scholarly journals Enhanced detection rate of typhoid fever among clinically suspected patients in a tertiary referral hospital in Dhaka, Bangladesh using nested polymerase chain reaction technology

2016 ◽  
Vol 41 (3) ◽  
pp. 138-143
Author(s):  
Shafinaz Khan ◽  
Ruhul Amin Mia ◽  
Sumaiya Khatun
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Culture ◽  
Typhoid Fever ◽  
Nested Pcr ◽  
Salmonella Typhi ◽  
Chain Reaction ◽  
Tertiary Referral Hospital ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Pcr Targeting

A nested polymerase chain reaction (PCR) specific for Salmonella enterica subspecies enteric serovar Typhi was used for the detection of the pathogen in blood. This study was done during the period of March 2013 to February 2014. A total of 80 clinically suspected cases of typhoid fever were included in the study. Blood was collected from all participating individuals. Nested PCR targeting the flagellin gene (fliC) of Salmonella Typhi & blood culture were done for each of the cases. The positivity rate of PCR & blood culture was 70%& 20% respectively. The positivity rate of PCR was significantly higher than blood culture (P< 0.05). With the nested PCR, S. Typhi DNAs were detected from blood specimens of 67.2% (43/64) patients among the suspected typhoid fever cases on the basis of clinical features but with negative cultures. We conclude that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases in an endemic country like Bangladesh.

scholarly journals Comparison of results obtained by widal agglutination test & polymerase chain reaction among clinically suspected typhoid fever cases

2015 ◽  
Vol 30 (2) ◽  
pp. 46-50
Author(s):  
Shafinaz Khan ◽  
Md Ruhul Amin Miah ◽  
Shammin Haque ◽  
Chowdhury Rafia Naheen
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Culture ◽  
Typhoid Fever ◽  
False Negative ◽  
Salmonella Typhi ◽  
Widal Test ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Positive Results ◽  
Prior Antibiotic Treatment

The diagnosis of typhoid fever currently depends on isolation of Salmonella Typhi from blood. The sensitivity of blood culture is very low due to prior antibiotic treatment which is a common practice in Bangladesh. The sensitivity of blood culture also decreases at later stage of the disease. Widal test is the most utilized test in Bangladesh next to blood culture because it is inexpensive, less invasive. But the result of the test is controversial due to false negative & false positive results in some cases.  In this study, a recently introduced polymerase chain reaction-based technique (which has 100% specificity for S. Typhi) was compared with widal test among 80 clinically suspected typhoid fever cases.  Among 80 cases, the respective figures of positivity for PCR & widal test were 70% & 43.75% respectively.  It can be concluded that PCR based technique is more sensitive & much superior to widal for diagnosis of typhoid fever. DOI: http://dx.doi.org/10.3329/bjpp.v30i2.22683 Bangladesh J Physiol Pharmacol 2014; 30(2): 46-50

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.

scholarly journals In SituReverse Transcriptase-Nested Polymerase Chain Reaction to Identify Intracellular Nucleic Acids without the Necessity of Dnase Pretreatment and Hybridisation

2001 ◽  
Vol 22 (3) ◽  
pp. 151-158 ◽  
Author(s):  
Mario Menschikowski ◽  
Margot Vogel ◽  
Rolf Eckey ◽  
Gerd Dinnebier ◽  
Werner Jaross
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acids ◽  
Nested Pcr ◽  
In Situ Hybridisation ◽  
Target Sequence ◽  
Chain Reaction ◽  
Multiple Control ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain

In the present study a protocol of in situ reverse transcriptase‐nested polymerase chain reaction (in situ RT‐nested PCR) was examined based on the following modifications. (i) To exclude false positive signals caused by “DNA repair mechanisms” and “endogenous priming”, a two‐step PCR was applied after reverse transcription. The first step was performed in the presence of extrinsic primers and unlabeled nucleotides with a maximum of PCR cycles possible without destroying the cell morphology. The second step consisted of only one annealing/elongation reaction, the target sequence marked by addition of digoxigenin‐labeled nucleotides and intrinsic primers. (ii) In order to prevent amplifications of genomic DNA nested primer pairs were applied crossing intron sequences. (iii) To minimize the diffusion of PCR products in cells, the extrinsic primers were extended with complementary 5′‐tails. This approach results in the generation of high molecular weight concatamers during PCR cycles. By applying this protocol, immunostainings specific for phospholipase A2 of type IIA mRNA were exclusively detectable in the cytoplasm of HepG2 hepatoma cells, which were used as a model system, whereas the nuclei were unstained. Multiple control experiments yielded completely negative results. These data suggest that the in situ RT‐nested PCR, which in comparison to the method of in situ RT‐PCR‐in situ‐hybridisation is simpler and less time‐consuming, can be used as an alternative approach to identify intracellular nucleic acids.

Application of Nested Polymerase Chain Reaction to Detection of Salmonella in Poultry Environment

2002 ◽  
Vol 65 (8) ◽  
pp. 1227-1232 ◽  
Author(s):  
TONGRUI LIU ◽  
KAREN LILJEBJELKE ◽  
ELIZABETH BARTLETT ◽  
CHARLES HOFACRE ◽  
SUSAN SANCHEZ ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nested Pcr ◽  
Virulence Gene ◽  
Pcr Primers ◽  
Processing Plant ◽  
Chain Reaction ◽  
Chi Square ◽  
Nested Polymerase Chain Reaction ◽  
Secondary Enrichment ◽  
Polymerase Chain

Isolation of Salmonella from environmental and processing-plant poultry samples requires the sampling of large numbers of areas within the poultry house or plant. Subsequently, the required number of samples necessitates a large volume of work for a microbiology laboratory, especially when the protocol requires the inclusion of a delayed secondary enrichment for the isolation of Salmonella. This study examined the use of the polymerase chain reaction (PCR) to identify those secondary enrichments containing Salmonella. The unique Salmonella virulence gene invA was chosen as the target for the development of a nested PCR because of its uniform distribution among Salmonella serotypes. The use of nested PCR primers increased the sensitivity of detection 100-fold, resulting in the detection of as few as four cells. There was a strong, statistically significant positive correlation between PCR and culture results as determined by chi-square (P &lt; 0.001) and kappa (k = 0.915; excellent agreement) tests. Using PCR to screen primary enrichments for presumptive Salmonella contamination, we improved our efficiency at isolating Salmonella upon secondary enrichment by 20%, and no false negatives were observed. This method will not only validate the use of secondary enrichment procedures but also reduce costs and manpower required for the surveillance of Salmonella.

scholarly journals Epidemiology of epizootic lymphangitis of carthorses in northern Ethiopia using conventional diagnostic methods and nested polymerase chain reaction

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Birhanu Hadush ◽  
Molla Michaelay ◽  
Habtamu Taddele Menghistu ◽  
Nigus Abebe ◽  
Abreha Tesfaye Genzebu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Clinical Examination ◽  
Nested Pcr ◽  
Control Measure ◽  
Genetic Material ◽  
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. It is one of the most important diseases of equines in Ethiopia, causing significant economic loss, particularly in the livelihood of carthorse owners. To date there is neither effective 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 this country. The aim of this study was to investigate epidemiology of EL in northern Ethiopia, using the conventional methods as well as nested polymerase chain reaction (PCR). Results The presence of HCF genetic material was confirmed in 44% (84/191) of the carthorses. Subclinical infection was observed in 18.2% (22/121) of the apparently healthy carthorses. Considering the nested PCR as a gold standard, sensitivity and specificity of clinical examination were 74% and 92.5%, 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 observed between the nested PCR and clinical examination. Conclusions This study demonstrated widespread occurrence of EL in northern Ethiopia, and the advantage of the nested PCR in detecting infection of HCF, even before the clinical symptoms became apparent.

scholarly journals Performance of a Multiplex Nested Polymerase Chain Reaction in Detecting 7 Pathogens Containing DNA in Their Genomes Associated With Congenital Infections

2019 ◽  
Vol 144 (1) ◽  
pp. 99-106
Author(s):  
Lidia Yamamoto ◽  
Antonio G. Amorim Filho ◽  
Joelma A. Queiroz ◽  
Mario H. B. de Carvalho ◽  
Jonatas C. Rodrigues ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nested Pcr ◽  
Detection Rate ◽  
Simultaneous Detection ◽  
Immunoglobulin M ◽  
Fisher Exact Test ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Congenital Infections ◽  
Polymerase Chain

Context.— Infections are the leading cause of perinatal and infant mortality in low-income and low-resource countries, which have a higher prevalence of infections. Definitive diagnosis of congenital and perinatal infections is largely dependent upon the results of laboratory tests. Objective.— To develop a multiplex nested polymerase chain reaction (PCR) technique for the simultaneous detection of 7 pathogens containing DNA in their genomes in suspected cases of congenital infection. Design.— Eligible participants were pregnant women with positive immunoglobulin M antibodies raised to one of the pathogens in the prenatal serologic screening, associated or not with fetal ultrasound abnormalities or positive fetal serology. Neonates whose mothers did not attend prenatal care were included when they presented with symptomatology and laboratory parameters suggestive of infection. The detection rate of the multiplex nested PCR was compared with maternal, fetal, and neonatal serology, as well as placental immunohistochemistry and noncommercial amplifications. Results.— Of 161 suspected cases, the multiplex nested PCR detected 60 (37.3%), whereas the tests available in hospital laboratories detected 13 of 60 (21.7%) of the cases detected by the multiplex nested PCR, demonstrating a 4.6 times higher detection rate for the multiplex nested PCR (Fisher exact test, P &lt; .001). Positive amplifications were to Toxoplasma gondii (32 cases), cytomegalovirus (14 cases), parvovirus B19 (5 cases), and adenovirus (5 cases). In 4 cases, 2 pathogens were simultaneously detected. All types of biological matrices were suitable for amplification. Sequencing of multiplex nested PCR products confirmed the molecular findings. Conclusions.— The multiplex nested PCR significantly increased the number of diagnosed congenital infections. Given the scarcity of DNA recovered from amniotic fluid and some neonatal samples, this multiplex nested PCR allows the simultaneous detection of 7 pathogens associated with congenital infections in a reliable, faster, cost-effective, and more sensitive way.

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