|Year : 1997 | Volume
| Issue : 8 | Page : 265-269
Haemagglutination system for the simultaneous detection of LPS and anti LPS antibodies of S.typhi
Sanjay Shukla, DS Chitnis
Department of Microbiology & Immunology, Choitram Hospital and Research Centre, Indore-452 001, India
Department of Microbiology & Immunology, Choitram Hospital and Research Centre, Indore-452 001
|How to cite this article:|
Shukla S, Chitnis D S. Haemagglutination system for the simultaneous detection of LPS and anti LPS antibodies of S.typhi. Indian J Med Sci 1997;51:265-9
| ¤ Introduction|| |
With the continued high endemicity of enteric fever in developing countries like India, the need of a new, rapid, simple and economical test is urgently felt. Recently many in-imunological Tests have been developed viz. ELISAs, ,,, CIEP,  COA+C,  HA, ,, and PCBs  etc. but none of them could be Proven useful for diagnosis at ground level because of various reasons.
In view of this, we developed a Haemagglutination system to detect the anti-LIPS antibodies and the same sys em was also used to detect the LPS antigen by inhibition process. Thus, a simultaneous detection of antigen and antibody was the aim of this study for the rapid and simple diagnosis of typiioid.
| ¤ Material and Methods|| |
(A) Extraction of LioopolysaccI,) ride (LPS) : The LPS was extrrctod from Salmonella More Details typhi 'O' 901 strain. The acetone dried cells were used for LPS extraction by Wes phal's hot puenol - water method. 
(B) Sensitization of Sheep Red blood cells : The formalin fixed sheep erythrocytes were sensitized with saponified LPS. 2.5% suspension of SRBCs in nor mal saline was mixed with saponified LPS (100 µg/mL). After incubation at 37°C for 1 h, the SRBCs were washed and finally a 0.5% suspension was preserved at 4°C with 1% sodium azide. 
Widal positive serum, LPS solution of known concentration and unsensitized S'RBSs (0.5%) were used as controls in this heemaggiutination system.
The test was standardized by checker board titration with different reagents and physical conditions. Finally, the following protoco'Is were used :
(i) Protocol for haemagglutination (Antibody detection)
50 µL SRBCs + 25 µL serum or control.
Incubation at room temperature for 1 h
(ii) Protocol for haemagglution inhibition (Antigen detection)
(a) Step-I : 25 µL of Widal positive serum (1:40) mixed with 25µL serum or culture supernate and kept at RT for 30 min. (b) Step-11 : 50µL sensitized SRBCs were added into the microwells containing antibody + test sample. Incubation at RT for 1 h.
1. Haemagglutination : The results were interpreted according to the appearance of "button" or "mat" in the microwell. The sample is positive if the mat appears in haemagglutination test.
2. Haemagglutination Inhibition : The LPS present in the test sample will neutralize the antibodies (antiLPS) present in the Widal positive antiserum in Step-I. Therefore, there will be no antibody to react with the sensitized SRBCs and in turn haemagglutination reaction will be inhibited. Thus, appearance of button in microwells i ans indication of positivity. In other words, if a sample gives positive haemagglutination it means it contains anti-LPS antibodies against S. typhi while a positive haemagglutination inhibition means that the sample contains LPS antigen of S. typhi. The standardized haemagglutination inhibition test showed a sensitivity in the range of 1.5625 µg/ mL LPS.
The following three groups were subjected to both the tests :(a) Healthy volunteers : Sera samples from 500 relative/voluntary blood donors at our blood bank were collected who did not give history of typhoid or TAB vaccination during recent two years. (b) Culture proven cases of typhoid : Thirty patients from whom the Si typhi was isolated in the blood culture were included in this group. (c) Suspected cases of typhoid : Patients who were suspected to be having typhoid on the basis of clinical features and in whom the blood culture was either negative or could not be a tempted. Sera were received from 96 such persons. Culture broth enrichment: Some representative gram negative organisms (approx. 2 µL) were included in the bottles containing 50 mL glucose broth medium. These culture bottles were incubated at 37° C. One mL broth was taken out from every bottle at hourly interval. These hourly aliquots were then boiled, spun and the supernatants were subjected to HAI test. Before subjecting these samples for antibody detection by HA, the cut off value was established by applying this test o 500 sera from normal persons. The cut off was fixed as mean 2 standard deviations HA+2SD value was 28.08. For ease in dilutions, a cut off titre of 40 or above was finally accepted as positive for interpretation.
| ¤ Results|| |
Among the 500 normal healthy controls, the presence of anti LPS antibodies by HA test was observed in 1.8% persons at the cut off 1:40. The positive haemagglutination was observed in only 18 out of 30 authentic typhoid cases while this was positive in 62.5% persons who were suspected to have typhoid. Thus, this anti LPS haemagglutination test showed a sensitivity of 60% and specificity 98.2%. The positive predictive value and negative predictive value were found to be 66.66%, 97.61% respectively. The haemagglutination inhibition test for the LPS detection in the same samples failed to pick up any positive sample even among the authentic typhoid patients. During our preliminary study [Table 2] the HAI test was applied to the culture supernatants for antigen detection. This tests could pick up the antigen just after 1 hour of inoculation of S. typhi into broth but none of the other organisms gave positive haemaggIuination inhibition.
| ¤ Discussion|| |
Blood culture and Widal test are the conventional techniques of the diagnosis of typhoid fever but they have many limitations. Blood culture is time consuming, facilities to carry out culture are available only in the larger laboratories in developing countries where typhoid is endemic. Further, the initiation of antibiotics prior to culture inhibits the bacterial growth. 
Serodiagnosis by Widal test is convenient and rapid but not reliable and controversies exist on its usefulness. 
We reported significant basal antibody levels in normal healthy population by Widal test. In our observations  Widal test showed a `false' positivity in 17.74% of normal population while the presently designed HA test was found to false positivity in only 1.8% among healthy persons. This is almost ten times less than the Widal test. This may be because Widal test uses dead cells of Si typhi and in turn detects antibodies against 0 and H antigens besides several other bacterial antigens. Some of the antigens can have crossreactions with other salmonellae and many other gram negative bacteria. [Table 1] shows comparative results of Widal and HA tests performed on sera samples of patients suspected to have typhoid and bacteriologically proved typhoid.
Our approach to detect the LPS antigen in serum through HAI failed to pick up even a single serum sample including the authentic typhoid cases Qadri et al  have reported the failure of their ELISA to detect the LPS in serum or urine even using monoclonal antibodies. The sensitivity of our assay (HAI) was in the range of 1.5625 µg/mL and the sensitivity of Qadri et al's ELISA was 10 ng/mL. Therefore, the amount of soluble LPS yin serum or urine must be in the range of nanogram to picogram. However, many workers have demonstrated the presence of soluble antigen in serum or urine using various techniques. ,,, Since none of them had a sensitivity less than 10 ng/mL, it is difficult to accept their results.
An alternative approach is the detection of antigen after enrichment of sample through culture.  The haemagglutination inhibition was observed just after one hour of inoculation of the sample for enrichment in to the broth. Also, the HAI test showed a 100% specificity when the culture supernates of the other gram negative organisms were subjected. The cross reactivity was not found even with the other salmonellae.
Haemagglutination system is simple and the reagents can be prepared in laboratories. The other tests which detected the LPS in culture broths need, not only costly reagents but expensive infrastructure and expertise and therefore can be useful only in larger laboratories. With this view, the HA system developed at our end is advantageous over the other tests being economical, quick and performable anywhere. Like others, this test also faces the problem of prior antibiotic therapy or the bcatericidal action of blood itself which renders bacteria not to grow in the culture medium and the enrichment cannot be achieved.
| ¤ Summary|| |
In view of the limitations of Widal test for the diagnosis of typhoid, haemagglutination test using sensitized sheep red blood cells was designed at our laboratory. The test gave only 1.8% positivity at 1:40 dilution among 500 normal persons. Eighteen of the 30 culture proven cases gave the HA test positive while the positivity was 62.5%, among suspected cases of typhoid. Thus, the anti LPS haemagglutination test showed a sensitivity of 60% and specificity of 98.2%. The positive predictive value and negative predictive value were 66.66% and 96.7% respectively.
The haemagglutination inhibition test was also developed for the detection of LPS antigen of S. typhi in the serum samples of typhoid cases and could detect 1.5625 µg/ mL of S. typhi LPS antigen but failed to detect LPS in the sera of bacteriologically proven cases of typhoid. However, it could detect the growth of simulated blood cultures of S. typhi within one hour of inoculation and did not give any cross reactions with other bacterial cultures.
The data suggest that the haemaggluination test could be a good adjunct for Widal test and the haemagglutination inhibition test could help the early detection of S. typhi in culture.
| ¤ Acknowledgement|| |
The authors are thankful to Dr. NS Bhagwanani, Medical Director, Choithram Hospital & Research Centre and the management for their support in this work.
| ¤ References|| |
|1.||Quadri A, Ghosh S, Prakash K et al. Sandwich Enzyme Immuno assays for the detection of Salmonella typhi. J Immuno Assay 1990;11:251-270. |
|2.||Chaicumpa W, Ruangkunaporn Y, Burr D et al. Diagnosis of typhoid fever by detection of Salmonella typhi antigen in urine. J Clin Microbiol 1992;30:2513-2515. |
|3.||Rao J, Raghunath M, Singh N. Rapid diagnosis of typhoid fever by a new ELISA test with porin antigens. Indian J Med Microbiol 1993; 11:191-196. |
|4.||Appassakij H, Bunchuin N, Sara sombath S et al. Enzyme linked Immunosorbent Assay for the detection of Salmonella typhi protein antigen. J Clin Microbiol 1987;25: 273-277. |
|5.||Gupta AK, Rao KM. Simultaneous detection of Salmonella typhi antigen and antibody i n serum by counterimmunoelectrophoresis for an early and rapid diagnosis of typhoid fever. J Immunol Methods 1979;30:349-353. [PUBMED] |
|6.||Chatterjee PP, Mohan M, Talwar V et al. Evaluation of coagglutination test for diagnosis of typhoid fever in children. Indian J Med Res 1988:87:157-160. |
|7.||Xi W, Ni X, Lu T et al Early diagnosis of typhoid fever using monoclonal antibody, Chin Med Sci J 1993;8:123-124. |
|8.||Petchclai B, Ausavarunguirun R, Manatsathit S. Passive haemagglutinationn test for enteric fever. J Clin Microbiol 1987;25:138-141. |
|9.||Rai GP, Zachariah K, Shrivastava S. Comparative efficacy of indirect haemagglutination test, indirect fluorescent antibody test and enzyme linked immunosorbent assay in serodiagnosis of typhoid fever. J Trop Med Hyg 1989;92131-434. |
|10.||Song JH, Cho H, Park MY et al. Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction. J Clin Microbiol 1993:31:1439-1443. |
|11.||Westphal 0, Jann K. Bacterial lipopolysaccharides. Extraction with phenol-water and further application of the procedure. Methods Carbohyder Chem 1965:5:83. |
|12.||Moudgil KD, Singh AK. Agglutination methods. In : A handbook of practical and Clinical Immunology. vol 1 2nd Ed. Talwar GP, Gupta SK (eds). pp 194-208 CBS publishers & Distributors, New Delhi, 1993. |
|13.||Shukla S, Patel B. Chitnis DS. 100 years of Widal test and its reappraisal in an endemic area. Indian .l Med Res 1997:105:53-57. |
[Table 1], [Table 2]