Optimization of a molecular method for the diagnosis of canine babesiosis

Babesiosis is a hemolytic disease caused by protozoans of the genus Babesia (Apicomplexa). This disease occurs worldwide and is transmitted by ticks to a variety of mammals, including humans. The objective of the present study was to optimize a molecular approach for the detection of a fragment of 18S rDNA of Babesia canis, Babesia vogeli, Babesia rossi or Babesia gibsoni based on a single semi-nested Polymerase Chain Reaction (PCR), and compare the efficiency of this approach with that of a simple PCR protocol. To this end, 100 blood samples collected from dogs with suspected hemoparasite infections were analyzed. A comparison of the results of simple PCR and semi-nested PCR indicated a highly significant difference (p value = 0.0000). While only five (5%) of the samples tested positive using the simple protocol, 22 (22%) were positive using the snPCR technique. The results of this study reinforce the findings of previous studies, which have demonstrated the greater sensitivity of tests based on nested or semi-nested PCR. Therefore, to avoid false-negative results due to low levels of parasitemia, we suggest the preferential use of this protocol in epidemiological studies of canine babesiosis, particularly those that require reliable estimates of the prevalence of infection.

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Three highly sensitive "bedside" serum and urine tests for pregnancy compared

Clinical Chemistry ◽

10.1093/clinchem/36.9.1686 ◽

1990 ◽

Vol 36 (9) ◽

pp. 1686-1688 ◽

Cited By ~ 10

Author(s):

H Christensen ◽

H H Thyssen ◽

O Schebye ◽

A Berget

Keyword(s):

Paired Comparison ◽

False Negative ◽

Diagnostic Sensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Negative Results ◽

False Negative Results ◽

Significant Difference ◽

Comparison Of The Results ◽

Urine And Serum

Abstract We examined three enzyme-linked immunosorbent assay (ELISA) kits for human choriogonadotropin (hCG) (pregnancy tests) for use with urine and serum samples: the Tandem Icon II hCG Urine and Tandem Icon II hCG Serum, the NovoClone Target hCG Test, and the Abbott TestPacks hCG-urine and hCG-serum. Paired comparison of the results from each kit indicated that the NovoClone Target assay showed significantly lower diagnostic sensitivity (P less than 0.05) than did the Tandem Icon II or Abbott TestPack, both for urine and for serum samples. None of the products demonstrated any significant difference (P greater than 0.05) in diagnostic specificity, but the NovoClone Target kit showed several serious false-negative results with both urine and serum. Paired testing of urine kits vs serum kits also showed no significant differences (P greater than 0.05) in diagnostic sensitivity or specificity. We found the Abbott kits to be the most convenient to use and to read.

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The underestimated issue of non-reproducible cardiac troponin I and T results: case series and systematic review of the literature

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2020-1564 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Julien Favresse ◽

Jean-Louis Bayart ◽

Damien Gruson ◽

Sergio Bernardini ◽

Aldo Clerico ◽

...

Keyword(s):

Systematic Review ◽

Clinical Judgment ◽

Troponin I ◽

Troponin T ◽

Case Reports ◽

False Negative ◽

Case Series ◽

Elevated Alkaline Phosphatase ◽

Negative Results ◽

Significant Difference

Abstract Cardiac troponins (cTn) are the preferred biomarkers for the evaluation of myocardial injury and play a key role in the diagnosis of acute myocardial infarction (MI). Pre-analytical or analytical issues and interferences affecting troponin T and I assays are therefore of major concern given the risk of misdiagnosis. False positive troponin results have been related to various interferences including anti-troponin antibodies, heterophilic antibodies, or elevated alkaline phosphatase level. On the other hand, false negative results have been reported in the case of a large biotin intake. These interferences are characterized with erroneous but reproducible troponin results. Of interest, non-reproducible results have also been reported in the literature. In other words, if the sample is reanalyzed a second time, a significant difference in troponin results will be observed. These interferences have been named “fliers” or “outliers”. Compared to the biotin interference that received major attention in the literature, troponin outliers are also able to induce harmful clinical consequences for the patient. Moreover, the prevalence of outliers in recent studies was found to be higher (0.28–0.57%) compared to the biotin interference. The aim of this systematic review is to warn clinicians about these non-reproducible results that may alter their clinical judgment. Four case reports that occurred in the Clinique of Saint-Luc Bouge are presented to attest this point. Moreover, we aimed at identifying the nature of these non-reproducible troponin results, determining their occurrence, and describing the best way for their identification.

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Discussing the Efficacy and Safety of Covid-19 Vaccine Available in India- A Mini Review

Journal of University of Shanghai for Science and Technology ◽

10.51201/jusst/21/11924 ◽

2021 ◽

Vol 23 (11) ◽

pp. 330-343

Author(s):

Yogendra Shrestha ◽

◽

Jeet Bahadur Moktan ◽

Renukaradhya Chitti ◽

Shiv Kumar Yadav ◽

...

Keyword(s):

Disease Transmission ◽

Neutralizing Antibodies ◽

False Negative ◽

Emergency Situation ◽

Epidemiological Studies ◽

Self Awareness ◽

Negative Results ◽

Wide Range ◽

The Government ◽

Restricted Use

Background: Many variants detected after Wuhan-Hu-1 reference which were able to develop the resistance against the neutralizing antibodies induced by vaccine and may cause false negative results in diagnostic test. Novel variant B1.617 was detected in India and the Covid-19 cases hiked to its maximum; forcing the government towards approving a new vaccine for restricted use in emergency situation to cover a maximum population. Aims: This review looks at the efficacy, safety, and economical aspects of vaccines that have been authorized in India. Materials and methods: Wide-ranging assessment and analysis of accessible resources on online database. Results: The rAd26-s & rAd5-s demonstrate high efficacy as well as safety, followed by BBV152 and AZD1222. Various combinations of the vaccines with different platforms or vectors may induce wide range of immunity than a specific one. As per economical aspect, AZD1222 is more economical than the other two currently approved in India. Conclusion: There is a lack of clear end point to measure efficacy of the vaccine so the epidemiological studies with huge number of populations is required which may predict the perfect endpoint for efficacy measurement. Until then, inoculation with locally accessible vaccines and self-awareness about disease transmission prevention are the main options for reducing fatalities, protecting the health-care system, and eventually disease control.

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Effect of Muscle Activation on the Gravity Stress View in Unstable Weber B Ankle Fractures

Foot & Ankle International ◽

10.1177/1071100720938668 ◽

2020 ◽

Vol 41 (11) ◽

pp. 1342-1346

Author(s):

Kimberly K. Broughton ◽

Caroline Williams ◽

Christopher P. Miller ◽

Kristen Stupay ◽

John Y. Kwon

Keyword(s):

Muscle Activation ◽

False Negative ◽

Ankle Fractures ◽

Published Data ◽

Neutral Position ◽

Distal Fibula ◽

Level Of Evidence ◽

Negative Results ◽

False Negative Results ◽

Significant Difference

Background: In the setting of apparently isolated distal fibula fractures, the gravity stress view (GSV) is a validated method to determine mortise stability. There is currently no published data evaluating whether dynamic muscle activation can reduce an unstable mortise. If patients with instability can overcome gravity, resultant images could yield false-negative results. The goal of this investigation was to determine if patient effort can influence medial clear space (MCS) measurements in proven unstable bimalleolar-equivalent ankle fractures. Methods: Patients presenting with Weber B fibula fractures were assessed for mortise stability using the GSV. If the GSV demonstrated instability based on MCS widening >4 mm, 3 additional views were performed: GSV with an assistant maintaining the ankle in a neutral position; GSV with the patient actively dorsiflexing to neutral; and GSV with the patient actively dorsiflexing and supinating the foot. Twenty-four consecutive patients met inclusion criteria, with a mean age of 48.7 (range, 22-85) years. Fifteen patients (62.5%) were female and 9 (37.5%) were male. The laterality was evenly divided. Results: The mean MCS was 5.8 ± 2.0 6.0 ± 2.6, and 6.2 ± 2.7 mm for the manual assist, active dorsiflexion, and active supination radiograph measurement groups, respectively ( P = .434). Only 5 of 24 subjects had any measurable decrease in their MCS with active supination, with a maximum change of 1.2 mm. The remainder of the patients had an increase in MCS ranging from 0.1 to 4.0 mm. Conclusion: There was no significant difference between measurement states indicating that muscle activation is unlikely to yield a false-negative result on GSV. Mortise instability, secondary to deep deltoid injury in the presence of gravity stress, is unlikely to be actively overcome by dynamic stabilizers, supporting the validity and specificity of the GSV. Level of Evidence: Level III, prospective study.

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Can Patient Effort Influence Sensitivity & Specificity of the Gravity Stress View? Assessing the Potential for False Negative Results

Foot & Ankle Orthopaedics ◽

10.1177/2473011420s00146 ◽

2020 ◽

Vol 5 (4) ◽

pp. 2473011420S0014

Author(s):

Kimberly K. Broughton ◽

Christopher P. Miller ◽

Caroline Williams ◽

Kristen L. Stupay ◽

John Y. Kwon

Keyword(s):

Muscle Activation ◽

Stress Test ◽

False Negative ◽

Ankle Fractures ◽

Measurement Tool ◽

Neutral Position ◽

Distal Fibula ◽

Negative Results ◽

False Negative Results ◽

Significant Difference

Category: Ankle; Trauma Introduction/Purpose: In the setting of apparently isolated distal fibula fractures, the radiographic gravity stress view (GSV) has been validated as a reliable method to determine mortise stability. Based on previous studies demonstrating that plantarflexion can increase the measured medial clear space (MCS), patients are often asked to actively hold their ankle in a neutral position. However, it has not been studied whether dynamic muscle activation in the form of attempted dorsiflexion and/or supination can reduce and realign an unstable mortise. If these efforts can overcome gravity, resultant images could lead to a false negative result and missed diagnosis of instability. The goal of the present investigation is to determine if active effort by the patient can influence MCS measurements in proven unstable bimalleolar-equivalent ankle fractures. Methods: Eighteen consecutive patients with apparently isolated distal fibula fractures (Weber-B type) identified on initial nonweightbearing radiographs were assessed for mortise stability with a standard, unassisted GSV. If the radiograph demonstrated MCS widening > 4 mm, 3 additional views were performed: GSV with an assistant manually maintaining the ankle in a neutral position; GSV with the patient actively dorsiflexing the ankle to the same neutral position; and GSV with the patient actively dorsiflexing and supinating the foot to the same neutral position. All radiographs were digitally obtained and measured using a DICOM measurement tool. The MCS was measured by the senior author (JYK) who was blinded to the nature of the ankle position. Statistical analysis of the data was then conducted. Results: Of the eighteen patients who met inclusion criteria, the mean age was 48.9 years (range: 22-85, SD=18.9). Twelve patients (66%) were female and six (33%) were male. The laterality was evenly split with nine patients presenting with a right ankle injury and nine left ankles. The data was non-parametric; therefore, a Friedman’s test was utilized for analysis between the different MCS measurements per patient. There was no statistically significant difference in the measured MCS in any of the three tested scenarios, χ2(2)=4.261, p=0.119. Only 5 of 18 subjects had any measurable decrease in their MCS when asked to supinate their foot. Of these 5, the maximum reduction was 1.21mm. The remainder of patients had an increase in MCS ranging from 0.1 to 4.0mm. Conclusion: This investigation supports the notion that the gravity stress test is unlikely to yield false negative results when patients attempt to actively maintain a neutral ankle position during imaging. There was a non-significant inverse trend indicating that dynamic effort may in fact further displace the mortise as indicated by an increased MCS in several study participants, rather than reduce it. Thus, one can conclude that despite voluntary effort, patients with bimalleolar-equivalent ankle fractures are unlikely able to overcome the effect of gravity on the unstable mortise.

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Frequency of Chlamydia trachomatis Infection in Spontaneous Abortion of Infertile Women During First Pregnancy Referred to Tabriz University of Medical Sciences by Nested PCR Method in 2015

International Journal of Women s Health and Reproduction Sciences ◽

10.15296/ijwhr.2019.87 ◽

2018 ◽

Vol 7 (4) ◽

pp. 526-230

Author(s):

Mehdi Haghdoost ◽

Sanaz Mousavi ◽

Mehdi Khanbabayi Gol ◽

Majid Montazer

Keyword(s):

Spontaneous Abortion ◽

Nested Pcr ◽

Control Method ◽

Transmitted Disease ◽

Infertility Treatment ◽

P Value ◽

Medical Sciences ◽

Infertile Women ◽

Significant Difference ◽

Pcr Method

Objectives:Chlamydia trachomatis, as the main cause of bacterial sexually transmitted disease (STD), can lead to serious complications such as spontaneous abortion. Therefore, the aim of this study was to determine the frequency of C. trachomatis infection in spontaneous abortion of infertile women who referred to Tabriz University of Medical Sciences during the first pregnancy by means of nested polymerase chain reaction (PCR) method in 2015. Materials and Methods: The present descriptive, cross-sectional study was performed in the infertility clinics of Tabriz University of Medical Sciences from March 21, 2015 to March 19, 2016. A total of 120 infertile women were selected by the convenience sampling method. The specimens were prepared by the Dacron swab after four rotations in the endocervix and discharged into the specific transport medium of C. trachomatis. The DNA extraction was then performed by AccuPrep genomic DNA kit and the DNA was extracted until performing the PCR at -20° C. Next, nested PCR was conducted in 2 rounds and the final product of PCR was agar -2% gel electrophoresis. After entering the data in SPSS, the chi-square test was used to examine the role of factors influencing C. trachomatis infection and a P value less than 0.05 was considered significant. Results: The incidence of C. trachomatis infection in women with spontaneous abortion was 16.66%. In addition, there was a significant difference between the infected and non-infected groups regarding employment (P<0.04), birth control method (P<0.03), and the number of sexual intercourses per week (P<0.001). Conclusions: The prevalence of C. trachomatis in women who became pregnant with infertility treatment and spontaneous abortion was high in this study. Thus, nested PCR is considered an appropriate method for the diagnosis of C. trachomatis and it is essential for pregnant women who experience pregnancy with infertility treatment.

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Improving of the Nested PCR for Detection of Bovine Leukemia Virus

Mikrobiolohichnyi Zhurnal ◽

10.15407/microbiolj83.03.056 ◽

2021 ◽

Vol 83 (3) ◽

pp. 56-65

Author(s):

L.M. Ishchenko ◽

◽

V.V. Nedosekov ◽

V.D. Ishchenko ◽

O.Yu. Kepple ◽

...

Keyword(s):

Internal Control ◽

Nested Pcr ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

False Negative ◽

Envelope Gene ◽

Rt Pcr ◽

Second Stage ◽

Negative Results ◽

False Negative Results

Enzootic bovine leukosis caused by a bovine leukemia virus has a significant economic impact and is reported in World Organization for Animal Health(OIE). Aim. The purpose of our work was to improve the nested polymerase chain reaction (PCR) recommended by the OIE conducting it second-stage in real-time (RT) PCR. Such modification does not require the stage of gel electrophoresis and consequently reduces contamination risks and prevents false positive results. Methods. Primers that are recommended by the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (OIE) were used for the first amplification stage. For the second stage of the proposed modification of nested PCR, the primers and probe were designed based on the alignment of the sequences envelope gene of different isolates of bovine leukemia virus including Ukrainian isolates. Amplification of the internal control was carried out for the second stage to prevent false negative results. Results. Comparative studies of 48 blood samples for bovine leukemia virus identification by a proposed nested RT-PCR, nested PCR recommended by the protocol of the OIE, and RT-PCR were conducted. The sample panel included both positive and negative samples. A 100% match of the results of the bovine leukemia virus presence in nested PCR proposed by the OIE and in our proposed nested RT-PCR was obtained. Comparative analysis of results that were obtained using the RT-PCR and the proposed nested RT-PCR showed that false-negative results in 5 samples and 3 doubtful results that require retesting were obtained by use of RT-PCR. The interpretation of the results using nested RT-PCR is more efficient than RT-PCR since the cycle threshold value of positive samples obtained using RT-PCR was in the range of 24–40 cycles, whereas in the case of nested RT-PCR using, the value of Ct was in the range of 4–20 cycles. Conclusions. Proposed nested PCR modification includes the combination of the OIE recommendation about nested PCR and the reduction of the risk of contamination by conducting the second stage in RT-PCR. Results of approbation of proposed nested RT-PCR give a reason to recommend it for the identification of bovine leukemia virus.

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Combined CTP- IGF-1 Score: A New Score for Assessment of Disease Severity in HCV Related Liver Cirrhosis

10.21203/rs.3.rs-1216787/v1 ◽

2022 ◽

Author(s):

Elsayed Ghannam ◽

Salah Rozaik ◽

Ramy Hasan Agwa ◽

Ahmed Marwan ◽

Mervat El-Sayed Mashaly ◽

...

Keyword(s):

Liver Cirrhosis ◽

Negative Predictive Value ◽

Predictive Value ◽

False Negative ◽

The Body ◽

P Value ◽

Study Group ◽

Negative Results ◽

False Negative Results ◽

Ctp Score

Abstract Background: Liver is the largest endocrine organ in the body. It is a key organ in insulin mediated metabolism, growth hormone and insulin like growth factors (IGF) pathway. Liver cirrhosis is the end result of many chronic diseases including hepatitis C virus infection. Child-Turcotte-Pugh (CTP) score is the standard used in assessment of hepatic reserve but it has its drawbacks in the form of subjective variables, hepatic encephalopathy and ascites. The aim of this work is to assess IGF-1 in patients with liver cirrhosis, correlate it with CTP score and assess value of modified combined CTP-IGF-1 score. Patients and Methods: 170 patients with CLD (liver cirrhosis) and 72 healthy controls in the study groups were enrolled in the study. All patients were subjected to thorough history, clinical examination and laboratory assessment. IGF-1 was measured and all patients were evaluated using CTP and CTP-IGF-1 scores. Results: IGF1 showed highly significant low values in the study group in comparison to controls (42.15 ± 27.976 and 66.31 ± 33.084 ng/ml respectively, p <0.001). It showed also highly significant negative correlation to CTP score in the study group (p < 0.001) with progressive decrements with CTP score stage progression where IGF1 levels were 48.32 ± 28.611, 40.28 ± 25.869 and 18.80 ± 15.953 ng/ml (mean ± standard deviation) in relation to CTP score groups A, B and C respectively (p value < 0.001). The combined CTP-IGF-1 score in comparison to the classic CTP score showed improved area under curve (0.848 and 0.854), sensitivity (71.2% and 88%), negative predictive value (41.7% and 53.7%), false negative results (49 and 19) and accuracy (75.73% and 83.98%) but decreased specificity (97.22% and 61.1%), positive predictive value (99.2% and 91.5%) and higher false positive results (1 and 14) respectively. Conclusion: IGF-1 show progressive decrements with progression of liver cirrhosis and is negatively correlated with CTP score. Addition of IGF-1 to CTP score to formulate combined score improves the AUC, sensitivity, negative predictive value and the accuracy of CTP score and decreases the false negative results.

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Comparative Efficacy of Different Mastitis Markers for Diagnosis of Sub-Clinical Mastitis in Cows

International Journal of Applied Sciences and Biotechnology ◽

10.3126/ijasbt.v2i2.10191 ◽

2014 ◽

Vol 2 (2) ◽

pp. 121-125 ◽

Cited By ~ 4

Author(s):

Anil Langer ◽

Sunanda Sharma ◽

Narendra Kumar Sharma ◽

DS Nauriyal

Keyword(s):

Electrical Conductivity ◽

False Negative ◽

Clinical Mastitis ◽

Mean Value ◽

P Value ◽

Paper Strip ◽

Bacterial Isolates ◽

True Negative ◽

Milk Samples ◽

Negative Results

Seven hundred ninety six milk samples from 266 quarters of 69 lactating cows were subjected to microbiological investigations for identification of pathogens. One hundred ninety bacterial isolates were recovered from 89 infected quarters, among these monomicrobial infection was found in 50 (56.2%) quarters, whereas, mixed infection was observed in 39 (43.8%) quarters. Bacterial isolates identified were Staph. chromogenes (49.47%), Staph. hyicus (21.1%), Staph. epidermidis (11.05%), Str. agalactiae (5.8%), Staph. aureus (4.2%), Staph. intermedius (3.1%), Enterobacter sp. (1.5%), Klebsiella sp., E. coli (1.05%), Micrococcus sp. (1.05%) and Serratia marcescens (0.52%). Milk samples from every quarter of each cow were also subjected to 6 mastitis marker tests named Somatic cell count (SCC), California mastitis test (CMT), electrical conductivity (EC) by EC-meter as well as by Hand-held mastitis detector, pH detection by impregnated paper strip and also by pH meter. Efficacy of mastitis markers for diagnosis of sub-clinical mastitis was determined by comparing results of mastitis marker tests with microbiological findings. Mean value of SSC in milk from healthy quarters was significantly lower (p than that in milk from infected quarters. Significantly higher (p value of SSC was observed in milk samples having coagulase positive staphylococci as compared to that in milk from quarter with coagulase negative pathogens. The mean electrical conductivity (EC) in milk samples from infected quarters was significantly higher (P<0.05) than that from healthy quarters. Numbers and percentages of samples showing true positive, true negative, false positive and false negative results with SSC, CMT, EC by EC-meter, EC by hand-held meter, pH by impregnated strips, pH by digital pH-meter tests were evaluated and compared. The sensitivity and specificity of impregnated pH paper strip, CMT, pH-meter test, SCC, electrical conductivity by EC-meter and the same by Hand-held mastitis detector were evaluated The compatibility between the results of SCC, impregnated pH paper strip, CMT, EC-meter, pH-meter, Hand-held mastitis detector and bacteriological culture examination (reference test) was found to be 64.4, 63.4, 61.5, 59, 59 and 53 respectively.DOI: http://dx.doi.org/10.3126/ijasbt.v2i2.10191Int J Appl Sci Biotechnol, Vol. 2(2): 121-125

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Detection of Echinococcus multilocularis in faeces by nested PCR with the use of diluted DNA samples

Polish Journal of Veterinary Sciences ◽

10.2478/pjvs-2014-0010 ◽

2014 ◽

Vol 17 (1) ◽

pp. 79-83 ◽

Cited By ~ 4

Author(s):

J. Karamon

Keyword(s):

Echinococcus Multilocularis ◽

Nested Pcr ◽

Dna Isolation ◽

False Negative ◽

Standard Samples ◽

Red Foxes ◽

Negative Results ◽

False Negative Results ◽

Faecal Samples ◽

Positive Results

AbstractThe aim of this study was to choose the optimal variant of PCR examination of faeces to detect Echinococcus multilocularis infection which would allow to reduce the influence of different inhibitors in faeces. The investigation was carried out by comparison of 3 different methods of DNA isolation from faeces and different DNA dilutions used in PCR. Thirty five intestines of red foxes were used. Small intestines were examined by the sedimentation and counting technique (SCT). Faeces were collected from the rectum for PCR and flotation. DNA were isolated with the use of 3 different methods. Two methods were dedicated for faeces: method 1 (M1) - for larger samples and method 2 (M2) - for standard samples. The third method, method 3 (M3), was not dedicated for faeces. DNA samples were tested by nested PCR in 6 variants: not diluted (1/1) and 5 diluted (1/2.5, 1/5, 1/10. 1/20, 1/40). E. multilocularis was found by SCT in 18 from 35 (51.4%) intestines. Taenia-type eggs were detected only in 20.0% of faecal samples. In PCR the highest number of positive results (45.7%) were obtained during examination of DNA isolated by M1 method, and then 40.0% and 34.3%, respectively, for M2 and M3. In some samples positive results in PCR were obtained only in diluted DNA. For example, 8 from 12 positive samples isolated by M3 method gave the PCR negative results in non-diluted DNA and positive only after dilution 1:2.5, 1:10 or 1:20. Also 3 samples isolated by methods dedicated for stool gave positive results only after DNA dilution. The investigation has revealed that in copro-PCR for detection of E. multilocularis infection additional using of diluted DNA (besides non diluted) can avoid false negative results causing by PCR inhibition. In the best method of DNA isolation (M1), the use of non diluted DNA sample together with diluted in proportion 1:10 seems to be optimal.

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