scholarly journals Evaluating Performance of Multiplex Real Time PCR for the Diagnosis of Malaria at Elimination Targeted Low Transmission Settings of Ethiopia.

2021 ◽  
Author(s):  
Mahlet Belachew ◽  
Mistire Wolde ◽  
Desalegn Nega ◽  
Bokretsion Gidey ◽  
Legessie Negash ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Real Time ◽  
Malaria Elimination ◽  
Real Time Pcr ◽  
Nucleic Acid Amplification ◽  
Molecular Testing ◽  
Pcr Assay ◽  
Blood Samples ◽  
Low Transmission ◽  
Highly Sensitive

Abstract Background: Malaria incidence has declined in Ethiopia in the past ten years. Current malaria diagnostic tests, including light microscopy and antigen-detecting rapid tests (RDTs) cannot reliably detect low-density infections. Studies have shown that nucleic acid amplification tests are highly sensitive and specific in detecting malaria infection. Thus, this study took place with the aim of evaluating the performance of multiplex real time PCR for the diagnosis of malaria using patient samples collected from health facilities located at malaria elimination targeted low transmission settings in Ethiopia. Methods: A health facility based cross sectional survey was conducted in selected malaria sentinel sites. Malaria suspected febrile outpatients referred to laboratory for malaria testing between December 2019 and March 2020 were enrolled into this study. Socio demographic information and capillary blood samples were collected from the study participants and tested at spot with RDTs. Additionally, five circles of dry blood sample (DBS) samples on Whatman filter paper and thick and thin smear were prepared for molecular testing and microscopic examination, respectively. Multiplex real time PCR assay was performed at EPHI malaria laboratory. The performance of multiplex real time PCR assay, microscopy and RDT for the diagnosis of malaria was compared and evaluated against each other.Results: Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as P. falciparum infection, 16 as P. vivax and 3 as mixed infections. Of the total samples, light microscopy detected 33 as Pf, 18 as PV and RDT detected 43 as Pf, 17 as PV, and one mixed infection. Using light microscopy as reference test, the sensitivity and specificity of multiplex real time PCR were 100% (95% CI [93-100]) and 83.2% (95% CI [77.6-87.9]), respectively. Using multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58% (95% CI [46.9-68.4] and 67% (95% CI [56.2-76.7]); and 100% (95% CI [98-100] and 98.9 (95% CI (96-99.9), respectively. Substantial level of agreement was reported between microscopy and multiplex real time PCR results with kappa value of 0.65. Conclusions: Multiplex real time PCR had an advanced performance in parasite detection and species identification on febrile patients’ samples than did microscopy and RDT in low malaria transmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria elimination program, particularly for community based epidemiological samples. Although microscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinical facilities.

scholarly journals Evaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia

2022 ◽  
Vol 21 (1) ◽  
Author(s):  
Mahlet Belachew ◽  
Mistire Wolde ◽  
Desalegn Nega ◽  
Bokretsion Gidey ◽  
Legessie Negash ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Real Time ◽  
Malaria Elimination ◽  
Real Time Pcr ◽  
Diagnostic Tests ◽  
Molecular Testing ◽  
Pcr Assay ◽  
Blood Samples ◽  
Low Transmission ◽  
Highly Sensitive

Abstract Background Malaria incidence has declined in Ethiopia in the past 10 years. Current malaria diagnostic tests, including light microscopy and rapid antigen-detecting diagnostic tests (RDTs) cannot reliably detect low-density infections. Studies have shown that nucleic acid amplification tests are highly sensitive and specific in detecting malaria infection. This study took place with the aim of evaluating the performance of multiplex real time PCR for the diagnosis of malaria using patient samples collected from health facilities located at malaria elimination targeted low transmission settings in Ethiopia. Methods A health facility-based, cross-sectional survey was conducted in selected malaria sentinel sites. Malaria-suspected febrile outpatients referred to laboratory for malaria testing between December 2019 and March 2020 was enrolled into this study. Sociodemographic information and capillary blood samples were collected from the study participants and tested at spot with RDTs. Additionally, five circles of dry blood spot (DBS) samples on Whatman filter paper and thick and thin smear were prepared for molecular testing and microscopic examination, respectively. Multiplex real time PCR assay was performed at Ethiopian Public Health Institute (EPHI) malaria laboratory. The performance of multiplex real time PCR assay, microscopy and RDT for the diagnosis of malaria was compared and evaluated against each other. Results Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as Plasmodium falciparum infection, 16 as Plasmodium vivax and 3 as mixed infections. Of the total samples, light microscopy detected 33 as P. falciparum, 18 as P. vivax, and RDT detected 43 as P. falciparum, 17 as P. vivax, and one mixed infection. Using light microscopy as reference test, the sensitivity and specificity of multiplex real time PCR were 100% (95% CI (93–100)) and 83.2% (95% CI (77.6–87.9)), respectively. Using multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58% (95% CI 46.9–68.4) and 67% (95% CI 56.2–76.7); and 100% (95% CI 98–100) and 98.9% (95% CI 96–99.9), respectively. Substantial level of agreement was reported between microscopy and multiplex real time PCR results with kappa value of 0.65. Conclusions Multiplex real-time PCR had an advanced performance in parasite detection and species identification on febrile patients’ samples than did microscopy and RDT in low malaria transmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria elimination programme, particularly for community based epidemiological samples. Although microscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinical facilities.

Evaluation of an automated and highly sensitive real-time PCR assay for CMV DNA in whole blood samples

2006 ◽  
Vol 36 ◽  
pp. S26
Author(s):  
C. Deback ◽  
S. Akhavan ◽  
S. Blanc-Perrel ◽  
F. Dupuis ◽  
S. Schaffer ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Pcr Assay ◽  
Blood Samples ◽  
Highly Sensitive ◽  
Whole Blood Samples

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

scholarly journals Detection of Aspergillus species in bone marrow transplant patients

2010 ◽  
Vol 4 (08) ◽  
pp. 511-516 ◽  
Author(s):  
Parisa Badiee ◽  
Abdolvahab Alborzi
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Bone Marrow Transplant ◽  
Real Time Pcr ◽  
Clinical Signs ◽  
Marrow Transplant ◽  
Aspergillus Species ◽  
Pcr Assay ◽  
Blood Samples ◽  
Transplant Patients

Introduction:  Invasive aspergillosis is a severe complication of cytotoxic chemotherapies and bone marrow transplantation (BMT). The aim of this study was to assess the utility of a real-time PCR assay for the early diagnosis of Aspergillus species in blood samples from BMT patients. Methodology: Blood specimens (n = 993) from patients (n = 82) scheduled for BMT were collected prior to transplant and for 100 days post transplantation.  The specimens were later tested using an Aspergillus-specific real-time PCR assay. Cultures of clinical samples, along with sonography and computerized tomographic scans, were performed as standard of care. Results: Aspergillus DNA was positive in 94 sequential blood samples from 13 patients with clinical and radiological signs of infection. Samples from three of these patients were PCR-positive for Aspergillus in the first week of admission, prior to transplantation. Four patients with aspergillosis were cured with antifungal agents and nine died. An additional 12 patients without clinical signs of infection were PCR-positive on one occasion each, while two patients with clinical signs of infection were PCR-negative. Compared to routine methods of aspergillosis diagnosis, the respective sensitivity, specificity, negative, and positive predictive values of the PCR method by patient were 86.6%, 82%, 96.5% and 52%. Conclusions: The results show that Aspergillus infections in the blood of bone marrow transplant patients can be dectected by PCR methods. Early detection of Aspergillus infections by PCR has the potential to positively impact patient mortality rate and provide cost savings to hospitals.

Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test

2018 ◽  
Vol 56 (7) ◽  
pp. 1133-1139 ◽  
Author(s):  
Hanah Kim ◽  
Mina Hur ◽  
Eunsin Bae ◽  
Kyung-A Lee ◽  
Woo-In Lee
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Nucleic Acid Amplification ◽  
Coefficient Of Determination ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Cobas Taqman ◽  
Limit Of Quantitation ◽  
Two Systems

Abstract Background: Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). Methods: Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0–1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). Results: Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). Conclusions: The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

scholarly journals Characterization of a universal screening approach for congenital CMV infection based on a highly-sensitive, quantitative, multiplex real-time PCR assay

PLoS ONE ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. e0227143
Author(s):  
Angela Nagel ◽  
Emmanouela Dimitrakopoulou ◽  
Norbert Teig ◽  
Peter Kern ◽  
Thomas Lücke ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Universal Screening ◽  
Cmv Infection ◽  
Pcr Assay ◽  
Screening Approach ◽  
Highly Sensitive ◽  
Congenital Cmv Infection ◽  
Congenital Cmv

Paradigm for diagnosing mycobacterial disease: direct detection and differentiation of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in clinical specimens using multiplex real-time PCR

2018 ◽  
Vol 71 (9) ◽  
pp. 774-780 ◽  
Author(s):  
Jeong-Uk Kim ◽  
Dae-Shick Ryu ◽  
Choong-Hwan Cha ◽  
Seon-Hee Park
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Predictive Value ◽  
Direct Detection ◽  
Positive Culture ◽  
Nucleic Acid Amplification ◽  
Mycobacterial Disease ◽  
Pcr Assay ◽  
Clinical Specimens

AimsMycobacterium tuberculosis and non-tuberculous mycobacteria (NTM) are clinically different, and the rapid detection and differentiation of M. tuberculosis complex (MTBC) and NTM is crucial for patient management and infection control. Given the slow growth of most pathogenic mycobacteria, nucleic acid amplification assays are excellent tools for direct identification of mycobacteria in clinical specimens. Recently, a multiplex real-time PCR assay was developed that can directly detect 20 mycobacterial species in clinical specimens. Here, we evaluated the diagnostic performance of the assay for diagnosing mycobacterial disease under routine laboratory conditions.MethodsA total of 3334 specimens collected from 1437 patients suspected of tuberculosis infection were subjected to acid-fast bacilli staining, conventional culture and the multiplex real-time PCR assay. To evaluate the sensitivity and specificity of the assay, the overall diagnosis of tuberculosis was defined by positive culture plus medical history, and the 2007 American Thoracic Society and Infectious Disease Society of America diagnostic criteria for NTM disease were applied.ResultsThe sensitivity, specificity, positive predictive value and negative predictive value were 87.5%, 99.6%, 96.1% and 98.5%, respectively, for the detection of MTBC isolates and 53.3%, 99.9%, 95.2%, and 98.9%, respectively, for detecting NTM isolates.ConclusionsThus, the assay can correctly differentiate between MTBC and NTM isolates in clinical specimens and would be a useful tool for the rapid differentiation of tuberculosis and NTM disease, despite its limited sensitivity for the diagnosis of NTM disease.

Sign in / Sign up

Export Citation Format

Share Document