scale
therapeutic failure of conventional treatment, pentavalent antimony (Sb), has emerged [3, 4].
Despite the associated adverse reactions and the need for
prolonged parenteral treatment, Sb has remained the traditional, first-line therapy for kala-azar throughout the world,
primarily because it is affordable and effective and is surely a
time-tested therapy [5]. Although low-level unresponsiveness to
This study was approved by the Ethics Committee of the Institute of
Medical Sciences, Banaras Hindu University, Varanasi, India, and informed
consent was obtained either from the subjects or from their parents or
guardians.
S.S. participated in designing the study, collecting the data, and writing
the paper and served as the principal investigator. D.K.M., M.K.S., V.P.S.,
S.S., A.M., and P.C.K.K. were involved in the care and clinical assessment
of patients and in the collection of data; H.W.M. collaborated on the design
of the study, was responsible for data interpretation, and helped with the
writing of the paper.
Reprints or correspondence: Dr. Shyam Sundar, 6, S.K. Gupta Nagar,
Lanka, Varanasi 211 005, India ([email protected]).
Clinical Infectious Diseases 2000; 31:1104–7
q 2001 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2000/3104-0038$03.00