Early diagnosis of Q fever: Detection of immunoglobulin M by radioimmunoassay and enzyme immunoassay

The use of a radioimmunoassay and an enzyme immunoassay for early diagnosis of Q fever is described, both of which are based on the IgM antibody-capture principle. A commercially available phase II antigen and a labeled, purified anti-Q-IgG of human origin were employed. With these tests Q fever antibodies were detected earlier in the course of infection than with the complement fixation test.     

Immunoblot detection of class-specific humoral immune response to outer membrane proteins isolated from Salmonella typhi in humans with typhoid fever

The studies reported here were undertaken to assess the ability of the outer membrane proteins (OMPs) of Salmonella typhi to induce a humoral immune response in humans with typhoid fever. OMPs were isolated with the nonionic detergent Triton X-100 and were found to be contaminated with approximately 4% lipopolysaccharide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns showed protein bands with molecular size ranges from 17 to 70 kilodaltons; the major groups of proteins were those that correspond to the porins and OmpA of gram-negative bacteria. Rabbit antiserum to OMPs or to S. typhi recognized OMPs after absorption with lipopolysaccharide. Sera from patients with typhoid fever contained immunoglobulin M antibodies which reacted with a protein of 28 kilodaltons and immunoglobulin G antibodies which reacted mainly with the porins, as determined by immunoblotting. These results indicate that the porins are the major immunogenic OMPs from S. typhi and that the immune response induced in the infection could be related to the protective status.     

Early diagnosis of typhoid fever by the detection of salivary IgA

BACKGROUND:/AIMS: Current diagnostic methods for typhoid fever have low sensitivity and specificity. This study aimed to develop an enzyme linked immunosorbent assay (ELISA) with greater sensitivity and specificity. METHODS: The ELISA was developed and evaluated on patients with acute typhoid infection, febrile controls, and healthy controls. A sequential study on patients with culture confirmed typhoid was also carried out to determine the time period of maximum sensitivity. RESULTS: The ELISA detected anti-Salmonella typhi lipopolysaccharide (LPS) salivary IgA antibodies. A six month follow up study of patients with culture confirmed typhoid fever showed that the test shows maximum efficiency during the second and third weeks of fever and enables detection of the acute infection during the early phase. CONCLUSIONS: This ELISA can detect typhoid fever during the early phase of infection and is most efficient during the second and third weeks of fever, the time at which patients normally present for treatment. Because the sensitivity of the assay is subsequently greatly reduced, it will be useful for the diagnosis of acute infection.     

Salmonella typhi VI antigen co-agglutination test for the rapid diagnosis of typhoid fever

A slide Co-agglutination test for the detection of Salmonella typhi Vi antigen in blood was evaluated for its efficiency in rapid diagnosis of Typhoid fever. The results were compared with conventional methods like Blood culture and Widal test. The test showed a sensitivity of 86.67% and specificity of 88.83% when compared with blood culture positivity or Widal titre above 160. This is a useful rapid diagnostic test for the early diagnosis of Typhoid fever.     

Detection of Salmonella typhi protein antigen in serum and urine: a value for diagnosis of typhoid fever in an endemic area

Using haemoculture as the gold standard, a double antibody sandwich ELISA for the detection of Salmonella typhi Barber protein antigen (BP) was compared with the Widal test. Specimens used were serum and urine obtained from normal healthy individuals and from patients with typhoid fever, paratyphoid fever, pyrexia caused by other bacteria and pyrexia with negative haemoculture. The ELISA for antigenuria gave a significantly higher sensitivity, specificity, accuracy and positive predictive value than the Widal test (p less than 0.05). The ELISA for antigenaemia gave a significantly higher sensitivity and positive predictive value only. All other values were not significantly different. The timing of specimen collection was critical for sensitivity in the ELISA for antigenaemia and antigenuria, and the best results could be obtained by carrying out both assays simultaneously. The clearance of BP from serum into urine occurred around 16 days after the onset of fever in one patient. In two patients, BP could be detected in sera up to 3 weeks after the onset of fever. In two patients, serum BP could still be detected although haemoculture was negative.     

Competitive enzyme immunoassay for antibodies to a 43,000-molecular-weight Francisella tularensis outer membrane protein for the diagnosis of tularemia.

Antibodies against a 43,000-molecular-weight Francisella tularensis outer membrane (OM) protein (43K protein) were measured in paired serum specimens from 23 patients with tularemia and matched against antibodies in sera from 25 patients with nontularemic infectious diseases and from 25 blood donors. Antibodies were measured by a competition enzyme-linked immunosorbent assay which tested the ability of human serum to compete with rabbit anti-43K protein antibodies for its binding to the F. tularensis OM coat. The sera from nontularemic patients and from blood donors, in dilutions of 1:16 and 1:64, respectively, showed no or very low levels of antibodies. All of the tularemia patients showed positive tests with the first, the second, or both of the serum specimens examined. For instance, with serum diluted 1:64, each of the serum specimens showed a sensitivity of 95.7% and a specificity of 96%. When used for antibody competition in Western blotting (immunoblotting), the rabbit anti-43K selectivity blocked the binding of human serum antibodies to the 43,000-molecular-weight protein. This protein was immunoaccessible in Formalin-killed F. tularensis. These data indicate an important role of the 43,000-molecular-weight OM protein in the immunobiology of tularemia and emphasize its potential usefulness as an antigen in serodiagnostic tests.     

Simultaneous detection of Salmonella typhi antigen and antibody in serum by counter-immunoelectrophoresis for an early and rapid diagnosis of typhoid fever

Serum samples were collected from 26 proved cases and 15 clinically suspected cases of typhoid fever and from 23 normal controls. The convalescent phase serum samples were obtained in 18 of the 26 proved cases of typhoid fever. Salmonella typhi antigen and antibody were detected simultaneously in the serum samples by counter-immunoelectrophoresis (CIE). The conventional Widal test was also performed to elicit S. typhi H and O agglutinins. Out of the 26 cases proved by culture examination, 25 were detected during early stages of the disease (24 positive for S. typhi antigen and 1 for antibody) by CIE. Out of 15 clinically suspected cases of typhoid fever, 4 were positive for S. typhi antigen and none positive for antibody. All the 18 convalescent phase serum samples were positive for antibody, but were negative for S. typhi antigen. None of the 23 normal controls gave any false positive reaction for either antigen or antibody. In contrast, the Widal test could detect only 1 out of 26 cases in the acute stage, and all the 18 cases where convalescent serum samples were obtained were positive for antibodies at diagnostic titres. None of the 15 clinically suspected cases and 23 normal controls were positive by the Widal test. The feasibility of using simultaneous S. typhi antigen and antibody detection by CIE in diagnosis of typhoid fever is discussed.     

Rapid diagnosis of severe Haemophilus influenzae serotype b infections by monoclonal antibody enzyme immunoassay for outer membrane proteins.

A highly sensitive and specific enzyme immunoassay (EIA) for the detection of Haemophilus influenzae serotype b antigens in body fluids and broth cultures was developed, with a polyclonal antibody directed against polyribose phosphate as the solid-phase reagent and a biotinylated monoclonal antibody directed against H. influenzae type b outer membrane protein as the liquid-phase reagent. H. influenzae type b antigens could be detected in broth cultures containing as little as 50 organisms per ml. The sensitivity and specificity of this system were compared with those of two commercial kits and counterimmunoelectrophoresis. The overall detection of H. influenzae type b antigens in clinical specimens collected from children infected with H. influenzae type b was as follows: with Phadebact, 86 and 86% in cerebrospinal fluid and urine specimens, respectively; with Bactigen, 86, 80, and 92%, with counterimmunoelectrophoresis, 78, 73, and 75%, and with biotin-avidin EIA, 100, 100, and 100% for cerebrospinal fluid, serum, and urine specimens, respectively. In the biotin-avidin EIA, no positive reactions were noted in specimens collected from patients infected with other bacteria or from patients without evidence of bacterial infection, whereas false-positive reactions were found by counterimmunoelectrophoresis and the commercial kits. These results suggest that this monoclonal antibody reacting with the outer membrane protein is more specific and sensitive than the conventional methods using polyclonal antisera for the detection of H. influenzae type b antigens during severe infections in children.     

Diagnosis of typhoid fever by detection of Salmonella typhi antigen in urine.

A monoclonal antibody specific for group D Salmonella antigen 9 was used in an indirect enzyme-linked immunosorbent assay (ELISA) for detecting the antigen in urine specimens collected from patients with clinical typhoid fever in Jakarta, Indonesia. The ELISA had a sensitivity of 95% in identifying patients in whom Salmonella typhi was isolated from hemocultures, 73% in patients in whom S. typhi was isolated from stool specimens, and 40% in patients in whom the organism was isolated from bone marrow cultures. Among patients in whom S. typhi was isolated from blood cultures, the ELISA had a sensitivity of 65% when a single urine specimen was examined and 95% when serially collected urine specimens were examined. A dot blot immunoassay performed on a nitrocellulose filter in parallel had a sensitivity of 85%, versus 83% for the plate ELISA in which S. typhi was isolated from blood, bone marrow, and/or stool specimens. Since S. typhi antigen is intermittently excreted in the urine of patients with typhoid fever, serially collected urine from patients with typhoid should be tested for antigen 9.     

Salmonella Typhi outer membrane protein STIV is a potential candidate for vaccine development against typhoid and paratyphoid fever

Enteric fever, caused by Salmonella enterica serovars, Typhi (S. Typhi) and Paratyphi (S. Paratyphi) is a major public health challenge for the developing nations. Globally, the disease affects ˜15-30 million individuals every year, resulting in >200,000 deaths. Multidrug-resistant S. Typhi H58 strain has emerged as the dominant circulating strain in a large part of the world and an extensively drug-resistant (XDR) subclade of the strain was recently reported. Many believe that vaccination of the susceptible populations is urgently needed and the best option to control the infection. However, the commercial live attenuated (Ty21a) vaccine is not recommended for children below six years of age while the Vi-polysaccharide-based vaccine has poor long-term efficacy against typhoid fever. Moreover, no vaccines are available against S. Paratyphi infection. Thus, a new formulation capable of providing long term protection against both the pathogens and safe for all age groups is immediately required. We show that recombinant, S. Typhi outer membrane protein STIV (rSTIV) is immunogenic in mice and elicits high serum titers of different immunoglobulin subtypes. STIV antibodies opsonize S. Typhi and S. Paratyphi A to promote antibody-dependent cellular cytotoxicity and complement-mediated lysis. Immunization with rSTIV also induces robust cell-mediated immunity, including antigen-specific T cell proliferation and cytotoxic T lymphocyte response. Finally, mice immunized with rSTIV are significantly protected against S. Typhi and S. Paratyphi A challenge, with reduced visceral bacterial load. Our results underscore the potential of rSTIV as a novel vaccine candidate for enteric fever.     

Duodenal isolation of Salmonella typhi by string capsule in acute typhoid fever.

Three of seven volunteers with acute typhoid fever had Salmonella typhi isolated from the duodenum using a string capsule device. The string capsule device provides a simple method for culturing S. typhi from the duodenal contents. Its possible use in typhoid carriers is discussed.     

Characterization of an outer membrane protein of Salmonella enterica serovar typhimurium that confers protection against typhoid

Typhoid caused by Salmonella enterica serovar Typhi remains a major health concern worldwide. The emergence of multidrug-resistant strains of Salmonella with increased virulence, communicability, and survivability leading to increased morbidity and mortality has further complicated its management. Currently available vaccines for typhoid have less-than-desired efficacy and certain unacceptable side effects, making it pertinent to search for new immunogens suitable for vaccine formulation. The outer membrane proteins (OMPs) of Salmonella have been considered possible candidates for conferring protection against typhoid. OMPs interface the cell with the environment, thus representing important virulence factors with a significant role in the pathobiology of gram-negative bacteria and bacterial adaptation. An OMP of Salmonella enterica serovar Typhimurium with an apparent molecular mass of 49 kDa that is highly immunogenic, evokes humoral and cell-mediated immune responses, and confers 100% protection to immunized rats against challenge with very high doses (up to 100 times the 50% lethal dose) of Salmonella enterica serovar Typhimurium has been identified. Further, very efficient clearance of bacteria from the reticuloendothelial systems of immunized animals was seen. This protein is recognized by the antibodies present in serum of typhoid patients. When sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel-eluted protein was further analyzed by high-performance liquid chromatography (HPLC) and two-dimensional electrophoresis, two polypeptides with the same molecular weight were resolved. These have different isoelectric points and gave two peaks with different retention times in reverse-phase HPLC. However, only one of the two bands interacted with patient serum. The immunogenicity studies (enzyme-linked immunosorbent assay and delayed-type hypersensitivity [DTH]) indicated that the immunoreactive protein evoked a strong immune response in rats. The N-terminal sequencing and analysis of the homology of this protein with sequences in the protein database of Salmonella resulted in a match with the N-terminal sequences of a protein in Salmonella enterica serovar Typhi (CT18 and Ty2 strains). The homology search further revealed it to be a hypothetical protein, whose gene had unidentified open reading frames in Salmonella serovar Typhi encoding 447 amino acid residues, corresponding to a molecular mass of 49 kDa. The nucleotide sequence of the encoding gene was deduced, and the gene was amplified by PCR using appropriate primers. An amplified 1.3-kb band was purified and sequenced to confirm its identity. These OMPs provide promising targets for the development of a candidate vaccine against typhoid.     

Alp, an arthropod-associated outer membrane protein of Borrelia species that cause relapsing fever

Borrelia hermsii and other relapsing fever (RF) species are noted for their highly polymorphic surface antigens, the variable major proteins (VMP). Less is known about other surface proteins of these pathogens in either their vertebrate reservoirs or arthropod vectors. To further characterize these proteins, we elicited antibodies against VMP-less cells, noted antibody reactions against whole cells and cell components, and then subjected selected antigens to mass spectroscopy for amino acid sequencing for comparison against a B. hermsii genome database. One of the derived monoclonal antibodies, H0120, agglutinated spirochetes, and in Western blot analyses, it bound to a 14-kDa protein of whole cells and their membrane fractions but not after protease treatment. A search of open reading frames of the B. hermsii genome with extracted peptides identified the 14-kDa protein with bha128, a 453-nucleotide gene of the 175-kb linear plasmid. The bha128 gene was synthesized and expressed in Escherichia coli. The protein product was bound by antibody H0120. Genes homologous to bha128 occur in the RF species Borrelia turicatae, B. duttonii, and B. recurrentis but not in Lyme disease Borrelia species or other organisms. The following findings indicated an association of BHA128, renamed Alp, with the tick environment: (i) Alp was produced at higher levels at 23°C than at 34 °C; (ii) almost all spirochetes in tick salivary glands were bound by the H0120 antibody, but only ~1% of spirochetes in the blood of infected mice were bound; and (iii) infected mice produced antibodies to several B. hermsii antigens but not detectably to native or recombinant Alp.     

Diagnosis of typhoid fever: detection of Salmonella typhi porins-specific antibodies by inhibition ELISA.

Porins are highly immunogenic outer membrane proteins of Salmonella. Sera from typhoid patients contained a high level of IgG antibodies directed to porins of Salm. typhi. Since porins are highly conserved proteins, anti-porins antibodies both from typhoid patients and healthy normals reacted with porins from several Gram-negative bacteria. Therefore, in order to improve the specificity of detecting Salm. typhi porins-specific antibodies, an inhibition ELISA was developed using enzyme-conjugated MoAbs (MP1 and MPN4) specific to Salm. typhi porins. Sera from typhoid patients with positive haemoculture (16 out of 17) inhibited the binding of MP1 to porins, thus showing a positive test for typhoid, whereas sera from patients with other Gram-negative bacterial infections (n = 7) and from healthy volunteers (66 out of 67) were found to be negative. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of this assay were 94.1, 98.7, 97.8, 94.1 and 98.7% respectively. The validity of our inhibition ELISA for typhoid was higher than that of the Widal test. The diagnosis of typhoid fever as early as 3 days after the onset of fever, using a single specimen is possible.     

Validation of an enzyme immunoassay for serodiagnosis of acute Q fever

An enzyme immunoassay was validated for the serodiagnosis of acute Q fever. Minimum positive tests were determined for both serial dilutions and a single dilution of patient sera. To establish the specificity of the test, 152 serum samples were tested from individuals with no evidence of pastCoxiella burnetii infection. Diagnostic titers were set at 128 for the IgM and IgG responses to phase I, at 512 for the IgM response to phase II and at 1,024 for the IgG response to phase IICoxiella burnetii. These titers gave a falsepositive rate of 1 %. Alternatively, testing a single dilution of sera (1:128) gave specificities ranging from 97.3 to 98.7 %. Tests with the greatest sensitivities, using serially diluted early convalescent-phase sera, were the IgM (84 %) and IgG (80 %) responses to phase IICoxiella burnetii. At a single serum dilution, 92 % of early convalescent sera had a positive IgG response to phase IICoxiella burnetii. With a high specificity and good sensitivity, the EIA can be used to diagnose acute Q fever with a single convalescent serum specimen. The duration of a positive response was greater than five years.     

Immune response to the iron-deprivation-induced proteins of Salmonella typhi in typhoid fever.

Iron starvation conditions limited the growth of Salmonella typhi, as evidenced by an increase in the lag phase of a culture and a decrease in the number of bacteria reached in the stationary phase. The analysis of the outer membrane of bacteria grown under these conditions identified new protein components with apparent molecular weights of 83,000, 78,000, and 69,000. The extent of induction of these proteins was regulated by increased iron deprivation. Immunoblot analysis showed that the serum of patients with typhoid fever exhibited an immunoglobulin G response to these iron-deprivation-induced proteins. The results of bioassays and DNA-DNA hybridization experiments indicated that pathogenic strains of S. typhi produced enterochelin but not aerobactin. Immunodetection with an anti-FepA antiserum confirmed that one of the induced proteins is the S. typhi analog of the Escherichia coli fepA gene product. These studies suggest a role for iron uptake in the pathogenesis of typhoid fever and confirm the immunogenicity of some of the outer membrane proteins of this pathogen.     

Toxocariasis: serological diagnosis by enzyme immunoassay.

An enzyme-immunoassay was developed to measure the concentration of serum antibody specific for the secretory antigens released by migrating toxocaral larvae. This technique was evaluated by testing sera from healthy UK adults, and from patients with and without toxocariasis. In 922 healthy adults, 2.6% were found to have elevated specific antibody levels. Elevated values were observed twice as frequently in males as in females but showed no significant regression with age between 20 and 65 years. Of 62 patients with non-toxocaral helminthic infections, all had antitoxocaral antibody levels within the range of values observed in healthy controls and had a mean level which was not significantly elevated. All of 13 patients with clinical toxocariasis had enzyme-linked immunosorbent assay (ELISA) antibody levels above the 100th percentiles of both the healthy population and the helminth-infected group and had a significantly high mean value (p less than 0.001) more than 12 times that of the healthy or infected controls. The high degree of sensitivity and specificity of the toxocariasis enzyme-immunoassay indicates that this new test should be useful in reference immunodiagnostic applications and in large-scale seroepidemiological surveys.