Anti-typhi immobilized MWCNT-PANI nano sensor for salmonella typhi detection
24 Jul 2013-pp 383-386
TL;DR: An electronic nano bio-sensor is developed to detect the presence of typhoid in the earlier stage with high accuracy as mentioned in this paper, the sensor is developed on a silicon substrate and Multi Walled Carbon Nano tube is aligned over it and coated with polyaniline.
Abstract: An electronic Nano bio-sensor is developed to detect the presence of typhoid in the earlier stage with high accuracy. The sensor is developed on a silicon substrate and Multi Walled Carbon Nano tube is aligned over it and coated with polyaniline. The antibody anti-typhi was immobilized on to the electrode to have a sensitive detection of the salmonella antigens from the samples. The resultant variation of resistance by the sensor indicates the detection of Salmonella Typhi in earlier stage and shows the accuracy of the sensor developed
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TL;DR: It is suggested that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases.
Abstract: A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella typhi in the blood specimens from patients with typhoid fever. Two pairs of oligonucleotide primers were designed to amplify a 343-bp fragment of the flagellin gene of S. typhi. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization by using a 32P-labeled 40-base probe internal to the amplified DNA. The nested PCR with two pairs of primers could detect 10 organisms of S. typhi as determined by serial dilutions of DNA from S. typhi. The peripheral mononuclear cells from 11 of 12 patients with typhoid fever confirmed by blood culture were positive for DNA fragment of the flagellin gene of S. typhi, whereas 10 blood specimens of patients with other febrile diseases were negative. With the nested PCR, S. typhi DNAs were detected from blood specimens of four patients with suspected typhoid fever on the basis of clinical features but with negative cultures. We suggest that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases.
206 citations
TL;DR: The Widal test was insensitive and displayed interoperator variability, and two rapid kits, TyphiDot and TUBEX, demonstrated promising results.
Abstract: Laboratory diagnosis of typhoid fever requires isolation and identification of Salmonella enterica serotype Typhi. In many areas where this disease is endemic, laboratory capability is limited. Recent advances in molecular immunology have led to the identification of sensitive and specific markers for typhoid fever and technology to manufacture practical and inexpensive kits for their rapid detection. We evaluated three commercial kits for serologic diagnosis of typhoid fever. Patients presenting with ≥ 4 days of fever were enrolled at two hospitals in Southern Vietnam. Cases were patients with serotype Typhi isolated from blood samples, and controls were patients with other laboratory-confirmed illnesses. Serotype Typhi isolates were confirmed and tested for antimicrobial susceptibility at the Pasteur Institute in Ho Chi Minh City. The Widal test was run at the hospitals and the Pasteur Institute. Sera were shipped frozen to the Centers for Disease Control and Prevention and tested by using Multi-Test Dip-S-Ticks, TyphiDot, and TUBEX to detect immunoglobulin G (IgG), IgG and IgM, and IgM, respectively. Package insert protocol instructions were followed. We enrolled 59 patients and 21 controls. The sensitivity and specificity findings were as follows: 89 and 53% for Multi-Test Dip-S-Ticks, 79 and 89% for TyphiDot, 78 and 89% for TUBEX, and 64 and 76% for Widal testing in hospitals and 61% and 100% for Widal testing at the Pasteur Institute. For all assays, the sensitivity was highest in the second week of illness. The Widal test was insensitive and displayed interoperator variability. Two rapid kits, TyphiDot and TUBEX, demonstrated promising results.
178 citations
Journal Article•
TL;DR: This zoonotic disease is present in all livestock systems and increased demand for dairy products accompanied with changing and intensified farming practices has raised the concern for increased spread and intensified transmission of this infection to the human population with increased risk of disease.
Abstract: Brucellosis is an important but neglected disease in India. This zoonotic disease is present in all livestock systems and increased demand for dairy products accompanied with changing and intensified farming practices has raised the concern for increased spread and intensified transmission of this infection to the human population with increased risk of disease. Brucellosis can be controlled by mass vaccination of livestock. Human brucellosis can be treated with a combination of antibiotics but is very difficult to diagnose and requires laboratory testing for confirmation. Only a few recent studies have addressed the prevalence and importance of brucellosis as a human disease problem in India. The disease may be overlooked and misdiagnosed because of the difficult diagnosis and the absence and lack of experience with laboratory testing. Alertness of medical staff is needed to recognize and diagnose the disease. Awareness of risk groups is needed to take appropriate preventive measures and to accept control measures.
141 citations
TL;DR: The new test (TUBEX) detects anti-Salmonella O9 antibodies in patients by inhibiting the binding between an anti-O9 IgM monoclonal antibody (MAb) conjugated to colored latex particles and S. typhilipopolysaccharide (LPS) conjugs to magnetic latex particles.
Abstract: Typhoid fever is caused by Salmonella typhi Detection of anti-S typhi antibodies in the patient is a useful diagnostic aid Among the various methods developed over the years for this purpose, the Widal test, based on bacterial agglutination, has remained the most widely used, even though it is neither specific nor sensitive Its popularity stems from the fact that it is simple to use and inexpensive We describe a new test which also uses a simple one-step procedure but is more rapid and accurate than the Widal The new test (TUBEX) detects anti-Salmonella O9 (both immunoglobulin M [IgM] and IgG) antibodies in patients by inhibiting the binding between an anti-O9 IgM monoclonal antibody (MAb) conjugated to colored latex particles and S typhi lipopolysaccharide (LPS) conjugated to magnetic latex particles The reactants are mixed in a specially designed microtube for 2 min, and the result is read based on the resultant color of the supernatant following forced sedimentation of the magnetic beads In the absence of inhibitory antibodies, there is a color change (from blue to red) due to cosedimentation of the indicator particles with the magnetic particles, whereas if these antibodies are present, they prevent such a change to a degree dependent on their concentration Preliminary examination of TUBEX using the anti-O9 MAb and irrelevant MAbs as inhibitors revealed the test to be specific and reproducible, with an analytical sensitivity of 16 micrograms per ml of antibody The reagents remained stable for at least 9 months when kept at 4 degrees C In the examination of 16 stored sera obtained from 14 patients with proven cases of typhoid fever and 78 serum samples from 75 subjects without typhoid fever, TUBEX was found to be 100% sensitive and 100% specific The nontyphoid group comprised 26 healthy blood donors, 30 antinuclear antibody (ANA)-negative patients, 9 ANA-positive patients, of whom 1 was positive for anti-DNA antibody, 4 typhus patients, and 6 septicemic patients In addition, the sera obtained from 11 patients clinically diagnosed as having typhoid fever were all positive in the test The TUBEX results correlated to some extent, albeit insignificantly (r = 038, P = 007), with those of an enzyme-linked immunoassay (ELISA) which used a similar detection format (inhibition) and reagents (S typhi LPS and anti-O9 antibody) TUBEX correlated very well with ELISAs which detected anti-S typhi LPS IgM (r = 058, P = 0003) or IgG (r = 054, P = 0006) antibodies from the typhoid patients There was no correlation with the Widal test The TUBEX test, if performed on slides (instead of tubes) or with soluble antigen (instead of antigen-conjugated magnetic beads), suffered significantly in sensitivity Direct agglutination tests using LPS-conjugated indicator particles performed either on slides or in microwells also failed to detect antibodies from the majority of typhoid patients Thus, TUBEX appears to be well designed and well suited for use in the laboratory or by the bedside as a simple, rapid aid to the routine diagnosis of typhoid fever
85 citations
TL;DR: Nested PCR using H1-d primers, which is specific for Salmonella enterica serovar Typhi, was compared to blood culture and the single-tube Widal test and indicates that nested PCR can be used as a gold standard to determine the cutoff titer of the Widal Test for diagnosis of typhoid fever.
Abstract: Typhoid fever is a systemic illness caused by Salmonella enterica serotype Typhi, and it is endemic in developing countries. Although blood culture is considered the gold standard for the diagnosis of typhoid fever, its results are often jeopardized due to prior inadequate doses of antimicrobials (10). The specific gene sequence of the bacterium, which can be amplified and detected specifically and rapidly by nested PCR (4, 13), is another important target, having the sensitivity of detecting even one bacterium in a given sample within a few hours (7). Moreover, detecting antibodies by the Widal test can also yield a diagnosis of typhoid fever. However, this test carries many shortcomings, as the sensitivity, specificity, and predictive values differ in different geographic areas (9, 11, 12, 15). For better utilization of this test, an appropriate cutoff titer must be determined for a particular geographic area in relation to the ideal gold standard test. Such a cutoff titer cannot be proposed until a proper gold standard test is determined.
This study was conducted in the University Hospital, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India. A total of 63 clinically suspected cases of typhoid fever, in the age group of 1 to 12 years, were included based on presentation with continuous high-grade fever, toxic appearance, splenomegaly (<3 cm), and constitutional symptoms. Patients of both sexes were included and were comprised of those attending the outpatient department and also hospitalized cases, irrespective of their prior antityphoid treatment. The control group consisted of 25 healthy children in the same age group without febrile illness the preceding 6 months. Appropriate amounts of blood were collected from each child for Widal tests, 2 to 8 ml of blood in brain heart infusion broth with sodium polyanethol sulfonate was collected for culture isolation, and 3 ml of blood in a container also holding citrate phosphate buffer (pH 7) was collected for the PCR-based study.
DNA extraction was done from whole-blood samples by using the phenol-chloroform method (13), with few modifications. Nested PCR was performed as described by Song et al. (13) and was modified according to Frankel (4). The reaction mixture for the first-round PCR contained 2.5 μl of 10× PCR buffer (MBI Fermentas, Hanover, Md.), 1.1 μl of (1.5 mM) MgCl2 (MBI Fermentas), 11 pmol of each primer ST1 and ST2 (QIAGEN Operon, Cologne, Germany), 1 μl of deoxynucleoside triphosphate mix (MBI Fermentas), 1 U of Taq DNA polymerase (MBI Fermentas), 10 μl of DNA template, and water to a final volume of 25 μl. The first-round amplification was carried out in a thermocycler (Biometra, Goettingen, Germany) under the following conditions: 40 cycles for 1 min and denaturation at 94°C, 1 min 15 s annealing at 57°C, and 1 min elongation at 72°C, with a final elongation step extended to 7 min.
The nested PCR master mix was the same as that of the first-round PCR, except it contained 21 pmol of each primer ST3 and ST4 and 4 μl of DNA template (1:6-diluted product of the primary cycle). Thermal cycling was carried out as described for first-round PCR, except that the annealing temperature was set to 63°C. To separate amplified products, 5 μl of solution was electrophoresed on a 1.5% agarose gel in TBE (Tris-borate-EDTA) buffer at 80 V for 1 h. The gels were stained with ethidium bromide, and the bands were visualized under UV light.
For isolation of the bacterium, the blood culture bottles (HiMedia, Mumbai, India) were incubated at 37°C for up to 7 days, with subcultures taken on alternate days. The isolates were identified by following standard methods (2). The Widal test was performed by using a colored antigen kit (Span Diagnostics, Surat, India).
For statistical analysis, positive predictive value (PV+), negative predictive value (PV−), likelihood ratio for a positive test result (LR+), and likelihood ratio for a negative test result (LR−) were calculated (5). A z test was applied to determine the significance between two proportions (3).
None of the afebrile healthy control subjects was positive for the bacterium by PCR and blood culture. However, one (4%) child of this group was observed to have an antibody titer against somatic (TO) antigen of 160. Of the 63 suspected cases, nested PCR was positive in 53 (84.1%) cases, the Widal test was positive in 41 (65.0%) cases at the cutoff titer 160 for O and/or H agglutinins, and positivity by blood culture was observed in 17 (26.9%) cases. The positivity of PCR was significantly higher (P < 0.01) than that of blood culture and Widal.
When the two most specific tests were evaluated in the suspected cases of typhoid fever that were positive by any of the three tests employed (n = 57), PCR was found to have sensitivity of 92.8% while blood culture had only 29.8% sensitivity. Few reports on the application of PCR in the diagnosis of typhoid fever from areas where it is endemic have shown lower sensitivity than that observed in the present study (1, 6, 8). The reasons for the better sensitivity found in the present study might be the collection of 3 ml of whole blood for DNA extraction in place of 1 ml and a slight modification of the recommended DNA extraction protocol (13). The collection of 3 ml of whole blood ensured the presence of at least 1 bacterium in the sample, based on the observation that in children below 15 years of age, the level of bacteremia ranged from <0.3 to 387 CFU/ml, with a median of 1 CFU of S. enterica serovar Typhi/ml of blood, of which a mean of 63% were intracellular (14). The negative predictive value (PV−) of PCR was found to be better than that of blood culture. As some of the cases that tested positive exclusively by the Widal test may not be true cases of typhoid, the observed PV− (60%) in this study may be less than the actual rate of positivity. For assigning a test to be of clinical utility, it is recommended that the LR+ and LR− of the test should be ≥10 and ≤0.1, respectively (5). In the present study, the LR+ and LR− of PCR was found to be ∞ and 0.07, respectively.
Considering PCR as the gold standard, the most appropriate diagnostic cutoff titer of TO and TH have been evaluated at the two different cutoff titers, i.e., 80 for O agglutinin, 160 for H agglutinin, and 160 for O and/or H agglutinins. It was found that specificity was significantly higher with the latter titer (Table (Table11).
TABLE 1.
Statistical evaluation of blood culture and a single Widal test (n = 53)
It is important to note that a total of 11 cases were negative by Widal at this titer but were positive exclusively by PCR, with a mean day of presentation of 5.3 days. There were five other cases which were PCR and culture positive, with a mean day of presentation of 3.7 days (Table (Table2).2). These observations suggest that the nested PCR assay can be used in the early diagnosis of typhoid fever, which will not only reduce morbidity, mortality, and acquisition of the carrier state but will also reduce the transmission of the disease. Further, it was observed that the Widal test seems to be relevant in the second week of illness at the proposed titer, as it failed to detect 28% of the total typhoid cases that presented in the first week of illness.
TABLE 2.
Status of PCR, blood culture, and Widal test and mean duration of illness in suspected cases of typhoid fever
It may, therefore, be concluded that nested PCR must be considered the gold standard test in the diagnosis of typhoid fever in order to determine the biostatistical parameters of Widal and other simpler serological tests to be used in field conditions.
65 citations
"Anti-typhi immobilized MWCNT-PANI n..." refers background in this paper
...Forme, "Value of a single tube widal test in diagonosis of typhoid fever" Journal of clinical microbiology, 1999, p:2882-2886 [11] 1....
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