Surveillance for typhoid fever in Matsuyama city during 1974-1981 and detection of Salmonella typhi in sewage and river waters

Eighty-two cases of typhoid fever were found in Matsuyama city in the period from 1974 to 1981. Seventy-six cases were found to be infected with Salmonella typhi other three with Salmonella paratyphi A, and the remaining three were diagnosed only clinically. The strains of S. typhi isolated from these patients showed such a variety of Vi-phage types as D1, D2, E1, M1, 53 and degraded Vi-positive strain (DVS). The concurrent survey of the city sewage and river waters for typhoid bacilli was conducted with total 578 samples taken therefrom. S. typhi was isolated from 120 of those samples. The Vi-phage types of the isolates were closely related with those of the isolates from the patients. The periodical examinations of the city sewage and the draining river may serve as a useful means for the controlling typhoid fever epidemics.     

Development of specific cellular immunoreactivity in typhoid fever.

Development of cellular immunoreactivity to Salmonella typhi and Salmonella paratyphi-A was studied by the leukocyte migration inhibition test in 9 patients with typhoid fever and in 2 patients with paratyphoid fever. Cellular reactivity could be demonstrated from the first days of the disease in all the subjects. The most pronounced migration inhibition was observed during the febrile period. It is suggested that specific cellular reactivity may play a pathogenetic role in typhoid fever.     

Immunofluorescent Identification of Salmonella typhi During a Typhoid Outbreak

Immunofluorescence and conventional bacteriological methods were compared for their ability to detect Salmonella typhi in 134 fecal specimens from 105 individuals associated with an outbreak of typhoid fever. Smears prepared from untreated fecal material (direct method) and after a preliminary incubation in selenite F broth (delayed method) were tested with an anti-Vi serum conjugated with fluorescein isothiocyanate. The delayed method was more sensitive than the direct method in detecting S. typhi. The delayed method was positive in 40 of 41 patients positive by culture methods, but gave positive or questionable reactions in 11 presumably uninfected individuals. The fluorescent-antibody test employing a Vi conjugate is a satisfactory screening procedure for detecting S. typhi, but all positives must be confirmed bacteriologically.     

Determination of O and Vi antigens in typhoid fever in the slide coagglutination reaction

Investigations with a view to the development and trial of a slide coagglutination test system for the detection of specific Salmonella typhi antigens have been made. As a result, diagnostic agents with sensitivity to group D Salmonella lipopolysaccharides and Vi-antigen, equal to 1 X 10(-5) to 1 X 10(-6) g/l, have been obtained. Specimens of saliva, urine and fecal filtrates from 61 adult patients with bacteriologically confirmed typhoid fever and 54 practically healthy persons have been studied. The coagglutination test has been positive with specimens from 90 +/- 4% of typhoid fever patients and, within the first 5 days of the disease, with those from 85 +/- 7% of such patients. The slide coagglutination test with saliva specimens has been found to be more informative than that with urine specimens. The data obtained in these investigations indicate that the slide coagglutination test is highly sensitive and specific, which offers good prospects for its use as a simple, economic and demonstrative method for the early tentative diagnosis of typhoid fever.     

Occurrence of Salmonella typhi and Salmonella paratyphi in Jordan

In order to justify the surveillance control system and hygiene policy in Jordan, this study evaluated the occurrence of diarrhoea during the period 1988-2000, focusing on cases caused by Salmonella typhi and Salmonella paratyphi. From January 1988 to December 2000, the number of notified diarrhoeal cases by the Ministry of Health in Jordan was 1,399,563 million. Other groups of patients confined to the Governorate of Amman was diagnosed at Al-Battikhi Medical Laboratories. One-way ANOVA and Least Significant Difference (LSD) were carried out for statistical analysis. The number of reported diarrhea cases was 1,399,563, 53.0% were males, and 47.0% were females, among them, 80.3% were < 20 years and 19.7%, were > 20 years. Out of 245,255 patients tested for S. typhi and S. pararyphi, positive stool culture were 1992 (0.6%). Out of these, 960 (48.2%) were males and 1,032 (51.8%) were females (P = 0.028). The highest incidence rate (10.8) was observed in the year 1993, while the lowest incidence rate (0.9) was found in year 2000. A significant difference (P < 0.001) was found between the number of S. typhi and S. Paratyphi cases and year. The seasonal variation was also found to be significant (P < 0.0001), with the summer period showing the highest incident rate. A significant difference (P < 0.001) was observed between number of typhoid and paratyphoid cases and districts. A significance difference between number of typhoid and paratyphoid cases with age and sex. The group most affected was school age and adolescence. The demographic situation plays an important role in reporting typhoid and paratyphoid cases, where there might be an urgent indication for a better surveillance control system on water resources and disposal systems. S. typhi and S. paratyphi antibiotics resistance pattern showed they were resistant to tetracycline (56.0%, 58.0%), ampicillin (45.0%, 48.0%), trimethoprim (43.0%, 47.0%), cephtazidime (12.0%, 13.5%) chloramphenicol (6.8%, 7.2%), gentamycin (3.0%, 4.0%) neomycin (2.1. 1.8%), calvulanic acid (augmentin (1.4%, 2.2%) and norofloxacin (0.92%, 1.1%). Susceptibility to amikacin, ciprofloxacin, cetfriaxone, ofloxacine, imepenim, cefixime and cefotaxime was 100.0%. The increase in percentage of antibiotic resistant strain might indicate a need for a further prescribing policy for treatment.     

Molecular cloning of a Salmonella typhi LT-like enterotoxin gene

Diarrhoea is a common event during typhoid fever; nevertheless, the possible participation of a diarrhoea-inducing enterotoxin has not been described (Roy et al., 1985). Recombinant bacteriophage lambda FDC1 was isolated from a genomic library of Salmonella typhi, the causal agent of typhoid fever, by screening with a probe for the B subunit gene of the heat-labile, cholera-like, Escherichia coli enterotoxin (LT). Lambda FDC1 codes for an enterotoxin that causes secretion in rat ileal loops, that elongates Chinese hamster ovary (CHO) cells, that is recognized by antibodies against LT, and does not bind in vitro to ganglioside GM1. These results should allow further studies towards elucidating a possible role for the S. typhi enterotoxin in the pathogenesis of typhoid fever.     

Prevalence of multi-drug resistant Salmonella typhi among clinically diagnosed typhoid fever patients in Lagos, Nigeria

A total of 635 clinically diagnosed typhoid fever patients were bled from three different health institutions in the metropolis of Lagos, Nigeria over a period of 15 months, May 1997 to July 1998. Out of the total blood cultured, 101 (15.9%) isolates of Salmonella species were isolated of which 68 (67.3%) were S. typhi, 17 (16.8%) and 16 (15.8%) were S. paratyphi A. and S. arizonae respectively. The overall isolation rate of S. typhi among patients is 10.7%, with most isolates 45.9% found among the severely-ill young adults, age group 16-30 years. All isolates were subjected to anti-microbial susceptibility testing using 12 different antibiotics: chloramphenicol, ampicillin, cotrimoxazole, gentamicin, colistin sulfate, nalidixic acid, nitrofurantoin, cefotaxime, tetracycline, streptomycin, ofloxacin and ciprofloxacin. All the S. typhi and S. paratyphi A isolates showed resistance to two or more of the 10 of 12 antibiotics tested particularly the 3-first-line antibiotics commonly used (chloramphenicol, ampicillin and cotrimoxazole) in the treatment of typhoid fever in Nigeria. No isolate showed resistance to ofloxacin and ciprofloxacin, however, nalidixic acid and gentamicin showed a moderate and appreciable inhibition to most of our isolates.     

Pathogenetic and immunological characteristics of different forms of the Salmonella typhi carrier state

Infectious granulomas with macrophages containing Salmonella typhi have been detected in the immune organs of the intestine of typhoid patients by means of morphological investigation techniques, immunofluorescent and electron microscopy. This suggests that typhoid granulomas form the basis of S. typhi primary carriership complicated by the relapses of this infection in cases of weakened cell-mediated immunity, which is proved by a decrease in the level of T-lymphocytes and by increased leukocyte migration index in relapses of typhoid fever and in S. typhi primary carriership. At the same time, the formation of S. typhi secondary carriership occurs in the process of the colonization of the altered organs and tissues of the body by S. typhi. This secondary carriership differs from the primary one by a number of pathogenetic signs. The detailed characterization of these two forms of S. typhi carriership is presented.     

Detection of Salmonella typhi by polymerase chain reaction

A rapid and sensitive method for detection of Salmonella typhi would help in preventing the spread of outbreaks and in clinical diagnosis. In order to develop unique PCR primers to detect Salm. typhi , ribosomal RNA genes from Salm. typhi (Rawlings) were cloned in pUC18. The resulting clone was confirmed by sequencing. The cloned DNA fragment contained the 5S, part of the 23S rRNA genes and the 5S-23S spacer region (EMBL/GenBank accession No. U04734).
It was expected that the 5S-23S spacer region is divergent unlike the highly conserved 23S+5S genes. This was confirmed by comparison with the rRNA gene sequences in the EMBL/GenBank database. A pair of PCR primers specific for Salm. typhi was obtained, based on this spacer region sequence. The specificity of this pair of primers was tested with 54 Salm. typhi strains (of 27 different phage types). All these Salm. typhi strains showed the positive 300 bp PCR product with this pair of primers. Six other Salmonella species as well as six other non- Salmonella bacteria were tested and none showed the 300 bp PCR product. The sensitivity of the detection level was 0·1 pg of pure Salm. typhi genomic DNA, or approximately 40 Salm. typhi cells in a spiked food sample. This pair of primers therefore has the potential for development into a diagnostic tool for the rapid diagnosis of typhoid fever.     

Transient inhibition of glucose utilization by erythrocytes during the acute stage of typhoid fever

The exact reason for hemolysis of glucose-6-phosphate dehydrogenase-deficient (G6PD) erythrocytes in patients with typhoid fever is unknown. Therefore, glucose utilization by normal and G6PD-deficient erythrocytes was measured during incubation with plasma of healthy controls as well as from patients in acute or recovery stages of typhoid fever. Glucose utilization in normal and G6PD-deficient erythrocytes significantly decreased compared to the controls when incubated with plasma of patients with acute typhoid fever, which normalized to the baseline after recovery from typhoid fever, suggesting an acquired alteration in G6PD enzyme properties by Salmonella typhi or its endotoxins.     

Development and evaluation of a PCR method for diagnosis of Salmonella enteric fever, based on DNA sequences of the hilA gene

Typically, diagnosis of enteric fever due to Salmonella spp. is by bacterial isolation from blood culture; however, the blood culture method is slow, not always available, and not informative in patients with antibiotic treatment. Salmonella spp. uses the hilA gene (component of the pathogenicity island I) to invade epithelial cells and produce infection. Using the hilA gene sequence a PCR test was designed to detect Salmonella in blood samples. The sensitivity (S), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV) of the PCR method were obtained by testing the blood samples from 34 patients with suspected of enteric fever. Presence of S. typhi was confirmed by blood culture. Blood samples were also tested from 35 patients with infections due to other non-Salmonella pathogens, again corroborated by blood culture (Klebsiella pneumoniae, 9; Serratia marcescens, 5; Escherichia coli, 4; Pseudomonas aeruginosa, 9; Providencia alcalifaciens, 4; Enterobacter cloacae, 4). Control samples were obtained from 150 healthy volunteers. The S, SP, PPV and NPV for the PCR method were all 100%. The lowest number of colony forming units/ml detected by PCR in blood samples was 10.     

Leukocyte migration inhibition by a specific antigen in human typhoid fever.

Cellular reactivity to heat-killed Salmonella typhi antigen was investigated by the leukocyte migration inhibition (LMI) method in 33 S. typhi infected patients and in 32 control persons. In the typhoid group a statistically significant LMI was observed as compared to the members of the control group. A correlation was found between the level of the cellular sensitivity and the time elapsed between onset of the disease and performance of the test. Previous typhoid vaccination had no influence on the LMI. No correlation was found between the agglutinin titres and the sensitivity demonstrated by the LMI test. The value of the method in studies of cellular immunity in typhoid fever is discussed.     

Demonstration of an antigenic protein specific for Salmonella typhi

Current studies were undertaken to determine the presence of a specific antigenic protein on the outer membrane of Salmonella typhi. Immunoblot analysis using sera from patients with fevers revealed that the 50 kD band was specifically recognized only by typhoid sera. The 50 kD band located on the outer membrane is protein by nature and is not a Vi (capsular), dH (flagellar), or O9 (somatic) antigen of S. typhi. These results indicate the usefulness of the specific antigen in the development of a serodiagnostic test for typhoid fever since antibodies of both the IgM and IgG class responses were obtained.     

Neurologic Manifestations Associated with an Outbreak of Typhoid Fever, Malawi - Mozambique, 2009: An Epidemiologic Investigation

Background

The bacterium Salmonella enterica serovar Typhi causes typhoid fever, which is typically associated with fever and abdominal pain. An outbreak of typhoid fever in Malawi-Mozambique in 2009 was notable for a high proportion of neurologic illness.

Objective

Describe neurologic features complicating typhoid fever during an outbreak in Malawi-Mozambique

Methods

Persons meeting a clinical case definition were identified through surveillance, with laboratory confirmation of typhoid by antibody testing or blood/stool culture. We gathered demographic and clinical information, examined patients, and evaluated a subset of patients 11 months after onset. A sample of persons with and without neurologic signs was tested for vitamin B6 and B12 levels and urinary thiocyanate.

Results

Between March – November 2009, 303 cases of typhoid fever were identified. Forty (13%) persons had objective neurologic findings, including 14 confirmed by culture/serology; 27 (68%) were hospitalized, and 5 (13%) died. Seventeen (43%) had a constellation of upper motor neuron findings, including hyperreflexia, spasticity, or sustained ankle clonus. Other neurologic features included ataxia (22, 55%), parkinsonism (8, 20%), and tremors (4, 10%). Brain MRI of 3 (ages 5, 7, and 18 years) demonstrated cerebral atrophy but no other abnormalities. Of 13 patients re-evaluated 11 months later, 11 recovered completely, and 2 had persistent hyperreflexia and ataxia. Vitamin B6 levels were markedly low in typhoid fever patients both with and without neurologic signs.

Conclusions

Neurologic signs may complicate typhoid fever, and the diagnosis should be considered in persons with acute febrile neurologic illness in endemic areas.     

Reaction and response of newborn guinea pigs to experimental Salmonella typhi infection

Newborn guinea pigs, orally infected with Salmonella typhi were examined at various intervals of time in order to determine bacterial distribution in tissues and to establish possible correlation with the clinical aspects manifested. Histopathological examination evidenced typical lesions in jejunum, ileum, caecum and especially in regional lymphatic tissues. Spleen, liver and mesenteric lymph nodes presented granulomatous lesions similar to those observed in in human typhoid fever. After oral administration, the animals reacted with anorexia, febrile reactions, bacteremia, diarrhoea, positive stool cultures, dehydration, lethargy and antibodies too were produced. Our results indicate that typhoid infection may be induced in newborn guinea pigs; the model may be used for an assessment of attenuated live typhoid vaccine control.     

An immunoblotting as a method for the diagnosis of typhoid fever

The patients' sera had been referred to the National Salmonella Centre for routine Widal serology. Sera were predominately from patients suspected of having been infected with Salmonella Typhi, but also included one serum from patient with typhoid fever who was culture positive for Salmonella Typhi. The immunoblotting procedure using Salmonella Typhi somatic (O=9,12 LPS) and flagellar (H=d) antigens was used for preliminary testing of selected patients sera previously evaluated by Widal agglutination assay as containing different levels of antibodies against O and/or H antigens of Salmonella Typhi. Following Chart et al., immunoblotting reactions were graded between 0 and 3, with 0 indicating an absence of antibody binding, and 3 where antibody binding was readily observed. Sera giving reaction of 2 or 3 were considered to be antibody positive for this study. Positive immunoblotting reaction to O=9,12 LPS antigen was obtained only with the serum of patient with typhoid fever. Presence of specific anti-LPS antibodies was also observed in two other patients' sera diluted 1:50, and in case of one of them also in dilution 1:200, but intensity of antigen-antibody reaction was under positive result criterion. The most other sera positive to O=9,12 antigen in law dilutions (1:50, 1:100) by Widal assay, showed the traces of non-specific reaction by immunoblotting. Presence of positive antigen-antibody reaction was indicated for five sera in dilution 1:50 when tested with the >55 kDa H=d flagellar protein subunit, including the serum of patient with typhoid fever. Only in this serum the high level of specific antibodies was detected also in dilution 1:200, what was not observed in case of the other four, which appeared negative. All the other sera were shown not to contain antibodies to flagella antigen. Although the presented results are preliminary and additional study of more sera of people infected with Salmonella Typhi is needed, it can be concluded after Chart et al., that an immunoblotting procedure incorporating O=9,12 LPS and flagellar H=d antigens is a useful method for providing serological evidence of infection with Salmonella Typhi. In our opinion it can serve as a rapid test for the diagnosis of typhoid fever.     

Molecular characterization of antibiotic resistance in clinical Salmonella typhi isolated in Ghana

Fifty-eight clinical Salmonella typhi strains isolated from patients suspected of suffering from typhoid fever were obtained at the Korle-Bu Teaching Hospital and the Noguchi Memorial Institute for Medical Research, both located in Ghana, Africa. Each isolate was examined for susceptibility to ampicillin, chloramphenicol, streptomycin, tetracycline, and trimethoprim/sulfamethoxazole by the disk diffusion assay. Five of the isolates were resistant to all five antibiotics while 10 isolates were resistant to ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole, which are considered 'first line' antibiotics in the treatment of typhoid fever. Thirty-four isolates were resistant to at least one of the antibiotics tested and 62% of these isolates possessed conjugable plasmids belonging to incompatibility group IncHI. Ninety percent of the conjugable plasmids conferred a multiple drug-resistant phenotype on the strains harboring them. Additionally, 14 strains contained plasmids that were transformable and six of them encoded multiple drug resistance. Our findings indicate that multiple drug resistance to the 'first line' antibiotics in S. typhi may be more prevalent in Africa than previously thought.     

Sensitivity of Moore sewer swabs for isolating Salmonella typhi.

Moore swabs (sewer swabs) have been used successfully to culture pathogenic organisms from wastewater. Sensitivity seems to depend on the size of the waterway sampled as well as the number of organisms present. In Santiago, Chile, we placed 24 swabs into the sewers draining the homes of 10 known chronic carriers of typhoid. Swabs were positive for Salmonella typhi in 5 of the 10 households (50%) and 6 of the 24 swabs placed (25%).     

Sensitivity of Moore sewer swabs for isolating Salmonella typhi

Moore swabs (sewer swabs) have been used successfully to culture pathogenic organisms from wastewater. Sensitivity seems to depend on the size of the waterway sampled as well as the number of organisms present. In Santiago, Chile, we placed 24 swabs into the sewers draining the homes of 10 known chronic carriers of typhoid. Swabs were positive for Salmonella typhi in 5 of the 10 households (50%) and 6 of the 24 swabs placed (25%).