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Molecular characterization of entamoeba histolytica tRNA genes
No full textM.Sc. (Microbiology)Department of MicrobiologyBACKGROUND: Entamoeba histolytica is a eukaryotic protozoan parasite responsible for the disease called amoebiasis. Amoebiasis is a major cause of morbidity and mortality in the developing world. It is well established that about 500 million people are infected worldwide, resulting in up to 100,000 deaths annually. It is not completely understood why some individuals once infected with E. histolytica, develop clinical amoebiasis while others remain asymptomatic. Very few studies have been conducted in order to determine the potential role of the parasite genomic features on the outcome of the infection. No studies have been done in South Africa to show how parasite genotypes play a role in determining the outcome of infection. Therefore, the present study determined the molecular characteristics of tRNA genes of E. histolytica in relation to the occurrence of diarrhea.
METHOD: In this study, patients were recruited from rural pnmary health care clinics in Giyani, Limpopo Province and private clinics in Pretoria, Gauteng Province. The participants were supplied with a consent form and their information was kept confidential. Diarrheal and non-diarrheal stool samples were collected. All the stool samples were observed under a light microscope for the presence of E. histolytica cysts and trophozoites. The Techlab E. histolytica II kit was used to detect the antigen against E. histolytica. Genomic DNA was extracted from 78 stool samples that were positive by ELISA using ZYMO RESEARCH fecal DNA mini Prep kit from inqaba biotech. A multiplex PCR protocol was used for the identification of E. histolytica. Specific primers for the different loci (NK, RR, AL, DA and STGA_D) of the tRNA genes of E. histolytica were used for genotyping. In this study, 15 stool samples from the 42 positive
samples identified by ELISA and confirmed by PCR were amplified for the NK locus using the tRNA specific primers NK-3 and NK-5. The genotyping as well as the data were analyzed using the Statistical Package for Social Sciences (SPSS for WINDOWS version 18.0) program in order to determine the potential implications of E. histolytica infection.
RESULTS: A total of 774 stool samples were collected and it was found that, out of 774 stool samples examined by wet mount microscopically 16.7% were infected with E. histolytica cysts and trophozoites. The TechLab ELISA based antigen detection kit specific only for E. histolytica in stool samples revealed that 10.1% were positive for E. histolytica. The highest prevalence of
E. histolytica was found in Pretoria with 10.5%, as compared to Giyani which was 5.4% and the difference was not statistically significant (X2= 1.491; P= 0.222). Entamoeba histolytica was more common in males (12%) than in females (8.4%), but the difference was not statistically significant (X2= 2.653; P= 0.103). Most of the participants who were infected were aged between 26-45 years with 21.2% followed by those who were in the age group 1-25 years with 16.8%. The least infected were of the age group 49-90 years with 8.2%. However, the difference was not statistically significant (X2= 3.341; P= 0.188). According to samples consistency, the highest prevalence was found in watery stool samples with 13.6%, followed by soft with 11.1% and the least was formed with 5.9%, but the difference was not statistically significant (X2= 5.781; P=
Forty-two samples showed positive for E. histolytica small-subunit rRNA gene. Nine (9) different banding patterns were obtained for NK locus. The ratio of the profile and the number of samples tested was therefore three profiles for every 5 samples. Two hundred base pairs was the most common band that occurred five times in different samples as compared to 150bp and
250bp that occurred twice. Other bands observed included 120bp, 300bp, 350bp, 500bp, 600bp and 750bp which occurred only once each. Out of the 42 positive samples identified by ELISA and confirmed by PCR the RR locus was amplified in 30 samples using the tRNA specific primers RR-3 and RR-5. This gave a success rate of 71%. The product sizes of 150, 200, 250, 300, 400,450, 500, 550, 600 and 750bp were obtained. It was observed that 150bp and 250bp occurred only once. A total of 16 profiles were obtained giving a ratio of 0.53.
Out of the 42 positive samples identified by ELISA and confirmed by PCR, the AL locus was amplified in 25 samples using the tRNA specific primers AL-3 and AL-5. This gave an amplification success of 59.5%. The product size of 150, 180 200, 220, 300, 350, 400 450, 500, 550, 600 and 1000bp were obtained. A total of 15 profiles were obtained for a ratio of 0.6. The band size of 200bp was seen in most of the samples, followed by 150, 180 220, 300, 350, 400,450, 500, 550, 600 and 1000bp. It was observed tr.at l 50bp and 550bp occurred only once.
Thirteen (31%) stool samples from the 42 positive samples identified by ELISA and confirmed by PCR were amplified for the DA locus using the tRNA specific primers DA-3 and DA-5. The product sizes of 150, 200, 280 300, 500 and 1200bp were obtained. One hundred and fifty base pair was seen in most of the samples, followed by 300 and 500bp bands. It was observed that bands of 200bp, 280bp, and l 200bp occurred only once.
ine (21%) stool samples from the 42 positive samples identified by ELISA and confirmed by PCR were amplified for the sTG -D locus using the tFNA specific primers STGA_D -3 and sTc - D -5. The product size of 150 180, 200, 220, 300, 350, 400,450, 500, 550, 600 and 1000bp were obtained. From the 9 samples that amplified for the sTGA_D locus, bands of 150, 200, 280 and
400bp was seen in most of the samples. It was observed that 100, 240, and 300bp occurred only once. All samples were from Pretoria.
CONCLUSION: The present study indicates that infection caused by E. histolytica was prevalent in diarrheal samples obtained from Pretoria. Entamoeba histolytica infection was more prevalent in males than in females, and in the age group of 26- 45 years. The possible cause of infection in the stools of patients in this study could possibly be due to various factors such as drinking water from unprotected source or by direct contact with infected animals. According to our results, icroscopy is a simple method and it should be combined with other methods such as ELI A and PCR for identification of the species to avoid false and/or insufficient diagnosis and treatment applications.
Loci K, RR, L, DA and STGA_D of t:ie tRNA genes identified in this study show promise as surrogate markers for prediction of infection outcome of Entamoeba histolytica. The findings of this study therefore suggest that further studies are needed to evaluate the prevalence heterogeneity and combination of virulence-related genes in E. histolytica infection as well as their association with diarrhea and non-diarrhea
Molecular detection and identification of Cryptosporidium species isolated from human and animal sources in Limpopo and Gauteng Provinces
No full textMSc (Microbiology)Department of MicrobiologyBackground: Diarrheal diseases constitute an important problem among children but also
among HIV positive patients particularly in developing countries such as South Africa.
Cryptosporidium infect humans and has been shown to be an important cause of infection
among different types of animals. Because of its small size, Cryptosporidium can easily go
through the water purification system and can easily become a cause of an epidemic.
Previous studies have shown that Cryptosporidium is an important cause of diarrhea in
Limpopo Province. However, very few studies have been conducted on the genetic diversity
of these organisms in the region. Therefore, the aim of this study was to detect and identify
the genetic diversity of Cryptosporidium species from humans and animals in Giyani situated
in the northern part of South Africa and Pretoria situated in the central part of the country.
Methodology: A total of 560 samples were collected from human and animals and were all
screened by microscopy using modified Ziehl-Neelsen staining technique. All the samples
were tested by Enzyme-Linked Immunosorbent Assay (ELISA) using the Cryptosporidium II
kits from Techlab, Virginia, USA. Positive samples from microscopy and ELISA were
examined by different PCR protocols including conventional PCR for amplification of
Cryptosporidium oocyst wall protein (COWP) region; Real-time PCR employing SYBR
Green detection format for amplification of 18S rRNA region; Real-time PCR employing
Hydrolysis probes detection format for amplification of SSU rRNA region; Real-time PCR
specific for amplification of C. hominis region and C. parvum region. Positive samples from
real-time PCR that gave clear bands on gel electrophoresis were sent for sequencing. The
sequences were analysed using Staden package software to edit the nucleotides, Bioedit and
MEGA6 software were used to align sequences and draw phylogenetic trees. The SPSS
software was used for statistical analysis.
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Results: The overall prevalence of Cryptosporidium as detected by ELISA method from the
samples collected from humans was 41.2% (239/580). The prevalence was higher from the
rural area 73.0% (159/218) compared to the urban area 22.1% (80/362) and the difference
was statistically significant (χ2 = 145.1; p = 0.0001). Due to the limited amount of samples,
only 134 ELISA-positive samples were tested using real-time PCR. Of these samples, 35.8%
(48/134) tested positive. Of 48 real-time positive samples 25 were successfully sequenced
and two different species (C. hominis and C. muris) were identified. Of all the sequences
obtained, one (4.0%) was C. muris and 20 (80%) were C. hominis isolated from rural area,
whereas 16.0% (4/25) were also C. hominis isolated from samples obtained from urban area.
Cryptosporidium was not associated with diarrhea in the present study.
A total of 85 samples were collected from animals (52 from cattle and 33 from goats) and of
these 4 (4.7%) were positive by microscopy and ELISA. All these samples were non
diarrheal. Conventional PCR also detected a similar number. Of these 4 positive samples, 1
was from a male goat, while the 3 others were obtained from female adult goats.
Real-time PCR detected 56.5% (48/85) positive samples. Only 12 of the 85 animal samples
were diarrheal and of these 4 were positive for Cryptosporidium. The prevalence of
Cryptosporidium infection was higher 68.4% (13/19) in male animals compared to female
animals 53.0% (35/66). The prevalence rates in cattle and goats were 55.8% (29/52) and
60.6% (20/33) respectively.
Of 48 real-time positive samples from animals, 12 (25.0%) were successfully sequenced and
two species (C. parvum and C. andersoni) were identified. Of these 6 were from cattle and
the other 6 were from goats. Out of the 12 samples 10 (83%) were C. parvum while 2 (17%)
were C. andersoni. Of the two C. andersoni, one was from a goat and one was from a cow.
Of the 10 C. parvum, 5 were from goats and 5 were from cattle.
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In conclusion, microscopy remains the low sensitive tool for the detection of
Cryptosporidium while real time PCR appeared to be far much more sensitive by detecting
more samples than all the three other methods combined. Closer to the real time PCR was
ELISA that detected also more samples compared to conventional PCR and microscopy.
The present study identified C. muris from humans’ samples in our area for the first time.
However, C. hominis remains the dominant species that infects humans in our area.
Cryptosporidium species was mostly found in samples from asymptomatic individuals. In
animals, C. parvum was the most commonly isolated organism while C. andersoni was
identified in our region for the first time as well and occurred in both goats and cattle.
Populations in the affected areas need to be made aware of the infections so that care should
be taken to avoid the spread of infection in water sources or in immunocompromised
individuals
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Venda Study Area
No full textThis is a Google Earth file with additional geographic information for the randomized controlled study on the effect of ceramic water filter and silver-impregnated porous ceramic water treatment (MadiDrop) on child health.
This is a part of a study reported in a manuscript by: Joshua N. Edokpayi, Elizabeth T. Rogawski, David M. Kahler, Courtney Hill, Catherine Reynolds, Emanuel Nyathi, James A. Smith, John O. Odiyo, Amidou Samie, Pascal Bessong, Rebecca Dillingham.
The authors acknowledge the tireless work of the community field workers who installed interventions and collected all of the survey data. The authors also acknowledge A. Gaylord, N. Khuliso, S. Mammburu, K. McCain and E. Stinger, who performed much of the water quality analysis and T. Singh, who supported the laboratory analysis for inorganic materials
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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