Characterization of the Plasmodium falciparum sarcoplasmic/endoplasmic reticulum Ca2+-ATPase gene in samples from Equatorial Guinea before implementation of artemisinin-based combination therapy

Plasmodium falciparum resistance to the primary drugs used for treatment of malaria has become the main obstacle to malaria control. Artemisinin combination therapies are the current treatment strategy, and it has been suggested that resistance to artemisinin derivatives may be related to mutations in the Plasmodium falciparum sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase ortholog of the mammalian sarco-endoplasmic reticulum Ca(2+) ATPase gene, known as the pfatp6 gene. Thus, the purpose of this study was to determine the prevalence of single-nucleotide polymorphisms (SNPs) in pfatp6. The presence of different SNPs was detected by polymerase chain reaction amplification of the pfatp6 gene, and then sequencing to identify all possible alleles of the gene. A total of 20 SNPs were detected, including eight SNPs that have not been previously described: K481R in Malabo; R801H on Annobon Island; and the synonymous SNPs a141t, c1788t, a2211g, t2739g, a2760c, and g2836a. The genotypic profile of pfatp6 in samples from Equatorial Guinea, may be a useful epidemiologic tool for monitoring local efficacy of artemisinin combination therapies.This study was supported by the Spanish Agency for International Development Cooperation and the Collaborative Research Network in Tropical Diseases.S

Evaluation of five diagnostic methods for Strongyloides stercoralis infection in Amhara National Regional State, northwest Ethiopia

Background: Strongyloides stercoralis is an intestinal parasite that can cause chronic infection, hyperinfection and/or a dissemination syndrome in humans. The use of techniques targeting ova fails to detect S. stercoralis, as only larvae of the parasite are excreted in faeces. Due to the absence of "Gold" standard diagnostic method for S. stercoralis, there is a paucity of reported data worldwide. Objective: This study aimed to evaluate the performance of diagnostic methods of S. stercoralis infection by taking the composite reference as a "Gold" standard. Methods: A cross-sectional study was conducted among 844 schoolchildren in Amhara Region, Ethiopia, from April to December 2019. Stool samples were collected and processed with formol-ether concentration technique (FECT), spontaneous tube sedimentation technique (STST), Baermann concentration technique (BCT), agar plate culture (APC) and real-time polymerase chain reaction (RT-PCR). Sensitivity, specificity, positive predictive value, and negative predictive value of each diagnostic method were computed against the composite reference. The agreements of diagnostic methods were evaluated by Kappa value at 95% CI. Results: The composite detection rate of S. stercoralis by the five diagnostic methods was 39.0% (329/844). The detection rate of the parasite from stool samples by FECT, STST, BCT, APC and RT-PCR was 2.0% (17/844), 4.0% (34/844), 10.2% (86/844), 10.9% (92/844) and 28.8% (243/844), respectively. The highest detection rate (37.8%; 319/844) of S. stercoralis was recorded by a combination of BCT, APC, and RT-PCR followed by a combination of STST, BCT, APC and RT-PCR (37.3%; 315/844). The sensitivity of FECT, STST, BCT, APC and RT-PCR against the composite reference was 5.2%, 10.3%, 26.4%, 28.0% and 73.9%, respectively. The diagnostic agreements of RT-PCR, APC, BCT, STST and FECT with the composite reference in detection of S. stercoralis were substantial (0.775), fair (0.321), fair (0.305), slight (0.123), and slight (0.062), respectively. Conclusion: RT-PCR detected the highest number of S. stercoralis infections. A combination of RT-PCR with APC and/or BCT better detected S. stercoralis from stool samples compared to other combinations or single diagnostic methods. Therefore, RT-PCR and combination of RT-PCR with APC and/or BCT diagnostic methods should be advocated for detection of S. stercoralis infection.The fund obtained from Bahir Dar University and Mundo Sano Foundation was used for data collection and laboratory detection purpose.N

High prevalence of Strongyloides stercoralis in school-aged children in a rural highland of north-western Ethiopia: the role of intensive diagnostic work-up

Background: Soil-transmitted helminthiases (hookworms, Ascaris lumbricoides and Trichuris trichiura) are extremely prevalent in school-aged children living in poor sanitary conditions. Recent epidemiological data suggest that Strongyloides stercoralis is highly unreported. However, accurate data are essential for conducting interventions aimed at introducing control and elimination programmes. Methods: We conducted a cross-sectional survey of 396 randomly selected school-aged children in Amhara region in rural area in north-western Ethiopia, to assess the prevalence of S. stercoralis and other intestinal helminths. We examined stools using three techniques: conventional stool concentration; and two S. stercoralis-specific methods, i.e. the Baermann technique and polymerase chain reaction. The diagnostic accuracy of these three methods was then compared. Results: There was an overall prevalence of helminths of 77.5%, with distribution differing according to school setting. Soil-transmitted helminths were recorded in 69.2%. Prevalence of S. stercoralis and hookworm infection was 20.7 and 54.5%, respectively, and co-infection was detected in 16.3% of cases. Schistosoma mansoni had a prevalence of 15.7%. Prevalence of S. stercoralis was shown 3.5% by the conventional method, 12.1% by the Baermann method, and 13.4% by PCR, which thus proved to be the most sensitive. Conclusions: Our results suggest that S. stercoralis could be overlooked and neglected in Ethiopia, if studies of soil-transmitted helminths rely on conventional diagnostic techniques alone. A combination of molecular and stool microscopy techniques yields a significantly higher prevalence. In view of the fact that current control policies for triggering drug administration are based on parasite prevalence levels, a comprehensive diagnostic approach should instead be applied to ensure comprehensive control of helminth infections.This study was funded by the Mundo Sano Foundation and the Network of Tropical Diseases Research Center (Red de Investigación cooperative de Enfermedades Tropicales-RICET)S

Epidemiology of intestinal helminthiases in a rural community of Ethiopia: Is it time to expand control programs to include Strongyloides stercoralis and the entire community?

Soil transmitted helminths are highly prevalent worldwide. Globally, approximately 1.5 billion people are infected with Ascaris lumbricoides, Trichuris trichiura or hookworm. Endemic countries carry out periodic mass treatment of at-risk populations with albendazole or mebendazole as a control measure. Most prevalence studies have focused on school aged children and therefore control programs are implemented at school level, not at community level. In this study, the prevalence of intestinal helminths, including Strongyloides stercoralis, was examined using a comprehensive laboratory approach in a community in north-western Ethiopia. A cross-sectional survey was conducted on 792 individuals ≥5 years old in randomly selected houses in a rural district. Stools were examined using three techniques: a formol-ether concentration, the Baermann technique and a real time polymerase chain reaction test (these last two specific for S. stercoralis). Statistical analyses were performed between two large age groups, children (≤14 years old) and adults (≥15 years old). The prevalence of helminths was 91.3%; (95% CI: 89.3-93.3%). Hookworm was the most prevalent, 78.7% (95% CI 75.6-81.4%), followed by S. stercoralis 55.7% (95% CI 52.2-59.1%). Co-infection with both was detected in 45.4% (95% CI 42.0-49.0%) of the participants. The mean age of hookworm-infected individuals was significantly higher than non-infected ones (p = 0.003). Also, S. stercoralis infection was significantly associated with age, being more prevalent in adults (p = 0.002). This is the highest prevalence of S. stercoralis detected in Ethiopia so far. Our results highlight the need of searching specifically for infection by this parasite since it usually goes unnoticed if helminth studies rely only on conventional diagnostic techniques, i.e. Kato-Katz. Moreover, the focus of these programs on children undermines the actual prevalence of hookworm. The adult population acts as a reservoir for both hookworm and S. stercoralis and this fact may negatively impact the current control programs in Ethiopia which only target treatment of school aged children. This reservoir, together with a lack of adequate water, sanitation and hygiene, increases the probability of re-infection in children. Finally, the high prevalence of S. stercoralis found calls for a comprehensive diagnostic approach in endemic areas in addition to a revision of control measures that is, adding ivermectin to current albendazole/mebendazole, since it is the drug of choice for S. stercoralis.This study was funded by the Mundo Sano Foundation and the Spanish Network on Tropical Diseases Research (Red de Investigación cooperativa de Enfermedades Tropicales-RICET RD12/0018/0001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Prevalence and Genetic Diversity of <i>Giardia duodenalis</i> and <i>Cryptosporidium</i> spp. among School Children in a Rural Area of the Amhara Region, North-West Ethiopia

<div><p>Backgroud</p><p><i>Giardia duodenalis</i> and <i>Cryptosporidium</i> spp. are enteric protozoan causing gastrointestinal illness in humans and animals. Giardiasis and cryptosporidiosis are not formally considered as neglected tropical diseases, but belong to the group of poverty-related infectious diseases that impair the development and socio-economic potential of infected individuals in developing countries.</p><p>Methods</p><p>We report here the prevalence and genetic diversity of <i>G</i>. <i>duodenalis</i> and <i>Cryptosporidium</i> spp. in children attending rural primary schools in the Bahir Dar district of the Amhara Region, Ethiopia. Stool samples were collected from 393 children and analysed by molecular methods. <i>G</i>. <i>duodenalis</i> was detected by real-time PCR, and the assemblages and sub-assemblages were determined by multilocus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of the parasite. Detection and identification of <i>Cryptosporidium</i> species was carried out by sequencing of a partial fragment of the small-subunit ribosomal RNA gene.</p><p>Principal Findings</p><p>The PCR-based prevalences of <i>G</i>. <i>duodenalis</i> and <i>Cryptosporidium</i> spp. were 55.0% (216/393) and 4.6% (18/393), respectively. A total of 78 <i>G</i>. <i>duodenalis</i> isolates were successfully characterized, revealing the presence of sub-assemblages AII (10.3%), BIII (28.2%), and BIV (32.0%). Discordant typing results AII/AIII and BIII/BIV were identified in 7.7% and 15.4% of the isolates, respectively. An additional five (6.4%) isolates were assigned to assemblage B. No mixed infections of assemblages A+B were found. Extensive genetic variation at the nucleotide level was observed within assemblage B (but no within assemblage A), resulting in the identification of a large number of sub-types. <i>Cryptosporidium</i> diversity was demonstrated by the occurrence of <i>C</i>. <i>hominis</i>, <i>C</i>. <i>parvum</i>, and <i>C</i>. <i>viatorum</i> in the population under study.</p><p>Conclusions</p><p>Our data suggest an epidemiological scenario with an elevated transmission intensity of a wide range of <i>G</i>. <i>duodenalis</i> genetic variants. Importantly, the elevated degree of genetic diversity observed within assemblage B is consistent with the occurrence of intra-assemblage recombination in <i>G</i>. <i>duodenalis</i>.</p></div

Median real-time PCR cycle threshold (Ct) values from <i>Giardia duodenali</i>s positive samples by age group in children aged 6–15 years in the Amhara Region, Ethiopia.

<p>Statistical significance (P < 0.01) is indicated by asterisks.</p

Diversity and frequency of single-nucleotide polymorphisms displayed by <i>Giardia duodenalis</i> sub-assemblage BIV isolates at the glutamate dehydrogenase locus (partial sequence between positions 80 to 467) identified in the present study.

<p>Novel sub-types were shown underlined. Transversion mutations were highlighted in bold. Point mutations inducing amino-acid substitutions were marked as superscript indicating the amino-acid change.</p

Diversity and frequency of single-nucleotide polymorphisms displayed by <i>Giardia duodenalis</i> assemblage B isolates at the beta giardin locus (partial sequence between positions 108 to 573) identified in the present study.

<p>Novel sub-types were shown underlined. Point mutations inducing amino-acid substitutions were marked as superscript indicating the amino-acid change.</p

Evolutionary relationships among assemblages of <i>G</i>. <i>duodenalis</i> at the <i>BG</i> locus inferred by a Neighbor-Joining analysis of the nucleotide sequence covering a 487-bp region (positions 108 to 573 of GenBank accession number AY072727) of the gene.

<p>The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 iterations) is indicated next to the branches. Bootstrap values lower than 50% were not displayed. The evolutionary distances were computed using the Kimura 2-parameter method. The rate variation among sites was modelled with a gamma distribution (shape parameter = 2). Filled circles represent B sequences from this study, whereas open circles indicate B sequences previously reported in human isolates from Ethiopia (see ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159992#pone.0159992.ref022" target="_blank">22</a>]).No outgroup taxa was used as beta-giardin is a <i>Giardia</i>-specific structural protein.</p

Sub-assemblage frequencies of <i>Giardia duodenalis</i> in children attending rural schools in the Bahir Dar district, Amhara Region, Ethiopia, 2013.

<p>Sub-assemblage frequencies of <i>Giardia duodenalis</i> in children attending rural schools in the Bahir Dar district, Amhara Region, Ethiopia, 2013.</p