scholarly journals Identification of the most common pathogenic bacteria in patients with suspected sepsis by multiplex PCR

2014 ◽  
Vol 8 (04) ◽  
pp. 461-468 ◽  
Author(s):  
Mohammad Reza Arabestani ◽  
Hossein Fazzeli ◽  
Bahram Nasr Esfahani
Keyword(s):  
Multiplex Pcr ◽  
Internal Control ◽  
Predictive Value ◽  
Pathogenic Bacteria ◽  
Target Genes ◽  
Clinical Samples ◽  
Coagulase Negative Staphylococci ◽  
Acinetobacter Baumanii ◽  
Suspected Sepsis ◽  
Microbiological Methods

Introduction: Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus spp., Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumanii have been found to be the most prevalent bacteremia-causing bacteria in patients with septicemia. Early detection of bloodstream infection (BSI) is crucial in the clinical setting. A multiplex PCR method for identification of these agents in clinical samples has been developed in parallel by conventional microbiological methods. Methodology: The target genes selected for each of the organisms were very specific for designing primers. Design of primers was done using Mega4, Allel ID6, Oligo6, and Oligo analyzer software. The test comprises a universal PCR from the 16S rDNA gene and multiplex PCR from the rpoB, gyrA, sss, and chromosome X (as an internal control). Results: The sensitivity and specificity for universal PCR and multiplex PCR in comparison with BC were 83.87% and 91.58%, and 74.19% and 91.58%, respectively. The positive predictive value (PPV) and the negative predictive value (NPV) for these two PCRs were 76.47% and 94.57%, and 74.19% and 91.58%, respectively. PCR failed to identify bacteria which were found conventionally in only 3.96% and 6.34% of the cases by universal and multiplex PCR (mostly bacteria not included in the PCR cassette). In 6.34% of the cases, multiplex PCR afforded identification of bacteria, but BC showed no bacteria in the sample. Conclusions: The multiplex PCR approach facilitates the detection of bacteremia in blood samples within a few hours. Rapid detection of bacteria by multiplex PCR appears to be a valuable tool, allowing earlier pathogen-adopted antimicrobial therapy in critically ill patients.

Rapid and Simultaneous Detection of Vibrio parahaemolyticus, Salmonella spp. and Escherichia coli in Fish by Multiplex PCR

2020 ◽  
Vol 15 (2) ◽  
pp. 53
Author(s):  
Radestya Triwibowo ◽  
Novalia Rachmawati ◽  
Dwiyitno Dwiyitno
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Internal Control ◽  
Vibrio Parahaemolyticus ◽  
Pathogenic Bacteria ◽  
Target Genes ◽  
Simultaneous Detection ◽  
Salmonella Spp ◽  
Fish Products ◽  
Confirmatory Assay

Pathogenic bacteria are commonly found as natural contaminants in seafood and fish products. Globally, several countries have been imposing strict regulations on the maximum levels of pathogens and consequently require microbial testing of pathogens before the products can be marketed. A culture-based method with biochemical assay has been widely used to detect pathogenic bacteria in food, despite its long and extensive process. Meanwhile, the alternative molecular-based method to overcome this problem, cannot differentiate between viable and nonviable cells, which may lead to underestimation. This study aimed to develop a multiplex PCR (mPCR) method as a confirmatory assay for the culture-based method to detect pathogens in fish products simultaneously. This method applied a pre-enrichment step to ensure the growth of low-level pathogens and the injured cells in the sample. The target genes were ToxR, InvA, and UidA for Vibrio parahaemolyticus, Salmonella spp. and Escherichia coli, respectively. This assay also amplified the 16S rDNA gene of bacteria as an internal control for the PCR reaction. By implementing liquid-based DNA extraction during analysis, the developed-mPCR was comparable to detect the targeted bacteria in artificially-contaminated samples. The method was more sensitive in naturally-contaminated samples, where the number of E. coli, Salmonella spp. and V. parahaemolyticus detected were 28, 7, and 22, respectively. While the conventional method only detected 26, 5, and 19 of the respective pathogens. With a relatively shorter time and lower operation cost, the mPCR method is potential as an alternative for the culture-based method.

scholarly journals Is Staphylococcus lugdunensis Significant in Clinical Samples?

2017 ◽  
Vol 55 (11) ◽  
pp. 3167-3174 ◽  
Author(s):  
Xavier Argemi ◽  
Yves Hansmann ◽  
Philippe Riegel ◽  
Gilles Prévost
Keyword(s):  
Pathogenic Bacteria ◽  
Clinical Manifestations ◽  
Clinical Samples ◽  
Opportunistic Pathogens ◽  
Coagulase Negative Staphylococci ◽  
Staphylococcus Lugdunensis ◽  
Content Type ◽  
Severe Infections ◽  
Microbiological Data

ABSTRACTThe implication of coagulase-negative staphylococci in human diseases is a major issue, particularly in hospital settings wherein these species often act as opportunistic pathogens. In addition, some coagulase-negative staphylococci such asS. lugdunensishave emerged as pathogenic bacteria, implicated in severe infections, particularly, osteoarticular infections, foreign-body-associated infections, bacteremia, and endocarditis.In vitrostudies have shown the presence of several putative virulence factors such as adhesion factors, biofilm production, and proteolytic factors that might explain clinical manifestations. Taken together, the clinical and microbiological data might change the way clinicians and microbiologists look atS. lugdunensisin clinical samples.

Development of multiplex PCR assay for rapid detection Listeria monocytogenes in clinical samples

2021 ◽  
Vol 30 (4) ◽  
pp. 20-26
Author(s):  
Le Thanh Huong ◽  
Ha Thi Phuong Mai ◽  
Hoang Thi Thu Ha ◽  
Nguyen Dong Tu ◽  
Bui Tien Sy ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Rapid Detection ◽  
Pathogenic Bacteria ◽  
Vulnerable Groups ◽  
Clinical Samples ◽  
Specific Gene ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Specificity And Sensitivity

Listeria monocytogenes is widely present in the natural environment. This bacteria can cause infections in both humans and animals. In humans, the most vulnerable groups to be infected with L. monocytogenes are the elderly, people with an impaired immune system and chronically illness, pregnant women, and newborn babies. The aim of this study was to develop a multiplex PCR assay for the rapid detection of L. monocytogenes in mock clinical samples. A pair of primers were designed for detection of L. monocytogenes based on prs, a Listeria genus specific gene, and hly, a hemolysin gene. The specificity of the primers were tested by using different L. monocytogenes strains and other common pathogenic bacteria. The results showed that L. monocytogenes strains were positive in the detection and other tested strains were negative in mock (spiked) clinical samples. The sensitivity of multiplex PCR assay was 102 CFU/ml per reaction. The specificity and sensitivity of multiplex PCR technology for detecting L. monocytogenes in mock (spiked) clinical samples were high, and the assay could be completed within 1.5 hours. Therefore, this established multiplex PCR provides a rapid and reliable method and will be useful for the detection of L. monocytogenes in mock clinical samples.

scholarly journals A Multiplex-PCR Method for Strain Identification and Detailed Phylogenetic Analysis of AY-Group Phytoplasmas

Plant Disease ◽  
2014 ◽  
Vol 98 (3) ◽  
pp. 299-305 ◽  
Author(s):  
Shigeyuki Kakizawa ◽  
Yoichi Kamagata
Keyword(s):  
Phylogenetic Analysis ◽  
Multiplex Pcr ◽  
16S Rdna ◽  
Pathogenic Bacteria ◽  
Target Genes ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Single Copy ◽  
Strain Identification ◽  
Specific Detection

Phytoplasmas are plant pathogenic bacteria that cause devastating losses in the yield of diverse crops worldwide. Specific detection and strain identification of phytoplasmas is important to prevent the spread of phytoplasma-induced diseases. Hence, methods to rapidly detect these organisms are important for pest control. Polymerase chain reaction (PCR) methods using phytoplasma-specific primers are widely used to detect phytoplasmas from infected plants and insects because they are highly sensitive, easily handled, and have a variety of analytical secondary applications. The phytoplasma 16S rDNA was widely used as a target of the PCR detection method; however, further target genes and more rapid methods have been required for more specific detection of phytoplasmas. Here, we developed a multiplex-PCR system to amplify several phytoplasma genes. We designed 36 primers, based on the genome sequence of ‘Candidatus Phytoplasma asteris’, to amplify 18 single-copy genes covering wide regions of the phytoplasma genome. Nine genes could be simultaneously amplified in a single PCR. This multiplex-PCR was applied to DNAs from 10 phytoplasma strains belonging to the AY-group, and different amplification patterns were obtained between strains, suggesting that this method would allow us to differentiate phytoplasmas at the strain level. Direct sequencing was also possible after the multiplex-PCR amplification by a modified sequencing method. Detailed phylogenetic analysis was performed using concatenated sequences, and evolutionary relationships among four Japanese isolates were revealed, where these strains could not be distinguished by their 16S rDNA. Thus, this multiplex-PCR system is useful for rapid strain identification and detailed phylogenetic analysis of phytoplasmas.

scholarly journals An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kean Hean Ooi ◽  
Mengying Mandy Liu ◽  
Jie Wen Douglas Tay ◽  
Seok Yee Teo ◽  
Pornchai Kaewsapsak ◽  
...  
Keyword(s):  
Positive Predictive Value ◽  
Internal Control ◽  
Predictive Value ◽  
Viral Genome ◽  
Clinical Samples ◽  
Diagnostic Assay ◽  
Healthy Individuals ◽  
Rna Purification ◽  
Rate Of Reaction ◽  
Hybrid Dna

AbstractExtensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.

scholarly journals Possible Bacteria Pathogens Found in the Internal Surface of Ladies Handbags in Umuahia, Abia State, South-Eastern Nigeria

2020 ◽  
pp. 66-73
Author(s):  
Nwankwo E.O. ◽  
Keyword(s):  
Pathogenic Bacteria ◽  
Culture Media ◽  
Bacterial Pathogens ◽  
Internal Surface ◽  
Health Concern ◽  
Diffusion Method ◽  
Biochemical Tests ◽  
Salmonella Spp ◽  
Coagulase Negative Staphylococci ◽  
Microbiological Methods

Bacteria including the pathogenic species have been isolated from fomites, these organisms are sometimes multidrug resistant and are of public health concern. It is therefore important to isolate and identify potential bacterial pathogens associated with the internal surface of ladies handbags, in Umuahia, Abia state. One hundred and forty swabbed samples were collected from the ladies hand bags in different groups of individuals which include; Nurses, civil servants, students and market women. Also the handbags from which the samples were collected includes: Leather, Cotton, Nylon and Polyester and velvet handbags. The bags were swabbed with sterile swab sticks and inoculated on different types of culture media and incubated at 37o C for 24 hours. Bacterial isolates were identified using standard microbiological methods including biochemical tests before subjecting isolates to different antimicrobial sensitivity test that was carried out by disc diffusion method. The following bacteria were isolated from the internal surface of the handbags, Coagulase Negative Staphylococci 6(2.6%), Escherichia coli 36(15.7%), Klebsiella spp. 14(6.1%), Staphylococcus aureus 49(21.3%), Bacillus spp. 48(20.9%), Pseudomonas aeruginosa 5(2.2%), Proteus spp. 5(2.2%), streptococcus spp. 31(13.5), Micrococcus spp. 20(8.7%), Salmonella spp. 3(1.3%) and Enterococcus faecalis 13(5.7%). Most of the isolates were sensitive to levofloxacin, gentamicin, norfloxacin, ciprofloxacin and resistant to ampiclox, chloramphenicol and erythromycin. Potentially pathogenic bacteria resistant to multiple antibiotics can be spread by hand contact from ladies handbags. Keywords: Bacterial pathogens, ladies handbags, antibiogram

scholarly journals Development of a multiplex PCR assay for the simultaneous and rapid detection of six pathogenic bacteria in poultry

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Zhihao Wang ◽  
Jiakun Zuo ◽  
Jiansen Gong ◽  
Jiangang Hu ◽  
Wei Jiang ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Pasteurella Multocida ◽  
Target Genes ◽  
Bacterial Pathogens ◽  
Clinical Samples ◽  
Test Results ◽  
Pcr Assay ◽  
Specific Test ◽  
Multiplex Pcr Assay ◽  
Specificity And Sensitivity

AbstractEscherichia coli, Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella spp. and Staphylococcus aureus are six bacterial pathogens of avian. However, these pathogens may cause many similar pathological changes, resulting in clinical isolates that are difficult to quickly and simultaneously detect and identify. Here, a multiplex polymerase chain reaction (m-PCR) assay is reported to rapidly identify targets genes (phoA, KMT1, ureR, toxA, invA, and nuc) of these six pathogens in clinical samples. Six pairs of specific primers were designed. The optimal reaction conditions, specificity, and sensitivity of the m-PCR assay were investigated. The results showed that betaine remarkably improved amplification of the target genes. Specific test results showed that all six pathogens were detected by the proposed m-PCR protocol without cross-amplification with viruses or parasites. Sensitivity test results showed that the m-PCR system could amplify the six target genes from bacterial genomes or cultures with template amounts of 500 pg or 2.8–8.6 × 103 colony forming units, respectively. Furthermore, the six bacterial pathogens isolated from the infected tissue samples were successfully identified. The proposed m-PCR assay is a useful tool to monitor and diagnose bacterial infection in birds with high specificity, sensitivity and throughput.

scholarly journals A Search for Identifying Aerobic Bacteria by Culture and Multiplex PCR in Market Eggs Causing Gastroenteritis and Enteric Fever in Bangladesh

2015 ◽  
Vol 1 (1) ◽  
pp. 1-7
Author(s):  
Jannatul Fardows ◽  
SM Shamsuzzaman
Keyword(s):  
Multiplex Pcr ◽  
Salmonella Enteritidis ◽  
Pathogenic Bacteria ◽  
Klebsiella Oxytoca ◽  
Enterobacter Aerogenes ◽  
Aerobic Bacteria ◽  
Salmonella Spp ◽  
Acinetobacter Baumanii ◽  
Source Of Infection ◽  
Egg Shells

To observe the chance of possible transmission of pathogenic bacteria from market egg to the community, potential pathogenic aerobic bacteria were detected from market eggs by culture and multiplex PCR. Egg shells and egg contents of 150 eggs collected from different markets of Dhaka city were tested. Total 145 (96.67%) egg shells yielded growth of bacteria, 23 (15.86%) of them were ESBL producers. Esch. coli was the most common (26.67%) bacteria and 7 (4.67%) were Salmonella spp. Other bacteria were Klebsiella pneumoniae (6.67%), Proteus vulgaris (3.33%), Proteus mirabilis (2%), Providencia rettgeri (15.33%), Providencia alkalifaciens (1.33%), Acinetobacter baumanii (8.67%), Citrobacter freundii (10%), Enterobacter aerogenes (6.67%) Klebsiella oxytoca (4.67%) and Pseudomonas aeruginosa (6.67%). By PCR, 15 (10%) Salmonella spp. was identified from egg shells and the most common serotype was Salmonella Enteritidis (53.33%). No bacteria were detected from egg contents. Most of the bacteria were sensitive to imipenem and colistin. All Salmonella serotypes were sensitive to chloramphenicol, imipenem, gentamicin, ciprofloxacin and ceftriaxone. In conclusion, it can be said that market eggs may be an important source of infection of many gram negative bacteria including Salmonella to the community.

scholarly journals Detection of porcine circoviruses in clinical specimens using multiplex PCR in Hubei, central China

2018 ◽  
Author(s):  
Keli Yang ◽  
Zuwu Jiao ◽  
Danna Zhou ◽  
Rui Guo ◽  
Zhengying Duan ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Infection Rate ◽  
Target Genes ◽  
Limit Of Detection ◽  
Central China ◽  
Clinical Samples ◽  
Monitoring And Control ◽  
Pcr Assay ◽  
Tissue Samples ◽  
Multiplex Pcr Assay

In order to detect and simultaneously discriminate PCV1, PCV2 and PCV3, a multiplex PCR assay was developed and used to detect clinical samples in this study. Each of target genes of PCV1, PCV2 and PCV3 was amplified using the designed primers, while no other porcine viruses genes were detected. The limit of detection of the assay was 10 copies/μL of PCV1, PCV2 and PCV3. The tissue samples from eight pig farms were detected using the multiplex PCR assay. The results showed that PCV1, PCV2 and PCV3 are co-circulating in central China. The PCV1, PCV2 and PCV3 singular infection rate was 52.4% (150/286), 61.2% (175/286) and 45.1% (129/286), respectively, while the PCV1 and PCV2 co-infection rate was 11.2% (32/286), the PCV1 and PCV3 co-infection rate was 5.9% (17/286), the PCV2 and PCV3 co-infection rate was 23.4% (67/286), and the PCV1, PCV2 and PCV3 co-infection rate was 1.7% (5/286), respectively, which were 100% consistent with the sequencing method and Real-time PCR methods. It proved that this multiplex PCR assay could be used as a differential diagnostic tool for monitoring and control of PCVs in the field. The results also indicate that the PCVs infection and their co-infection are severe in Hubei Province, Central China.

scholarly journals Dominance of Staphylococcus aureus in clinical specimens in Islami bank hospital, Rajshahi, Bangladesh

2017 ◽  
Vol 3 (7) ◽  
pp. 200
Author(s):  
M. Jakir Hossain ◽  
Muhammad Afser Siddiqi ◽  
M. Morsed Zaman Miah ◽  
Khairun Nahar Khan ◽  
Ahmed Imtiaj
Keyword(s):  
Staphylococcus Aureus ◽  
Pathogenic Bacteria ◽  
Health Workers ◽  
Small Sample Size ◽  
Small Sample ◽  
Clinical Samples ◽  
Coagulase Negative Staphylococci ◽  
Isolation And Identification ◽  
Mortality And Morbidity ◽  
High Incidence

<p class="abstract"><strong>Background:</strong> <em>Staphylococcus aureus </em>is a medicologically important pathogenic bacteria which is largely responsible for the thousands of human health hazzards. This study investigated the incidence of <em>Staphylococcus aureus </em>in various clinical samples from in- and outpatients attending Islami Bank Hospital, Rajshahi, Bangladesh.</p><p class="abstract"><strong>Methods:</strong> Clinical isolates from the hospital was confirmed as <em>Staphylococcus aureus </em>using standard bacteriological techniques. This study reports the isolation and identification of <em>Staphylococcus aureus</em>, coagulase negative staphylococci and catalase negative cocci in clinical samples at Islami Bank Hospital, Rajshahi, Bangladesh.  </p><p class="abstract"><strong>Results:</strong> Out of a total of 144 putative isolates of Staphylococci from urine, ear and wounds screened for <em>Staphylococcus aureus</em>, 87 of them were confirmed as <em>S. aureus</em>, 30 were coagulase negative staphylococci while 27 were catalase negative cocci. The high incidence of <em>S aureus </em>in this study compared to other staphylococci demonstrates the versatility and propensity of <em>S. aureus </em>to cause diseases.</p><strong>Conclusions:</strong> This is worrisome because of the high mortality and morbidity often associated with infections of this bacterium. It therefore calls for proper handling of specimen suspected to contain the organism or patients who might be at risk of infection. This is to avoid transmission to other patients and healthy individuals especially health workers as they might constitute vehicles for the spread of the organism. Further studies are recommended because of the small sample size in this study. This would help to establish whether this was peculiar to the Islami Bank Hospital Rajshahi, Bangladesh or wide spread in other hospitals in the country.

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