A prospective study of the importance of enteric fever as a cause of non-malarial febrile illness in patients admitted to Chittagong Medical College Hospital, Bangladesh

Chittagong Medical College Hospital (CMCH) is a 1000-bed teaching hospital in Chittagong Division of Bangladesh. It provides inpatient and outpatient medical, surgical, pediatric and gynecology services. Each year there are approximately 700,000 outpatient visits, 700,000 emergency department visits and 600,000 admissions (500,000 adults and 100,000 children). Bed occupancy for medical wards runs at more than 100 % with extra beds provided on a daily basis.

Chittagong is the second largest city of Bangladesh. It is located in southeastern Bangladesh with a population in the metropolitan area of over 6.5 million people. Chittagong has a tropical monsoon climate with average temperatures ranging between 21.6 and 30.2 °C and average humidity of 78 %. The climate varies from tropical monsoon climate from March to November and cool and dry winters from December to February [18].

Patients admitted to CMCH between 15^th January 2012 and 5^th July 2012 were considered for enrollment. Eligibility criteria were age more than 6 months, documented axillary temperature ≥38 °C, a reported history of fever less than 2 weeks, a negative malaria smear and written informed consent given by patient or by the parents or caregiver if a child (age <16 years). Each study day all the patients admitted to the “on-take” wards were reviewed and those with a history of fever of the appropriate duration and a negative malaria smear had their temperature measured on at least one occasion. Eligible patients were then asked if they consented to participate in the study. Demographic and clinical information was recorded on a case record form at the time of admission and during the course of hospitalization.

A final diagnosis was made by the study team based on clinical presentation, basic laboratory results and microbiology results. A diagnosis of probable enteric fever was considered if the patient presented with a febrile illness of >3 days duration and two or more of the following clinical symptoms: the presence of abdominal symptoms (abdominal pain, diarrhea, constipation, nausea or vomiting), a documented fever of ≥39 °C; hepatomegaly and/or splenomegaly; a low or normal white cell count; elevation of liver enzymes (aspartate transaminase, alanine transaminase) three times above the normal range; combined with a slow defervescence with ceftriaxone treatment (the standard antibiotic used for hospital admitted febrile patients with suspected enteric fever) or a clear typhoid complication and no alternative confirmed diagnosis was established.

Blood was taken for complete blood count, urea and creatinine, aspartate transaminase (AST), alanine transaminase (ALT), malaria smear, a single BactAlert® blood culture, and a sample for real time PCR assays and serology. Cerebrospinal fluid was taken at the discretion of the responsible physician in suspected central nervous system infection as part of routine practice. Usually only small volumes of CSF were taken, sufficient for a cell count and biochemistry but insufficient for any further investigations. EDTA whole blood, serum, and any residual CSF (where there was sufficient) were stored at −20 °C for later analysis by PCR and serology at the Oxford University Clinical Research Unit (OUCRU), Ho Chi Minh City, Vietnam.

Blood was taken for culture within 24 h of admission, when possible before antimicrobial therapy was started in hospital. A volume of blood, 5–12 mL in adults and 1–12 mL for children, was inoculated into an adult or pediatric BactAlert® blood culture bottle. The exact volume of blood added was determined by weighing the bottle before and after blood inoculation. They were incubated aerobically in a BactAlert® automated system (bioMérieux, Marcy l’Etoile, France) at 37 °C for 5 days. A Gram stained smear was prepared from the broth of bottles that were positive and which was also sub-cultured onto 5 % sheep blood agar and chocolate agar (Oxoid, Basingstoke, UK) incubated in a candle jar and MacConkey agar (Oxoid, Basingstoke, UK) incubated in air for 48 h. Bacterial isolates were identified by standard methods including biochemical test using API test strips (bioMérieux, Marcy l’Etoile, France) and agglutination with specific antisera (Biorad, Hemel Hempstead, UK) [19].

Antimicrobial susceptibility tests were determined using disc diffusion with results interpreted according to Clinical Laboratory Standards Institute (CLSI) guidelines [20]. Isolates of Salmonella were tested with chloramphenicol (30 μg), ampicillin (10 μg), trimethoprim-sulphamethoxazole (1.25/23.75 μg), ceftriaxone (30 μg), ciprofloxacin (5 μg), and nalidixic acid (30 μg). The minimum inhibitory concentration (MIC) was determined by E- test according to the manufacturer’s guidelines (AB Biodisk, Solna, Sweden) against ciprofloxacin, ceftriaxone and azithromycin. Salmonella breakpoints for ciprofloxacin were: susceptible ≤0.06 μg/mL; intermediate >0.06–≤0.5 μg/mL; and resistant ≥1.0 μg/mL. A cut-off for susceptibility of ≤16 μg/mL was used for azithromycin. Other Enterobacteriacae were additionally tested against ceftazidime (30 μg), imipenem (10 μg) and gentamicin (10 μg). Isolates non-susceptible to ceftriaxone or ceftazidime were tested for extended spectrum beta-lactamase (ESBL) activity by comparing inhibition zone sizes of cefpodoxime, ceftriaxone and ceftazidime with and without clavulanic acid with a difference of 5 mm or more indicating ESBL activity. Streptococcus pneumoniae was tested with oxacillin (1 μg). and Staphylococcus aureus was tested with penicillin (10U) and cefoxitin (30 μg) (resistance indicating meticillin resistance). Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 were used as control strains for these assays. All media and tests were subject to regular internal quality assessment. Bacterial isolates were stored on beads in glycerol at −80 °C and later transferred to the OUCRU, Vietnam for re-confirmation of their identification and susceptibility results.

The admission serum was analysed by ELISA for dengue NS1 antigen using a PanBio Kit (PanBio, Sinnamon Park, Australia) according to the manufacturer’s instructions.

DNA was extracted from the stored whole blood samples (2 mL in adults and 1 mL from children) with a QIAmp DNA mini kit (Qiagen, Manchester, UK). A real-time PCR for S.enterica Typhi and S.enterica Paratyphi was performed using 25 μL reactions containing 5 μL of extracted DNA targeting STY0201 (Putative fimbrial adhesion in S. Typhi CT18) or SSPA2308 (hypothetical protein in SPA AKU-12601) as previously described [21]. Probe-based real-time PCR was also performed on the DNA extracted from blood to detect Leptospira spp, R.typhi and O.tsustugamushi [22‐24]. Low-positive plasmid controls determined adequate detection limits of each assay.

Total nucleic acid was isolated from 100 μL of CSF specimens using the automated easyMAG® system (bioMérieux), and diagnostic PCRs were performed [25]. Four real-time PCR protocols were used for detection of S pneumoniae, H influenzae type B, Neisseria meningitidis, and Streptococcus suis. Real time-PCRs were used to detect herpes simplex virus 1 and 2, varicella zoster virus, enteroviruses (generic and 71-specific) [26], and human parechoviruses (generic).

This was an observational study intended to serve as the basis for future studies with larger sample sizes and/or interventions. The true incidence of patients presenting with enteric fever in the CMCH is unknown. With a sample of at least 250 patients we would be able to estimate a disease prevalence of 5 % with a confidence interval (CI) of ± 2.8 %.

Demographic and clinical features were described for the whole cohort and within the stratified age categories of <5 years, 5–15 years and ≥16 years. Continuous data were described using median and inter-quartile range and compared using the Mann Whitney U-test. Proportions were compared with the Chi-squared test or the Fisher’s exact test as appropriate. Age, sex, duration of illness prior to admission to hospital, and the clinical syndrome were evaluated for association with in-hospital death through a univariate analysis. A multivariate logistic regression model controlling simultaneously for the effects of confounding included variables associated with the outcome of death (p < 0.10) as well as a priori factors age, sex and duration of illness prior to admission. Analysis was performed using SPSS version 21 (SPSS inc, Chicago, USA). The dataset supporting the conclusions of this article is available from the corresponding author.

We enrolled 304 eligible febrile patients in this study. It was not possible to take blood from one patient and in three patients, who were blood culture negative, there was insufficient blood for PCR amplification. These four patients were excluded from this analysis. Of the 300 patients analyzed 156 were children (age ≤15 years) and 144 were adults. The median (interquartile range, range) age was 13.5 (5.0–31.0, 0.5–89) years and the median duration of illness before admission was five (IQR, 2–8; range, 1–14) days. A history of prior antimicrobial therapy was reported in 185 (61.7 %) of patients including 96 (61.5 %) of children and 89 (61.8 %) of adults.

A final clinical suspected or microbiologically confirmed diagnosis of enteric fever was made in 52 (17.3 %; 95 % Confidence Interval 13.5–22.0 %) patients. The other common clinical syndromes diagnosed were: lower respiratory tract infection in 48 patients (16.0 %), non-specific febrile illness in 48 patients (16.0 %), a CNS infection in 37 patients (12.3 %), urinary sepsis in 23 patients (7.7 %), upper respiratory tract infection in 21 patients (7.0 %), and diarrhea (including dysentery) in 21 patients (7.0 %). Malaria was clinically suspected in seven patients, despite a negative malaria smear, although all had received prior anti-malarial treatment. Two subsequently had a malaria positive rapid diagnostic test result. There were a 14 patients who fulfilled the clinical criteria for enteric fever but had a very rapid response to ceftriaxone treatment that was not typical for enteric fever. They were classified as another syndrome, most commonly as a non-specific febrile illness. The demographic features and clinical syndromes diagnosed in the patients according to age ranges are shown in Table 1.

The median (IQR) volume of blood cultured was 2.2 (1.7–2.9) mL in children <5 years; 5.2 (4.2–5.8) mL in children 5–9 years; 6.6 (5.5–9.9) in children 10–15 years; and 9.2 (8.4–10.3) in those ≥16 years. A microbiological diagnosis was confirmed in 57 (19 %) patients as outlined in Table 2. All of the positive blood cultures grew just one organism. Salmonella Typhi was most prevalent organism detected in this study and accounted for 34 cases (11.3 %; 95%CI 8.2–15.5 %). Nineteen (6.3 %) patients had a positive blood culture for S. Typhi, including three patients also PCR amplification positive for S.Typhi in blood. Fifteen (5.0 %) patients with a negative blood culture were PCR amplification positive for S. Typhi in blood. The diagnosis was therefore confirmed in 34/52 (65.4 %) of patients in whom the diagnosis of enteric fever was suspected. No patient was positive for Salmonella Paratyphi A by blood culture or by PCR. A Gram-negative bacillus was found in the blood culture of a patient clinically diagnosed as enteric fever and a Gram positive diplococcus was observed in the blood culture of an adult with pneumonia but neither grew on sub-culture. Two adults were PCR amplification positive for Rickettsia typhi and one was PCR amplification positive for Orientia tsutsugamushi. All patients were PCR amplification negative for Leptospirosis. A further two adults were positive for dengue NS1 antigen in the admission serum sample. In the 37 patients with a clinically diagnosed CNS infection there was sufficient CSF sample available for further examination in 12. These were positive in four children: by PCR for Neiserria meningitidis in two, Streptococcus pneumoniae in one and one was IgM positive for Japanese encephalitis virus.

Antimicrobial susceptibility results were available for 18 of the S. Typhi isolates. All 18 had intermediate susceptibility to ciprofloxacin, six were additionally multidrug-resistant (MDR) exhibiting resistance to chloramphenicol, ampicillin and co-trimoxazole but all were susceptible to ceftriaxone and azithromycin. Among the other Enterobacteriacae, one E.coli, one K.pneumoniae and one E.cloacae were resistant to ceftriaxone and Extended Spectrum Beta Lactamase (ESBL) positive. The E.coli and K.pneumoniae were additionally resistant to ciprofloxacin and the K.pneumoniae to gentamicin but all were susceptible to imipenem. The S.pneumoniae was penicillin susceptible and one of the two S. aureus was meticillin resistant.

A variety of empirical antimicrobials were employed for treatment. Most patients diagnosed with enteric fever were initially treated with ceftriaxone with a step down to oral azithromycin. The median duration of hospital stay was four (IQR 2–7, range 0–29) days.

A total of 29 (9.7 %) patients died during their hospital admission: 15/156 (9.6 %) of the children and 14/144 (9.7 %) adults. The mortality was 2/52 (3.8 %) patients with enteric fever; 5/48 (10.4 %) patients with lower respiratory tract infection; and 12/37 (32.4 %) patients with encephalitis/meningitis. There was no association with a history of antimicrobial use before hospital admission and mortality (p > 0.5). The association of age, sex, duration of illness before admission and the diagnosis of the four commonest clinical syndromes with fatal outcome are shown in Table 3. In a multivariate analysis including all of these variables a diagnosis of a CNS infection was independently associated with a fatal outcome (OR 6.81 (95 % CI 2.77–16.76; p < 0.001). Of the 12 patients with a CNS infection who died, sufficient CSF was available for pathogen detection in only two of which one was positive for S. pneumoniae.