Molecular Characterization of <i>Cryptosporidium spp</i>. among Children in Rural Ghana

<div><p>Background</p><p>The relevance of <i>Cryptosporidium</i> infections for the burden of childhood diarrhoea in endemic settings has been shown in recent years. This study describes <i>Cryptosporidium</i> subtypes among symptomatic and asymptomatic children in rural Ghana to analyse subtype-specific demographic, geographical, seasonal and clinical differences in order to inform appropriate control measures in endemic areas.</p><p>Methodology/Principal Findings</p><p>Stool samples were collected from 2232 children below 14 years of age presenting with and without gastrointestinal symptoms at the Agogo Presbyterian Hospital in the rural Ashanti region of Ghana between May 2007 and September 2008. Samples were screened for <i>Cryptosporidium spp</i>. by PCR and isolates were classified into subtypes based on sequence differences in the <i>gp60</i> gene. Subtype specific frequencies for age, sex, location and season have been determined and associations with disease symptoms have been analysed within a case-control study.</p><p><i>Cryptosporidium</i> infections were diagnosed in 116 of 2232 (5.2%) stool samples. Subtyping of 88 isolates revealed IIcA5G3 (n = 26, 29.6%), IbA13G3 (n = 17, 19.3%) and IaA21R3 (n = 12, 13.6%) as the three most frequent subtypes of the two species <i>C</i>. <i>hominis</i> and <i>C</i>. <i>parvum</i>, known to be transmitted anthroponotically. Infections peak at early rainy season with 67.9% and 50.0% of infections during the months April, May and June for 2007 and 2008 respectively. <i>C</i>. <i>hominis</i> infection was mainly associated with diarrhoea (odds ratio [OR] = 2.4; 95% confidence interval [CI]: 1.2–4.9) whereas <i>C</i>. <i>parvum</i> infection was associated with both diarrhoea (OR = 2.6; CI: 1.2–5.8) and vomiting (OR = 3.1; 95% CI: 1.5–6.1).</p><p>Conclusions/Significance</p><p>Cryptosporidiosis is characterized by seasonal anthroponotic transmission of strains typically found in Sub-Saharan Africa. The infection mainly affects young infants, with vomiting and diarrhoea being one of the leading symptoms in <i>C</i>. <i>parvum</i> infection. Combining molecular typing and clinical data provides valuable information for physicians and is able to track sources of infections.</p></div

Phylogenetic analysis.

<p>Phylogenetic analysis of <i>C</i>. <i>hominis</i> and <i>C</i>. <i>parvum</i> subtypes and six reference strains with their respective accession numbers using neighbour-joining analysis of the gylcoprotein 60 (<i>gp60</i>) gene. Values on branches are percentage bootstrap values using 1,000 replicates. Only bootstrap values greater than 50% are shown.</p

<i>Cryptosporidium parvum/hominis</i> and their association with gastrointestinal symptoms.

<p>OR: Age-adjusted odds ratios, with 95% confidence intervals (CI), calculated using the Mantel Haenszel method</p><p>^$for seven <i>Cryptosporidium spp</i>. positive children no clinical data was available.</p><p><i>Cryptosporidium parvum/hominis</i> and their association with gastrointestinal symptoms.</p

Parsimonious multivariate regressions model of characteristics positively associated with bacteremia in parasitemic children, stratified for parasite count.

<p>¹ OR (CI), odds ratio (95% confidence interval)</p><p>² Dehydration ≥ grade 1 (3–5%)</p><p>Parsimonious multivariate regressions model of characteristics positively associated with bacteremia in parasitemic children, stratified for parasite count.</p

Number of <i>Cryptosporidium parvum/homini</i>s cases by age and sex (n = 116).

<p>The majority of patients are below the age of three (87.1%, n = 101) years with 56.9% (n = 66) of cases being male. The mean age for male subjects (0.85; SD 0.90) is lower than for females (1.74; SD 2.36).</p

Observed prevalence (solid line) and predicted prevalence (dashed line) of intended hospital attendance by symptom along the travel distance to the nearest hospitals, Asante Akim North District, Ghana, 2008.

<p>Observed prevalence (solid line) and predicted prevalence (dashed line) of intended hospital attendance by symptom along the travel distance to the nearest hospitals, Asante Akim North District, Ghana, 2008.</p

Distribution of Cryptosporidium subtypes (n = 88) and percentage of all study participants infected with Cryptosporidium spp. (n = 2,322) over the study period.

<p>The proportion of infected patients peak during rainy seasons from May to July 2007 and April to July 2008. No clusters of specific subtype families are observed over time.</p

Test quality of CRP levels for the prediction of parasitemia, malaria and septicemia.

<p>Test quality of CRP levels for the prediction of parasitemia, malaria and septicemia.</p

CART – model for children between 2 and 12 months of age (N = 1304).

<p>¹ Number of patients with the respective combination of variables given by the branches of the decision tree. ² Number of patients positive for <i>P. falciparum</i> parasitaemia. ³ Odds Ratio for <i>P. falciparum</i> parasitaemia with the combination of variables in comparison to all other combinations.</p

Sensitivity and specificity of symptoms for prediction of <i>P. falciparum</i> parasitaemia in different age groups.

<p>Note: Percentage refers to the total number of patients within each age group.</p>a<p>ROF = <i>report of fever</i>;</p>b<p>EBT = <i>elevated body temperature</i>;</p>c<p>NRS = <i>no respiratory symptoms</i>.</p