Typhoid fever is becoming an ever increasing threat in the developing countries. Cultivation of bacteria and serology (especially Widal test) give unacceptable levels of false-negative and false-positive results, respectively.
The aims of present work is development of a PCR assay that can target specific multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. typhi) .(which has 100% specificity for Salmonella typhi) was compared with Widal test and Salmonella typhi IgG\IgM rapid test as well as Salmonella typhi IgG and IgM ELISA test during the first week of illness of 50 suspected cases of typhoid.
PCR primers for invasion, O, H and Vi antigen genes, invA, prt, and viaB were designed and used for the rapid detection of S. typhi by multiplex PCR.
The respective figures of positivity for PCR, Blood culture, Widal test and S. typhi IgG\IgM rapid test and Salmonella typhi IgG /IgM ELISA were 66%, 52%, 46%, 42% and 48% respectively. A control group of 20 healthy persons gave figures of 0%, 0%, 40%, 0% and 0% respectively.
The present study conclude that this PCR-based technique is not only absolutely specific, but also very sensitive and, therefore, much superior to Widal test, Blood culture S. typhi IgG\IgM rapid test and Salmonella typhi IgG/IgM ELISA tests for the early diagnosis of typhoid.Introduction
Typhoid (enteric) fever is still a common disease in many developing countries as well as it is a major health problem throughout the world. Globally, there are more than 21 million cases of typhoid each year, with more than 700,000 deaths. It is especially prevalent in developing countries. Early detection of the disease is very important for its control, but unfortunately, current definitive diagnostic tests are inadequate. [1,2,3].
Diagnosis is made by any blood culture, bone marrow or stool cultures and with the Widal test (demonstration of salmonella antibodies against antigens O-somatic and H-flagellar)., urine culture, rose-spot culture, duodenal string culture, ELISA and immunofluorescence. Widal test and blood culture remain the only universally practiced diagnostic procedures, becauother methods are either invasive, have failed to prove their utility, or are expensive [4].
Widal test is quite sensitive but has become highly nonspecific, poses some disadvantages in endemic areas. Previous exposure to S typhi or antigenically related Gram negative bacilli and vaccination against typhoid can result in raised titres in the absence of a current infection. In contrast, a poor antibody response to either the “O” or “H” antigen (or both) can occur in some patients. Hence, the Widal test often leads to confusion and, on occasions, to misdiagnosis of other febrile illnesses as typhoid fever [5,6].
Another shortcoming of the Widal test is that it becomes positive only in the second week of illness, so its value for early detection of the disease is limited [7].
Blood culture is positive in the first week but its utility is restricted by the verylow numbers of bacteria causing severe disease (which may be less than 10/mL). As a consequence, blood culture can detect only 40%-45% of cases, and even if antibiotic treatment has not been administered, the rate of detection is not more than 70%. Molecular techniques, as is the case in blood culture, target the pathogen itself (not antibodies produced against it), so they can be useful for the early detection of the disease [8]. Clinically suspected cases of typhoid who had had fever for less than three days and had not received any anti-typhoid therapy were included in this study.
While the blood culture tests though time- consuming is considered the gold standard in typhoid fever diagnosis [8], so the sensitivity of the MPCR assay with the given primer sets (By targeting three genes in Salmonella typhi), Widal test, Salmonella typhi IgG/IgM rapid test and Salmonella typhi IgG/IgM ELISA tests were performed on samples from these cases and next calculated and compared to the sensitivities of the blood culture test, which are the conventional means of diagnosis for typhoid fever, to evaluate their relative utility, specificity and sensitivity.
Materials and Methods
Patients
Fifty patients included in this study had fever for less than 3days, most of them are Widal positive, had had no anti-typhoid treatment, Members of both sexes representing all ages were included, all serum samples were collected from Mirjan educational Hospital during the summer of 2012. The control group constituted 20 healthy individuals with no recent history of fever.
Blood culture test. Patient blood samples (2.5 ml) were drawn and added to brain heart infusion medium. This medium was then incubated at 37°C for 24 h and later streaked onto MacConkey agar plate. If the colony did not ferment lactose, furtheroxidase test and slide agglutination test were done. The colonies that gave negative results by the oxidase test and a positive result by the slide agglutination test were further confirmed through biochemical tests where S. typhi is indole negative, and urease negative and does not ferment mannitol [9].
Widal test. Rapid slide test for qualitative in vitro determination of antibodies in serum against Salmonella Typhi O and H antigens and/or Salmonella Paratyphi A(H) and B(H) antigens were done using TyDAL kit (Arsitha Diatech) following the manufacturer’s instructions.
OnSite Typhoid IgG/IgM Combo Rapid Test-Cassette
The OnSite Typhoid IgG/IgM Combo Rapid Test (Biotech, USA) is a lateral flow immunoassay for the simultaneous detection and differentiation of anti- Salmonella typhi (S. typhi) IgG and IgM in human serum, plasma or whole blood. It is intended to be used as a screening test and as an aid in the diagnosis of infection with S. typhi [10].
Salmonella typhi IgG and IgM ELISA
S. typhi specific IgM and IgG antibodies were estimated according to the manufacturer instructions (Calbiotech, a life Science company), the positive IgG & IgM was calculated depend on finding the index value, more that 0.08 index value represent as positive cases.
DNA Extraction and Identification
One mL of blood containing 20 mM potassium EDTA as anticoagulant was centrifuged at 10,000 rpm for 5 minutes. Plasma was separated for serology. One mL of lysis buffer (0.2% Triton X100 in Tris HCl pH 8.0) was added to the pellet. The mixture was gently aspirated several times to effect hemolysis. The tube was centrifuged at 12,000 rpm for 6 minutes, the supernatant was discarded, and the procedure was repeated once. The pellet was washed once with distilled water. After the removal of the supernatant, the pellet was resuspended in 20-30 ?L of distilled water. The tubes were sealed, kept