Detection of Parasite-Specific DNA in Urine Sediment Obtained by Filtration Differentiates between Single and Mixed Infections of \u3cem\u3eSchistosoma mansoni\u3c/em\u3e and \u3cem\u3eS. haematobium\u3c/em\u3e from Endemic Areas in Ghana

Differential diagnosis of Schistosoma mansoni and S. haematobium, which often occur sympatrically in Africa, requires both urine and stool and the procedures are low in sensitivity. The standard diagnostic tests, such as Kato-Katz (KK) for S. mansoni eggs and presence of haematuria for S. haematobium both lack sensitivity, produce false-negative results and show reduced accuracy with decreasing intensity of infection. The need for a single diagnostic test with high sensitivity and specificity for both parasites is important as many African countries are implementing Mass Drug Administration (MDA) following recommendations of the World Health Organization (WHO). Eighty-six samples of urine sediment obtained by filtration were collected from a group of 5–23 years old people from an endemic area of southern Ghana. DNA was extracted from the urine sediment on filter paper from which a species-specific repeat fragment was amplified by polymerase chain reaction (PCR) with specific primers for S. mansoni and for S. haematobium. Additionally, all participants were tested by KK (stool) and dipstick for haematuria. Diagnostic parameters for all three tests were analyzed statistically. Amplification of species-specific DNA by PCR showed much higher sensitivity (99%–100%) and specificity (100%) compared to KK and haematuria (sensitivity: 76% and 30% respectively) for both schistosome species. The same pattern was observed when the data were stratified for age group and sex specific analysis. In addition PCR amplification detected DNA from 11 individuals infected with both parasites who were negative by KK and haematuria. This approach of detecting parasite specific DNA from either or both species in a single urine specimen is a practical advantage that avoids the need for two specimens and is more effective than standard tests including those based on serology. This promises to improve the effectiveness of surveillance of MDA control programs of schistosomiasis

Assessment of Dual Schistosome Infection Prevalence from Urine in An Endemic Community of Ghana By Molecular Diagnostic Approach

Schistosomiasis is an important Neglected Tropical Disease caused by blood parasites called schistosomes. In sub-Saharan Africa, two major human schistosomes, namely Schistosoma mansoni and S. haematobium, often occur sympatrically and is responsible for almost 90% of the affected 290 million people worldwide. We have utilized a highly sensitive and specific assay by amplifying species-specific cell-free repeat DNA fragments by polymerase chain reaction to detect either single or dual schistosome infection from a single urine sample from a broad age group. In this study, we have tested filtered urine samples collected from 163 individuals aged 3–63 years, mostly children (median age 10), to evaluate the prevalence of single and dual infections for S. mansoni and S. haematobium in Tomefa community in the Greater Accra region of Ghana. 40–50 mL of urine was filtered through a 12.5 cm Whatman # 3 filter paper in the field. The filter papers were dried, packed individually in sealable plastic bags with a desiccant, and shipped to Marquette University, where DNA was isolated and PCR amplification was carried out with species-specific primers. Disease prevalence was found to be 46.6% for S. mansoni and 48.5% for S. haematobium. Most importantly, 23.3% of participants had dual infections. All of the samples were detected without any cross amplification. The data was evaluated for four age groups and infection rate was highest for the age group of 3–12 years, with more S. haematobium infections than S. mansoni infections. We found a high prevalence of both S. haematobium and S. mansoni infection and a significant proportion of dual infection for the Tomefa community, which in most cases would be missed by traditional parasitological examination of urine or stool. Our highly sensitive and specific approach for detecting underlying multiple schistosome infections is an effective means to detect low intensity infections and would enhance the effectiveness of surveillance and Mass Drug Administration control programs of schistosomiasis

Detection of Parasite-Specific DNA in Urine Sediment Obtained by Filtration Differentiates between Single and Mixed Infections of <i>Schistosoma mansoni</i> and <i>S. haematobium</i> from Endemic Areas in Ghana

<div><p>Differential diagnosis of <i>Schistosoma mansoni</i> and <i>S. haematobium</i>, which often occur sympatrically in Africa, requires both urine and stool and the procedures are low in sensitivity. The standard diagnostic tests, such as Kato-Katz (KK) for <i>S. mansoni</i> eggs and presence of haematuria for <i>S. haematobium</i> both lack sensitivity, produce false-negative results and show reduced accuracy with decreasing intensity of infection. The need for a single diagnostic test with high sensitivity and specificity for both parasites is important as many African countries are implementing Mass Drug Administration (MDA) following recommendations of the World Health Organization (WHO). Eighty-six samples of urine sediment obtained by filtration were collected from a group of 5–23 years old people from an endemic area of southern Ghana. DNA was extracted from the urine sediment on filter paper from which a species-specific repeat fragment was amplified by polymerase chain reaction (PCR) with specific primers for <i>S. mansoni</i> and for <i>S. haematobium</i>. Additionally, all participants were tested by KK (stool) and dipstick for haematuria. Diagnostic parameters for all three tests were analyzed statistically. Amplification of species-specific DNA by PCR showed much higher sensitivity (99%–100%) and specificity (100%) compared to KK and haematuria (sensitivity: 76% and 30% respectively) for both schistosome species. The same pattern was observed when the data were stratified for age group and sex specific analysis. In addition PCR amplification detected DNA from 11 individuals infected with both parasites who were negative by KK and haematuria. This approach of detecting parasite specific DNA from either or both species in a single urine specimen is a practical advantage that avoids the need for two specimens and is more effective than standard tests including those based on serology. This promises to improve the effectiveness of surveillance of MDA control programs of schistosomiasis.</p></div

Effectiveness of KK, haematuria and PCR as diagnostic tests to distinguish single and mixed schistosome infection from Ghana across different age groups and for both genders.

<p>This table tests the reproducibility of result between tests by segregating into groups based on age or sex.</p><p>*Positive  =  Number of positives by each test out of total number of samples had been analyzed.</p><p>**Negative  =  Number of negatives by each test out of total number of samples had been analyzed.</p

Detailed numerical distribution of single and mixed schistosome parasite infected people from Ghana diagnosed by KK, haematuria and PCR^*.

<p>* Important findings by PCR are highlighted. Areas not applicable remain blank.</p

Demographic and clinico-pathological parameters of participants.

*<p>Ultrasound Finding: Please see materials and methods for details.</p>**<p>57 subjects include 49 cases and 8 controls. No bladder damage was observed in controls by ultrasound examination.</p

Areas under the receiver operator characteristic curves (AUC-ROC) for the ability of <i>RASSF1A</i>, <i>TIMP3</i> and combination of <i>RASSF1A</i> and<i>TIMP3</i> to predict severe bladder damage associated with <i>S. haematobium</i> infection.

<p>The AUC- ROC of <i>RASSF1A</i>, <i>TIMP3</i> and combination of <i>RASSF1A</i>and<i>TIMP3</i> were 67.84%, 63.73% and 77.55% respectively.</p

Univariate and multivariate logistic regression analysis of <i>RASSF1A</i> and <i>TIMP3</i> genes.

<p>Univariate and multivariate logistic regression analysis of <i>RASSF1A</i> and <i>TIMP3</i> genes.</p

Promoter methylation status of <i>RASSF1A, TIMP3</i> and <i>MGMT</i> genes in the urine sediments of patients with <i>S. haematobium</i> infection.

*<p>non severe = all with grade 0, grade 1 or grade 2 bladder damage.</p>1<p>severe = all with grade 3 bladder damage.</p