scholarly journals Use of polymerase chain reaction in verification and differential diagnosis of babesiosis pathogens

2020 ◽  
Vol 11 (4) ◽  
pp. 563-567
Author(s):  
I. I. Torianyk ◽  
O. M. Tymchenko ◽  
M. O. Ostapets ◽  
N. A. Chygyrynska ◽  
S. I. Pokhyl ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Objective Assessment ◽  
Fatal Outcome ◽  
High Sensitivity ◽  
Blood Parasites ◽  
Chain Reaction ◽  
Negative Results ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Sensitivity Specificity

Today, Babesia is recognized as one of the most common blood parasites in the world, which in terms of the number of cases of invasion is second only to trypanosomes (the causative agent of African trypanosomiasis and Chagas’ disease). These microorganisms can cause parasitism in erythrocytes and hematopoietic organs. They cause an infectious process, the clinical course of which can vary from asymptomatic, subclinical, mild or moderate influenza-like forms – to severe progressive disease (fulminant form) with fatal outcome. Thus, the latter determines the significant burden of babesia for the leading branches of medicine, veterinary medicine and the economy as a whole. The presented work is devoted to the study of the prospects for verification of babesiosis causative agents by the polymerase chain reaction (PCR) method. Blood, erythrocyte suspension, homogenized tick-carriers of babesiosis, culture of Babesia spp. were used as research material (samples). In order to obtain an objective assessment, the PCR-diagnostics method was used in two formats – standard and multiplex (multi-primer). Multiple PCR testing of multiplex format using primers in model samples containing cells of different species of Babesia (B. microti, B. divergens, B. bovis, B. canis), allowed us to establish the level of reproducibility of the results of such studies, which ranged 94.6–96.4%, to determine the level of PCR sensitivity of the multiplex format for detection/identification of human pathogenic babesia (B. microti, B. divergens and B. venatorum). It is established that the advantages of the PCR-diagnostic method of babesiosis pathogens in the samples of the studied biomaterial were: speed of research (2–4 hours); high sensitivity, specificity, reproducibility of Babesia detection results, prospects of species identification, differentiation with apicomplex spores (Plasmodium falciparum, Toxoplasma). In view of the above, the PCR method is recommended for use in cases of persistent suspicion of babesiosis infection (in cases of negative results of microscopic/cytological studies, to identify asymptomatic, subclinical and chronic forms of babesiosis, verification of active invasion in seropositive individuals and for Babesia species and their differentiation).

scholarly journals Deteksi Plasmodium falciparum dengan menggunakan metode real-time polymerase chain reaction di daerah Likupang dan Bitung

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
David C. Tooy ◽  
Janno B. Bernadus ◽  
Angle Sorisi
Keyword(s):  
Polymerase Chain Reaction ◽  
Plasmodium Falciparum ◽  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Negative Results ◽  
Pcr Method ◽  
Polymerase Chain

Abstract: Malaria is one of the most important parasitic disease which is caused by Plasmodium spp. There are approximately 1,2 billion people in the world with high risk of getting malaria. Plasmodium falciparum (P. falciparum) is the cause of tropical malaria or falciparum malaria, and is responsible for most of the mortality rate. Currently, real-time polymerase chain reaction (RT-PCR) is being studied as an alterative of conventional malarian examination. Mangold et al reported that RT-PCR have 94.1% sensitivity and 100% specificity compared to microscopic examination in detecting P. falciparum. The aim of this research is to detect the presence of P. falciparum using RT-PCR in Likupang and Bitung region. This research were using descriptive design to find out the capability of real-time PCR method to detect P. falciparum in Likupang dan Bitung region. The researcher have examined 71 samples which are fulfill the research sample’s criteria. Postive results of P. falciparum found in 18 samples (25,3%) and negative results in 53 samples (74,6%) of total 71 samples with using RT-PCR. No positive results were found in samples from Likupang. There are positive result of P. falciparum in samples from Bitung. It is concluded that RT-PCR method can detect the presence of P. falciparum from the samples obtained from Likupang and Bitung based on the presence of its DNA. This detection efford is done by using 18S rRNA as target gene and ajust specific temperature on the RT-PCR instrument.Keywords: Plasmodium falciparum, Real-time Polymerase Chain Reaction (PCR), DetectionAbstrak: Malaria merupakan salah satu penyakit penting yang disebabkan oleh parasit Plasmodium spp. Kira-kira 1,2 miliar penduduk dunia memiliki risiko tinggi untuk mendapat malaria. Di Indonesia sendiri, terdapat 343.527 kasus terkonfirmasi dan 45 kematian karena malaria. Plasmodium falciparum (P. Falciparum) merupakan penyebab dari malaria tropika atau malaria falsiparum, dan bertanggung jawab atas sebagian besar angka mortalitas. Saat ini Real-Time Polymerase Chain Reaction (RT-PCR) telah banyak diteliti sebagai alternatif dari pemeriksaan malaria. Mangold dkk melaporkan bahwa real-time PCR memiliki nilai sensitivitas 94,1% dan nilai spesifisitas 100% terhadap pemeriksaan mikroskopis dalam mendeteksi P. falciparum. Penelitian bertujuan untuk mendeteksi P. falciparum dengan menggunakan RT-PCR di daerah Likupang dan Bitung. Penelitian ini menggunakan rancangan penelitian deskriptif untuk mengetahui kemampuan metode real-time PCR dalam mendeteksi P. falciparum di daerah Likupang dan Bitung. Tujuan penelitian ini ialah untuk mendeteksi keberadaan P. falciparum dengan menggunakan metode real-time PCR di daerah Likupang dan Bitung. Peneliti memeriksa 71 sampel darah yang memenuhi kriteria sampel penelitian. Hasil positif P. falciparum ditemukan pada 18 sampel (25,3 %) dan hasil negatif pada 53 sampel (74,6 %) dari total 71 sampel dengan menggunakan RT-PCR. Tidak ditemukannya hasil positif P. falciparum pada sampel dari Likupang. Ditemukan hasil positif P. falciparum pada sampel dari Bitung. Simpulan: Metode RT-PCR dapat mendeteksi P. falciparum berdasarkan keberadaan DNA-nya pada sampel yang diperoleh dari daerah Likupang dan Bitung. Deteksi ini berhasil dilakukan dengan menggunakan 18S rRNA sebagai gen target dan pengaturan suhu tertentu pada instrument RT-PCR.Kata kunci: P. falciparum, Real-time Polymerase Chain Reaction (PCR), Detection

scholarly journals Sensitivity of polymerase chain reaction for detection of known aliquots of Trypanosoma cruzi in the blood of mice: an in vitro study

2002 ◽  
Vol 35 (5) ◽  
pp. 487-490 ◽  
Author(s):  
Rozália F. Campos ◽  
Juracy B. Magalhães ◽  
Eliana A.G. Reis ◽  
Mitermayer G. Reis ◽  
Sonia G. Andrade
Keyword(s):  
Polymerase Chain Reaction ◽  
Trypanosoma Cruzi ◽  
In Vitro Study ◽  
High Sensitivity ◽  
Positive Control ◽  
Chain Reaction ◽  
Negative Results ◽  
Polymerase Chain ◽  
Positive Controls

To evaluate the sensitivity of polymerase chain reaction (PCR) to reveal known number of trypomastigote in the blood of mice, three separate experiments were done. First: To eight samples of 500mul of normal mice blood, one aliquot of 1, 2, 3, 4, 5, 10, and 50 trypomastigotes respectively, were added. Second and third: 10 aliquots with 1 and 10 with 2 trypomastigotes were added to samples of 500mul of normal mice blood. Positive control: 500mul of blood containing 100,000 trypomastigotes. For kDNA minicircles amplification by PCR the primers:S35 and S36 were used. PCR revealed products of 330 b.p in the positive controls. When only one sample with the aliquots of 1 or 2 trypomastigotes was examined, results were negative; results were positive with aliquots of 3 to 50 trypomastigotes. In the 2nd and 3rd experiments, 9/10 aliquots with one parasite and 9/10 with 2 trypomastigotes were positive revealing a high sensitivity of this reaction. In conclusion, the presence of one single parasite in 500mul of blood, is enough for a positive PCR. This method could be used as a complement to the various parasitological cure tests in treated mice, when low volumes of blood are individually examined.

scholarly journals Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material.

1996 ◽  
Vol 44 (10) ◽  
pp. 1205-1207 ◽  
Author(s):  
A Dakhama ◽  
V Macek ◽  
J C Hogg ◽  
R G Hegele
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Housekeeping Gene ◽  
Chain Reaction ◽  
Viral Genomes ◽  
Negative Results ◽  
Beta Actin ◽  
Target Nucleic Acid ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain

The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification of a housekeeping gene such as beta-actin. In the present study we used specific oligonucleotide primers previously designed to amplify both the genomic DNA and the mRNA transcript from paraffin-embedded tissue. These products have predicted sizes of 250 BP and 154 BP, respectively, but our results showed that PCR amplification only (without reverse transcription) unexpectedly generated the 154-BP product. Further investigation of the nature of this product demonstrated that it originated from the amplification of DNA, not RNA. We conclude that the 154-BP product generated by these primers cannot be exclusively considered as beta-actin RNA product and should not be used to assess successful extraction of RNA, to ascertain its integrity, or to normalize for the total amount of RNA assayed by RT-PCR from paraffin-embedded tissue.

scholarly journals UTILITY OF ANTIGEN DETECTION TEST AND POLYMERASE CHAIN REACTION IN THE DIFFERENTIATION OF TUBERCULOUS AND NON-TUBERCULOUS MYCOBACTERIA

2017 ◽  
Vol 10 (6) ◽  
pp. 289
Author(s):  
Yogita Singh ◽  
Raji Vasanth ◽  
Shrikala Baliga ◽  
Dhanashree B
Keyword(s):  
Polymerase Chain Reaction ◽  
False Negative ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Chain Reaction ◽  
College Hospital ◽  
Medical College ◽  
Negative Results ◽  
Polymerase Chain ◽  
Mycobacterium Spp

Objectives: Cultivation and identification of mycobacteria to species level remains difficult and time-consuming. Hence, easy and rapid diagnostic methods are necessary for the differentiation of Mycobacterium tuberculosis (MTB) from non-tuberculous mycobacteria (NTM). The present study aims to detect and differentiate MTB from NTM isolated from clinical samples by immunochromatographic test (ICT) and polymerase chain reaction (PCR). Methods: Over a period of 1 year, clinical samples (n=496) received from suspected cases of TB, at the Department of Microbiology, Kasturba Medical College Hospital, Mangalore were cultured to isolate Mycobacterium spp. Identification of all the isolates was done by conventional biochemical technique, ICT, and PCR. Results: Among the 496 samples processed, 49 (9.87%) were acid-fast bacilli smear positive and 59 (11.89%) samples showed the growth of Mycobacterium spp. Among these, 10 were rapid growers, 49 were slow-growing mycobacteria, out of which 30 were MTB as identified by conventional biochemical reaction. Out of 59 Mycobacterial isolates subjected to ICT for the detection of MPT 64 antigen, only 28 were identified as MTB. However, all the 30 isolates were correctly identified as MTB by PCR. Conclusion: Hence, PCR is essential for rapid differentiation of non-tuberculous Mycobacterium from MTB. False negative results seen with immunochromatographic MPT 64 antigen assay could be due to mutations within the mpt64 gene. Further studies are necessary to characterize these PCR-positive and immunochromatographic assay negative MTB isolates.

scholarly journals Molecular diagnosis and biochemical studies of tick-borne diseases (anaplasmosis and babesiosis) in Aberdeen Angus Cattle in New Valley, Egypt

2020 ◽  
Vol 13 (9) ◽  
pp. 1884-1891
Author(s):  
Nani Nasreldin ◽  
Rania M. Ewida ◽  
Hatem Hamdon ◽  
Yasser F. Elnaker
Keyword(s):  
Oxidative Stress ◽  
Polymerase Chain Reaction ◽  
Molecular Techniques ◽  
Babesia Bovis ◽  
Blood Parasites ◽  
Chain Reaction ◽  
Infected Female ◽  
Angus Cattle ◽  
Polymerase Chain ◽  
Female Cattle

Background and Aim: Anaplasmosis and babesiosis are tick-borne diseases that threaten livestock production with subsequent considerable economic losses. This study was conducted to diagnose Anaplasma and Babesia infection using molecular techniques in imported Aberdeen Angus cattle imported from Uruguay to El-Kharga Oasis in New Valley, Egypt, and to investigate the effects of disease on some serum biochemical and oxidative stress parameters. Materials and Methods: Blood samples were collected from 31 cattle, 21 diseased and ten apparently normal, of varying ages and sex. The blood was used for the preparation of blood smears, polymerase chain reaction assay, and separation of serum for biochemical investigation. The experimental production farm at the Faculty of Agriculture, New Valley University, was infested with ticks and variable clinical manifestations during the period from December 2017 to March 2018. One calf died of a suspected blood parasite infection. Results: The blood film examination revealed infection by blood parasites in 21 samples. Anaplasma marginale and Babesia bovis were identified in 12 and 14 samples, respectively. A total of 14 samples were examined by polymerase chain reaction (PCR) to make these identifications. Biochemical parameters showed significantly elevated serum alanine aminotransferase, aspartate aminotransferase, total bilirubin (T. Bil), and urea in blood from parasite-infected female cattle and male calves compared with controls. Increased serum total protein, globulin, and creatinine were recorded only in infected female cattle. The blood glucose level was significantly decreased in infected female cattle and male calves compared with controls. Furthermore, albumin and albumin/globulin ratio was significantly reduced in the infected female cattle. Oxidative stress profiles of infected animals showed a significant increase in serum nitric oxide and malondialdehyde, and both total antioxidant capacity and reduced glutathione (GSH) were significantly reduced in comparison with control animals. Conclusion: The incidence of A. marginale and B. bovis infection is high in imported Aberdeen Angus cattle in New Valley Province. PCR methods provide a short-term assessment of disease. An extensive epidemiological survey, employing serology together with molecular genetic methods, monitoring of abundance and distribution of tick vectors, availability of vaccination programs, and tracking of animal transport is also needed for control of blood parasites.

scholarly journals Diversity ofLeptospiraspp. in Rats and Environment from Urban Areas of Sarawak, Malaysia

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Chai Fung Pui ◽  
Lesley Maurice Bilung ◽  
Kasing Apun ◽  
Lela Su’ut
Keyword(s):  
Polymerase Chain Reaction ◽  
Water Samples ◽  
Urban Areas ◽  
Environmental Samples ◽  
Soil Samples ◽  
Chain Reaction ◽  
Prevalence Studies ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Soil And Water

Various prevalence studies onLeptospirain animals and humans, as well as environmental samples, had been conducted worldwide, including Malaysia. However, limited studies have been documented on the presence of pathogenic, intermediate, and saprophyticLeptospirain selected animals and environments. This study was therefore conducted to detectLeptospiraspp. in rats, soil, and water from urban areas of Sarawak using the polymerase chain reaction (PCR) method. A total of 107 rats, 292 soil samples, and 324 water samples were collected from April 2014 to February 2015. PathogenicLeptospirawas present in 5.6% (6/107) of rats, 11.6% (34/292) of soil samples, and 1.9% (6/324) of water samples. IntermediateLeptospirawas present in 2.7% (8/292) of soil samples and 1.9% (6/324) of water samples. SaprophyticLeptospirawas present in 10.3% (11/107) of rats, 1.4% (4/292) of soil samples, and 0.3% (1/324) of water samples. From this study, 76Leptospiraspp. were isolated. Based on DNA sequencing, the dominantLeptospiraspp. circulating in urban areas of Sarawak are pathogenicLeptospira noguchii, intermediateLeptospira wolffiiserovar Khorat, and saprophyticLeptospira meyeri, respectively. Overall, this study provided important surveillance data on the prevalence ofLeptospiraspp. from rats and the environment, with dominant local serovars in urban areas of Sarawak.

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