Mycobacterial infections—tuberculosis (TB), bovine tuberculosis (bTB), and Johne’s disease (JD)—are major infectious diseases of both human and animals. Methods presently in use for diagnosis of ...mycobacterial infections include bacterial culture, nucleic acid amplification, tuberculin skin test, interferon-γ assay, and serology. Serological tests have several advantages over other methods, including short turn-around time, relatively simple procedures, and low cost. However, current serodiagnostic methods for TB, bTB and JD exhibit low sensitivity and/or specificity. Recent studies that have aimed to develop improved serodiagnostic tests have mostly focused on identifying useful species-specific protein antigens. A review of recent attempts to improve diagnostic test performance indicates that the use of multiple antigens can improve the accuracy of serodiagnosis of these mycobacterial diseases. Mycobacteria also produce a variety of species-specific nonprotein molecules; however, only a few such molecules (e.g., cord factor and lipoarabinomannan) have so far been evaluated for their effectiveness as diagnostic antigens. For TB and bTB, there has been recent progress in developing laboratory-free diagnostic methods. New technologies such as microfluidics and “Lab-on-Chip” are examples of promising new technologies that can underpin development of laboratory-free diagnostic devices for these mycobacterial infections.
BACKGROUND: The use of serological assays for diagnosis of bovine tuberculosis (TB) has been intensively studied and use of specific antigens have aided in improving the diagnostic accuracy of the ...assays. In the present study, we report an in-house enzyme linked immunosorbent assay (ELISA), developed by using ethanol extract of Mycobacterium bovis (M. bovis). The assay, named (ethanol vortex ELISA EVELISA), was evaluated for detection of anti- M. bovis antibodies in the sera of cattle and white-tailed deer. METHODS: By using the EVELISA, we tested sera obtained from two species of animals; cattle (n = 62 uninfected, n = 40; naturally infected, n = 22) and white-tailed deer (n = 41 uninfected, n = 25; naturally infected, n = 7; experimentally infected, n = 9). To detect species specific molecules, components in the ethanol extract were analyzed by thin layer chromatography and western blotting. RESULTS: Among the tested animals, 77.2% of infected cattle and 87.5% of infected deer tested positive for anti- M. bovis antibody. There were only minor false positive reactions (7.5% in cattle and 0% in deer) in uninfected animals. M. bovis -specific lipids and protein (MPB83) in the ethanol extract were detected by thin layer chromatography and western blotting, respectively. CONCLUSION: The results warrant further evaluation and validation of EVELISA for bovine TB diagnosis of traditional and alternative livestock as well as for free-ranging animal species.
In the year 2007, a total of 200 faecal samples comprising of 100 samples each from cattle and buffaloes from different locations of Bikaner, Rajasthan were analyzed to confirm the presence of ...gastrointestinal parasitic infection. Twenty four (12.00%) samples were found positive for strongyle eggs. Eleven per cent cattle and 13 per cent buffaloes were found to be positive for gastrointestinal helminthosis. The prevalence in cattle varied from 9.09 to 12.50 in different locations. Prevalence range was slightly higher in buffaloes which ranged between 10.52 to 14.81. The estimation of EPG count for Strongyle species in cattle range between 200-1000, with an average of 504.00+245.41. This range was 200-1400 with an average of 684.61+350.82 in buffaloes.
Johne's disease or paratuberculosis is one of the most economically important diseases of the livestock. Most of the economic losses associated with paratuberculosis are related to decreased milk ...production, reduced fertility and higher rates of culling. Understanding the immunology of the disease is very important for better understanding of the interplay between the host and the causative agent, Mycobacterium avium subsp. paratuberculosis (MAP). After uptake of MAP by macrophages residing in host's intestinal tissue, two possible scenarios may emerge; MAP may be destroyed or may establish persistent infection within the macrophages. If MAP persists in the infected macrophage, it continuously modulates adaptive immune responses of the animal. In this short review we describe the host-pathogen interactions in Johne's disease and highlights potential protective mechanisms in order for future design of more effective diagnostic method and vaccine.
Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium ssp. paratuberculosis (MAP), is one of the most economically important diseases of dairy cattle. Control of JD could be ...achieved by good herd management practices, and diagnosis; however, this approach has been hampered by the low sensitivity of currently available enzyme-linked immunosorbent assay (ELISA) tests. In our previous study, we developed a sensitive serum ELISA test, ethanol-vortex enzyme-linked immunosorbent assay (EVELISA), using ethanol extract of MAP. The objective of this study is to demonstrate that the EVELISA can be used for detection of anti-MAP antibodies in milk samples. In this study, we tested and optimized concentrations of antigen, milk, and secondary antibody for better differentiation of milk samples of cattle with MAP infections from those of cattle in JD-free herds. We evaluated five environmental mycobacteria as absorbents of cross-reactive antibodies in milk and found that the mycobacteria had no significant effect on EVELISA results. Using the optimized conditions, a total of 57 milk samples from Holstein dairy cattle (37 animals found positive on the fecal polymerase chain reaction test and 20 animals from JD-free herds) were tested for anti-MAP antibody in milk by using the EVELISA method. The average of ELISA values in the JD-positive milk samples (mean±SD=0.355±0.455) was significantly higher than that in the JD-negative milk samples (mean±SD=0.071±0.011). These results warrant further studies for evaluation and validation of the EVELISA for milk testing of cattle for JD.
Abstract
Background
Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has principally been performed through the use of real-time reverse-transcription polymerase ...chain reaction testing. Results of such tests can be reported as cycle threshold (Ct) values, which may provide semi-quantitative or indirect measurements of viral load. Previous reports have examined temporal trends in Ct values over the course of a SARS-CoV-2 infection.
Methods
Using testing data collected during a prospective household transmission investigation of outpatient and mild coronavirus disease 2019 cases, we examined the relationships between Ct values of the viral RNA N1 target and demographic, clinical, and epidemiological characteristics collected through participant interviews and daily symptom diaries.
Results
We found that Ct values are lowest (corresponding to a higher viral RNA concentration) soon after symptom onset and are significantly correlated with the time elapsed since onset (P < .001); within 7 days after symptom onset, the median Ct value was 26.5, compared with a median Ct value of 35.0 occurring 21 days after onset. Ct values were significantly lower among participants under 18 years of age (P = .01) and those reporting upper respiratory symptoms at the time of sample collection (P = .001), and were higher among participants reporting no symptoms (P = .05).
Conclusions
These results emphasize the importance of early testing for SARS-CoV-2 among individuals with symptoms of respiratory illness, and allow cases to be identified and isolated when their viral shedding may be highest.
Abstract
Background
To better understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shedding and infectivity, we estimated SARS-CoV-2 RNA shedding duration, described participant ...characteristics associated with the first negative rRT-PCR test (resolution), and determined if replication-competent viruses was recoverable ≥10 days after symptom onset.
Methods
We collected serial nasopharyngeal specimens from 109 individuals with rRT-PCR–confirmed COVID-19 in Utah and Wisconsin. We calculated viral RNA shedding resolution probability using the Kaplan-Meier estimator and evaluated characteristics associated with shedding resolution using Cox proportional hazards regression. We attempted viral culture for 35 rRT-PCR–positive nasopharyngeal specimens collected ≥10 days after symptom onset.
Results
The likelihood of viral RNA shedding resolution at 10 days after symptom onset was approximately 3%. Time to shedding resolution was shorter among participants aged <18 years (adjusted hazards ratio aHR, 3.01; 95% confidence interval CI, 1.6–5.6) and longer among those aged ≥50 years (aHR, 0.50; 95% CI, .3–.9) compared to participants aged 18–49 years. No replication-competent viruses were recovered.
Conclusions
Although most patients were positive for SARS-CoV-2 for ≥10 days after symptom onset, our findings suggest that individuals with mild to moderate COVID-19 are unlikely to be infectious ≥10 days after symptom onset.
The majority of participants with mild to moderate COVID-19 continued to shed SARS-CoV-2 from the nasopharynx ≥10 days after symptom onset. However, we did not recover any replication-competent virus from 35 rRT-PCR–positive nasopharyngeal specimens collected ≥10 days after symptom onset.
Limited data exist on severe acute respiratory syndrome coronavirus 2 in children. We described infection rates and symptom profiles among pediatric household contacts of individuals with coronavirus ...disease 2019.
We enrolled individuals with coronavirus disease 2019 and their household contacts, assessed daily symptoms prospectively for 14 days, and obtained specimens for severe acute respiratory syndrome coronavirus 2 real-time reverse transcription polymerase chain reaction and serology testing. Among pediatric contacts (<18 years), we described transmission, assessed the risk factors for infection, and calculated symptom positive and negative predictive values. We compared secondary infection rates and symptoms between pediatric and adult contacts using generalized estimating equations.
Among 58 households, 188 contacts were enrolled (120 adults; 68 children). Secondary infection rates for adults (30%) and children (28%) were similar. Among households with potential for transmission from children, child-to-adult transmission may have occurred in 2 of 10 (20%), and child-to-child transmission may have occurred in 1 of 6 (17%). Pediatric case patients most commonly reported headache (79%), sore throat (68%), and rhinorrhea (68%); symptoms had low positive predictive values, except measured fever (100%; 95% confidence interval CI: 44% to 100%). Compared with symptomatic adults, children were less likely to report cough (odds ratio OR: 0.15; 95% CI: 0.04 to 0.57), loss of taste (OR: 0.21; 95% CI: 0.06 to 0.74), and loss of smell (OR: 0.29; 95% CI: 0.09 to 0.96) and more likely to report sore throat (OR: 3.4; 95% CI: 1.04 to 11.18).
Children and adults had similar secondary infection rates, but children generally had less frequent and severe symptoms. In two states early in the pandemic, we observed possible transmission from children in approximately one-fifth of households with potential to observe such transmission patterns.
Abstract
Background
The evidence base for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is nascent. We sought to characterize SARS-CoV-2 transmission within US households and estimate ...the household secondary infection rate (SIR) to inform strategies to reduce transmission.
Methods
We recruited patients with laboratory-confirmed SARS-CoV-2 infection and their household contacts in Utah and Wisconsin during 22 March 2020–25 April 2020. We interviewed patients and all household contacts to obtain demographics and medical histories. At the initial household visit, 14 days later, and when a household contact became newly symptomatic, we collected respiratory swabs from patients and household contacts for testing by SARS-CoV-2 real-time reverse-transcription polymerase chain reaction (rRT-PCR) and sera for SARS-CoV-2 antibodies testing by enzyme-linked immunosorbent assay (ELISA). We estimated SIR and odds ratios (ORs) to assess risk factors for secondary infection, defined by a positive rRT-PCR or ELISA test.
Results
Thirty-two (55%) of 58 households secondary infection among household contacts. The SIR was 29% (n = 55/188; 95% confidence interval CI, 23%–36%) overall, 42% among children (aged <18 years) of the COVID-19 patient and 33% among spouses/partners. Household contacts to COVID-19 patients with immunocompromised conditions and household contacts who themselves had diabetes mellitus had increased odds of infection with ORs 15.9 (95% CI, 2.4–106.9) and 7.1 (95% CI: 1.2–42.5), respectively.
Conclusions
We found substantial evidence of secondary infections among household contacts. People with COVID-19, particularly those with immunocompromising conditions or those with household contacts with diabetes, should take care to promptly self-isolate to prevent household transmission.
The current serological diagnostic method can be time consuming and labor intensive, which is not practical for on-site diagnosis and screening of infectious diseases. Capacitive bioaffinity ...detection using microelectrodes is considered as a promising label-free method for point-of-care diagnosis, though with challenges in sensitivity and the time “from sample to result.” With recent development in AC electrokinetics (ACEK), especially in dielectrophoresis (DEP), we are able to develop an ACEK enhanced capacitive bioaffinity sensing method to realize simple, fast and sensitive diagnosis from serum samples. The capacitive immunosensor presented here employs elevated AC potentials at a fixed frequency for impedimetric interrogation of the microelectrodes. According to prior work, such an AC signal is capable of inducing dielectrophoresis and other ACEK effects, so as to realize in-situ enrichment of macromolecules at microelectrodes and hence accelerated detection. Experimental study of the ACEK-enhanced capacitive sensing method was conducted, and the results corroborate our hypothesis. The capacitive sensing responses showed clear frequency dependence and voltage-level dependency, which supports the hypothesis that ACEK aids the antigen–antibody binding, and these dependencies were used to optimize our detection protocol. Our capacitive sensing method was shown to work with bovine sera to differentiate disease-positive samples from negative samples within 2min, while conventional immunoassay would require multiple processing steps and take hours to complete. The results showed high accuracy and sensitivity. The detection limit is found to reach 10ng/ml in 2min. The ACEK-enhanced capacitive immunosensor is a platform technology, and can be employed to detect any combination of probe (e.g. antigen) and analyte (e.g. serum antibody) in a small volume of bodily fluids.
•AC electrokinetics (ACEK) enhanced capacitive immunosensing can achieve significantly accelerated affinity assay by incorporating ACEK effects into capacitive sensing.•AC potential at an optimized frequency over microelectrodes will perform macromolecule enrichment and capacitive monitoring of binding process simultaneously.•Specific proteins (e.g. antibodies and biomarkers) in clinical specimens were detected within 2 minutes from sample to result.•ACEK-enhanced capacitive immunosensing can be applied to specifically detect various types of protein-protein interactions.
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