Nested PCR targeting intergenic spacer (IGS) in genotyping of Giardia duodenalis isolated from symptomatic and asymptomatic infected Egyptian school children

2016 ◽  
Vol 116 (2) ◽  
pp. 763-771 ◽  
Author(s):  
Eman M. Hussein ◽  
Ola A. Ismail ◽  
Amira B. Mokhtar ◽  
Samer E. Mohamed ◽  
Rania M. Saad
Keyword(s):  
School Children ◽  
Nested Pcr ◽  
Giardia Duodenalis ◽  
Intergenic Spacer ◽  
Pcr Targeting

scholarly journals Molecular Characterization of Giardia duodenalis in Children and Adults Sampled in Algeria

2020 ◽  
Vol 9 (1) ◽  
pp. 54
Author(s):  
Salem Belkessa ◽  
Daniel Thomas-Lopez ◽  
Karim Houali ◽  
Farida Ghalmi ◽  
Christen Rune Stensvold
Keyword(s):  
Real Time ◽  
Molecular Characterization ◽  
Real Time Pcr ◽  
Intestinal Parasites ◽  
Giardia Duodenalis ◽  
Specific Pcr ◽  
Stool Samples ◽  
Pcr Targeting ◽  
Assemblage B

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

scholarly journals Detection and partial genetic characterisation of novel avi- and siadenoviruses in racing and fancy pigeons (Columba livia domestica)

2016 ◽  
Vol 64 (4) ◽  
pp. 514-528 ◽  
Author(s):  
Mónika Z. Ballmann ◽  
Balázs Harrach
Keyword(s):  
Sequence Analysis ◽  
Dna Sequence ◽  
Dna Polymerase ◽  
Nested Pcr ◽  
Columba Livia ◽  
Hexon Gene ◽  
Polymerase Gene ◽  
Viral Dna ◽  
Phylogeny Analysis ◽  
Pcr Targeting

Up to now, only a single adenovirus (AdV) isolate seemingly specific for pigeons, hence named pigeon AdV-1 (PiAdV-1), has been characterised at DNA sequence level. In the present work, the prevalence and diversity of AdVs occurring in domestic pigeon were examined by a survey performed on randomly collected samples using a very efficient, consensus nested PCR targeting the viral DNA polymerase gene. The newly detected viruses were characterised by sequencing and phylogeny analysis. Amplification of additional genome fragments was attempted by the use of several other PCR methods aiming at the hexon gene. During a 4-year survey, samples from dead or live, healthy pigeons originating from 27 lofts were examined in Hungary. Almost 50% of the samples (48 out of 97) proved to be positive for AdV. Sequence analysis revealed the presence of four hitherto unknown pigeon AdV types. PiAdV-1 was also identified in one sample. Two novel viruses named PiAdV-2 and -3 were found to belong to the genus Aviadenovirus, and two other novel types (PiAdV-4 and -5) to the genus Siadenovirus. This is the first report on the occurrence of siadenoviruses in birds belonging to the order Columbiformes. Approximately two-thirds of the PiAdV-2 genome was sequenced and analysed.

scholarly journals Molecular Detection and Characterization of Borrelia garinii (Spirochaetales: Borreliaceae) in Ixodes nipponensis (Ixodida: Ixodidae) Parasitizing a Dog in Korea

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 289 ◽  
Author(s):  
Seung-Hun Lee ◽  
Youn-Kyoung Goo ◽  
Paul John L. Geraldino ◽  
Oh-Deog Kwon ◽  
Dongmi Kwak
Keyword(s):  
Molecular Detection ◽  
Single Gene ◽  
Protein A ◽  
Intergenic Spacer ◽  
Surface Protein ◽  
23S Rrna ◽  
Borrelia Garinii ◽  
Intergenic Spacer Region ◽  
Individual Level ◽  
Pcr Targeting

The present study aimed to detect and characterize Borrelia spp. in ticks attached to dogs in Korea. Overall, 562 ticks (276 pools) attached to dogs were collected and tested for Borrelia infection by PCR targeting the 5S-23S rRNA intergenic spacer region (rrf-rrl). One tick larva (pool level, 0.4%; individual level, 0.2%) was confirmed by sequencing Borrelia garinii, a zoonotic pathogen. For molecular characterization, the outer surface protein A (ospA) and flagellin genes were analyzed. Phylogenetic ospA analysis distinguished B. garinii from B. bavariensis, which has been recently identified as a novel Borrelia species. On the other hand, phylogenetic analysis showed that single gene analysis involving rrf-rrl or flagellin was not sufficient to differentiate B. garinii from B. bavariensis. In addition, the B. garinii-infected tick was identified as Ixodes nipponensis by sequencing according to mitochondrial 16S rRNA and the second transcribed spacer region. To our knowledge, this is the first study to report the molecular detection of B. garinii in I. nipponensis parasitizing a dog in Korea. Continuous monitoring of tick-borne pathogens in ticks attached to animals is required to avoid disease distribution and possible transmission to humans.

Prevalence of Salmonella typhi among Patients with Febrile Illness in Rural and Peri-Urban Populations of Vellore District, as Determined by Nested PCR Targeting the Flagellin Gene

2010 ◽  
Vol 14 (2) ◽  
pp. 107-112 ◽  
Author(s):  
Balaji Nandagopal ◽  
Sathish Sankar ◽  
Karthikeyan Lingesan ◽  
Kumarasekharan Chandrasekharan Appu ◽  
Baby Padmini ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Febrile Illness ◽  
Salmonella Typhi ◽  
Flagellin Gene ◽  
Urban Populations ◽  
Pcr Targeting

scholarly journals Comparison and diagnosis of Entamoeba in stool sample from rural community of Nepal

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Sandeep Thapa
Keyword(s):  
Stool Sample ◽  
Entamoeba Histolytica ◽  
Rural Community ◽  
Microscopic Examination ◽  
Nested Pcr ◽  
Health Knowledge ◽  
Intestinal Parasites ◽  
Intestinal Parasitosis ◽  
Microbiological Methods ◽  
Pcr Targeting

ObjectiveTo find out the prevalence of Entamoeba species in rural community of Nepal.The purpose of the study is to evaluate Nested PCR, microscopic examination and Elisa assay for detection and differentiation of Entamoeba species.IntroductionNepal being a developing country has many health problems, which escalate in intensity at different times of the year or in epidemic form.Amebiasis is one of the infectious diseases that is highly seen in rural area of Nepal caused by Entamoeba species ((1, 2). Recent reports show that open defecation, drinking untreated water, unsanitary habits and lack of basic health knowledge cause higher mortality and morbidity in our country.E. histolytica is an anaerobic pathogenic parasitic. However, E. dispar and E.moshkovskii exits as non-pathogenic. Likewise, E. histolytica, E. dispar and E. moshkovskii are morphologically identical but genetically distinct species (3).MethodsA total of 270 faecal sample were collected from south eastern terai region of Nepal after the informed consent form. The samples were processed by direct wet smear and formalin ethyl acetate concentration technique (4). Eventually, microscopic examination were performed for the detection of Entamoeba species along with other intestinal parasites. Furthermore, enzyme immunoassay were executed to detect antigens of E. histolytica through ELISA. Additionally, microscopically positive samples for Entamoeba species cysts were further characterized using a Nested- PCR targeting 16S-like ribosomal RNA gene (5). The PCR generate amplicons which was subjected to 2% agarose gels electrophoresis and visualized under UV transilluminator.Results8.52% of the total collected samples were microscopically positive for Entamoeba cysts either singly or in combination with other intestinal parasites. Likewise, among 270 stool sample, viral diarrheal was most significant form of diarrhoea found in 76.67% of patients. Among different organisms, As. Lumbricoids and E. histolytica, G. lambia and H. nana were identified in most of the patients accounting for 11.11%, 8.52%, 2.59% and 1.11% respectively. However, Lumbricoids, G. lambia, Tenia solium and E. histolytica were present in an individual patient while two patient was found with both As. Lumbricoids and G. lambia. Among several symptoms, diarrhoea seems to be the common symptoms infecting all of the patients which is followed by fever and vomiting which accounts for 55.1 % and 46.2% correspondingly. Whereas, nausea appears to be the least common symptoms infecting only 14.4% of patients.Subsequently, 56 cases were PCR positive, 51 cases were ELISA positive whereas 47 were found to be positive by microscopy.ConclusionsMolecular techniques are indeed promising tools for epidemiological studies, particularly in discriminating the pathogenic from the non-pathogenic species of the Entamoeba species. This study reports a new nested multiplex PCR strategy for detection and differentiation of E. histolytica, E. dispar and E. moshkovskii which is highly rapid, specific and sensitive which is useful for proper diagnosis, immunological assay and drug testing.References1. Chaudhary M, Maharjan M. ( 2014). Association of Anaemia with Parasitic Infection in Pregnant Women Attending Antenatal Clinic at Koshi Zonal Hospital. Neplalese Journal of Zoology, 2(1):1-7.2. Tandukar S, Ansari S, Adhikari N, Shrestha A, Gautam J, Sharma B, et al (2013). Intestinal parasitosis in school children of Lalitpur district of Nepal. BMC research notes, 6(1):449.3. Clark C, Espinosa Cantellano M, Bhattacharya A. Entamoeba histolytica: an overview of the biology of the organism (2013). Amebiasis: World Scientific, 1-45.4. Dhanabal J, Selvadoss PP, Muthuswamy K(2014). Comparative study of the prevalence of intestinal parasites in low socioeconomic areas from South Chennai, India. Journal of parasitology research.5. Van den Bossche, D., Cnops, L., Verschueren, J., & Van Esbroeck, M. (2015). Comparison of four rapid diagnostic tests, ELISA, microscopy and PCR for the detection of Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in feces. Journal of microbiological methods, 110, 78-84.

scholarly journals Prevalence and Molecular Characterization of Giardia duodenalis in Calves in Turkey

2017 ◽  
Vol 45 (1) ◽  
pp. 6
Author(s):  
Mehmet Gultekin ◽  
Kerem Ural ◽  
Nuram Aysul ◽  
Adnam Ayan ◽  
Canberk Balikci ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Nested Pcr ◽  
Giardia Duodenalis ◽  
Pcr Assay ◽  
Giardia Infection ◽  
Western Turkey ◽  
Aegean Region ◽  
Animal Populations ◽  
Faecal Samples

Background: Giardia duodenalis (G. duodenalis) is an ubiquitous, flagellated intestinal protozoan with major public health significance worldwide. Limited data are available on the epidemiology of G. duodenalis in dairy cattle from Turkey. Determining the zoonotic potential of the Giardia infection requires molecular characterization. The aim of the present study was to investigate the prevalence and to molecularly characterize G. duodenalis in calves less than three months of age in Aydin, Aegean region of Turkey.Materials, Methods & Results: The study was conducted on different dairy farms in the south-western part of the Turkey, Aegean Region, Aydin. A total of 198 Holstein Friesian calves less than three months of age, of both sexes were enrolled into the study. Faecal samples from each calf were collected manually from the rectum using a disposable latex glove. The consistency of collected samples was recorded as diarrhoeic or non-diarrhoeic.  Diagnosis of G. duodenalis infection was made microscopically by detection of cysts in the faecal samples. One hundred and sixteen (58.5%) of the 198 faecal samples were diarrheic. Giardia cysts were found in 27 (23.28%) of the diarrheic samples and in 8 (9.76%) of nondiarrheic samples (P < 0.05). The overall prevalence of giardiosis in calves was determined as 17.67%. The prevalence of Giardia genotypes was identified by DNA sequence analysis of the beta-giardin gene for every PCR positive sample. The beta-giardin nested PCR assay was revealed assemblage A and sub-genotype A3 was detected in all of 35 samples (100%).Discussion: The highest prevalence of Giardia infection in calves is reported at the age between 1 and 6 months, and the prevalence shows decreased rate from the age of 6 months. The present study was conducted in Aydin, a province of south-western Turkey in the Aegean Region, and the overall prevalence from a total of 198 dairy calves was 17.67%. The prevalence rate in calves with diarrhoea was higher and reached up to 23.28%, whereas it was 9.76% in non-diarrhoeic calves. A prevalence study with molecular characterization of G. duodenalis isolates in cattle has not yet been reported from Turkey. Molecular studies have shown that mostly assemblage E predominates in cattle, but recent studies denoted that assemblage A is increasingly being detected and might be more widespread than expected before. In the present study, Giardia positive samples identified with a beta-giardin nested PCR assay. The sub-genotype A3 was identified in all samples. The same sub-genotype was identified in human and dog samples from different countries. Furthermore, sub-genotype A3 was found in humans and dogs from Turkey. In this context, results of the present study suggested an important role of calves as potential reservoirs of human infections in Turkey. In conclusion, epidemiological data revealed that G. duodenalis infection is frequent in calves with diarrhoea in Aydin, Turkey. The presence of the potentially zoonotic sub-genotype A3 and high prevalence of Giardia infection in diarrheic calves indicated the importance of treatment and necessary preventative measures. Further studies in human and animal populations living in this region are warranted regarding the zoonotic epidemiology of Giardia duodenalis.

scholarly journals Molecular survey ofBartonella henselaeandBartonella clarridgeiaein pet cats across Japan by species-specific nested-PCR

2017 ◽  
Vol 145 (13) ◽  
pp. 2694-2700 ◽  
Author(s):  
S. SATO ◽  
H. KABEYA ◽  
A. NEGISHI ◽  
H. TSUJIMOTO ◽  
K. NISHIGAKI ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Bartonella Henselae ◽  
23S Rrna ◽  
Cat Scratch Disease ◽  
Internal Transcribed Spacer Region ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Species Specific ◽  
Pcr Targeting ◽  
Eastern Japan

SUMMARYCats are known to be the main reservoir forBartonella henselaeandBartonella clarridgeiae, which are the agents of ‘cat-scratch disease’ in humans. In the present study, we investigated the prevalence of the twoBartonellaspecies on 1754 cat bloods collected from all prefectures in Japan during 2007–2008 by a nested-polymerase chain reaction (PCR) targeting the 16S–23S rRNA internal transcribed spacer region. Overall,BartonellaDNA was detected in 4·6% (80/1754) of the cats examined. The nested-PCR showed that 48·8% (39/80) of the positive cats were infected withB. henselaemono-infection, 33·8% (27/80) withB. clarridgeiaemono-infection and 17·5% (14/80) were infected with both species. The prevalence (5·9%; 65/1103) ofBartonellainfection in the western part of Japan was significantly higher than that (2·3%; 15/651) of eastern Japan (P< 0·001). Statistical analysis of the cats examined suggested a significant association betweenBartonellainfection and FeLV infection (OR = 1·9; 95% CI = 1·1–3·4), but not with FIV infection (OR = 1·6; 95% CI = 1·0–2·6).

Molecular detection of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in school children at the Thai-Myanmar border

2021 ◽  
Author(s):  
Chantira Sutthikornchai ◽  
Supaluk Popruk ◽  
Aongart Mahittikorn ◽  
Dumrongkiet Arthan ◽  
Ngamphol Soonthornworasiri ◽  
...  
Keyword(s):  
School Children ◽  
Molecular Detection ◽  
Giardia Duodenalis ◽  
Enterocytozoon Bieneusi ◽  
Cryptosporidium Spp

scholarly journals Molecular Epidemiology of Bovine Babesiosis in Punjab, Pakistan

2021 ◽  
Vol 49 ◽  
Author(s):  
Asif Masih ◽  
Azhar Rafique ◽  
Farhat Jabeen ◽  
Shabana Naz
Keyword(s):  
Risk Factors ◽  
Nested Pcr ◽  
Dairy Industry ◽  
Summer Season ◽  
Babesia Bovis ◽  
Economic Losses ◽  
Molecular Evidence ◽  
Conventional Pcr ◽  
Associated Risk Factors ◽  
Pcr Targeting

Background: Babesiosis is endemic in Pakistan and is one of the most important bovine diseases that causes huge economic losses and high mortality in young animals. This disease is transmitted by a protozoan parasite babesia which belongs to genus Babesia (Apicomplexa: Piroplasmida: Babesiidae). This disease is very much prevalent in summers followed by rainy season because humid environment is favorable for the growth of these parasites. An epidemiological and molecular study was conducted to unveil the prevalence and associated risk factors of Babesia bigemina (B. bigemina) and Babesia bovis (B. bovis) in selected districts i.e., Faisalabad, Toba Tek Singh and Jhang of Punjab, Pakistan.Materials, Methods & Results: A total of 518 (Cattle = 360, Buffalo = 158) blood samples were collected. The samples were analyzed by polymerase chain reaction (PCR) and nested PCR (n-PCR) targeting apocytochrome b-genes (CYTb). Chi-square test for univariate analysis was used to analyze the data. The overall prevalence in summer based upon microscopic analysis was 20.55% (37/180) and 13.92% (11/79) in cattle and buffaloes respectively and in winter was 8.80% (16/180), 5.06% (4/79)) in cattle and buffaloes respectively. The samples were further analyzed through conventional PCR (c-PCR) and nested PCR (nPCR). The overall results of conventional PCR in summer showed that 72 cows and buffaloes were infected with babesiosis. The conventional PCR based results of summer showed that prevalence of babesiosis was 29.44% (53/180) in cows and 24.05% (19/79) buffaloes. The results of cPCR during the winter season showed that 12.77% (23/180) and 13.92% (11/79) buffaloes were positive for babesiosis. The overall results of conventional PCR in winter showed that 34/259 cows and buffaloes were infected with babesiosis. On the other hand, the nested PCR results of summer season showed that the prevalence of babesiosis in cows was 32.22% (58/180) and 29.11% (23/79) in buffaloes. In total, 81 cows and buffaloes were infected with babesiosis during summer season. The nPCR results of winter showed that 15% (27/180) cows and 20.25% (16/79) buffaloes were infected with babesiosis. In total, 43 cows and buffaloes were infected with babesiosis. The results have shown that sensitivity of n-PCR is more as compared to conventional PCR. This study is the first molecular evidence of B. bigemina and B. bovis and its associated risk factors in Punjab province, Pakistan.Discussion: Dairy sector in Pakistan is one of the fastest growing sectors. Despite of remarkable growth, dairy industry is facing many problems one of them is tick borne diseases (TBDs). TBDs are more prevalent in tropical and subtropical areas of the world and leads to huge economic losses to dairy industry in terms of decreased milk, meat and wool production. Babesiosis is characterized by increased fever, decreased production, poor quality wool, anemia, hemoglobinuria, paleness of mucous membrane. The risk factors analysis of summer and winter data revealed that, adult animals were more prone to babesiosis (24.00%) [P = 0.032] and (8.50%) [P = 0.048]. In both seasons (summer and winter), females were more infected with babesiosis (20.19% and 8.17%) [P = 0.049 and P =0.021] as compared to males, high prevalence in females was might be due to that females were reared for longer period of time. Babesiosis was more occurred in non-cemented floor system (26.01% and 13.51%) [P = 0.028 and P = 0.044] in summer and winter, respectively. Disease was found more prevalent in closed housing system in summer and winter (27.27% and 10.93%) [P = 0.043 and P = 0.034] as compared to open housing. Weak animals were more infected with babesiosis (30.84%) [P = 0.045] and (12.80%) [P = 0.042] in summer and winter, as compared to healthy ones. The animals with high tick infestations were more suffered with babesia infection (25.49% and 13.34%) [P = 0.036 and P = 0.003] in both seasons as compared to less tick burden. Keywords: apocytochrome gene, babesiosis, bovine, nPCR, PCR, season.

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