Diagnostic Value of Multiplex Polymerase Chain Reaction in Detection of Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia from Sepsis in Pediatrics

2021 ◽  
Vol 15 ◽  
Author(s):  
Sara Galeb ◽  
Maysaa El Sayed Zaki ◽  
Raghdaa Shrief ◽  
Rasha Hassan ◽  
Mohamed Anies
Keyword(s):  
Polymerase Chain Reaction ◽  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Multiplex Pcr ◽  
Stenotrophomonas Maltophilia ◽  
Multiplex Polymerase Chain Reaction ◽  
Diagnostic Value ◽  
Blood Cultures ◽  
Chain Reaction ◽  
Polymerase Chain

Background: Proper identification of the causative organism in pediatric sepsis is crucial for early diagnosis and prevention of septic shock and organ failure. Objectives: The present study aimed to evaluate the multiplex Polymerase Chain Reaction (PCR) to detect Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia from positive blood cultures for these pathogens isolated from children, with hospital-acquired sepsis compared to the conventional biochemical reactions for identification of these organisms. Methods: This study was a cross-sectional study performed on 100 isolates from pediatric blood cultures, including Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The study also included 100 isolates of Escherichia coli as a negative control. All isolates were identified by API 20NE and the multiplex PCR, with primers specific to the 3 tested bacteria. Results: Multiplex PCR was positive in 96% of isolates, and 4 isolates had negative results. False positive results were reported with three E. coli strains. Multiplex PCR identified all the isolates of Acinetobacter baumannii, 29 isolates of Pseudomonas aeruginosa, and 27 isolates of Stenotrophomonas maltophilia. Compared to the biochemical identification, the diagnostic value of the multiplex PCR revealed 96.04% sensitivity, 96.9% specificity, 97.00%, positive predictive value, 96.00% negative predictive value, and 96.50% accuracy. Conclusion: The present study highlights the diagnostic value of multiplex PCR to identify Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia from positive blood cultures. Multiplex PCR was sensitive, specific, and accurate. The accuracy differs according to the organisms, with 100% accuracy for Acinetobacter baumannii.

Development of a Multiplex Polymerase Chain Reaction Assay for Detection and Differentiation of Staphylococcus aureus in Dairy Products†

2001 ◽  
Vol 64 (5) ◽  
pp. 664-668 ◽  
Author(s):  
SUDHIR TAMARAPU ◽  
JOHN L. McKILLIP ◽  
MARYANNE DRAKE
Keyword(s):  
Staphylococcus Aureus ◽  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Dairy Products ◽  
Multiplex Polymerase Chain Reaction ◽  
Skim Milk ◽  
Cheddar Cheese ◽  
Polymorphism Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

A multiplex polymerase chain reaction (PCR) assay was developed for the detection and differentiation of enterotoxigenic Staphylococcus aureus in dairy products. A solvent extraction procedure was successfully modified for extraction of S. aureus DNA from 10 ml of artificially contaminated skim milk or 20 g cheddar cheese. Primers targeting the enterotoxin C gene (entC) and thermostable nuclease gene (nuc) were used in the multiplex PCR. PCR products were confirmed using restriction fragment length polymorphism analysis. DNA was consistently quantified and amplified by uniplex PCR from 10 CFU/ml of S. aureus in skim milk or 10 CFU/20 g cheddar cheese. The sensitivity of the multiplex PCR was 100 CFU/ml of skim milk or 100 CFU/20 g cheddar cheese. The developed methodology allows presumptive identification and differentiation of enterotoxigenic S. aureus in less than 6 h.

scholarly journals Molecular identification by multiplex polymerase chain reaction of Pasteurella multocida in cattle and buffaloes in Baghdad

2014 ◽  
Vol 38 (1) ◽  
pp. 99-106
Author(s):  
Ihab G. M. AL-Shemmari
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Pasteurella Multocida ◽  
Type B ◽  
Chain Reaction ◽  
Blood Samples ◽  
Nasal Swabs ◽  
Polymerase Chain ◽  
B 31

The aim of this study was to identify pasteurella multocida and their types by PCR in cattle’s and buffaloesi bagdad from March to August 2012 on 204 animals , including 102 cattle and 102 buffaloes at slaughter houses from Baghdad .Blood samples and nasal swaps were collected , before slaughtering and lung tissues of slaughtered animal , and from 54 clinically suspected cases of pasteurellosis , including 27 bovines ,and 27 buffaloes the samples taken included blood and nasal swabs . Pasteurellamultocida were isolated from 94 animals include 49 cattle 45 buffaloes. The typing of the isolates by multiplex PCR for genotyping Pasteuerllamultocida revealed 93 isolates of type B , 31 from cattle and 62 from buffaloes ,and 81 isolates of type A , 55 from cattle and 26 from buffaloes .

scholarly journals Application of a Multiplex Polymerase Chain Reaction Assay for the Simultaneous Confirmation of Listeria monocytogenes and Other Listeria Species in Turkey Sample Surveillance†

2002 ◽  
Vol 65 (5) ◽  
pp. 780-785 ◽  
Author(s):  
IRENE V. WESLEY ◽  
KAREN M. HARMON ◽  
JAMES S. DICKSON ◽  
ANN RAMOS SCHWARTZ
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Listeria Species ◽  
Multiplex Pcr Assay ◽  
Polymerase Chain

A multiplex polymerase chain reaction was developed to simultaneously identify Listeria monocytogenes and species of the genus Listeria. Two sets of primers were used, with the first amplifying a 938-bp region of the 16S rRNA gene that is highly conserved in all Listeria species and the second amplifying a 174-bp region of the listeriolysin (hlyA) gene of L. monocytogenes. Thus, isolates of Listeria spp. yield a single 938-bp product, whereas L. monocytogenes isolates yield both the 938-bp product and a 174-bp product. The specificity of the assay was verified with all six Listeria species and 11 serotypes of L. monocytogenes, as well as nonrelated bacteria. The multiplex PCR assay was used to determine the incidence of Listeria spp., especially L. monocytogenes, in mechanically separated turkey samples (n = 150 samples). L. monocytogenes strains were selected by using the University of Vermont two-step enrichment protocol and plating to selective Palcam agar. The multiplex PCR assay was used for verification of presumptive Listeria colonies. Approximately 38% of mechanically separated turkey samples (57 of 150) yielded L. monocytogenes; an additional 18% of these samples (27 of 150) harbored other Listeria spp. Fifty-one percent (29 of 57) of the L. monocytogenes isolates were of serogroup 1, 44% (25 of 57) were of serogroup 4, and 2% (1 of 57) were assigned to serogroups other than 1 and 4.

scholarly journals DETECTION OF Leishmania (Viannia) IN Nyssomyia neivai AND Nyssomyia whitmani BY MULTIPLEX POLYMERASE CHAIN REACTION, IN SOUTHERN BRAZIL

2014 ◽  
Vol 56 (5) ◽  
pp. 391-395 ◽  
Author(s):  
Herintha Coeto Neitzke-Abreu ◽  
Kárin Rosi Reinhold-Castro ◽  
Mateus Sabaini Venazzi ◽  
Regiane Bertin de Lima Scodro ◽  
Alessandra de Cassia Dias ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Natural Infection ◽  
Epidemiological Studies ◽  
Leishmania Infection ◽  
Southern Brazil ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Sensitivity Specificity

Sandflies transmit pathogens of leishmaniasis. The natural infection of sandflies by Leishmania (Viannia) was assessed in municipalities, in the state of Paraná, in Southern Brazil. Sandflies were collected with Falcão and Shannon traps. After dissection in search of flagellates in digestive tubes and identification of the species, female sandflies were submitted to the Multiplex Polymerase Chain Reaction (multiplex PCR) for detection of the fragment of the kDNA of Leishmania (Viannia) and the fragment from the IVS6 cacophony gene region of the phlebotomine insects. The analysis was performed in pools containing seven to 12 guts from females of the same species. A total of 510 female sandflies were analyzed, including nine Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai, and 268 Nyssomyia whitmani. Although none of the females was found naturally infected by flagellates through dissection, the fragment of DNA from Leishmania (Viannia) was shown by multiplex PCR in one sample of Ny. neivai (0.46%) and three samples of Ny. whitmani (1.12%). It was concluded that Ny. neivai and Ny. whitmani are susceptible to Leishmania infection, and that multiplex PCR can be used in epidemiological studies to detect the natural infection of the sandfly vector, because of its sensitivity, specificity and feasibility.

A Multiplex Polymerase Chain Reaction Assay for Rapid Detection and Identification of Escherichia coli O157:H7 in Foods and Bovine Feces†

2000 ◽  
Vol 63 (8) ◽  
pp. 1032-1037 ◽  
Author(s):  
PINA M. FRATAMICO ◽  
LORI K. BAGI ◽  
TIZIANA PEPE
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Ground Beef ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain ◽  
Bovine Feces

A multiplex polymerase chain reaction (PCR) assay was designed to simplify detection of Escherichia coli O157:H7 and to identify the H serogroup and the type of Shiga toxin produced by this bacterium. Primers for a plasmid-encoded hemolysin gene (hly933), and chromosomal flagella (fliCh7; flagellar structural gene of H7 serogroup), Shiga toxins (stx1, stx2), and attaching and effacing (eaeA) genes were used in a multiplex PCR for coamplification of the corresponding DNA sequences from enterohemorrhagic E. coli (EHEC) O157:H7. Enrichment cultures of ground beef, blue cheese, mussels, alfalfa sprouts, and bovine feces, artificially inoculated with various levels of E. coli O157:H7 strain 933, were subjected to a simple DNA extraction step prior to the PCR, and the resulting amplification products were analyzed by agarose gel electrophoresis. Sensitivity of the assay was ≤1 CFU/g of food or bovine feces (initial inoculum level), and results could be obtained within 24 h. Similar detection levels were obtained with ground beef samples that underwent enrichment culturing immediately after inoculation and samples that were frozen or refrigerated prior to enrichment. The multiplex PCR facilitates detection of E. coli O157:H7 and can reduce the time required for confirmation of isolates by up to 3 to 4 days.

Evaluation of an Enzyme-Linked Immunosorbent Assay, Direct Immunofluorescent Filter Technique, and Multiplex Polymerase Chain Reaction for Detection of Escherichia coli O157:H7 Seeded in Beef Carcass Wash Water†

1998 ◽  
Vol 61 (8) ◽  
pp. 934-938 ◽  
Author(s):  
PINA M. FRATAMICO ◽  
TERENCE P. STROBAUGH
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Wash Water ◽  
Enzyme Linked Immunosorbent Assay ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

In commercial beef processing, carcasses are customarily washed with water to remove physical and microbial contamination. Assaying the water that is shed from the carcasses after washing is a convenient method to determine whether the carcass is contaminated with Escherichia coli O157:H7 or other bacterial pathogens. E. coli O157:H7 was inoculated into carcass wash water at various levels and the bacteria were then concentrated by filtration. After collection of bacteria in the filter units, the nylon membranes were cut out and placed in tubes containing growth medium, and the tubes were mixed vigorously to dislodge the bacteria from the membranes. Prior to enrichment, samples were removed for testing by a multiplex polymerase chain reaction (PCR) and a direct immunofluorescent filter technique (DIFT). The remaining samples were subjected to 4-h enrichment culturing at 37°C, after which aliquots were removed for testing by multiplex PCR, DIFT, and an enzyme-linked immunosorbent assay (ELISA). Following 4-h enrichment culturing, E. coli O157:H7 was detected in wash water samples initially inoculated with ca. 100, 0.1, and 1 CFU/ml by ELISA, DIFT, and multiplex PCR, respectively. Testing of the wash water using the ELISA and the DIFT can be accomplished in less than 8 h. On the basis of these results, assaying carcass wash water by ELISA, DIFT, or multiplex PCR can be useful for detection of E. coli O157:H7 beef carcass contamination and can potentially be employed to identify carcasses for further processing to inactivate the organism.

scholarly journals Identification of Listeria spp. strains isolated from meat products and meat production plants by multiplex polymerase chain reaction

2015 ◽  
Vol 4 (4) ◽  
Author(s):  
Roberta Mazza ◽  
Francesca Piras ◽  
Daniela Ladu ◽  
Miriam Putzolu ◽  
Simonetta Gianna Consolati ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Meat Products ◽  
Clinical Samples ◽  
Meat Production ◽  
Chain Reaction ◽  
Processing Plants ◽  
Contaminated Food ◽  
Polymerase Chain

Listeriosis is a foodborne disease caused by <em>Listeria monocytogenes</em> and is considered as a serious health problem, due to the severity of symptoms and the high mortality rate. Recently, other <em>Listeria</em> species have been associated with disease in human and animals. The aim of this study was to develop a multiplex polymerase chain reaction (PCR) in order to simultaneously detect six Listeria species (<em>L. grayi</em>, <em>L. welshimeri</em>, <em>L. ivanovii</em>, <em>L. monocytogenes</em>, <em>L. seeligeri</em>, <em>L. innocua</em>) in a single reaction. One hundred eighteen <em>Listeria</em> spp. strains, isolated from meat products (sausages) and processing plants (surfaces in contact and not in contact with meat), were included in the study. All the strains were submitted to biochemical identification using the API Listeria system. A multiplex PCR was developed with the aim to identify the six species of <em>Listeria</em>. PCR allowed to uniquely identify strains that had expressed a doubtful profile with API Listeria The results suggest that the multiplex PCR could represent a rapid and sensitive screening test, a reliable method for the detection of all <em>Listeria</em> species, both in contaminated food and in clinical samples, and also a tool that could be used for epidemiological purposes in food-borne outbreaks. A further application could be the development of a PCR that can be directly applied to the pre-enrichment broth.

scholarly journals Role of multiplex polymerase chain reaction in diagnosing tubercular meningitis

2017 ◽  
Vol 9 (02) ◽  
pp. 145-147 ◽  
Author(s):  
Anupam Berwal ◽  
Kiran Chawla ◽  
Shashidhar Vishwanath ◽  
Vishnu Prasad Shenoy
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Predictive Value ◽  
Multiplex Polymerase Chain Reaction ◽  
Extrapulmonary Tuberculosis ◽  
Smear Microscopy ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Sensitivity Specificity

AbstractTuberculous meningitis (TBM) is one of the most serious manifestations of extrapulmonary tuberculosis. Timely and accurate diagnosis provides a favorable prognosis in patients with TBM. The study evaluated the use of multiplex polymerase chain reaction (PCR) in the diagnosis of TBM. A study was conducted on 74 patients clinically suspected with TBM. The cerebrospinal fluid (CSF) specimens were processed for smear microscopy, middle brook 7H9 culture, and multiplex PCR using primers directed against IS6110 gene and 38 kD protein for detection of Mycobacterium tuberculosis. The results were analyzed to assess the role of multiplex PCR in the diagnosis of TBM. A total of 26 (35.1%) patients were diagnosed with TBM. Microscopy was negative in all while culture was positive in two cases only. Comparing with clinical diagnosis and CSF adenosine deaminase levels of ≥ 10 U/L, multiplex PCR showed sensitivity, specificity, positive predictive value, and negative predictive value of 71.4%, 89.6%, 83.3%, and 81.2%, respectively, in the diagnosis of TBM.

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