Molecular Characterization of Cryptosporidium Species and Giardia duodenalis from Symptomatic Cambodian Children

Background: In a prospective study, 498 single faecal samples from children aged under 16 years attending an outpatient clinic in the Angkor Hospital for Children, northwest Cambodia, were examined for Cryptosporidium oocysts and Giardia cysts using microscopy and molecular assays. Methodology/Principal Findings: Cryptosporidium oocysts were detected in 2.2% (11/498) of samples using microscopy and in 7.7% (38/498) with molecular tests. Giardia duodenalis cysts were detected in 18.9% (94/498) by microscopy and 27.7% (138/498) by molecular tests; 82% of the positive samples (by either method) were from children aged 1–10 years. Cryptosporidium hominis was the most common species of Cryptosporidium, detected in 13 (34.2%) samples, followed by Cryptosporidium meleagridis in 9 (23.7%), Cryptosporidium parvum in 8 (21.1%), Cryptosporidium canis in 5 (13.2%), and Cryptosporidium suis and Cryptosporidium ubiquitum in one sample each. Cryptosporidium hominis and C. parvum positive samples were subtyped by sequencing the GP60 gene: C. hominis IaA16R6 and C. parvum IIeA7G1 were the most abundant subtypes. Giardia duodenalis was typed using a multiplex real-time PCR targeting assemblages A and B. Assemblage B (106; 76.8% of all Giardia positive samples) was most common followed by A (12.3%) and mixed infections (5.1%). Risk factors associated with Cryptosporidium were malnutrition (AOR 9.63, 95% CI 1.67–55.46), chronic medical diagnoses (AOR 4.51, 95% CI 1.79–11.34) and the presence of birds in the household (AOR 2.99, 95% CI 1.16–7.73); specifically C. hominis (p = 0.03) and C. meleagridis (p<0.001) were associated with the presence of birds. The use of soap was protective against Giardia infection (OR 0.74, 95% CI 0.58–0.95). Conclusions/Significance: This is the first report to describe the different Cryptosporidium species and subtypes and Giardia duodenalis assemblages in Cambodian children. The variety of Cryptosporidium species detected indicates both anthroponotic and zoonotic transmission in this population. Interventions to improve sanitation, increase hand washing after defecation and before preparing food and promote drinking boiled water may reduce the burden of these two parasites

Intestinal Parasite Infections in Symptomatic Children Attending Hospital in Siem Reap, Cambodia

Background Infections with helminths and other intestinal parasites are an important but neglected problem in children in developing countries. Accurate surveys of intestinal parasites in children inform empirical treatment regimens and can assess the impact of school based drug treatment programmes. There is limited information on this topic in Cambodia. Methods In a prospective study of intestinal parasites in symptomatic children attending Angkor Hospital for Children, Siem Reap, Cambodia, April-June 2012, samples were examined by microscopy of a direct and concentrated fecal sample. Two culture methods for hookworm and Strongyloides stercoralis were employed when sufficient sample was received. Demographic, clinical and epidemiological data were collected. Principal Findings We studied 970 samples from 865 children. The median (inter-quartile range) age of the children was 5.4 (1.9-9.2) years, 54% were male. The proportion of children with abdominal pain was 66.8%, diarrhea 34.9%, anemia 12.7% and malnutrition 7.4%. 458 parasitic infections were detected in 340 (39.3%) children. The most common parasites using all methods of detection were hookworm (14.3%), Strongyloides stercoralis (11.6%) and Giardia lamblia (11.2%). Giardia lamblia was most common in children aged 1-5 years, hookworm and Strongyloides stercoralis were more common with increasing age. Hookworm, Strongloides stercoralis and Giardia lamblia were more common in children living outside of Siem Reap town. In a multivariate logistic regression increasing age was associated with all three infections, defecating in the forest for hookworm infection, the presence of cattle for S. stercoralis and not using soap for handwashing for G. lamblia. Conclusions/Significance This study confirms the importance of intestinal parasitic infections in symptomatic Cambodian children and the need for adequate facilities for laboratory diagnosis together with education to improve personal hygiene and sanitation

Demographic, clinical and epidemiological details of all of the children studied.

<p>The results are separated by age group and only significant results are shown. The full list of demographic, clinical and epidemiological details are described in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123719#pone.0123719.s001" target="_blank">S1 Table</a>.</p><p>^aPercentage of those positive by age group (either ≤28 days, 29 days-1 year, 1–5 years, 6–10 years and 11–16 years). The numbers in superscripts are those missing per age group or overall.</p><p>*Anemia is based on attending doctors’ clinical decision rather than clinical hemoglobin levels as no patients had clinical anemia using CBC. results.</p><p>Demographic, clinical and epidemiological details of all of the children studied.</p

Comparison of methods for presence of faecal parasites (see Table 2 for the overall numbers).

<p>^aAPC is agar plate culture</p><p>Kappa statistic for comparisons between APC and charcoal agar</p><p>^b0.14 for culture of hookworm and</p><p>^c0.65 for culture of <i>S</i>. <i>stercoralis</i> for all samples positive for each parasite</p><p>Comparison of methods for presence of faecal parasites (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123719#pone.0123719.t002" target="_blank">Table 2</a> for the overall numbers).</p

Significant univariate and multivariate risk factor analyses for infections caused by the three most common clear parasites (hookworm, <i>Strongyloides stercoralis</i> and <i>Giardia lamblia</i>).

<p>Significant univariate and multivariate risk factor analyses for infections caused by the three most common clear parasites (hookworm, <i>Strongyloides stercoralis</i> and <i>Giardia lamblia</i>).</p

Presence of polyparasitism.

<p>^aPercentage of positive patients divided by total number of consented patients</p><p>Presence of polyparasitism.</p

Gastrointestinal parasites identified in fecal samples from children attending the Angkor Hospital for Children, April–June 2012.

<p>(%) Percentage of total examined.</p><p>^aNumber of patients positive (disease episodes).</p><p>^bPercentage of disease episode by age (total number of disease episodes/number in age group*100).</p><p>*331 samples examined for <i>Cryptosporicium</i> species and <i>Cyclospora cayetanesis</i> using ZN stains and</p><p>**Four samples were culture positive, however the parasite species could not be determined</p><p>Gastrointestinal parasites identified in fecal samples from children attending the Angkor Hospital for Children, April–June 2012.</p

Flow chart detailing the study participants.

<p>Flow chart detailing the study participants.</p

Comparison of methods for presence of faecal parasites^a (see Table 2 for the overall numbers).

<p>^aFor comparison, the 15 samples for which there was insufficient sample for formalin concentration were excluded hence numbers are not as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123719#pone.0123719.t002" target="_blank">Table 2</a></p><p>^bFC is the formalin concentration method</p><p>Comparison of methods for presence of faecal parasites<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123719#t004fn001" target="_blank">^a</a> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123719#pone.0123719.t002" target="_blank">Table 2</a> for the overall numbers).</p

Detection of <i>Cryptosporidium</i> spp. and <i>Giardia duodenalis</i>.

<p>Detection of <i>Cryptosporidium</i> spp. and <i>Giardia duodenalis</i>.</p