Indian Pediatrics 1999; 36:65-68 

Latex Agglutination Test (LAT) for the Diagnosis of Typhoid Fever

Rohini Kalhan
I. Kaur
R.P. Singh
H.C. Gupta

Typhoid fever is a serious public health problem in developing countries and remains an important cause of morbidity and mortality with 21 million cases and more than 700,000 deaths reported annually(1). The clinical presentation of typhoid is non- specific and varies from uncomplicated high grade fever to serious complications such as encephalopathy, peritonitis, perforation and hemorrhage.

Laboratory diagnosis is important for instituting early treatment and avoiding serious complications. The conventional methods being used for diagnosis are bacterial culture and serology. Hemoculture (Blood Culture) for Salmonella typhi is the most concurrent proof of diagnosis of typhoid fever(2) with isolation rate varying from 40-80%. Bacteriological isolation of Salmonella typhi (S.typhi) takes 3-4 days and this delay in initiating therapy might prove fatal, specially in young children.

Serological tests for typhoid fevers are reported to be non-specific, poorly standardized and confusing. The utility of Widal test, in endemic areas is stil1 controversial(3). Recently some new methods for rapid diagnosis of typhoid fever based on monoclonal antibody(4) and PCR (Polymerase chain reaction)(5) are being investigated. For these reasons a simple and rapid confirmatory test for antigen detection is required to diagnose typhoid fever. A study was conducted to standardize Latex Agglutination Test (LAT) for antigen detection in the blood culture broth to decrease the time taken for S. typhi isolation and increase sensitivity or detection of S. typhi from blood culture broth.

Subjects and Methods

The subjects for study were taken from children attending the Pediatric Outpatient Clinic of UCMS and GTB Hospital, Delhi, India. Six hundred and thirty eight children of either sex below 12 years of age with complaints of fever were assessed clinically for the cause of fever. History of associated symptoms like loss of appetite, pain abdomen, constipation, diarrhea and altered sensorium was taken. Complete physical examination specially recording weight, pulse, coated tongue, toxic look, hepatomegaly and splenomegaly was done. Among these blood was taken from 156 children suspected to have typhoid fever based on history and physical examination and inoculated into blood culture broth and plain tubes for sera separation. Included in the study were 100 children with blood culture isolation of Salmonella typhi or high widal titer.

The various groups were: (i) Study Group: Group I-Fifty children confirmed by culture isolation of Salmonella typhi (confirmed typhoid cases); and (ii) Control Groups: (a) Group II-Fifty febrile controls selected from pediatrics ward where cause other than S. typhi has been established; (b) Group III-Fifty afebrile healthy controls that were siblings of the children admitted in pediatric ward for any reason with no history of fever and TAB vaccination in the last one year; and (c) Group IV-50 children with high widal titer (TO or TH ≥ 128) in single sera sample. Cut of titer decided according to basal antibody level in this area(3,6). (Unpublished Data based on S, typhi 'O' and 'H' antibody determined in 1828 healthy children in East Delhi. in 1990).

Blood culture isolation and identification for S. typhi was done by conventional method using Sodium Taurocholate broth and subcultures done on 24 h, 48 hand 7 days of incubation. Widal test was done using commercially prepared colored antigens (Span Diagnostics Pvt. Ltd., Surat, India) by tube agglutination method on serum samples:

Latex Agglutination Test (LAT)

Raising of Antisera and Separation of Immunoglobulins

Ultrasonic lysate of S. typhi 901(0) strain was used as antigen to raise antiserum in rabbits and to check the specificity of the antisera by gel diffusion method(7). The anti serum was cross-absorbed with E. coli and Pseudomonas(7,8) and the protein content was estimated(9).. An aqueous suspension 10% solid content of Latex polystyrene particles (Sigma International, St. Louis, USA), average size 0.8 u, was diluted 1:10 to make 1 % suspension and then sensitized with anti- serum(10).

Checker Board Titration

For standardization a checker board was put up using a range of immunoglobulin concentration, 25-200 mg/ml, to coat latex particles to detect optimum concentration of immunoglobulin required to sensitize the latex particles to detect. minimum amount of antigen and give maximum sensitivity.

Test Procedure

To 20 ml of Blood Culture broth, 20 ml of sensitized latex particles were added on black plastic slides and mixed with applicator stick. Twenty ml of S. typhi inoculated broth was used as a positive control and 20 ml of uninoculated broth was used as negative control. After manual rotation of slide for 2 minutes, agglutination was recorded visually and results reported according to a 0-4+ scale(11); 2+ or greater agglutination was taken as positive reaction.

Results

This study included 100 children suffering from typhoid fever among which 50 were confirmed by blood culture isolation and 50 were possible typhoid fever based on high widal titer (TO or TH ≥128). One hundred children; 50 with febrile illness confirmed to be other than typhoid and 50 normal healthy children were used as negative controls. With respect to age and sex distribution, the study and control groups were similarly distributed with maximum number in older age group (9-12 yrs). The male to female ratio was approximately 2:1.

In Groups I and IV, fever was the presenting symptom in all the children. Gastrointestinal tract disturbances like diarrhea and abdominal pain were present in 20% and 28%, respectively. Altered sensorium and suspected enteric encephalopathy was seen in II %. Most cases had fever, which was high grade and continuous in character (70%). The typical stepladder pattern was not seen. After fever, hepatomegaly and splenomegaly was noted in 64% cases.

LAT, which could detect 900 ng/ml of antigen as observed in checker board titration, was positive in all 50 children from Group I who had positive blood' culture and in 38 children from Group IV who were culture negative and had high Widal titer positive (Table I). LAT was positive in 4 children in Group II and none in Group III (Table I). Using blood culture positive cases as true positive and children in Groups II and III as true negative, the test had a sensitivity of 100% (95% CL 91.1,100) and specificity of 96% (95% CI 89.5, 98.7).
 

TABLE I

Comparison of Blood Culture and LAT for Diagnosis of Typhoid Fever.

	total	Blood culture positive for S. typhi	LAT positive
Group I	50	50	50
Group II	50	0	0
Group III	50	0	4
Group IV	50	0	38

LA T was able to detect antigen in 46 cases in 24 h while 4 became positive after 48h (in all 38 in Group IV in 24 h) compared to blood culture in which S. typhi was isolated in 42 cases in 48 hand 8 cases in 72 h.

Discussion

The diagnosis of typhoid fever is difficult based on clinical history and examination alone due to the non-specificity of the clinical manifestations. Delay in therapy may result in major complications like enteric perforation with peritonitis, encephalopathy and intestinal hemorrhage. Unnecessary use of antibiotics in febrile illness which are not typhoid may result in emergence of antibiotic resistant strains of bacteria. Thus, definitive and early diagnosis of typhoid fever is necessary.

In the present study, the diagnostic value of LA T for detecting S. typhi antigen was evaluated in blood culture broth from 100 children with confirmed or possible typhoid fever and 100 controls. S. typhi could be isolated from blood of 50% patients' only as most of the patients (68%) presented between 7 to 10 days of illness. S. typhi could not be isolated from blood in 50% of patients even though the sample was collected during 1-2 weeks of illness, possibly due to prior antibiotic administration.

We used LAT for detection of antigen in culture broth and could detect antigen in all the S. typhi positive culture broth. These results are comparable to those reported by others(12). In addition, the test Was positive in 38 out of 50 culture negative broth in which Widal was positive (single high titer). The test is known to be able to. detect killed bacteria and soluble antigen in the absence of viable organisms(13). In 12 patients with possible typhoid fever, antigen could not be detected by LAT, possibly because the patients presented after 2 weeks of illness 'when antigen level decreases in the blood(14). Since the specificity of the test in true negative cases was high, we believe that the patients with LAT positive test butnegative cultures represent true cases of typhoid fever.

LAT could detect S. typhi antigen in 92% of culture broth of Group I after 24 hand 8%" more after 48 h in Group I and in all 100% positive cases in Group IV, thus saving 1-2 days which are required for confirming positivity of blood culture. Four false positive re- actions were seen in Group II cases suffering from S. paratyphi A and E. coli infections. This could be because of sharing of antigenic determinants.

In conclusion, LAT was found to be significantly sensitive (100%). and specific (96%) and could detect 76% more cases in Group IV (possible typhoid cases). Thus, LAT can be used for rapid diagnosis of typhoid fever though it cannot replace conventional blood culture required for isolation of organism to report the antibiotic sensitivity.

 

 References

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