Klebsiella pneumoniae bloodstream infections at a South African children’s hospital 2006–2011, a cross-sectional study

Background Klebsiella pneumoniae (KP) is a significant paediatric bloodstream pathogen in children. There is little data from Africa. In this study we describe the epidemiology of multi-drug resistant Klebsiella pneumoniae bloodstream infection (KPBSI) at Red Cross War Memorial Children’s Hospital, Cape Town, South Africa. Methods We conducted a retrospective cross-sectional study of KPBSI from 1 January 2006 to 31 December 2011 using conventional descriptive and inferential statistical methods. Results Of 410 hospitalised children with laboratory confirmed KPBSI, 339 (83 %) were caused by extended-spectrum β-lactamase (ESBL) producing isolates. The median age (IQR) was 5.0 (2–16) months, 212 (51.7 %) were male, 82 (20 %) were HIV-infected, and 241 (58.8 %) were moderately or severely underweight. The infection was hospital-acquired or healthcare-associated in 389 (95 %) children and community-acquired in 21 (5 %) children. Significant risk factors for ESBL-KPBSI included cephalosporin exposure in the 12 months prior to the KPBSI, adjusted risk ratio (aRR) 1.18 (95 % CI: 1.06–1.31); HIV infection, aRR 1.14 (1.04–1.25), and intravenous infusions for more than 3 days before the KPBSI, aRR 1.15 (95 % CI: 1.04–1.28). A total of 109 (26.6 %) children died within 30 days of the KPBSI, their median age was four (IQR 1–11) months. The median (IQR) time between KPBSI and death was three (1–9) days. HIV-infection, aRR 2.44(95 % CI: 1.59–3.74); skin erosions at the time of KPBSI, aRR 2.15 (95 % CI: 1.54–3.00); being in PICU at the time of the KPBSI, aRR 1.64 (95 % CI: 1.03–2.61) or needing PICU admission after developing KPBSI, aRR 1.72 (95 % CI: 1.10–2.70) were significant risk factors for death. Conclusion ESBL-producing KP is an important cause of laboratory confirmed bloodstream infection in hospitalised children and is associated with high mortality.


Background
Klebsiella pneumoniae (KP) is a significant bloodstream pathogen resulting in major treatment and cost challenges for health institutions [1]. There is little data on children from Africa where the effects of socioeconomic inequalities and inadequate healthcare delivery remain challenging and influence the outcome of infectious diseases.
Over the last few years anecdotal observation suggested that ESBL-producing KP frequently caused invasive infection at our hospital. We therefore reviewed laboratoryconfirmed KPBSI from 1 January 2006 to 31 December 2011 to document the extent of the problem at our hospital. The specific objectives of the study were to describe the clinical presentation of KPBSI, risk factors associated with ESBL-KPBSI, antibiotic susceptibility patterns of the KP isolates and KPBSI mortality including factors associated with in-patient mortality.

Study design and setting
This hospital based retrospective case folder review was conducted at Red Cross War Memorial Children's Hospital (RCWMCH), Cape Town, Western Cape province, South Africa. RCWMCH is a 282-bedded public children's hospital for children aged 0-13 years; it is also an academic teaching hospital of the University of Cape Town and is one of two local tertiary referral units that service 1.5 million children <14 years of age in the Western Cape. An average of 3500 new children per month (range 3000 to 6000 per month) are evaluated in its medical emergency unit, and the total number of hospital admissions peaks at 23 000 children per annum. There is a 20-bedded multidisciplinary PICU, an 18-bed burn unit, 13-bed oncology unit, 60 mixed surgical beds, 58 general paediatric beds; 29-bed cardiology and tracheostomy ward, 29 specialty medical beds, 10-bed trauma unit and 42 short stay medical beds. Dedicated newborn care is available at other hospitals in Cape Town where obstetric care is provided. Admission numbers peak during diarrhoeal seasonal months (December to April) and overcrowding commonly occurs throughout the hospital. The main entry points into the hospital are via the medical emergency or trauma units.

Study population
Most of the children admitted to RCWMCH come from homes in low socioeconomic, peri-urban settlements in the Western Cape Province. The Medical Microbiology laboratory of the National Health Laboratory Service (NHLS) identified and forwarded an electronic list of Klebsiella pneumoniae bloodstream isolates identified between 1 January 2006 and 31 December 2011 for children admitted to RCWMCH.

Data collection
Data were collected from paper-based medical records including patient demographics, admission diagnosis, HIV and nutrition status, age, date of current hospital admission, underlying medical condition, previous medical history, antibiotic history, site of KPBSI, potential risk factors for ESBL-KPBSI and mortality, length of hospital stay and outcome.

Laboratory procedures
All microbiology testing was conducted at the Groote Schuur National Health Laboratory Service (NHLS) microbiology laboratory. The laboratory used the BACTEC™ 9240 automated blood culture system (Becton Dickinson, Sparks, Maryland. For blood culture bottles flagging positive with mono-morphic Gram-negative bacilli seen on Gram stain, a locally validated method of direct inoculation into the automated Vitek®2 system (bioMérieux, Inc, France) was employed, [11] using Vitek® ID-GNB and AST-N133 cards for identification and susceptibility testing respectively. This direct method was supplemented, where necessary with repeat testing from bacterial colonies subcultured onto agar plates, using either Vitek, disk diffusion or E-test (bioMérieux, Marcy l'Etoile, France) methods. The following antibiotics were tested: ampicillin, co-amoxyclav, piperacillin-tazobactam, cefuroxime, cefoxitin, ceftriaxone, ceftazidime, cefepime, ertapenem, meropenem, imipenem, ciprofloxacin, gentamicin, amikacin, cotrimoxazole, tigecycline and colistin. Susceptibility results were interpreted according to the Clinical Laboratory Standards Institute (CLSI) criteria for the relevant years [12]. ESBLs were detected by the Vitek® 2 Advanced Expert system or by the double disk synergy method if using disk diffusion testing. Despite the changes to reporting of cephalosporin susceptibility in ESBL-producing organisms introduced by CLSI in 2010, the laboratory, in line with the contemporary national practice, continued to report ESBL-producing Enterobacteriaceae as resistant to all cephalosporins. Since ESBL production was not explicitly reported on the laboratory information system (LIS) due to limitations in the design and setup of the interface, ESBL production was assumed if Klebsiella pneumoniae isolates were reported to the clinicians (on the LIS) as resistant to all cephalosporins.

Hospital-acquired Klebsiella pneumoniae Blood Stream infection (HA-KPBSI)
Klebsiella pneumoniae blood stream infection detected 48 h or more after hospital admission and not incubating at the time of hospitalisation [13].

Healthcare-associated (HCA)-KPBSI
Klebsiella pneumoniae blood stream infection detected within 48 h of hospital admission in children who have had contact with the healthcare service including admission to an intermediate care facility during the 12-month period preceding hospitalisation. An intermediate facility provides step-down care for children recovering from acute illness, for a maximum duration of 6 weeks [13].

Community-acquired (CA)-KPBSI
Klebsiella pneumoniae blood stream infection detected within 48 h of hospital admission without previous contact with the healthcare service.

HIV infection
A positive HIV DNA PCR result confirmed by either a HIV RNA PCR or repeat HIV DNA PCR test, in any child < 18 months old, or 2 positive serological test results (HIV ELISA or HIV Rapitest) or a positive HIV DNA PCR result confirmed by either a HIV RNA PCR or repeat HIV DNA PCR test, in a child > 18 months old were considered HIV-infected [14].

Unknown HIV status
Any infant of child where there was no record of HIV testing at the NHLS laboratory database and whose mother's HIV status was unknown.

Severe immunosuppression
A CD4 percentage and / or an absolute CD4 count below the age defined range in an HIV-infected child, using the 2006 WHO definitions [15].

Nutritional status
Moderate and severe underweight were defined as weightfor-age z score (WAZ) between −2 and −3 standard deviations (SD) below the median World Health Organisation (WHO) growth reference standards, and a WAZ < −3 SD respectively.

Skin erosion
A burn wound or another form of skin loss (e.g. skin loss as a consequence of severe napkin dermatitis) documented in the medical or nursing notes.

Data analysis
Clinical data were extracted retrospectively from the hospital folders. Antibiotic sensitivity results were obtained from the NHLS microbiology database. The data were analysed using STATA Statistical software, release 11, (College Station, Texas, USA). Proportions were depicted as percentages. Continuous variables were tested for normality and mean and standard deviation (SD) or median and interquartile range (IQR) used to describe the data as appropriate. The Kruskal-Wallis equality of populations' rank test or Wilcoxon rank-sum tests for independent samples were used to compare all continuous variables. Factors associated with the outcome risk of ESBL-infection or mortality were explored by univariate analysis. Generalised linear models using Poisson regression with robust error variance were used to estimate risk ratios and 95 % confidence intervals after controlling for potential confounding factors. A two sided significance level of p < 0.05 was used in all calculations. Infection risk was calculated per 1000 hospital admissions. Children hospitalised with CA-KP BSI were younger, compared to those with HA-or HCA-KPBSI, median age of 1.5 (IQR 0.7-3.8) months versus 5.5 (IQR 1.9-16.2) months, p-value = 0.01.

Characteristics of the study population
The median age was 5.0 months (IQR 2 to 16 months), 198 were male and 212 female (Table 1). Two hundred and forty one (58.8 %) children were moderately or severely underweight-for-age (UWFA).
Throughout the study period, HA-KPBSI caused by ESBL-producing isolates predominated (Table 2). Among children with CA-KPBSI, the number of ESBLproducing isolates remained low. Furthermore, there was a 47 % reduction in ESBL-KPBSI events between 2010 and 2011, from 63 to 37.

Spectrum of KP bloodstream infection
At the time of the KPBSI 70 % of children (284/405) experienced fever i.e., temperature of ≥38.0°C. Although there was a wide range of clinical manifestations at the time of KPBSI, 80 % of the children either presented without a definable clinical focus or with pneumonia (Table 3).   Table 5.
Of the 82 HIV-infected children, 38 (46.3 %) were on cotrimoxazole prophylaxis prior to the KPBSI and 96.3 % of their isolates (79/82) were ESBL-producing organisms. All (38/38) isolates of HIV-infected children on cotrimoxazole prophylaxis were resistant to cotrimoxazole as well as 37/39 (97 %) isolates of those who were not on cotrimoxazole prophylaxis. In 29 children with malignancy or aplastic anaemia, 20/29 (70 %) isolates were sensitive to piperacillin and/or amikacin.

Antibiotic therapy
Data on antibiotic therapy were available for 398 children. Antibiotic therapy was considered appropriate if the laboratory reported the organism as susceptible to the specific antibiotic and suboptimal if the organism was reported as being intermediately susceptible or resistant. Empiric antibiotic therapy was appropriate in 307/398 (77.    (Table 7).

Discussion
In this hospital based retrospective study we have described laboratory-confirmed Klebsiella pneumoniae bloodstream infections in children admitted to RCWMCH from 1 January 2006-31 December 2011. This is the first  Multivariable model adjusted for gender, age, HIV infection, underweight-for-age, mechanical ventilation, patient in the PICU at the time of the BSI, continuous IV infusion for more than 3 days before the BSI, indwelling urinary catheter, major surgery before the BSI, Previous KP infection within last 12 months, skin erosions and cephalosporin exposure during last 12 months before the BSI. aRR adjusted risk ratio ESBL-KP extended-spectrum-β-lactamase-producing Klebsiella pneumoniae, BSI bloodstream infection a RR risk ratio; b PICU: paediatric intensive care unit; c A major surgical procedure included a laparotomy (emergency or elective), a thoracotomy, a skin graft or a craniotomy; Risk ratio 95 % confidence intervals that do not cross the null value of 1 are shown in bold comprehensive analysis of Klebsiella pneumoniae bloodstream infections in hospitalised children at a dedicated children's hospital in Sub-Saharan Africa. This study showed that ESBL-Klebsiella pneumoniae is an important cause of HA-and HCA-BSI and is associated with high mortality. Notably 68.9 % of the children were less than 12 months old. In the only other large published paediatric case series on KPBSI that involved 57 American children, infancy was also the predominant age-category i.e., 68 % (n = 38/57) of the children were less than 12 months [10]. Young children, particularly infants, are not fully immunocompetent and more susceptible to bacterial infections including KPBSI. Young age and immunological immaturity may also contribute to the delayed diagnosis of sepsis [16]. A high prevalence of moderate and severe underweight, an indicator of malnutrition was documented. On univariate analysis moderate or severe underweight-for-age was more frequent in children with ESBL-KPBSI (209/ 339, 61.7 %) compared to those with non-ESBL KPBSI (32/71, 45.1 %); p = 0.01. Malnutrition is known to be associated with widespread micronutrient deficiencies, immune dysfunction, and susceptibility to infectious complications [17].
The study population was representative of the children living in the Western Cape region, coming mainly from peri-urban, low socio-economic sub-districts. National figures show that the incidence of under-weight-for-age (UWFA) in children <5 varied from 16/1000 (2012) to 22/ 1000 (2009) and that the incidence of severe acute malnutrition was 4-5/1000 (2009-2012) [18]. The 2011 national Census established that 40 % of 5 million South African children <5 years-of-age were UWFA and that there was a high incidence of stunting (26.9 %) in children <3 years-of age [19,20]. Concomitant HIV infection is known to be a significant contributor to malnutrition and was present in 20 % of the study children.
At the time of the positive blood culture 30 % (126/ 410) of the study children did not experience fever and 50.2 % had no definable clinical focus. When a focus was present this was commonly in the lungs. In a parallel study on Staphylococcus aureus bloodstream infection at our hospital, 33 % of children had no identifiable source and, where one was identified, the lungs were the most common focus (22 %) [21]. In another paediatric study describing BSIs caused by of Gram-negative pathogens including KP, the absence of a clear clinical focus occurred in 68 % of the study subjects [6]. Unlike a child presenting from home with a CA-BSI where the clinical manifestations precipitate presentation to a health facility, HA-BSI may manifest with non-specific signs leading to delay in recognising active infection. Furthermore, the decision to take blood cultures were made by the caring clinicians based on clinical indications; given the retrospective study design no attempt was made to interrogate the possibility of blood culture contamination.
In the current study KPBSI was primarily a hospitalacquired infection caused by ESBL-producing isolates. Community-acquired infection was uncommon, particularly community-acquired infection caused by ESBLproducing isolates. These findings are similar to previous studies describing Gram-negative BSI in children [6,22]. Prior cephalosporin exposure increased the risk for ESBL-KPBSI, confirming findings of previous paediatric studies [2][3][4][5]. Two additional statistically significant risk factors were identified. The risk of HIV infection probably relates to the underlying immunodeficiency, particularly as 50 % of the HIV-infected children were not on ART at the time of acquiring KPBSI. The risk of continuous intravenous infusion for more than 3 days before the BSI was diagnosed may result from poor infection control practice and / or contamination of the infused  fluid during intravenous drug administration. Published studies have implicated these factors, but no paediatric study has previously shown a statistical relation between BSI and prior intravenous infusion on multivariate analysis [23,24]. At the time of the study, empiric antibiotic therapy at RCWMCH for CA-BSI was ampicillin plus gentamicin and for HA-and HCA-BSI, piperacillin-tazobactam plus amikacin. These empiric regimens were also applied to children who had malignancy or aplastic anaemia. The study findings suggest that these empiric antibiotic combinations would not have been effective against 22.9 % of non-susceptible isolates, intermediate or resistant, in patients with KPBSI. Once antibiotic susceptibility results were made available to the attending clinicians these patients were started on effective antibiotic therapy, and the resultant delay did not significantly affect 30-day mortality. However, the low cover for HA-and HCA-BSI suggest that more effective empiric antibiotic therapy using a carbapenem should be considered. This suggestion concurs with previous studies [3,5]. During the study period there was full access to all classes of appropriate antibiotics that were reported by the NHLS microbiology laboratory including ertapenem, imipenem and meropenem.
There was a notable surge and decrease in the risk of ESBL-producing KP HA-infection over 2009 and 2010. The impact of a South African measles epidemic starting in the Western Cape Province in 2009, peaking during a diarrhoeal surge season in March and April of 2010 and then abating in November of the same year was associated with severe pressure on beds and subsequent overcrowding in all wards of the hospital, this may have accounted for the surge seen. The falling trend in the risk thereafter at the hospital may well also reflect some success of the preventive strategy package driven by the hospital infection control unit e.g., increased awareness of the importance of hand hygiene, training on how to perform hand hygiene and an effort to reduce overcrowding in certain key areas of the hospital.
The impact of Gram-negative bacteraemia is significant in children with reduced immunity, including those with cancer, and access to carbapenem antibiotics is important particularly as the control of sepsis during febrile neutropenic episodes is time-critical.
The high 30-day fatality rate of 26.6 % and with 52.3 % of the deaths occurring within 3 days of the positive blood culture result were consistent with previous paediatric studies of Gram-negative BSI including KP where mortality ranged from 13 to 36 % [2,3,[8][9][10] and a bacteraemia study in rural Kenya in which 70.5 % of deaths occurred within 3 days of positive blood culture [25]. In the present study HIV infection, being in the PICU at the time of BSI or needing PICU admission and the presence of skin erosions were significant risk factors for death. Previous paediatric studies have identified a different spectrum of independent risk factors for death including the presence of an ESBL-producing organism, shock on admission to hospital and more than 48-h delay in initiating appropriate antibiotic therapy, and in neonates infectious complications after the onset of BSI and pulmonary hypertension. In the present study, mortality was higher in children who received suboptimal empiric antibiotic therapy (35.3 % vs 26.4 %). Although this difference appeared clinically relevant, it did not achieve statistical significance, partly because the analysis was underpowered on post-hoc evaluation i.e., power of 36 % at an alpha of 0.05.