Clinical and epidemiological use of nested PCR targeting the repetitive element IS 1111 associated with the transposase gene from Coxiella burnetii

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.

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

Parasitology Research ◽

10.1007/s00436-016-5347-0 ◽

2016 ◽

Vol 116 (2) ◽

pp. 763-771 ◽

Cited By ~ 7

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

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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

Molecular Diagnosis & Therapy ◽

10.1007/bf03256360 ◽

2010 ◽

Vol 14 (2) ◽

pp. 107-112 ◽

Cited By ~ 6

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

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

Online Journal of Public Health Informatics ◽

10.5210/ojphi.v11i1.9853 ◽

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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Molecular detection of Coxiella burnetii infection in aborted samples of domestic ruminants in Iran

PLoS ONE ◽

10.1371/journal.pone.0250116 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0250116

Author(s):

Ashraf Mohabati Mobarez ◽

Mohammad Khalili ◽

Ehsan Mostafavi ◽

Saber Esmaeili

Keyword(s):

Coxiella Burnetii ◽

Real Time Pcr ◽

Molecular Detection ◽

Q Fever ◽

Domestic Animal ◽

Domestic Ruminant ◽

Domestic Ruminants ◽

Pcr Targeting ◽

Different Parts

Background Coxiella burnetii is the causative agent of Q fever which is a highly infectious zoonotic disease. C. burnetii has become one of the most important causes of abortion in livestock, which can lead to widespread abortions in these animals. There are very limited studies on the prevalence of C. burnetii infection in cases of animal abortion in Iran. The aim of this study was to investigate the occurrence of C. burnetii in ruminant abortion samples in Iran. Methods Abortion samples from cattle, sheep and goats were collected from different parts of Iran and were tested using Real-time PCR targeting the IS1111 element of C. burnetii. Results In this study, 36 samples (24.7%) of the 146 collected samples were positive for C. burnetii. The prevalence of C. burnetii was 21.3% (20 of 94 samples) in sheep samples. Also, 10 of 46 cattle samples (21.7%) were positive. All six goat abortion samples were positive for C. burnetii. Conclusions The findings of the study demonstrate that C. burnetii plays an important role in domestic ruminant abortions in Iran, suggesting that more attention should be paid to the role of C. burnetii in domestic animal abortions by veterinary organizations. The risk of transmitting the infection to humans due to abortion of animals should also be considered.

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

Epidemiology and Infection ◽

10.1017/s0950268817001601 ◽

2017 ◽

Vol 145 (13) ◽

pp. 2694-2700 ◽

Cited By ~ 3

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).

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Molecular Epidemiology of Bovine Babesiosis in Punjab, Pakistan

Acta Scientiae Veterinariae ◽

10.22456/1679-9216.111565 ◽

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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Assessment of Microscopic detection of Malaria with Nested Polymerase Chain Reaction in War-torn Federally Administered Tribal Areas of Pakistan

10.21203/rs.3.rs-148296/v1 ◽

2021 ◽

Author(s):

Muhammad Faisal Nadeem ◽

Aamer Ali Khattak ◽

Adnan Yaqoob ◽

Usman Ayub Awan ◽

Nadia Zeeshan

Keyword(s):

Diagnostic Accuracy ◽

Sensitivity And Specificity ◽

Mixed Infection ◽

Microscopic Examination ◽

Nested Pcr ◽

Venous Blood ◽

Pcr Analysis ◽

Microscopic Detection ◽

Tribal Areas ◽

Pcr Targeting

Abstract Background: Diagnostic accuracy of malaria is critical for early treatment, control, and elimination of malaria, especially in war-affected malaria endemic areas. Microscopic detection of Plasmodium species has been the gold standard in remote malaria-endemic regions. However, the diagnostic accuracy is still questioned, especially in discriminating mixed and submicroscopic parasitic levels. This study was designed to evaluate the diagnostic performance of microscopic examination against nested PCR analysis in war-torn malaria-endemic Federally Administered Tribal Areas (FATA) of Pakistan. Methods: Venous blood samples were collected from symptomatic patients for microscopic examination and nested PCR analysis from January 2016 - December 2016 from five Agencies (Bajaur, Mohmand, Khyber, Orakzai and Kurram Agency) and four Frontier Regions (Peshawar, Kohat, Bannu, and Dera Ismail Khan Frontier Region) of FATA. Malaria-positive isolates were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes) for speciation. Results: Among enrolled participants, 762 were found positive for malaria parasite on microscopic examination of the blood film. P. vivax was found in 623, P. falciparum in 132 and 7 were diagnosed with mixed infection (P. vivax and P. falciparum coinfection). Nested PCR detected Plasmodium infection in 679 samples (523 P. vivax, 121 P. falciparum, and 35 mixed infections). Compared with microscopy, the sensitivity of nested PCR was 98.94%, and specificity was 98.27%, while the sensitivity and specificity of slide microscopy 89.34% and 87.99% respectively. Conclusion: The conventional microscopy method has low sensitivity to detect mixed infection as compared to nested PCR. High sensitivity and specificity observed in nested PCR makes this molecular tool a useful technique for monitoring, controlling, and eliminating malaria-endemic regions.

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Endocardite infecciosa por coxiella burnetti em válvulas cardíacas humanas congeladas em um hospital de referência em Brasília após sua detecção: continuação de uma linha de pesquisa

Programa de Iniciação Científica - PIC/UniCEUB - Relatórios de Pesquisa ◽

10.5102/pic.n0.2019.7612 ◽

2021 ◽

Author(s):

Pedro Lemgruber Xavier Mattoso Pavie ◽

Camila De Carvalho Gallo Pereira ◽

Fabíola Fernandes dos Santos Castro

Keyword(s):

Coxiella Burnetii ◽

Nested Pcr ◽

Distrito Federal ◽

Triton X

Coxiella burnetii é uma bactéria de morfologia cocobacilo, gram negativa, estritamente intracelular, do gênero Rickettsia causadora de zoonoses em mamíferos. Está envolvida na patogênese da febre Q e, na maioria das vezes, desencadeia um quadro agudo que tende a ser subclínico (60% dos casos) ou autolimitado. Sabe-se que a febre Q está associada a 5% das ocorrências desta afecção cardíaca, como também está entre as principais causadoras de endocardite com cultura bacteriana negativa. O diagnóstico da endocardite passa por cultura de sangue, excisão de tecido da válvula cardíaca ou dos êmbolos, PCR e sorologia. Para se confirmar o diagnóstico é necessário que haja a detecção microbiana por cultura ou PCR com um perfil sorológico correspondente e, ainda, ausência de indícios de infecção. O objetivo desse trabalho foi verificar a prevalência de endocardites por Coxiella burnetii dentre endocardites com culturas negativas em válvulas cardíacas humanas congeladas em um hospital de referência em Brasília. Inicialmente, foram selecionadas novas 25 amostras de valvas cardíacas humanas, além das 20 anteriormente processadas, que se encontram congeladas a 20º C negativos, acondicionadas em frascos individuais estéreis, no Instituto de Cardiologia do Distrito Federal-ICDF, nas quais se utilizou metodologia com princípios moleculares de detecção desenvolvida no PIC 2018/2019. O material genético é extraído a partir de aproximadamente 200 mg de tecido originário de valvas cardíacas que são colocadas em tubos plásticos de 1,5 mL e macerados mecanicamente com pistilo de vidro durante 2 minutos. O tampão de extração utilizado é 10 mM Tris-HCl pH 8,0, 1 mM EDTA pH 8,0 e 0,3% Triton X-100 sendo posteriormente usados fenol/ clorofórmio/álcool isoamílico (25:24:1) no processo de extração do DNA. Em seguida, realiza-se nested-PCR com consequente amplificação do material genético. A extração de DNA produz, em média, 40 ng/ul de DNA total. Esses DNA`s são então diluídos e usados nas reações de nested-PCR. A primeira reação de PCR gera fragmentos de tamanho esperado de 485 pb. Submetendo-se as amostras ao segundo ciclo de PCR deve-se obter fragmentos de tamanho esperado de 260 pb. Das 20 amostras de DNA testadas (incluídas as mesmas do PIC 2018-2019), 7 amostras de válvulas cardíacas foram identificadas como positivas para a presença de DNA da bactéria Coxiella burnetti. As demais amostras não resultaram em amplificação, sendo consideradas como negativas quanto à presença do DNA dessa bactéria. Em conclusão, a suspeita dos pesquisadores se confirmou pelos resultados obtidos gerando esperança com a expectativa de resultados futuros nas demais amostras congeladas disponíveis. No segundo semestre de 2019, as 20 amostras incialmente preparadas, contando as do PIC 2018-2019, foram analisadas, os materiais solicitados e novas bibliografias buscadas. Em 2020, não foi possível o preparo de novas valvas, que estão congeladas, e nem a realização das pesquisas, já que que os laboratórios do UniCEUB permanecem sem acesso e o funcionamento do laboratório da EMBRAPA está restrito e sobrecarregado com outras demandas, devido ao período paralisado pelo isolamento social. Infelizmente, não foi possível concluir a pesquisa da maneira esperada, mesmo havendo os materiais comprados e as valvas disponíveis.

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