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

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.

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

10.21203/rs.3.rs-572301/v1 ◽

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.

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

Journal of Clinical Virology ◽

10.1016/s1386-6532(06)80794-8 ◽

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

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Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0002000 ◽

2013 ◽

Vol 7 (1) ◽

pp. e2000 ◽

Cited By ~ 127

Author(s):

Tomas Duffy ◽

Carolina I. Cura ◽

Juan C. Ramirez ◽

Teresa Abate ◽

Nelly M. Cayo ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Real Time ◽

Real Time Pcr ◽

Satellite Dna ◽

Analytical Performance ◽

Pcr Assay ◽

Blood Samples ◽

Taqman Probes

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A new highly sensitive single-tube nested real-time PCR assay: Clinical utility in perinatal HIV-1 diagnosis

Journal of Virological Methods ◽

10.1016/j.jviromet.2021.114273 ◽

2021 ◽

Vol 297 ◽

pp. 114273

Author(s):

Matias Moragas ◽

Marcelo D. Golemba ◽

Andrea Mangano

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clinical Utility ◽

Pcr Assay ◽

Single Tube ◽

Perinatal Hiv ◽

Highly Sensitive ◽

Hiv 1

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

Journal of Food Protection ◽

10.4315/0362-028x-70.5.1080 ◽

2007 ◽

Vol 70 (5) ◽

pp. 1080-1087 ◽

Cited By ~ 63

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.

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Detection of Aspergillus species in bone marrow transplant patients

The Journal of Infection in Developing Countries ◽

10.3855/jidc.807 ◽

2010 ◽

Vol 4 (08) ◽

pp. 511-516 ◽

Cited By ~ 12

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.

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

PLoS ONE ◽

10.1371/journal.pone.0227143 ◽

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

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Detection and species identification of microsporidial infections using SYBR Green real-time PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.026781-0 ◽

2011 ◽

Vol 60 (4) ◽

pp. 459-466 ◽

Cited By ~ 29

Author(s):

Spencer D. Polley ◽

Samuel Boadi ◽

Julie Watson ◽

Alan Curry ◽

Peter L. Chiodini

Keyword(s):

Real Time ◽

Species Identification ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Enterocytozoon Bieneusi ◽

Sybr Green ◽

Pcr Assay ◽

Real Time Analysis ◽

Highly Sensitive ◽

Stool Samples

Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample)−1, whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 Enterocytozoon bieneusi, one Encephalitozoon cuniculi and two separate Pleistophora species infections.

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Development of a duplex real-time PCR assay for the differentiation of Blastomyces dermatitidis and B. gilchristii and a retrospective study of blastomycosis in New York (2005-2019)

10.1101/2020.08.11.20172932 ◽

2020 ◽

Author(s):

Mitchell Kaplan ◽

YanChun Zhu ◽

Julianne V Kus ◽

Lisa McTaggart ◽

Vishnu Chaturvedi ◽

...

Keyword(s):

New York ◽

Retrospective Study ◽

North America ◽

Real Time ◽

Real Time Pcr ◽

Blastomyces Dermatitidis ◽

Pcr Assay ◽

Rare Finding ◽

Clinical Specimens ◽

Highly Sensitive

Blastomycosis due to Blastomyces dermatitidis and B. gilchristii is a notable cause of respiratory mycoses in North America with recurrent outbreaks. We developed a highly sensitive, specific, and reproducible Taqman duplex real-time PCR assay for the differentiation of B. dermatitidis and B. gilchristii. The new assay permitted retrospective analysis of Blastomyces cultures (2005 to 2019), and primary clinical specimens (2013-2019) from NY patients. Blastomyces dermatitidis was the causal agent for the majority of 38 cases while B. gilchristii was implicated in five cases; a rare finding reported from New York. The duplex real-time PCR assay will be useful for further understanding of ecology and epidemiology of blastomycosis caused by B. dermatitidis and B. gilchristii.

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