2.1.3. Bases Teóricas
2.1.3.12. El derecho a la identidad
Robert J ten Hove, Jaco J Verweij, Tim Schuurman, Miriam Kooistra, Lieke Möller and Lisette van Lieshout
ABSTRACT
In this study the prevalence of Cryptosporidium hominis and Cryptosporidium
parvum is determined retrospectively in stool samples of 1914 patients attending
their general practitioner with gastro-intestinal symptoms in a period of approximately seven months. Microscopic examination aimed at Cryptosporidium
detection was requested by general practitioners 21 times and found positive in 13 samples. All samples were tested by real-time PCR analysis and revealed 80 cases positive for C. hominis / C. parvum. Subsequent species specific PCR analysis showed
C. hominis in 70% of cases and C. parvum in 17.5%. In the remaining 12.5% of cases no amplification product was observed. Children under age of 14 years held 70% of all Cryptosporidium infections. The highest prevalence was observed the month September. In this month 30% of all children under aged of 5 years were infected, mainly with C. hominis. The high C. hominis / C. parvum prevalence’s observed in this survey emphasizes the need for a sensitive high throughput system for
The protozoan Cryptosporidium is an enteric parasite found world wide among a broad variety of hosts (Fayer, 2005). Infection is typically acute and self-limiting, but symptoms can be severe in children and immune compromised persons. In The Netherlands Cryptosporidium hominis / Cryptosporidium parvum has a prevalence of about 2-3% in cases with gastroenteritis, and about 0.1% in the general population (De Wit et al., 2001a). Detailed epidemiological data on zoonotic potential and transmission routes of C. hominis and C. parvum in The Netherlands is limited. A recent study of Wielinga et al. describes the genetic diversity of Cryptosporidium in humans and farm animals from samples gathered over a period of three years at a number of laboratories located throughout The Netherlands (Wielinga et al., 2007). Samples were selected based on microscopic detection of the oocysts. However, in The Netherlands specific diagnostic procedures such as the modified acid-fast staining are not routinely used on each submitted stool sample. Because of the additional work involved, many laboratories perform this staining only if cryptosporidiosis is suspected by the general practitioner (GP). In a previous study 41 (4.3%) Cryptosporidium infections were revealed after molecular screening of 950 Dutch patients visiting their GP because of gastro-intestinal complaints (Ten Hove et al., 2007). In the current study an extended group of fecal-DNA samples collected from the same target population was further analysed on the prevalence of
C. hominis and C. parvum in relation to age and the season of sample collection. DNA of 1914 faecal samples was initially obtained as part of a study designed to evaluate the efficacy of molecular diagnosis for bacterial infections in patients with gastro-intestinal complaints (Schuurman et al., 2007a) and part of samples were also used to compare the molecular diagnosis of intestinal parasitic infections with microscopy (i.e. care as usual) (Ten Hove et al., 2007). The faecal samples from patients were submitted between 27th of June 2005 and 25th of January 2006 to the Laboratory for Infectious Diseases, Groningen, The Netherlands. For all samples the GPs requested bacterial cultures and for 1437 of those also microscopic analysis was requested for diagnosis of intestinal helminthes- and protozoan infections. During the same period, samples were also submitted to the laboratory for the detection of intestinal parasites only. These samples, however, were not included in the initial study (Schuurman et al., 2007a) and were therefore not available for screening with real-time PCR. Faecal DNA extraction was performed as described by Schuurman et al. (Schuurman et al., 2007b) and screened with multiplex real-time PCR for presence of Entamoeba histolytica, Giardia lamblia and C. parvum / C. hominis (Verweij et al., 2004a; Ten Hove et al., 2007). C. parvum / C. hominis species were subsequently differentiated with conventional PCR using primer sequences depicted from Morgan
et al (Morgan et al., 1997) to assess contribution of the zoonotic C. parvum infections during late summer when most cases of cryptosporidiosis are expected.
During the period of approximately seven months, C. hominis / C. parvum infections were detected with real-time PCR among 80 (4.2%) patients with gastro-intestinal complaints (range Ct-values: 24.5 – 42.3; median: 32.1). During the study period microscopic examination for Cryptosporidium was specifically requested by GPs 21 times and was found positive in 13 samples. In figure 3.1 the difference in percentages of C. hominis / C. parvum infections are compared by age groups. The vast majority of C. hominis / C. parvum infections occur in children under age of 14 years which contain 56 of all 80 detected C. hominis / C. parvum infections. Species specific PCR showed C. hominis in 70% of cases and C. parvum in 17.5% of cases. No PCR amplification product was observed in the remaining 12.5% of cases. This can be explained by the high Ct-values (range: 34.2 – 42.3; median 39.0) of these cases which indicate that the number of DNA copies are too low to be detected on ethidium-bromide stained agarose gel. The monthly prevalence of C. hominis is presented by a hyper curved shape with its peak in September whereas C. parvum
prevalence’s remains more or less constant (figure 3.2).
Figure 3.1. Distribution of Cryptosporidium hominis,Cryptosporidium parvum and undetermined Cryptosporidium species among patients (n = 1913) classified in age- groups. The age from one patient was not recorded.
Figure 3.2. Seasonal distribution of Cryptosporidium hominis, Cryptosporidium parvum and unspecified Cryptosporidium species detected in patients (n = 1914).
The outcomes of this study show that current mode of detection greatly underestimates the actual prevalence of C. hominis / C. parvum. In the month of September, even 30 % of all children under age of five with gastro-intestinal complaints showed to be infected with C. hominis / C. parvum. The increased prevalence in September was not contributed to increased number of samples from children; throughout the study the monthly percentage of samples originating from children under age of five remained around 11% ± 2SD. Screening for
Cryptosporidium should be recommended as a standard procedure in stool examination, in particular for children. This is also emphasised by the rate of
C. hominis / C. parvum infections detected in samples that were send to the laboratory without even a request for parasitological analysis. Of 477 stool samples that came in with a request only for bacteriology, C. hominis / C. parvum was still detected in 11 (2.3%) cases.
Outbreaks of C. hominis infections are often associated with recreational swimming and with contaminated drinking water whereas C. parvum is associated more with zoonotic transmission during the kidding period of live stock animals in early spring (McLauchlin et al., 2000). To investigate the role of C. parvum in the general
Cryptosporidium prevalence, molecular screening of patients should continue through spring season with special attention to agricultural activities nearby residential locations (Lake et al., 2007). The relationship between C. parvum isolates can be investigated further by combining acquired demographic data with sub-
genotype characterisation of Cryptosporidium isolates from stool samples of humans and live-stock animals (Grinberg et al., 2008).
The high Cryptosporidium prevalence’s observed in this survey emphasizes the need for a sensitive high throughput system for Cryptosporidium detection in routine diagnostic laboratories. Implementation of standard molecular screening will reveal
Cryptosporidium infections more accurately, recognize outbreaks more rapidly and