A comparison of PCR and ELISA methods to detect different stages of Plasmodium vivax in Anopheles arabiensis

Abstract Background In characterizing malaria epidemiology, measuring mosquito infectiousness informs the entomological inoculation rate, an important metric of malaria transmission. PCR-based methods have been touted as more sensitive than the current “gold-standard” circumsporozoite (CSP) ELISA. Wider application of PCR-based methods has been limited by lack of specificity for the infectious sporozoite stage. We compared a PCR method for detecting the parasite’s mitochondrial (mt) cytochrome oxidase I (COX-I) gene with ELISA for detecting circumsporozoite protein for identification of different life stages of the parasite during development within a mosquito. Methods A PCR-based method targeting the Plasmodium mt COX-I gene was compared with the CSP ELISA method to assess infectivity in Anopheles arabiensis colony mosquitoes fed on blood from patients infected with Plasmodium vivax. Mosquitoes were tested at six post-infection time points (days 0.5, 1, 6, 9, 12, 15). The head and thorax and the abdomen for each specimen were tested separately with each method. Agreement between methods at each infection stage was measured using Cohen’s kappa measure of test association. Results Infection status of mosquitoes was assessed in approximately 90 head/thorax and 90 abdomen segments at each time point; in total, 538 head/thorax and 534 abdomen segments were tested. In mosquitoes bisected after 0.5, 1, and 6 days post-infection (dpi), the mt COX-I PCR detected Plasmodium DNA in both the abdomen (88, 78, and 67%, respectively) and head/thorax segments (69, 60, and 44%, respectively), whilst CSP ELISA detected sporozoites in only one abdomen on day 6 post-infection. PCR was also more sensitive than ELISA for detection of Plasmodium in mosquitoes bisected after 9, 12, and 15 dpi in both the head and thorax and abdomen. There was fair agreement between methods for time points 9–15 dpi (κ = 0.312, 95% CI: 0.230–0.394). Conclusions The mt COX-I PCR is a highly sensitive, robust method for detecting Plasmodium DNA in mosquitoes, but its limited Plasmodium life-stage specificity cannot be overcome by bisection of the head and thorax from the abdomen prior to PCR. Thus, the mt COX-I PCR is a poor candidate for identifying infectious mosquitoes. Graphical Abstract

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A Comparison of PCR and ELISA Methods to Detect Different Stages of Plasmodium Vivax in Anopheles Arabiensis.

10.21203/rs.3.rs-589001/v1 ◽

2021 ◽

Author(s):

Allison L Hendershot ◽

Endashaw Esayas ◽

Alice C. Sutcliffe ◽

Seth R. Irish ◽

Endalamaw Gadisa ◽

...

Keyword(s):

Plasmodium Vivax ◽

Post Infection ◽

Anopheles Arabiensis ◽

Life Stage ◽

Malaria Epidemiology ◽

Highly Sensitive ◽

Pcr Method ◽

Infection Stage ◽

I Gene ◽

Time Point

Abstract Background: In characterizing malaria epidemiology, measuring mosquito infectiousness informs the entomological inoculation rate, an important metric of malaria transmission. PCR-based methods have been touted as more sensitive than the current “gold-standard” circumsporozoite (CSP) ELISA. Wider application of PCR-based methods has been limited by lack of specificity for the infectious sporozoite stage. We compared a PCR method for detecting the parasite’s cytochrome oxidase I (COX-I) gene with ELISA for detecting circumsporozoite protein for identification of different life stages of the parasite during development within a mosquito. Methods: A PCR-based method targeting the Plasmodium COX-I gene was compared with the CSP ELISA method to assess infectivity in Anopheles arabiensis colony mosquitoes fed on blood from patients infected with Plasmodium vivax. Mosquitoes were tested at six post-infection timepoints (days 0.5, 1, 6, 9, 12, 15). Head and thoraces, and abdomens for each specimen were tested separately with both methods. Agreement between methods at each infection stage was measured using Cohen’s Kappa measure of test association.Results: Infection status of mosquitoes was assessed in approximately 90 head and thoraces and 90 abdomens at each time point; in total 538 head and thoraces and 534 abdomens were tested. In mosquitoes bisected after 0.5, 1-, and 6-days post-infection, the COX-I PCR detected Plasmodium DNA in both the abdomens (88%, 78%, and 67%, respectively) and head and thoraces (69%, 60%, and 44%, respectively) whilst CSP-ELISA detect sporozoites in only 1 abdomen on day 6 post-infection. PCR was also more sensitive than ELISA for detection of Plasmodium in mosquitoes bisected after 9-, 12-, and 15-days post-infection in both the head and thoraces and abdomens. There was fair agreement between both methods for time points 9 -15 days post-infection (κ = 0.312, 95% CI: 0.230 – 0.394). Conclusion: The COX-I PCR is a highly sensitive, robust method for detecting Plasmodium DNA in mosquitoes, but its limited Plasmodium life-stage specificity cannot be overcome by bisection of the head and thorax from the abdomen prior to PCR. Thus, the COX-I PCR is a poor candidate for identifying infectious mosquitoes.

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A fast and highly sensitive blood culture PCR method for clinical detection of Salmonella enterica serovar Typhi

Annals of Clinical Microbiology and Antimicrobials ◽

10.1186/1476-0711-9-14 ◽

2010 ◽

Vol 9 (1) ◽

pp. 14 ◽

Cited By ~ 34

Author(s):

Liqing Zhou ◽

Andrew J Pollard

Keyword(s):

Blood Culture ◽

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhi ◽

Highly Sensitive ◽

Clinical Detection ◽

Pcr Method

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Evaluation of Putative Type II Toxin-Antitoxin Systems and Lon Protease Expression in Shigella flexneri Following Infection of Caco-2 Cells

Archives of Clinical Infectious Diseases ◽

10.5812/archcid.98625 ◽

2020 ◽

Vol 15 (3) ◽

Author(s):

Erfan Kheradmand ◽

Shabnam Razavi ◽

Malihe Talebi ◽

Mahmood Jamshidian

Keyword(s):

Cell Line ◽

Post Infection ◽

Shigella Flexneri ◽

Fold Increase ◽

Expression Level ◽

Bacterial Invasion ◽

P Value ◽

Lon Protease ◽

Expression Levels ◽

Pcr Method

: Shigella flexneri causes bacillary dysentery in developing countries. Due to recent reports regarding antimicrobial resistance in human S. flexneri, finding alternative therapeutics is of vital importance. Toxin-antitoxin (TA) systems have recently been introduced as antimicrobial targets owing to their involvement in bacterial survival in stress conditions and “persister” cell formation. In this study, the presence of four TA loci were studied in S. flexneri ATCC 12022. The presence of genes coding for the identified TA loci and Lon protease were confirmed by the PCR method using specific primers. Caco-2 cell lines were then infected with this standard strain, and 8 and 24 h post-infection, expression levels of genes coding for the studied TA loci, and Lon protease were evaluated using a real-time PCR method. Expression of mazF, GNAT (Gcn5-related N-acetyltransferase), yeeU, pfam13975, and Lon genes showed 5.4, 9.8, 2.3, 2.7, and 13.8-fold increase, respectively, 8 h after bacterial invasion of the Caco-2 cell line. In addition, the expression of the aforementioned genes showed 4.8, 10.8, 2.3, 3.7, and 16.8-fold increase after 24 h. The GNAT and lon genes showed significantly higher expression levels compared to the control (P value < 0.05). However, the increase in the expression level of yeeU was the same at 8 h and 24 h post-infection. In addition, mazF expression level showed a slight decrease at 24 h compared to 8h post-infection. Genes coding for GNAT and Lon protease showed a significantly higher expression after invading the Caco-2 cell line. Therefore, targeting GNAT or Lon protease can be taken into consideration for finding novel antimicrobial drug strategies. The exact functions and mechanisms of TA systems in S. flexneri isolates are suggested to be experimentally determined.

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Highly Sensitive Single Molecular Array Immunoassay Measurement of t-Tau, NF-L, GFAP, and UCH-L1 Biomarkers in Acute Concussion/Mild Traumatic Brain Injury (mTBI) Patient Serum and Saliva Samples

Neurology ◽

10.1212/01.wnl.0000581020.28808.bd ◽

2019 ◽

Vol 93 (14 Supplement 1) ◽

pp. S19.3-S20

Author(s):

Ahmed Chenna ◽

Christos Petropoulos ◽

John Winslow

Keyword(s):

Traumatic Brain Injury ◽

Brain Injury ◽

Mild Traumatic Brain Injury ◽

Limited Information ◽

Post Injury ◽

Time Points ◽

Highly Sensitive ◽

Biomarker Analysis ◽

T Tau ◽

Control Samples

ObjectiveTo determine if t-Tau, NF-L, GFAP and UCH-L1 protein biomarkers are elevated in early time points of acute concussion/mild traumatic brain injury patient serum and saliva, relative to control samples.Backgroundt-Tau, NF-L, GFAP and UCH-L1 levels have been reported to increase in cerebral spinal fluid (CSF) and blood following head trauma within 24 hours or longer, and are candidate diagnostic and prognostic biomarkers of concussion and mild to moderate TBI. However, limited information exists on the relationship between these biomarkers at short time points post-injury, and detectability in saliva of mTBI patients.Design/MethodsBiomarker analysis of serum from a total of 120 participants, derived from two independent sample groups consisting of 60 concussion/mTBI patients each, with blood collected within 1-4 hr and 8-16 hr post-injury, respectively, was compared with 30 healthy control sera. Saliva samples were collected after 8-16 hr post-injury from a n = 30 subset of the same patients. Quanterix Simoa 4-plex immunoassay was used for highly sensitive measurements of these biomarkers.ResultsMedian levels of NF-L, GFAP and UCH-L1 were significantly higher in independent sets of patient serum samples (n = 60 each), both at early (1–4 hr) and later (8–16 hr) time points post-mTBI/concussion, relative to control samples (n = 30) (p < 0.0001, = 0.0001, <0.0001, respectively). Low levels of t-Tau are detected, but are significantly elevated post-concussion relative to controls (p = 0.0001). Significant correlations were observed between levels of t-Tau and UCH-L1, NF-L and GFAP, and t-Tau and GFAP in both post-injury time-point groups, and between NF-L and UCH-L1 levels in the 8-16 hr group. The four biomarkers were detected in saliva from concussion/mTBI patients (n = 30).ConclusionsThis study supports the utility of ultra-sensitive multiplex immunoassays to detect increases in CNS proteins at high sensitivity in serum and saliva within 1-4 and 8-16 hr of concussion/mTBI.

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Riemerella anatipestifer infection in ducks induces IL-17A production, but not IL-23p19

Scientific Reports ◽

10.1038/s41598-019-49516-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Rochelle A. Flores ◽

Cherry P. Fernandez-Colorado ◽

Fahmida Afrin ◽

Paula Leona T. Cammayo ◽

Suk Kim ◽

...

Keyword(s):

Mrna Expression ◽

Proinflammatory Cytokines ◽

Post Infection ◽

Proinflammatory Cytokine ◽

Th17 Cells ◽

Critical Role ◽

Splenic Lymphocytes ◽

Expression Levels ◽

Time Points ◽

Mrna Expression Analysis

Abstract R. anatipestifer (RA) is one of the most harmful bacterial pathogens affecting the duck industry, and infection is associated with the production of proinflammatory cytokines, including IL-17A. Another proinflammatory cytokine, IL-23, is critical for the development of Th17 cells, which produce IL-17. However, IL-23 roles have not been studied in this infection. Here, we describe the identification and mRNA expression analysis of duck IL-23p19 (duIL-23p19) in splenic lymphocytes and macrophages stimulated with killed RA and in spleens of RA-infected ducks. Expression of duIL-23p19 transcript identified in this study was relatively high in livers of healthy ducks and was upregulated in mitogen-activated splenic lymphocytes as well as in splenic lymphocytes and macrophages stimulated with killed RA. In spleens of RA-infected ducks, expression levels of duIL-23p19 transcript were unchanged at all time points except on days 4 and 7 post-infection; however, duIL-17A and IL-17F expression levels were upregulated in both spleens of RA-infected ducks and splenic lymphocytes and macrophages stimulated with killed RA. In sera collected at 24 h after this infection, duIL-23p19 expression levels were unchanged, whereas IL-17A significantly upregulated. These results suggest that IL-23p19 does not play a critical role in the IL-17A response in early stages of RA-infected ducks.

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Rapid identification and differentiation of fungal DNA in dermatological specimens by LightCycler PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.45779-0 ◽

2004 ◽

Vol 53 (12) ◽

pp. 1207-1214 ◽

Cited By ~ 29

Author(s):

Ralf Gutzmer ◽

Susanne Mommert ◽

Uta Küttler ◽

Thomas Werfel ◽

Alexander Kapp

Keyword(s):

Skin Diseases ◽

Rapid Identification ◽

Diagnostic Information ◽

Clinical Samples ◽

Melting Points ◽

Number Of Patients ◽

Highly Sensitive ◽

Fungal Dna ◽

Pcr Method ◽

Primer Sets

The aim was to develop a LightCycler PCR method for the rapid detection and differentiation of fungal DNA in dermatological specimens such as skin scales and skin swabs. LightCycler PCR assays were established for seven primer sets specific for fungal DNA. For each primer set LightCycler melting points were defined by amplification of DNA from 21 fungi and sensitivity was determined by amplification of serial dilutions of fungal DNA. A protocol was established that allows detection and differentiation of mould and yeast DNA with one highly sensitive PCR reaction by assessment of LightCycler melting points. Two subsequent LightCycler PCR reactions and one RFLP reaction allowed the differentiation of dermatophytes and non-dermatophyte moulds and the subclassification of yeasts. Analysis of clinical samples from 38 patients with fungal skin diseases provided conclusive new diagnostic information in 9/38 cases (23.7 %) by this PCR protocol that was not equally provided by direct microscopy and mycological culture. Thus the LightCycler PCR protocol established here represents a rapid diagnostic tool that aids in the diagnosis of fungal skin disease in a substantial number of patients.

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Comparison of parasitological, immunological and molecular methods for evaluation of fecal samples of immunosuppressed rats experimentally infected with Strongyloides venezuelensis

Parasitology ◽

10.1017/s0031182015001298 ◽

2015 ◽

Vol 142 (14) ◽

pp. 1715-1721 ◽

Cited By ~ 2

Author(s):

LEILANE A. CHAVES ◽

ANA LÚCIA R. GONÇALVES ◽

FABIANA M. PAULA ◽

NEIDE. M. SILVA ◽

CLÁUDIO V. SILVA ◽

...

Keyword(s):

Post Infection ◽

Pcr Amplification ◽

Conventional Polymerase Chain Reaction ◽

Diagnostic Value ◽

Diagnostic Methods ◽

Enzyme Linked Immunosorbent Assay ◽

Fecal Samples ◽

Infection Time ◽

Time Points ◽

Strongyloides Venezuelensis

SUMMARYDefinitive diagnosis of strongyloidiasis in humans is typically achieved by detection of larvae in fecal samples. However, limitations on sensitivity of parasitological methods emphasize the need for more robust diagnostic methods. The aim of this study was to compare the diagnostic value of three methods: eggs per gram of feces (EPG), coproantigen detection by enzyme linked immunosorbent assay (ELISA), and DNA detection by conventional polymerase chain reaction (PCR). The assays were performed at 0 and 5, 8, 13, 21 and 39 days post-infection (dpi) using fecal samples from experimentally infected immunocompetent and immunosuppressed rats. In immunocompetent rats, eggs were detected in feces on days 5, 8 and 13 dpi; coproantigen detection and PCR amplification were successful at all post-infection time points (5, 8, 13, 21 and 39 dpi). In immunosuppressed rats, eggs were detected at 5, 8, 13 and 21; coproantigen detection and PCR amplification were successful at all post-infection time points. In conclusion, these results suggest that coproantigen detection and PCR may be more sensitive alternatives to traditional methods such as EPG for diagnosis of Strongyloides venezuelensis infection.

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Maps and missing malaria – if in doubt request a blood film

Acute Medicine Journal ◽

10.52964/amja.0275 ◽

2013 ◽

Vol 12 (1) ◽

pp. 18-20

Author(s):

Victoria Adewole ◽

◽

Sarah Logan ◽

Ramsay Singer ◽

Ruth Kinson ◽

...

Keyword(s):

Plasmodium Vivax ◽

Clinical Picture ◽

Vivax Malaria ◽

Endemic Area ◽

Delayed Diagnosis ◽

Blood Film ◽

Malaria Endemic Area ◽

Malaria Epidemiology ◽

Plasmodium Vivax Malaria ◽

The Uk

The severe sequelae of infection from the conventionally termed ‘benign’ forms of malaria are being increasingly recognised, and delayed diagnosis and treatment lead to worse outcomes. The clinical picture can be non-specific and malaria epidemiology is constantly changing, presenting challenges for the acute clinician. The most critical step in the diagnosis of patients presenting in the UK is the clinician’s awareness of the disease and its key presenting features. We describe a case of Plasmodium vivax malaria in a young man who presented with fever and diarrhoea, who had never travelled to a recognised malaria-endemic area.

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