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

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

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

Gastrointestinal Infections and Diarrheal Disease in Ghanaian Infants and Children: An Outpatient Case-Control Study

<div><p>Introduction</p><p>Diarrheal diseases are among the most frequent causes of morbidity and mortality in children worldwide, especially in resource-poor areas. This case-control study assessed the associations between gastrointestinal infections and diarrhea in children from rural Ghana.</p><p>Methods</p><p>Stool samples were collected from 548 children with diarrhea and from 686 without gastrointestinal symptoms visiting a hospital from 2007–2008. Samples were analyzed by microscopy and molecular methods.</p><p>Results</p><p>The organisms most frequently detected in symptomatic cases were <i>Giardia lamblia</i>, <i>Shigella</i> spp./ enteroinvasive <i>Escherichia coli</i> (EIEC), and <i>Campylobacter jejuni</i>. Infections with rotavirus (adjusted odds ratio [aOR] = 8.4; 95% confidence interval [CI]: 4.3–16.6), <i>C</i>. <i>parvum/hominis</i> (aOR = 2.7; 95% CI: 1.4–5.2) and norovirus (aOR = 2.0; 95%CI: 1.3–3.0) showed the strongest association with diarrhea. The highest attributable fractions (AF) for diarrhea were estimated for rotavirus (AF = 14.3%; 95% CI: 10.9–17.5%), <i>Shigella</i> spp./EIEC (AF = 10.5%; 95% CI: 3.5–17.1%), and norovirus (AF = 8.2%; 95% CI 3.2–12.9%). Co-infections occurred frequently and most infections presented themselves independently of other infections. However, infections with <i>E</i>. <i>dispar</i>, <i>C</i>. <i>jejuni</i>, and norovirus were observed more often in the presence of <i>G</i>. <i>lamblia</i>.</p><p>Conclusions</p><p>Diarrheal diseases in children from a rural area in sub-Saharan Africa are mainly due to infections with rotavirus, <i>Shigella</i> spp./EIEC, and norovirus. These associations are strongly age-dependent, which should be considered when diagnosing causes of diarrhea. The presented results are informative for both clinicians treating gastrointestinal infections as well as public health experts designing control programs against diarrheal diseases.</p></div

Frequencies (per cent) of gastrointestinal organisms identified in stool samples sorted by species group and frequency of occurrence.

<p>Missing values (not considered in percentages):</p><p>^a225;</p><p>^b224,</p><p>^c218,</p><p>^d602,</p><p>^e213,</p><p>^f217</p><p>^$identified via Polymerase Chain Reaction (PCR)</p><p>^§identified via ELISA</p><p>Frequencies (per cent) of gastrointestinal organisms identified in stool samples sorted by species group and frequency of occurrence.</p

Crude odds ratio (OR), age-adjusted OR (aOR), and age-stratified OR on associations between gastrointestinal infections and diarrhea, sorted by aOR.

<p>Abbreviations: OR, odds ratio; CI, confidence interval; aOR, age-adjusted odds ratio.</p><p>^§Combined age groups to avoid empty cells in cross-tabulation.</p><p>Crude odds ratio (OR), age-adjusted OR (aOR), and age-stratified OR on associations between gastrointestinal infections and diarrhea, sorted by aOR.</p

Age-adjusted relative risk of the occurrences of selected gastrointestinal co-infections in all children and for cases and controls separately.

<p>Abbreviations: aRR, age-adjusted risk ratio; CI, confidence interval; NA: not applicable due to low frequency.</p><p>Age-adjusted relative risk of the occurrences of selected gastrointestinal co-infections in all children and for cases and controls separately.</p

Proportions of case and control children by age group infected by various organisms.

<p>All organisms detected in more than 50 samples are shown. Median ages in months (IQR) at the time of infection are shown in brackets.</p