Salmonella blood stream infections in a tertiary care setting in Ghana

Background Despite the clinical significance of Salmonella infections, surveillance data worldwide remains limited and is more so exemplified by the lack of reports from Africa especially in eastern, central and western Africa. This study reports on Salmonella serotypes as significant causes of blood stream infections (BSI) and multidrug antibiotic resistance at Korle-Bu Teaching Hospital in Accra, Ghana. Methods Antibiogram patterns, seasonal variations in disease incidence and predisposing factors for infection with Salmonella serotypes were analyzed retrospectively over a 4-year period from January 2010 to December 2013. Blood cultures were processed with BACTEC 9240 blood culture system. Speciation was done with BBL Crystal Enteric/Nonfermenter identification system®, and with slide agglutination using specific antisera. Antimicrobial susceptibility testing was carried out by the Kirby-Bauer disc diffusion method according to Clinical and Laboratory Standard Institute guidelines. Results We report a 6.5% (n = 181/2768) prevalence of Salmonella bacteraemia at the Korle-Bu Teaching Hospital; with a preponderance of non-typhoidal salmonellae (NTS) over typhoidal salmonella (TS) (n = 115/181, 63.5% versus n = 66/181, 36.5%; P-value <0.002). Children under 5 years bore the brunt of the disease (n = 93/174, 53.4%). Resistance to ciprofloxacin (n = 1/127, 0.7%), amikacin (n = 3/81, 3.7%), and cefotaxime (n = 6/99, 6.1%) remained low, despite high levels of multidrug resistant Salmonella phenotypes (n = 81/181, 44.2%). In multivariate analysis, and among patients with Salmonella BSI, those < 1 year old had reduced risk of non-typhoidal infections [Odds ratio, 0.51; 95% confidence interval (95% CI), 0.16-0.92, P-value 0.021]. Similarly, patients with cefuroxime resistant strans were at increased risk of having multidrug resistant Salmonella BSI (OR, 8.97; 95% CI, 3.62-24.15; P-value, 0.001). Conclusions Salmonellae, predominantly NTS, account for a reasonable low proportion of positive blood cultures in our tertiary care setting; but with significant multidrug resistant phenotypes and low ciprofloxacin and cefotaxime resistance. Electronic supplementary material The online version of this article (doi:10.1186/s12879-014-0697-7) contains supplementary material, which is available to authorized users.


Background
Invasive salmonellosis remains a global public health problem. Global estimates in 2000 showed that typhoid fever accounted for 21, 000 000 illnesses and 216, 000 deaths, whilst 5, 412 744 illnesses were attributable to paratyphoid fever [1]. The epidemiology of invasive salmonellosis varies worldwide. In Europe, non-typhoidal Salmonella (NTS) mainly Salmonella Enteritidis predominate but are rarely associated with systemic disease other than diarrhoea except for severely immunocompromised patients) [2]. In South East Asia, the predominant organism is Salmonella Typhi [1,3], whilst in Africa NTS predominates [1,[4][5][6][7][8]. The disease is common in developing countries and concomitant with poor public health and low socio economic indices [9,3]. Thus the bulk of the disease burden is seen in South East Asia, Africa and Latin America [3]. Its' epidemiology is also affected by seasonal variations [2,10]. In India peak incidence of Salmonella Typhi occurs between April-June (dry season) followed by July-September (monsoon season). In Africa the epidemiology of invasive salmonellosis has been linked with malaria infections [4,8,11,12].
In Ghana, there is paucity of epidemiological data on invasive salmonellosis with a few studies suggesting high disease burden in children. A recent report found invasive non-typhoidal salmonellosis in 10% of hospitalized children [5], whilst another study documented a prevalence of 57% in infant bactereamia [4].
Salmonella species are increasingly evolving antimicrobial resistance to several commonly used antimicrobial agents. This phenomenon which started with a report of chloramphenicol resistant Salmonella Typhi from India in 1972 [13] has increased to the extent that multi-drug resistant strains are now circulating globally [14]. This is the result of indiscriminate use of antibiotics in areas of high transmission or endemicity [14]. The problem of MDR Salmonella increases the challenge in the management of the disease in endemic regions by increasing morbidity and mortality as well as cost of treatment.
Surveillance data worldwide remains limited (2), and is more so exemplified by the lack of data from Africa especially in eastern, central and western Africa (1). Such data is relevant in decision making by public health officials for disease prevention and control programmes (1). In Ghana the situation is no different. In this study, we have documented the epidemiology of invasive salmonellosis in a tertiary hospital setting in Ghana. Our primary outcomes were to report the prevalence, circulating serotypes, antimicrobial resistance patterns, seasonal variations in disease incidence and some predisposing factors for this infection.

Study setting
The retrospective study was conducted in Korle-Bu Teaching Hospital (KBTH), a 2000-bed tertiary teaching hospital with about 200 admissions per day [15]. The hospital covers all medical specialties and provides referral healthcare services to an estimated population of 24 million Ghanaians. The central outpatient department records about 29,757 patient turnout per month [15]. The bacteriology unit of the Microbiology Department of KBTH processes over 40,000 clinical cultures annually.

Study design
From January 2010 through December 2013, we studied all clinical bloodstream isolates of salmonellae collected at the bacteriology unit of the Central Microbiology Laboratory in KBTH. Two sampling approaches were used. First, all Salmonella isolates recovered from blood cultures of patients visiting the microbiology laboratory of KBTH were characterized and analysed. Second, we reviewed laboratory records of all patients with Salmonella BSI for microbiological data. Salmonella species were selected based on the following criteria: (i) confirmed as the causative agent of the infection for which blood cultures were performed, and (ii) identified as first isolate per patient within study period. Multiple isolates per patient were considered only if antibiogram was different than previous, and their bacteraemia episodes were more than 3 months separate.

Patients' record review
To provide accurate information, patients and isolates data were abstracted in the following two steps, (i) manual work through of laboratory records, and (ii) physicianassisted medical reconciliation of data. Data were retrospectively reviewed and compared. When bacteraemic episodes with Salmonella were identified, we categorized those with typhoid and non-typhoid isolates. Data were retrospectively reviewed and compared. Subsequently, univariate and multivariate analysis were conducted to compare patients with Salmonella BSI caused by typhoidal strains to their counterparts with non-typhodal isolates using patients' data from laboratory records as independent predictor variables. Similar analysis were also conducted to compare a second case group comprising patients with BSI caused by MDR Salmonella to those with non-MDR Salmonella BSI. Each patient was included only once for each outcome. Patients' groups were compared regarding the following: demographics (age, gender), sickle cell disease, month and year of infection as well as patients' hospital and assigned department of care. We also collected data on variables related to infections: types of Salmonella species and antibiogram.

Specimen, culture and identification
During the study period, and as part of hospital routing care, 23,708 patients submitted blood cultures for bacteriological investigations. Speceimens were processed with BACTEC 9240 blood culture system (Becton Dickinson, NJ, USA) according to manufacturer's instructions. A total of 2,768 blood cultures were positive for various infections. Subcultures were made on blood, chocolate and MacConkey agar; and were incubated aerobically at 37°C for 20 hours. Typical lactose non-fermenting colonies were speciated with standard bacteriologic reactions and BBL Crystal Enteric/Nonfermenter identification system® (Becton Dickinson, NJ, USA). Salmonellae were confirmed with slide agglutination using specific antisera (Himedia Laboratories, India).

Statistics
Data from laboratory investigations were captured into Microsoft ACCESS to generate a database, and exported into Statistical Package for Social Sciences (SPSS, Version 20.0) for data editing and statistical analyses. Continuous data were compared using student's t-test, analyses of variance (ANOVA) or Mann-Whitney U test, (respectively for normalized and non-parametric distributions), with point estimates of statistical significance indicated by with 2 tailed P-values <0.05. Categorical data were compared across study parameters using Chi-square with Maracuilo's post hoc tests for multiple comparisons, or the Fisher's exact test. Correlations were assessed with Pearson coefficient (r) or Spearman's rho (r s ) or their coefficient of determination (r 2 or r s 2 ) where appropriate. Univariate comparisons were computed with Chi-square tests and unadjusted Odds ratios (OR) at 95% confidence interval (CI). From univariate analyses, variable with a P-value <0.05 were analysed in a multivariate logistic regression models to identify independent risk factors. Predictive accuracy of the models was evaluated by Hosmer and Lemeshow goodness-of-fit test with P-value >0.05 suggesting that the model predicts accurately on average. The area under the ROC (Receiver Operating Characteristic) Curve > 0.7 was used to analyse the discriminatory capability of bactereamia with typhoidal salmonella or MDR salmonellae versus their respective controls.

Ethical considerations
Ethical approval was not required as the study was regarded as part of routine surveillance measures for infection control by the Ethical and Protocol Review Committee of University of Ghana Medical School, College of Health Sciences. Considering the retrospective nature of the study, we could not obtain patients consent for use of clinical data. Nevertheless, on receipt of isolates and patients' data, we de-identified all patients to ensure anonymity. We also allotted arbitrary numbers to all isolates assigned to the study.

Cefuroxime resistant phenotypes
As a crude measure to determine beta-lactamase producing phenotypes, we examined the resistance pattern of the salmonellae to cefuroxime (30 μg) ( Table 1). The 4-year prevalence of cefuroxime resistance was similar among TS and NTS (n = 11/66, 16.7% versus n = 21/115, 18.2% respectively; P-value = 0.786). Marascuilo's comparisons showed homogeneity in the proportion of cefuroxime resistant strains across the four yearly populations of typhoidal salmonellae (P-value = 0.1115). Conversely, although we recorded significant difference (P-value = 0.0161) in the proportion of cefuroxime resistance for the yearly populations of non-typhoidal salmonellae, no difference between any two particular years was significant (marascuilo's post hoc > 0.05 for all pairwise comparisons). For TS and NTS, the Spearman's ranked test showed no correlations between cefuroxime prevalence and study years [TS, r s (2) = −0.6, P-value = 0.438; NTS, r s (2) = −0.8; P-value = 0.231].
Seasonal variations in prevalence of salmonellae Figure 1 shows the monthly distribution of TS and NTS over a four-year study period. There was seasonality for TS, but not NTS, with rainfall pattern. The peak seasons for TS bactereamia were observed in March though July and from September through November; and coincided with the two rainy seasons in Ghana -April to July, and September to November (Figure 1). For instance, the highest TS prevalence peaks were observed in March (2.2%) and May (2.0%) for 2010; and in May (2.3%) for 2011. April (2.2%), May (2.75%) and August (2.2%) recorded the highest bactereamic episodes in 2012; whereas Sepetember (1.6%) and November (1.6%) accounted for the highest prevalence in 2013. Altogether, non-typhoidal salmonellae (NTS) appeared to be more fairly distributed across the months and over the years. For the particular years under study, we sought to examine the correlation between months and the number of salmonellae isolated. The number of TS recovered in 2013, unllike the other years, showed some significant degree of covariation with months; and the direction of covariation was positive. More so, 51.9% of the variance in the number of TS was coupled with advancing months (r s = 0.799; r s 2 = 0.519; P-value = 0.001). On the contrary, no significant trend in correlation was observed between the number of nontyphoidal Salmonella (NTS) and months of isolation for any particular year.

Antibiotic susceptibility results
The in vitro susceptibility results of salmonellae to potentially useful antimicrobial agents are shown in Table 2. Overall, ampicillin, chloramphenicol and tetracycline were the least effective antibiotics with over 70% of TS and NTS resistant to each antimicrobial. For TS, no resistance was found to cefotaxime and the ciprofloxacin; whereas gentamicin, amoxicillin/clavulanate and amikacin were respectively ineffective against 4.1% (n = 1/24), 8.3% (n = 1/12) and 9.1% (n = 1/11) of the isolates. Similarly, NTS were mostly susceptible to the ciprofloxacin (n = 134/135, 99.2%), amikacin (n = 70/71, 98.6%), cefotaxime (n = 93/99, 93.9%), and gentamicin (n = 84/93, 90.3%). The percentage resistance between TS and NTS were similar for all tested antibiotics except cotrimoxazolewith a smaller percentage of TS than NTS being resistant to this antimicrobial (TS, 15.5%, n = 27/174 versus NTS, 62.1%, n = 54/87; P-value = 0.002). Chloramphenicol was the only antibiotic to which salmonellae resistance appears to be increasing; we noted a positive correlation between time over 4 years and percentage of NTS resistant to this antimicrobial (r s = +1, P-value = 0.0001).

Age distribution of salmonellae strains
In Figure 2, we examined the age-specific incidence of TS and NTS across study patients using locally weighted scatter plot smoothing (LOWESS) fit lines. Two observations are noteworthy. First, the results show similarities in the incidence of TS and NTS with age. Second, the occurrence of Salmonella bacteraemia show contrasting patterns for patients at extremes of ages. Specifically, TS and NTS bactereamias increased with age and were highest in children below 5 years, but declined gradually over ages 5 through 32, remaining fairly constant among the over 33 to 72-year-olds. A Pearson Product-moment Correlation was run to determine the relationship between incidence of Salmonella bactereamia and age. Whereas a negative correlation was observed between incidence of TS and age, only 21.5% of the incidence variance was accounted for by increases in patients' age (r = −0.464; r 2 = 0.2153; P-value = 0.0297). In contrast, no demonstrable measure of covariation was recorded between NTS and age (r = −0.1795; r 2 = 0.0322; P-value = 0.2613). Overall, the highest number of culture positive cases for TS and NTS was observed in children 1 year of age (TS, n = 7/66, 10.6%; NTS, n = 20/115, 17.4%), and the least from amongst adults >33 years.

Predictors of Salmonella bactareamia
In  Monthly variations (X-axis) in Salmonella infections for each year are reported as percentage prevalence (Y-axis); prevalence for TS and NTS infections are respectively shown with dotted and plain lines. We observed a seasonal trend for TS, but not NTS, with rainfall pattern. The peak seasons for TS bactereamia were observed in March though July and from September through November; and coincided with the two rainy seasons in Ghana -April to July, and September to November.

Discussion
When poor public health prevails in low socioeconomic settings, it is natural to speculate that it will have an impact on the health status of the people. This may manifest itself as an increase in the incidence of diseases, including Salmonella infections. Over the last decade, several studies have demonstrated the clinical significance of salmonellae causing infections in hospitals. Despite this rise, there are very few reports from West Africa, and Ghana in particular, on the occurrence of Salmonella infections. To our knowledge this is the first study conducted in Ghana across children and adult populations to document the prevalence, circulating serotypes, antimicrobial resistance patterns, and seasonal variations in disease incidence, as well as predisposing factors for blood stream infections with salmonellae. Our study reveals an overall low prevalence (6.5%) of Salmonella bactereamia among patients in KBTH from January 2010 through December 2013. Approximately 64% (n = 115/181) of the isolates were non-typhoidal Salmonella. Our finding of salmonellae in KBTH is lower compared with that documented elsewhere in Ghana (50%) [18], but higher than those reported in other salmonellae-affected institutions in India (0.69%) [10], and Tanzania (4.7%) [8]. The Korlebu Teaching Hospital from which these isolates were recovered serves as a referral centre for primary and secondary health care facilities mostly from the Southern sector of Ghana. Thus most of these patients are likely to have been treated with prior antibiotics which is likely to reduce the culture yields.
There was a significant (P = 0.0002) prevalence of NTS (4.1%) compared to TS (2.4%) over the study period. The predominance of NTS over TS could be explained in part by the preponderance of children in our study population (86.8%, n = 151/174). In 2002, the estimated prevalence of TS (27.6%) predominated over that of NTS (40.7%) in patients with bacterial bloodstream infections in rural hospitals in Ghana [18]. The figures were lower (NTS, 18.8%; TS, 31.3%) 7 years later in the same study, but still higher for TS contrary to the results described in this study. In our study most patients were children, while patients who had their diagnostic samples collected in the previous rural study were primarily adults with a reported interquartile range of 26 years. Using comparable methodology, our finding is much closer to the 59% NTS and 25% TS previously reported among bacteraemia children in this institution [19]. In a Tricube kernel smooth fit line with 50% of points to fit. The results show similarities in the incidence of TS and NTS with age. Overall, TS and NTS increased with age and were highest in children below 5 years, but declined gradually over ages 5 through 32, remaining fairly constant among the over 33 to 72-year-olds.  [20]. We cannot directly compare our figures with those observed in that study because they did not assess NTS; nevertheless, they seem to provide a similar qualitative picture, in that the incidence of typhoidal fever is low in children. Elsewhere in Africa, NTS has been shown to be a prominent cause of blood stream infections in children [7,[21][22][23]. The interrelationship between severe malaria, NTS, and HIV infection has been described [24]. However, a much more consistent association has been suggested between bactereamia due to NTS and severe malaria [24,25]. In this study, we are unable to determine the proportion of patients with HIV. But given that children in Ghana are prone to develop malaria due to the endemicity of the disease in this region, our finding is perhaps be more explained by the association of malaria with NTS. Our study shows that TS and NTS bactereamia increased with age and were highest in children below 5 years, but declined gradually after the age of five years. This is corroborated by other studies in Ghana [20]. The high prevalence of Salmonella bloodstream infections in children under five could be explained by the relative poor hygiene observed by children within this group. We also observed that children under 1 year were less likely to be infected with TS and were less often infected with MDR strains; this is at variance with another study in Ghana which showed that the frequency of TS was low in children <2 years [20].
Peak isolation of Salmonella bloodstream infections occurred between the periods of March and November. This period marks the rainy season in Ghana [26], a time when floods occur leading to contamination of water bodies and an increase in the risk of developing Salmonella infections. Similar associations have been found in India [10] and Malawi [23] where the increase in incidence of invasive Salmonella infections has been associated with the rainy season.
Overall resistance of NTS and TS to ampicillin, tetracycline and chloramphenicol was over 70%. Such high resistance rates of Ghanaian salmonellae for ampicillin, chloramphenicol and tetracycline have been recently described by Marks et al. [27]. There are other reports in Ghana where high resistance of salmonellae to these agents have been documented [5,18,20]. High resistance of salmonellae to antimicrobial agents have also been documented elsewhere in subsaharan Africa [23,28,27]. In this current study, resistance of NTS and TS to ciprofloxacin and cefotaxime (recommended agents for treatment in Ghana) [29] was very low compared to reports from India where high resistance of about 13% to ciprofloxacin have been documented [30]. The low resistance ciprofloxacin and cefotaxime in Ghana have been coroborrated by others [18,20,31]. The relatively recent introduction of these agents as treatment options for invasive Salmonellosis may have contributed to such low resistance. Morover, resistance to these agents on the African continent is not widespread but is said to be an emerging problem [27].
There are some potential limitations of this study that should be discussed briefly. Blood stream infections from Korle-Bu were identified as including TS and NTS which constituted 6.5% of blood culture positive isolates. Whereas this may reflect the relative incidence of organisms in a Ghanaian tertiary care setting biased by its referral policies, we were unable to determine if these were nosocomial incidents or community acquired infections because of insufficient data on inpatient and outpatient status. Another issue worth mentioning is the unavailability of data on nalidixic acid screening for the detection of reduced susceptibility of fluoroquinolone. To test for in vivo fluoroquinolone resistance, in vitro nalidixic acid is more appropriate. Quinolone-therapy will fail in spite of apparent ciprofloxacin sensitivity if first resistance mutations have occurred leading to in-vitro nalidixic acid resistance. Note also that by being retrospective, some patients had been stratified with predetermined definitions to which we were unable to fully assess clinical history for correlations that might contribute to the risk of TS or NTS infections. Patient clinical outcome data would have been very helpful in the contextual interpretation of the multi-drug resistant strain infections. Despite these shortcomings, our findings offer baseline information needed to create the awareness of salmonellae blood stream infections in Ghanaian hospitals, and also the need for surveillance and control.

Conclusions
In conclusion, we report a 6.5% prevalence of Salmonella bacteraemia at the Korle-Bu Teaching Hospital; with a preponderance of NTS over TS. Children under five bear the brunt of the disease. Resistance to ciprofloxacin and cefotaxime, the recommended antimicrobial agents for treatment remains low; although multi drug resistance to other agents is high. In Ghana where Salmonella infections are endemic, it is important to maintain surveillance of the disease to allow for appropriate and timely interventions when required.