scholarly journals First detection and molecular identification of the zoonoticAnaplasma caprain deer in France

2018 ◽  
Author(s):  
Maggy Jouglin ◽  
Barbara Blanc ◽  
Nathalie de la Cotte ◽  
Katia Ortiz ◽  
Laurence Malandrin
Keyword(s):  
Nested Pcr ◽  
Red Deer ◽  
Diagnostic Methods ◽  
Sequencing Analysis ◽  
Wild Fauna ◽  
Tick Transmission ◽  
Pcr Targeting ◽  
Swamp Deer ◽  
Anaplasma Capra ◽  
Rrna Sequences

AbstractCervids are known to be reservoir of zoonotic tick-transmitted bacteria. The aim of this study was to perform a survey in a wild fauna reserve to characterizeAnaplasmaspecies carried by captive red deer and swamp deer. Blood from 59 red deer and 7 swamp deer was collected and analyzed over a period of two years. A semi-nested PCR that targets the23SrRNA was performed to detect and characteriseAnaplasmaspp. and determine zoonotic species presence.Anaplasma phagocytophilumwas identified in 14/59 deer (23.7%) but not in swamp deer. Few sequences could not be assigned to any particular species based on the23SrRNA sequences. Nested PCR targeting16SrRNA,gltAandgroELgenes and sequencing analysis detected a recently reported zoonotic species,Anaplasma caprain red deer as well as in swamp deer. This is the first reporting of the tick-borne zoonotic bacteriumA. caprain France, a species otherwise described only in China and Japan, in goats, sheep, deer and japanese serows. Even if this bacterium may have been introduced in the Park with infected imported animals, its local epidemiological cycle through tick transmission seems possible as locally born deer were found infected. Diagnostic methods, especially molecular ones, should take into account the potential infection of animals and humans with this species.

scholarly journals Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods

2018 ◽  
Vol 27 (1) ◽  
pp. 60-65 ◽  
Author(s):  
Vinícius da Silva Camargo ◽  
Bruna Nicoleti Santana ◽  
Elis Domingos Ferrari ◽  
Alex Akira Nakamura ◽  
Walter Bertequini Nagata ◽  
...  
Keyword(s):  
Real Time ◽  
Malachite Green ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
18S Rrna ◽  
Diagnostic Methods ◽  
Negative Staining ◽  
Cryptosporidium Spp ◽  
Genotype Iii ◽  
Pcr Targeting

Abstract This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.

scholarly journals Leishmaniasis Caused by Leishmania major on the Glans Pe-nis: A Case Report

2019 ◽  
Author(s):  
Mahdi MOSAYEBI ◽  
Mehdi MOHEBALI ◽  
Aliasghar FARAZI ◽  
Mohammad Reza SHIRZADI ◽  
Davood AKHLAGHI ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Leishmania Major ◽  
Leishmania Species ◽  
Diagnostic Methods ◽  
Glans Penis ◽  
Sequencing Analysis ◽  
Serological Tests ◽  
Liver Size ◽  
Abdominal Ultrasonography ◽  
Leishmanin Skin Test

Cutaneous leishmaniasis (CL) is one of the prevalent parasitic diseases in Iran principally caused by two species, Leishmania major and L. tropica. Here, we present a rare case of a congenital form of CL around the glans penis from the central part of Iran in 2017. A 24-yr-old male patient from the central part of Iran presented with biennial ulceration of the glans penis. Diagnostic methods included physical and preclinical examination, microscopic observation, leishmanin skin test (LST), and serological tests including direct agglutination test (DAT). Nested PCR and sequencing analysis were used on the positive smears for confirmation of CL and Leishmania species identification. The preclinical results were normal, and no anti-Leishmania antibodies were detected in the peripheral blood of the patient using DAT. In abdominal ultrasonography, the spleen and liver size were normal. LST was positive (≥5 mm) after 72 h, and a few amastigote forms of Leishmania sp. were demonstrated under light microscopy. L. major was confirmed using nested PCR and sequencing analysis. The patient responded to oral administration of miltefosine (2.5 mg/kg/d) for 28 days. To the best of our knowledge, genital CL due to L. major has not been previously reported from Iran.

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.

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 Cryptosporidium spp. during chemotherapy: a cross-sectional study of 94 patients with malignant solid tumor

2021 ◽  
Vol 41 (5) ◽  
pp. 293-298
Author(s):  
Mehmet Karabey ◽  
Hüseyin Can ◽  
Tülay Öncü Öner ◽  
Mert Döşkaya ◽  
Sedef Erkunt Alak ◽  
...  
Keyword(s):  
Sample Size ◽  
Real Time ◽  
Solid Tumors ◽  
Real Time Pcr ◽  
Tertiary Care ◽  
Diagnostic Methods ◽  
Cross Sectional ◽  
Cryptosporidium Spp ◽  
Stool Samples ◽  
Pcr Targeting

BACKGROUND: Cryptosporidium spp . is a protozoan parasite that infects many vertebrate animals, including humans. Since Cryptosporidium spp . can cause chronic life-threatening diarrhea and severe malabsorption in immunocompromised patients, we investigated the prevalence of this parasite among patients undergoing chemotherapy for malignant solid tumors. OBJECTIVE: Investigate the prevalence of Cryptosporidium spp . in stool samples. DESIGN: Cross-sectional. SETTING: Tertiary care. PATIENTS AND METHODS: Stool samples were collected from adult patients with malignant solid tumors receiving chemotherapy and diarrhea. Cryptosporidium spp . prevalence was determined using Ziehl–Neelsen staining, ELISA, and real-time PCR targeting of the COWP gene. MAIN OUTCOME MEASURE: The prevalence of Cryptosporidium spp . in patients undergoing chemotherapy for malignant solid tumors. SAMPLE SIZE: 94 RESULTS: The prevalence was 2.1% (2/94), 5.3% (5/94), and 5.3% (5/94) as detected by Ziehl–Neelsen staining, real-time PCR and ELISA, respectively. The prevalence reached 8.5% (8/94) using all results obtained from the three methods. Among eight positive stool samples, four were positive by at least two different methods (Ziehl–Neelsen staining-ELISA or ELISA-real-time PCR) whereas the remaining four were positive by either ELISA or real-time PCR. CONCLUSION: These findings show the risk of cryptosporidiosis in cancer patients and the necessity to use at least two diagnostic methods during the diagnosis of cryptosporidiosis to reach more accurate and trustworthy results. LIMITATIONS: Further studies with a larger sample size are recommended. CONFLICT OF INTEREST: None.

Epidemiology of Epizootic Lymphangitis of cart horses in Northern Ethiopia Using Conventional diagnostic Methods and Nested Polymerase Chain Reaction

2019 ◽  
Author(s):  
Birhanu Hadush Abera ◽  
Molla Michaelay ◽  
Habtamu Taddele ◽  
Nigus Abebe ◽  
Abrha Tesfay ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Clinical Examination ◽  
Roc Curve ◽  
Nested Pcr ◽  
Control Measure ◽  
Diagnostic Methods ◽  
Northern Ethiopia ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain

Abstract Background: Epizootic lymphangitis (EL), caused by Histoplasma capsulatum variety farciminosum (HCF) is a contagious chronic disease of equines characterized by development of nodular lesions in the lymph nodes, lymphatic vessels and skin. This disease is the most important diseases of equines in Ethiopia causing a significant economic loss, particularly cart pulling equines. Todate there is no sound diagnostic nor control measure implemented in the country. Furthermore, there is a shortage of data on the epidemiology of the disease in different regions of the country including northern Ethiopia. This study was conducted to investigate the epidemiology of EL in northern Ethiopia using the conventional methods and the nested polymerase chain reaction (PCR). Methods: A total of 191 cart-horses were enrolled and used as sources of pus and blood samples. The blood was used for the extraction of the DNA of HCF from buffy coat for nested PCR while the pus samples were cultured on Sabourauds Dextrose Agar for isolation. Statistical Package for Social Sciences (SPSS) version 21 was used for data analysis by applying logistic regression, receiver operating characteristic (ROC) curve and Cohen’s kappa coefficient test. In addition, the level of agreement between the clinical examination and the nested PCR was evaluated. Results: Infection with HCF was confirmed in 44% (84/191) of the horses using nested PCR. Subclinical infection was observed in 18.18% (22/121) of the apparently healthy horses. Considering nested PCR as a gold standard, the sensitivity and specificity of the clinical examination were 74% and 95%, respectively while the area under the ROC curve (AUR) was 0.83 (95% confidence interval, 0.77, 0.896). Moreover, a moderate (k=0.675) agreement was observed between the nested PCR and clinical examination.Conclusions: The findings of the present study showed the wide spread occurrence of EL in northern Ethiopia and the advantage of the nested PCR in detecting of the infection of HCF even before the clinical symptoms are apparent.

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

An evaluation of the usefulness of invasive and non-invasive methods used to diagnose Helicobacter spp. infections in dogs

2017 ◽  
Vol 20 (3) ◽  
pp. 491-499 ◽  
Author(s):  
M. Jankowski ◽  
J. Spużak ◽  
K. Kubiak ◽  
K. Glińska-Suchocka ◽  
M. Biernat
Keyword(s):  
Nested Pcr ◽  
Gastric Biopsy ◽  
Diagnostic Methods ◽  
Rapid Urease Test ◽  
Pcr Assay ◽  
Non Invasive ◽  
Biopsy Specimens ◽  
Urease Test ◽  
Invasive Method ◽  
Pcr Method

AbstractThe aim of this study was to assess the suitability of invasive and non-invasive methods used to diagnose Helicobacter spp. in the stomachs of dogs. The study was carried out on 30 dogs of both sexes and different breeds, between one and 15 years old. A histopathologic examination, a microbiological culture, a rapid urease test, a direct bacteriological preparation and a nested PCR assay were carried out. Gastric Helicobacter spp. was identified in gastric biopsy specimens from 16 (53.3%) dogs using direct bacteriological preparation, in four (13.3%) dogs based on a culture, in 23 (76.6%) dogs using the rapid urease test and in 21 (70,0%) dogs based on a histopathological assessment of the biopsy specimens. The nested PCR of the gastric biopsy specimens revealed gastric Helicobacter spp. in all the dogs (100%). A saliva PCR assay revealed gastric Helicobacter spp. in 23 (76.6%) dogs, while stool PCR revealed the bacterium in seven (23.3%) dogs. We found that invasive methods were more accurate than non-invasive methods in detecting a Helicobacter spp. infection in dogs. In addition, the nested PCR method used to evaluate the gastric mucosal biopsy specimens was the most accurate test for detecting Helicobacter spp. It was further found that the PCR-based saliva assay was the best non-invasive method for detecting Helicobacter spp. However, taking into consideration that most of the diagnostic methods used to detect this bacterium have drawbacks, at least two diagnostic methods should be used to detect Helicobacter spp. as is done in human medicine.

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.

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