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Cryptosporidium infections in Denmark, 2010-2014

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Cryptosporidium infections in Denmark, 2010-2014

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The incidence of cryptosporidiosis in Denmark is unknown. Here, we present the number of cases detected in the 2010-2014 period along with data on species and subtypes. Complete national data retrieved from the Danish Microbiology Database and Statens Serum Institut (SSI) comprised test results on cryptosporidia detected by microscopy or polymerase chain reaction (PCR) between 1 January 2010 and 30 April 2014. Samples that tested positive at the SSI were submitted to species and subtype analysis by conventional PCR and sequencing of ribosomal and gp60 genes, respectively. A total of 689 Cryptosporidium-positive stool samples were submitted by 387 patients. Limiting case episodes to two months (60 days), a total of 388 case episodes representing 387 patients were identified. Cryptosporidiosis was most common among infants and toddlers. Moreover, a peak in incidence was observed among younger adults aged 23-24 years. In 43 Cryptosporidium-positive faecal samples, identification was performed to species and subtype level. Cryptosporidium parvum was found in 34 samples, C. hominis in eight, and C. meleagridis in one sample; C. parvum subtypes IIaA15G2R1 (n = 10) and IIaA16G3R1 (n = 5) were predominating. Cryptosporidia are a significant cause of diarrhoea in Denmark. Outbreaks may not be detected due to continued use of diagnostic tests of limited sensitivity and due to lack of surveillance. With molecular methods now being introduced in many Danish laboratories, we propose establishing national surveillance of cryptosporidiosis. not relevant. not relevant.
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Dan Med J 62/5 May 2015 DANISH MEDICAL JOURNAL 1
ABSTRACT
IntroductIon: The incidence of cryptosporidiosis in Den-
mark is unknown. Here, we present the number of cases
detected in the 2010-2014 period along with data on spe-
cies and subtypes.
Methods: Complete national data retrieved from the Dan-
ish Microbiology Database and Statens Serum Institut (SSI)
comprised test results on cryptosporidia detected by mi-
croscopy or polymerase chain reaction (PCR) between 1
January 2010 and 30 April 2014. Samples that tested posi-
tive at the SSI were submitted to species and subtype analy-
sis by conventional PCR and sequencing of ribosomal and
gp60 genes, respectively.
results: A total of 689 Cryptosporidium-positive stool sam-
ples were submitted by 387 patients. Limiting case episodes
to two months (60 days), a total of 388 case episodes repre-
senting 387 patients were identified. Cryptosporidiosis was
most common among infants and toddlers. Moreover, a
peak in incidence was observed among younger adults aged
23-24 years. In 43 Cryptosporidium-positive faecal samples,
identification was performed to species and subtype level.
Cryptosporidium parvum was found in 34 samples, C. homi-
nis in eight, and C. meleagridis in one sample; C. parvum
subtypes IIaA15G2R1 (n = 10) and IIaA16G3R1 (n = 5) were
predominating.
conclusIon: Cryptosporidia are a significant cause of diar-
rhoea in Denmark. Outbreaks may not be detected due to
continued use of diagnostic tests of limited sensitivity and
due to lack of surveillance. With molecular methods now
being introduced in many Danish laboratories, we propose
establishing national surveillance of cryptosporidiosis.
FundIng: not relevant.
trIal regIstratIon: not relevant.
Cryptosporidiosis is an infection caused by single-celled
parasites of the Cryptosporidium genus. Infected pa-
tients may present with watery diarrhoea lasting for up
to weeks [1, 2]. Even in otherwise healthy individuals,
pronounced symptoms may arise. The parasites are pri-
marily transmitted through contaminated drinking or
recreational water; however, infection may occasionally
arise through contaminated food or contact to animals
or infected individuals [3, 4].
1-10 oocysts (spores) are sufficient to establish in-
fection. The cryptosporidia invade and multiply in the
gut epithelium, which is usually accompanied by devel-
opment of symptoms; oocysts are shed in the faeces
throughout this period. The longevity of oocyst shedding
varies. Oocysts are environmentally resilient and may
survive for days even in chlorinated water; meanwhile,
ultraviolet (UV) irradiation and boiling of water is effi-
cient in terms of limiting waterborne transmission.
Cryptosporidiosis is self-limiting within 1-3 weeks in
otherwise healthy individuals, whereas immunosup-
pressed patients may develop chronic diarrhoea with
prolonged oocyst shedding. An effective treatment regi-
men remains to be identified, although some effect of
nitazoxanide, a nitrothiazolyl-salicylamide derivative,
has been reported [5, 6].
On a global scale, cryptosporidia are a frequent
cause of outbreaks. In Milwaukee, USA, an outbreak in
1993 caused by contaminated drinking water affected
more than 400,000 people [7]. In 2010, approximately
27,000 people became infected in Östersund, also
through contaminated drinking water [8], and water-
borne outbreaks are quite common in Sweden [4, 8-11].
In Sub-Saharan Africa and other regions, cryptosporidio-
sis is one of the most common and serious causes of
diarrhoea-related morbidity and mortality in infants and
toddlers [12].
The first known outbreak in Denmark was recorded
at Hvidovre Hospital in 1989 [13], and in 2005 an out-
break was described that affected employees in a large
Danish company due to oocyst-contaminated raw car-
rots served in the company’s canteen [3]. Since then, mi-
nor outbreaks have been described related to the han-
dling of calves, a significant reservoir of Cryptosporidium
parvum infection. C. parvum is possibly the species most
commonly associated with cryptosporidiosis in humans;
another common species is primate-adapted C. hominis.
Zoonotic transmission involving other species, including
C. meleagridis, C. canis, and C. felis, is seen occasionally
[9, 14]. C. parvum infection is associated with more se-
vere symptoms than cases of cryptosporidiosis caused
by C. hominis [9]. Multiple subtypes and subtype vari-
ants have been described for several species, and mole-
cular typing is critical to successful outbreak investiga-
tions.
Contrary to the situation in our neighbouring coun-
tries, no national surveillance of cryptosporidiosis exists
in Denmark. Hence, the incidence of the infection is un-
known, and no guidelines as to which patients should be
Cryptosporidium infections in Denmark, 2010-2014
Christen Rune Stensvold1, Steen Ethelberg2, Louise Hansen2, Sumrin Sahar1, Marianne Voldstedlund2, Michael Kemp3,
Gitte Nyvang Hartmeyer3, Erik Otte4, Anne Line Engsbro5, Henrik Vedel Nielsen1 & Kåre Mølbak2
ORIGINAL
ARTICLE
1) Laboratory of
Parasitology,
Department of
Microbiology and
Infection Control,
Statens Serum Institut
2) Department of
Infectious Disease
Epidemiology,
Statens Serum Institut
3) Department of
Clinical Microbiology,
Odense University
Hospital
4) Department of
Clinical Microbiology,
Aarhus University
Hospital
5) Department of
Clinical Microbiology,
Hvidovre Hospital,
Denmark
Dan Med J
2015;62(5):A5086
2 DANISH MEDICAL JOURNAL Dan Med J 62/5 May 2015
suspected of and tested for cryptosporidiosis are avail-
able. The aim of this article is to increase awareness of
cryptosporidia as a cause of diarrhoea. Based on cur-
rently available national data, we summarised the num-
ber of cases detected between January 2010 and April
2014, and generated preliminary data that indicate
which species account for cryptosporidiosis detected in
Denmark. By introducing sensitive diagnostic methods
and by increasing the focus on cryptosporidia as a po-
tential cause of diarrhoea, a more complete and accu-
rate picture of the incidence and transmission patterns
will be revealed.
METHODS
Data from the Danish Microbiology Database and
molecular typing
Data were retrieved from the Danish Microbiology Data-
base (MiBa) [15, 16], which receives laboratory test re-
sults from all microbiology departments in Denmark.
Data comprised test results on cryptosporidia detected
by microscopy or polymerase chain reaction (PCR) be-
tween 1 January 2010 and 30 April 2014. As the national
reference laboratory for parasitology at Statens Serum
Institut (SSI) did not comply with the national standard
protocol for transferring electronic microbiological re-
ports to MiBa during the first two years of the study,
data from this laboratory were extracted directly from
the SSI database and merged with the MiBa data set to
obtain complete nationwide data.
Samples identified as positive by the SSI were fur-
thermore submitted to species and subtype analysis by
conventional PCR and sequencing of ribosomal (SSU
rRNA) and gp60 genes, respectively, as assays for gp60-
based subtyping are now available for the three most
common species, C. parvum, C. hominis and C. melea-
gridis [17]. Data were standardised and analysed using
Stata v. 13. Results are indicated as number of cases, a
case-episode being defined as an individual with one or
more positive stool samples, excluding multiple positive
test results obtained within a 60-day period.
Trial registration: not relevant.
RESULTS
Incidence and distribution
From January 2010 to April 2014, a total of 689 Crypto-
sporidium-positive stool samples had been submitted by
387 patients, several patients testing positive more than
once. Limiting case-episodes to two months (60 days),
only one patient had more than one episode. Hence, a
total of 388 case-episodes representing 387 patients
were identified, including 210 episodes in females and
178 episodes in males. Analysing the patient age distri-
bution (Figure 1), it appears that cases of cryptosporidi-
osis were most common among infants and toddlers.
Moreover, a peak in incidence was observed among
younger adults aged 23-24 years. In older adults and
seniors, only few positive samples were seen.
Among children less than five years old, boys were
more prone to developing infection than girls, whereas
in adolescents and younger adults, more cases of crypto-
sporidiosis were found among females than males
(Figure 2). In terms of seasonal variation, there was a
general trend towards identifying more cases in late
FIGURE 1
Age distribution of the 388 episodes of cryptosporidiosis
detected from January 2010 to April 2014 in Denmark.
0
0 20 40 60
Age (years)
80
5
10
15
20
n
Dan Med J 62/5 May 2015 DANISH MEDICAL JOURNAL 3
summer and autumn, which is in agreement with obser-
vations from other countries [18].
GP60 data
For 43 Cryptosporidium-positive faecal samples analysed
from 2010 to 2014, parasites were identified to species
and subtype level. C. parvum was found in 34 samples,
C. hominis in eight, and C. meleagridis was identified in a
sample from a patient with a recent history of kidney
transplantation. With regard to C. parvum, we observed
a relatively high number of two subtypes that are com-
mon in calves in Scandinavia, IIaA15G2R1 (n = 10) and
IIaA16G3R1 (n = 5). Some of the affected individuals
were veterinarians who had been handling calves prior
to developing the infection.
DISCUSSION
Diagnostic considerations
Traditional parasitology uses microscopy of Ziehl-Neel-
sen (ZN)-stained smears of faecal concentrates to detect
cryptosporidia or commercial kits based on antigen de-
tection. In Denmark, DNA-based methods are being in-
troduced in several routine clinical microbiology labora-
tories for detection of parasites such as Cryptosporidium
spp. and Giardia intestinalis. A study including 889 pro-
spective and random faecal samples identified a total of
16 cases of cryptosporidiosis by real-time PCR, while no
cases were detected by microscopy of ZN-stained faecal
smears [19]. At SSI, a genus-specific real-time PCR is em-
ployed to enable the detection of species other than C.
hominis and C. parvum. Even when real-time PCR is
used, one of two samples from an infected patient may
test negative, and it is recommended to test at least two
samples to reduce the risk of overlooking cases.
In Denmark, no national guidelines are available re-
garding the referral of samples for Cryptosporidium test-
ing. In Halland, Sweden, PCR has been implemented in
the routine screening of patients with diarrhoea regard-
less of type of diarrhoea and region of exposure (do-
me stic or travel-related). Interestingly, Halland has the
highest incidence of cryptosporidiosis seen throughout
Sweden, 17/100,000 (2013; [20]). Meanwhile, in Den-
mark, testing for cryptosporidiosis is primarily per-
formed in cases of travel-associated or persisting diar-
rhoea, or in cases of diarrhoea in immunocompromised
individuals. Otherwise healthy individuals with acute
diarhoea and no history of travelling are therefore not
tested for cryptosporidiosis on a routine basis.
Preliminary research data from the SSI indicate that the
incidence of cryptosporidiosis in patients with acute
diarrhoea acquired in Denmark is comparable to the in-
cidence among patients suspected of cryptosporidiosis,
including patients with chronic and/or travel-associated
diarrhoea. Based on observations from Halland and the
SSI, ongoing studies will address whether testing for
Cryptosporidium should be implemented consistently in
the routine screening of stool samples from patients
with diarrhoea acquired in Denmark.
The samples that were subtyped had been collected
over a period of five years. Therefore, the probability of
an unrecognised outbreak of, e.g., IIaA15G2R1 is small,
though still possible.
Six out of eight cases of C. hominis infection were
registered in the winter season. In Sweden, the vast ma-
jority of C. hominis cases are imported [9].
The typing data may indicate that mostly sporadic
cases or cases related to exposure to calves are detected
in Denmark. A few cases of travel-associated C. hominis
are seen, and cases in which the disease is particularly
debilitating (as in infants and toddlers) are likely to be
FIGURE 2
40
n
30
20
10
Age (years)
0
Female
0-4
5-9
10-14
15-19
20-24
25-29
30-34
35-39
40-44
45-49
50-54
55-59
60-64
65-69
70-74
45-79
80-84
85-89
Male
Distribution of gender and age (five-year intervals)
among 388 episodes of cryptosporidiosis detected from
January 2010 to April 2014.
4 DANISH MEDICAL JOURNAL Dan Med J 62/5 May 2015
identified, which again may explain the relatively higher
incidence among infants and toddlers. However, since
cryptosporidiosis is probably under-diagnosed, these
data should be interpreted with caution.
Moving towards surveillance
For several reasons, cryptosporidiosis is likely under-
diagnosed in Denmark. Most often cryptosporidiosis is a
self-limiting disease with only few patients seeking med-
ical assistance. Furthermore, there is often a low index
of suspicion of cryptosporidiosis and specific examina-
tion of faeces for Cryptosporidium species is rarely re-
quested. Finally, some methods currently in use for the
detection of cryptosporidia in patient samples have only
a low or moderate sensitivity. The disease burden may
therefore be heavier than anticipated, and the chances
of detecting outbreaks are not optimal.
It is not very likely that the risk of waterborne out-
breaks is as high in Denmark as in countries such as
Sweden and England. In Denmark, surface water is used
as a source of drinking water only to a very limited ex-
tent, which reduces the risk of domestic zoonotic trans-
mission. However, the general lack of awareness in
Denmark explains why the outbreak in 2005 was de-
tected late [3], since testing for cryptosporidia was per-
formed only after the exclusion of bacteria and virus as
potential outbreak causes.
Since sensitive DNA-based tests are currently being
introduced into routine diagnostic clinical microbiology
laboratories in Denmark, it is now relevant to implement
national surveillance. MiBa was established in 2010 by
combined efforts of the regional clinical microbiology
laboratories and the SSI with a view to developing a
state-of-the-art digital surveillance system. If general
awareness regarding cryptosporidia as a potential cause
of diarrhoea is increased, it will be possible to efficiently
monitor infections in real-time, which will enable fast
and targeted action to contain and manage suspected
outbreaks as a cause of exposure to contaminated water
and foods, or infected animals.
CONCLUSION
In Denmark as well as in other countries, cryptosporidia
constitute an important pathogen causing diarrhoeal
disease in otherwise healthy children and adults. Out-
breaks are frequently seen in our neighbouring countries
and are also likely to occur in Denmark although prob-
ably less frequently. However, outbreaks, if they exist,
would likely not be easily recognised because of lack of
awareness, lack of national guidelines on testing and be-
cause of the continued use of diagnostic methods with
limited sensitivity. As more sensitive methods are being
introduced in diagnostic laboratories, we propose estab-
lishing national surveillance of cryptosporidiosis.
CORRESPONDENCE: Christen Rune Stensvold, Infektionsepidemiologi,
Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark.
E-mail: run@ssi.dk
ACCEPTED: 25 March 2015
CONFLICTS OF INTEREST: Disclosure forms provided by the authors are
available with the full text of this article at www.danmedj.dk
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... C. hominis and C. parvum are internationally the most commonly species infecting humans. [12,13] . Humans can acquire cryptosporidium infections through several transmission routes such as person to person transmission, zoonotic transmission, food borne transmission and waterborne transmission [14] .A single oocyst is sufficient to cause infection and disease [15,16] . ...
... C. hominis and C. parvum are internationally the most commonly species infecting humans. [17,18] . Humans can acquire cryptosporidium infections through several transmission routes such as person to person transmission, zoonotic transmission, food borne transmission and waterborne transmission [19] .A single oocyst is sufficient to cause infection and disease [20] . ...
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... C. hominis and C. parvum are internationally the most commonly species infecting humans. [11,12] . Humans can acquire cryptosporidium infections through several transmission routes such as person to person transmission, zoonotic transmission, food borne transmission and waterborne transmission [13] .A single oocyst is sufficient to cause infection and disease [14,15] . ...
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... C. hominis and C. parvum are internationally the most commonly species infecting humans. [12,13] . Humans can acquire cryptosporidium infections through several transmission routes such as person to person transmission, zoonotic transmission, food borne transmission and waterborne transmission [14] .A single oocyst is sufficient to cause infection and disease [15,16] . ...
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Chapter
According to the World Health Organisation, cryptosporidiosis is a global diarrhoeal disease affecting millions of individuals; it is the second most common cause of infantile death in developing countries and is increasingly identified as an emerging cause of morbidity and mortality worldwide. The disease is also extremely severe in livestock, causing profuse diarrhoea and considerable economic losses in farmed young animals. Given the lack of effective treatment (absence of vaccines and effective drugs) and the limited understanding of the causative parasite, cryptosporidiosis represents a major challenge in the battle against global diarrhoeal diseases. Currently, there are 45 described Cryptosporidium species infecting a whole spectrum of animals. In this book chapter we will present an overview of the parasite, focusing on its taxonomic status, its morphology, its prevalence and transmission. We will review both cell biological and molecular techniques currently used to investigate the biology of this parasite and we will introduce the new state-of-the-art techniques that have been established by several laboratories in the field. With the development of these new technologies, we will be able to further understand the unique biology of Cryptosporidium and its role in health and disease of its host.
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SUMMARY Most human cases of cryptosporidiosis are caused by Cryptosporidium parvum or Cryptosporidium hominis, but the use of molecular diagnostic methods has revealed that several other less common species or genotypes can also be involved. Here, we describe two unusual causes of cryptosporidiosis, one being the recently described species Cryptosporidium viatorum and the other Cryptosporidium chipmunk genotype I. Two Swedish patients who were infected with C. viatorum had travelled to Kenya and Guatemala, respectively, and two others had been infected with Cryptosporidium chipmunk genotype I in Sweden. None of these four patients were immunocompromised, and all four showed classical symptoms of cryptosporidiosis. We performed extensive molecular characterization, including analysis of four loci. The two C. viatorum isolates were found to differ slightly at the 70-kDa heat shock protein locus, which may indicate a local geographical variation in this species that has previously been described exclusively on the Indian subcontinent.
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Background: Diarrhoeal diseases cause illness and death among children younger than 5 years in low-income countries. We designed the Global Enteric Multicenter Study (GEMS) to identify the aetiology and population-based burden of paediatric diarrhoeal disease in sub-Saharan Africa and south Asia. Methods: The GEMS is a 3-year, prospective, age-stratified, matched case-control study of moderate-to-severe diarrhoea in children aged 0-59 months residing in censused populations at four sites in Africa and three in Asia. We recruited children with moderate-to-severe diarrhoea seeking care at health centres along with one to three randomly selected matched community control children without diarrhoea. From patients with moderate-to-severe diarrhoea and controls, we obtained clinical and epidemiological data, anthropometric measurements, and a faecal sample to identify enteropathogens at enrolment; one follow-up home visit was made about 60 days later to ascertain vital status, clinical outcome, and interval growth. Findings: We enrolled 9439 children with moderate-to-severe diarrhoea and 13,129 control children without diarrhoea. By analysing adjusted population attributable fractions, most attributable cases of moderate-to-severe diarrhoea were due to four pathogens: rotavirus, Cryptosporidium, enterotoxigenic Escherichia coli producing heat-stable toxin (ST-ETEC; with or without co-expression of heat-labile enterotoxin), and Shigella. Other pathogens were important in selected sites (eg, Aeromonas, Vibrio cholerae O1, Campylobacter jejuni). Odds of dying during follow-up were 8·5-fold higher in patients with moderate-to-severe diarrhoea than in controls (odd ratio 8·5, 95% CI 5·8-12·5, p<0·0001); most deaths (167 [87·9%]) occurred during the first 2 years of life. Pathogens associated with increased risk of case death were ST-ETEC (hazard ratio [HR] 1·9; 0·99-3·5) and typical enteropathogenic E coli (HR 2·6; 1·6-4·1) in infants aged 0-11 months, and Cryptosporidium (HR 2·3; 1·3-4·3) in toddlers aged 12-23 months. Interpretation: Interventions targeting five pathogens (rotavirus, Shigella, ST-ETEC, Cryptosporidium, typical enteropathogenic E coli) can substantially reduce the burden of moderate-to-severe diarrhoea. New methods and accelerated implementation of existing interventions (rotavirus vaccine and zinc) are needed to prevent disease and improve outcomes. Funding: The Bill & Melinda Gates Foundation.
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SUMMARY This study describes the epidemiology and symptoms in 271 cryptosporidiosis patients in Stockholm County, Sweden. Species/genotypes were determined by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) of the Cryptosporidium oocyst wall protein (COWP) and 18S rRNA genes. Species were C. parvum (n=111), C. hominis (n=65), C. meleagridis (n=11), C. felis (n=2), Cryptosporidium chipmunk genotype 1 (n=2), and a recently described species, C. viatorum (n=2). Analysis of the Gp60 gene revealed five C. hominis allele families (Ia, Ib, Id, Ie, If), and four C. parvum allele families (IIa, IIc, IId, IIe). Most C. parvum cases (51%) were infected in Sweden, as opposed to C. hominis cases (26%). Clinical manifestations differed slightly by species. Diarrhoea lasted longer in C. parvum cases compared to C. hominis and C. meleagridis cases. At follow-up 25-36 months after disease onset, 15% of the patients still reported intermittent diarrhoea. In four outbreaks and 13 family clusters, a single subtype was identified, indicating a common infection source, which emphasizes the value of genotyping for epidemiological investigations.