scholarly journals Assessment of Microscopic detection of Malaria with Nested Polymerase Chain Reaction in War-torn Federally Administered Tribal Areas of Pakistan

2021 ◽  
Author(s):  
Muhammad Faisal Nadeem ◽  
Aamer Ali Khattak ◽  
Adnan Yaqoob ◽  
Usman Ayub Awan ◽  
Nadia Zeeshan
Keyword(s):  
Diagnostic Accuracy ◽  
Sensitivity And Specificity ◽  
Mixed Infection ◽  
Microscopic Examination ◽  
Nested Pcr ◽  
Venous Blood ◽  
Pcr Analysis ◽  
Microscopic Detection ◽  
Tribal Areas ◽  
Pcr Targeting

Abstract Background: Diagnostic accuracy of malaria is critical for early treatment, control, and elimination of malaria, especially in war-affected malaria endemic areas. Microscopic detection of Plasmodium species has been the gold standard in remote malaria-endemic regions. However, the diagnostic accuracy is still questioned, especially in discriminating mixed and submicroscopic parasitic levels. This study was designed to evaluate the diagnostic performance of microscopic examination against nested PCR analysis in war-torn malaria-endemic Federally Administered Tribal Areas (FATA) of Pakistan. Methods: Venous blood samples were collected from symptomatic patients for microscopic examination and nested PCR analysis from January 2016 - December 2016 from five Agencies (Bajaur, Mohmand, Khyber, Orakzai and Kurram Agency) and four Frontier Regions (Peshawar, Kohat, Bannu, and Dera Ismail Khan Frontier Region) of FATA. Malaria-positive isolates were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes) for speciation. Results: Among enrolled participants, 762 were found positive for malaria parasite on microscopic examination of the blood film. P. vivax was found in 623, P. falciparum in 132 and 7 were diagnosed with mixed infection (P. vivax and P. falciparum coinfection). Nested PCR detected Plasmodium infection in 679 samples (523 P. vivax, 121 P. falciparum, and 35 mixed infections). Compared with microscopy, the sensitivity of nested PCR was 98.94%, and specificity was 98.27%, while the sensitivity and specificity of slide microscopy 89.34% and 87.99% respectively. Conclusion: The conventional microscopy method has low sensitivity to detect mixed infection as compared to nested PCR. High sensitivity and specificity observed in nested PCR makes this molecular tool a useful technique for monitoring, controlling, and eliminating malaria-endemic regions.

scholarly journals Comparison and diagnosis of Entamoeba in stool sample from rural community of Nepal

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Sandeep Thapa
Keyword(s):  
Stool Sample ◽  
Entamoeba Histolytica ◽  
Rural Community ◽  
Microscopic Examination ◽  
Nested Pcr ◽  
Health Knowledge ◽  
Intestinal Parasites ◽  
Intestinal Parasitosis ◽  
Microbiological Methods ◽  
Pcr Targeting

ObjectiveTo find out the prevalence of Entamoeba species in rural community of Nepal.The purpose of the study is to evaluate Nested PCR, microscopic examination and Elisa assay for detection and differentiation of Entamoeba species.IntroductionNepal being a developing country has many health problems, which escalate in intensity at different times of the year or in epidemic form.Amebiasis is one of the infectious diseases that is highly seen in rural area of Nepal caused by Entamoeba species ((1, 2). Recent reports show that open defecation, drinking untreated water, unsanitary habits and lack of basic health knowledge cause higher mortality and morbidity in our country.E. histolytica is an anaerobic pathogenic parasitic. However, E. dispar and E.moshkovskii exits as non-pathogenic. Likewise, E. histolytica, E. dispar and E. moshkovskii are morphologically identical but genetically distinct species (3).MethodsA total of 270 faecal sample were collected from south eastern terai region of Nepal after the informed consent form. The samples were processed by direct wet smear and formalin ethyl acetate concentration technique (4). Eventually, microscopic examination were performed for the detection of Entamoeba species along with other intestinal parasites. Furthermore, enzyme immunoassay were executed to detect antigens of E. histolytica through ELISA. Additionally, microscopically positive samples for Entamoeba species cysts were further characterized using a Nested- PCR targeting 16S-like ribosomal RNA gene (5). The PCR generate amplicons which was subjected to 2% agarose gels electrophoresis and visualized under UV transilluminator.Results8.52% of the total collected samples were microscopically positive for Entamoeba cysts either singly or in combination with other intestinal parasites. Likewise, among 270 stool sample, viral diarrheal was most significant form of diarrhoea found in 76.67% of patients. Among different organisms, As. Lumbricoids and E. histolytica, G. lambia and H. nana were identified in most of the patients accounting for 11.11%, 8.52%, 2.59% and 1.11% respectively. However, Lumbricoids, G. lambia, Tenia solium and E. histolytica were present in an individual patient while two patient was found with both As. Lumbricoids and G. lambia. Among several symptoms, diarrhoea seems to be the common symptoms infecting all of the patients which is followed by fever and vomiting which accounts for 55.1 % and 46.2% correspondingly. Whereas, nausea appears to be the least common symptoms infecting only 14.4% of patients.Subsequently, 56 cases were PCR positive, 51 cases were ELISA positive whereas 47 were found to be positive by microscopy.ConclusionsMolecular techniques are indeed promising tools for epidemiological studies, particularly in discriminating the pathogenic from the non-pathogenic species of the Entamoeba species. This study reports a new nested multiplex PCR strategy for detection and differentiation of E. histolytica, E. dispar and E. moshkovskii which is highly rapid, specific and sensitive which is useful for proper diagnosis, immunological assay and drug testing.References1. Chaudhary M, Maharjan M. ( 2014). Association of Anaemia with Parasitic Infection in Pregnant Women Attending Antenatal Clinic at Koshi Zonal Hospital. Neplalese Journal of Zoology, 2(1):1-7.2. Tandukar S, Ansari S, Adhikari N, Shrestha A, Gautam J, Sharma B, et al (2013). Intestinal parasitosis in school children of Lalitpur district of Nepal. BMC research notes, 6(1):449.3. Clark C, Espinosa Cantellano M, Bhattacharya A. Entamoeba histolytica: an overview of the biology of the organism (2013). Amebiasis: World Scientific, 1-45.4. Dhanabal J, Selvadoss PP, Muthuswamy K(2014). Comparative study of the prevalence of intestinal parasites in low socioeconomic areas from South Chennai, India. Journal of parasitology research.5. Van den Bossche, D., Cnops, L., Verschueren, J., & Van Esbroeck, M. (2015). Comparison of four rapid diagnostic tests, ELISA, microscopy and PCR for the detection of Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in feces. Journal of microbiological methods, 110, 78-84.

scholarly journals Fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum clinical isolates collected from unrest tribal agencies of Pakistan

2023 ◽  
Vol 83 ◽  
Author(s):  
M. F. Nadeem ◽  
N. Zeeshan ◽  
A. A. Khattak ◽  
U. A. Awan ◽  
A. Yaqoob
Keyword(s):  
Plasmodium Falciparum ◽  
Nested Pcr ◽  
Resistant Mutant ◽  
Small Subunit ◽  
Clinical Isolates ◽  
Chloroquine Resistance ◽  
Mortality And Morbidity ◽  
Blood Smears ◽  
Tribal Areas ◽  
Pcr Targeting

Abstract Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.

scholarly journals Field validation of a magneto-optical detection device (Gazelle) for portable point-of-care Plasmodium vivax diagnosis

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0253232
Author(s):  
Hugo O. Valdivia ◽  
Priyaleela Thota ◽  
Greys Braga ◽  
Leonila Ricopa ◽  
Keare Barazorda ◽  
...  
Keyword(s):  
Diagnostic Accuracy ◽  
Sensitivity And Specificity ◽  
Nested Pcr ◽  
Diagnostic Tests ◽  
Point Of Care ◽  
Malaria Diagnosis ◽  
Final Analysis ◽  
Rapid Diagnostic Tests ◽  
Field Validation ◽  
Pcr Assay

A major challenge for malaria is the lack of tools for accurate and timely diagnosis in the field which are critical for case management and surveillance. Microscopy along with rapid diagnostic tests are the current mainstay for malaria diagnosis in most endemic regions. However, these methods present several limitations. This study assessed the accuracy of Gazelle, a novel rapid malaria diagnostic device, from samples collected from the Peruvian Amazon between 2019 and 2020. Diagnostic accuracy was compared against microscopy and two rapid diagnostic tests (SD Bioline and BinaxNOW) using 18ssr nested-PCR as reference test. In addition, a real-time PCR assay (PET-PCR) was used for parasite quantification. Out of 217 febrile patients enrolled and tested, 180 specimens (85 P. vivax and 95 negatives) were included in the final analysis. Using nested-PCR as the gold standard, the sensitivity and specificity of Gazelle was 88.2% and 97.9%, respectively. Using a cutoff of 200 parasites/μl, Gazelle’s sensitivity for samples with more than 200 p/uL was 98.67% (95%CI: 92.79% to 99.97%) whereas the sensitivity for samples lower than 200 p/uL (n = 10) was 12.5% (95%CI: 0.32% to 52.65%). Gazelle’s sensitivity and specificity were statistically similar to microscopy (sensitivity = 91.8, specificity = 100%, p = 0.983) and higher than both SD Bioline (sensitivity = 82.4, specificity = 100%, p = 0.016) and BinaxNOW (sensitivity = 71.8%, specificity = 97.9%, p = 0.002). The diagnostic accuracy of Gazelle for malaria detection in P. vivax infections was comparable to light microscopy and superior to both RDTs even in the presence of low parasitemia infections. The performance of Gazelle makes it a valuable tool for malaria diagnosis and active case detection that can be utilized in different malaria-endemic regions.

scholarly journals Optimal Range of Fecal Calprotectin for Predicting Mucosal Healing in Patients with Inflammatory Bowel Disease: A Systematic Review and Meta-Analysis

2021 ◽  
pp. 1-11
Author(s):  
Bing-Jie Xiang ◽  
Min Jiang ◽  
Ming-Jun Sun ◽  
Cong Dai
Keyword(s):  
Inflammatory Bowel Disease ◽  
Diagnostic Accuracy ◽  
Likelihood Ratio ◽  
Sensitivity And Specificity ◽  
Bowel Disease ◽  
Meta Analysis ◽  
Positive Likelihood Ratio ◽  
Fecal Calprotectin ◽  
Mucosal Healing ◽  
Inflammatory Bowel

<b><i>Objective:</i></b> Fecal calprotectin (FC) is a promising marker for assessment of inflammatory bowel disease (IBD) activity. However, the utility of FC for predicting mucosal healing (MH) of IBD patients has yet to be clearly demonstrated. The objective of our study was to perform a meta-analysis evaluating the diagnostic accuracy of FC in predicting MH of IBD patients. <b><i>Methods:</i></b> We systematically searched the databases for studies from inception to April 2020 that evaluated MH in IBD. The methodological quality of each study was assessed according to the Quality Assessment of Diagnostic Accuracy Studies checklist. The extracted data were pooled using a summary receiver operating characteristic curve model. Random-effects model was used to summarize the diagnostic odds ratio, sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio. <b><i>Results:</i></b> Sixteen studies comprising 1,682 ulcerative colitis (UC) patients and 4 studies comprising 221 Crohn’s disease (CD) patients were included. The best performance of FC for predicting MH in UC was at cut-off range of 60–75 μg/g with area under the curve (AUC) of 0.88 and pooled sensitivity and specificity of 0.87 and 0.79, respectively. The pooled sensitivity and specificity values of cutoff range 180–250 μg/g for predicting MH in CD were 0.67 and 0.76, respectively. The AUC of 0.79 also revealed improved discrimination for identifying MH in CD with FC concentration. <b><i>Conclusion:</i></b> Our meta-analysis has found that FC is a simple, reliable noninvasive marker for predicting MH in IBD patients. FC cutoff range 60–75 μg/g appears to have the best overall accuracy in UC patients.

Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

2021 ◽  
pp. 030098582199156
Author(s):  
Alexandra N. Myers ◽  
Unity Jeffery ◽  
Zachary G. Seyler ◽  
Sara D. Lawhon ◽  
Aline Rodrigues Hoffmann
Keyword(s):  
Polymerase Chain Reaction ◽  
Diagnostic Accuracy ◽  
Molecular Techniques ◽  
Fungal Culture ◽  
Chain Reaction ◽  
Identification Of Fungi ◽  
Fungal Dna ◽  
Polymerase Chain ◽  
Panfungal Pcr ◽  
Pcr Targeting

Molecular techniques are increasingly being applied to stained cytology slides for the diagnosis of neoplastic and infectious diseases. Such techniques for the identification of fungi from stained cytology slides have not yet been evaluated. This study aimed to assess the diagnostic accuracy of direct (without nucleic acid isolation) panfungal polymerase chain reaction (PCR) followed by sequencing for identification of fungi and oomycetes on stained cytology slides from dogs, cats, horses, and other species. Thirty-six cases were identified with cytologically identifiable fungi/oomycetes and concurrent identification via fungal culture or immunoassay. Twenty-nine controls were identified with no cytologically or histologically visible organisms and a concurrent negative fungal culture. Direct PCR targeting the internal transcribed spacer region followed by sequencing was performed on one cytology slide from each case and control, and the sensitivity and specificity of the assay were calculated. The sensitivity of the panfungal PCR assay performed on stained cytology slides was 67% overall, 73% excluding cases with oomycetes, and 86% when considering only slides with abundant fungi. The specificity was 62%, which was attributed to amplification of fungal DNA from control slides with no visible fungus and negative culture results. Direct panfungal PCR is capable of providing genus- or species-level identification of fungi from stained cytology slides. Given the potential of panfungal PCR to amplify contaminant fungal DNA, this assay should be performed on slides with visible fungi and interpreted in conjunction with morphologic assessment by a clinical pathologist.

Diagnostic accuracy of serum inflammatory markers in late fracture-related infection

2018 ◽  
Vol 100-B (12) ◽  
pp. 1542-1550 ◽  
Author(s):  
J. van den Kieboom ◽  
P. Bosch ◽  
J. D. J. Plate ◽  
F. F. A. IJpma ◽  
R. Kuehl ◽  
...  
Keyword(s):  
Diagnostic Accuracy ◽  
Sensitivity And Specificity ◽  
Inflammatory Markers ◽  
Meta Analysis ◽  
Diagnostic Value ◽  
Mixed Effects Models ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Erythrocyte Sedimentation ◽  
Serum Inflammatory Markers

Aims To assess the diagnostic value of C-reactive protein (CRP), leucocyte count (LC), and erythrocyte sedimentation rate (ESR) in late fracture-related infection (FRI). Materials and Methods PubMed, Embase, and Cochrane databases were searched focusing on the diagnostic value of CRP, LC, and ESR in late FRI. Sensitivity and specificity combinations were extracted for each marker. Average estimates were obtained using bivariate mixed effects models. Results A total of 8284 articles were identified but only six were suitable for inclusion. Sensitivity of CRP ranged from 60.0% to 100.0% and specificity from 34.3% to 85.7% in all publications considered. Five articles were pooled for meta-analysis, showing a sensitivity and specificity of 77.0% and 67.9%, respectively. For LC, this was 22.9% to 72.6%, and 73.5% to 85.7%, respectively, in five articles. Four articles were pooled for meta-analysis, resulting in a 51.7% sensitivity and 67.1% specificity. For ESR, sensitivity and specificity ranged from 37.1% to 100.0% and 59.0% to 85.0%, respectively, in five articles. Three articles were pooled in meta-analysis, showing a 45.1% sensitivity and 79.3% specificity. Four articles analyzed the value of combined inflammatory markers, reporting an increased diagnostic accuracy. These results could not be pooled due to heterogeneity. Conclusion The serum inflammatory markers CRP, LC, and ESR are insufficiently accurate to diagnose late FRI, but they may be used as a suggestive sign in its diagnosis.

scholarly journals The Sensitivity and Specificity of a Red Blood Cell Agglutination D-Dimer Assay for Venous Thromboembolism When Performed on Venous Blood

2002 ◽  
Vol 162 (2) ◽  
pp. 217 ◽  
Author(s):  
Sanjeev D. Chunilal ◽  
Patrick A. Brill-Edwards ◽  
Pamela B. Stevens ◽  
Jody P. Joval ◽  
Joanne A. McGinnis ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Blood Cell ◽  
Sensitivity And Specificity ◽  
Red Blood Cell ◽  
Venous Blood ◽  
Cell Agglutination ◽  
D Dimer

scholarly journals PRe-hospital Evaluation of Sensitive TrOponin (PRESTO) Study: multicentre prospective diagnostic accuracy study protocol

BMJ Open ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. e032834 ◽  
Author(s):  
Abdulrhman Alghamdi ◽  
Eloïse Cook ◽  
Edward Carlton ◽  
Aloysius Siriwardena ◽  
Mark Hann ◽  
...  
Keyword(s):  
Chest Pain ◽  
Hospital Admission ◽  
Diagnostic Accuracy ◽  
Blood Sample ◽  
Acute Coronary Syndromes ◽  
Venous Blood ◽  
Prehospital Setting ◽  
Registration Number ◽  
Coronary Syndromes ◽  
Rule Out

IntroductionWithin the UK, chest pain is one of the most common reasons for emergency (999) ambulance calls and the most common reason for emergency hospital admission. Diagnosing acute coronary syndromes (ACS) in a patient with chest pain in the prehospital setting by a paramedic is challenging. The Troponin-only Manchester Acute Coronary Syndromes (T-MACS) decision rule is a validated tool used in the emergency department (ED) to stratify patients with suspected ACS following a single blood test.We are seeking to evaluate the diagnostic accuracy of the T-MACS decision aid algorithm to ‘rule out’ ACS when used in the prehospital environment with point-of-care troponin assays. If successful, this could allow paramedics to immediately rule out ACS for patients in the ‘very low risk’ group and avoid the need for transport to the ED, while also risk stratifying other patients using a single blood sample taken in the prehospital setting.Methods and analysisWe will recruit patients who call emergency (999) ambulance services where the responding paramedic suspects cardiac chest pain. The data required to apply T-MACS will be prospectively recorded by paramedics who are responding to each patient. Paramedics will be required to draw a venous blood sample at the time of arrival to the patient. Blood samples will later be tested in batches for cardiac troponin, using commercially available troponin assays. The primary outcome will be a diagnosis of acute myocardial infarction, established at the time of initial hospital admission. The secondary outcomes will include any major adverse cardiac events within 30 days of enrolment.Ethics and disseminationThe study obtained approval from the National Research Ethics Service (reference: 18/ES/0101) and the Health Research Authority. We will publish our findings in a high impact general medical journal.Trial registration numberRegistration number: ClinicalTrials.gov, study ID: NCT03561051

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