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lactate analyzer confirmed by molecular typing.
journal
of Hospital infection ( I 999) 4 I : 2 I 9-222
Serrcltio marcescens pseudobacteraemia in neonates associated with a contaminated blood glucose/lactate analyzer confirmed by molecular typing. TJ. Neal*, J.E. CorkiIl*,
K.J. Bennettt,
and C.W.Yoxall$
*Department o f Medical Microbiology, Royal Liverpool University Hospital, fDepartment of C/inical Chemistry, Alder Hey Childrens Hospital, Liverpool, and #Department ofhediatrics, Liverpool Womens Hospital.
Summary:
Three episodes of Serratia mavcescens pseudobacteraemia occurred on a neonatal intensive care unit. Following the first two cases, one full term and one pre-term infant, the source was identified as a glucose/lactate analyzer. Blood culture and environmental isolates of the organisms involved were indistinguishable when subjected to pulsed-field gel electrophoresis of Spe 1 digests and PCR ri b o t yping. Failure to recognize pseudobacteraemia in neonates results in inappropriate therapy for the individual and increased antibiotic pressures on the unit. Attention to the possibility of cross infection when using automated analyzers is required to minimize the risks of true or pseudoinfection to patients. Kq)
words: Pseudobacteraemia;
neonate;
molecular
typing.
Introduction Infants requiring neonatal intensive care are particularly susceptible to nosocomial infection as a consequence of immature defence mechamonitoring and therapeutic nisms, invasive pr0cedures.l Diagnosis of infection in the neonate may be difficult as classical signs and
Received 8 October 1998; revised manuscript accepted 2.5 November 1998 Address correspondence to: TJ Neal, Department of Medical Microbiology 8th Floor Duncan Building Royal Liverpool University Hospital, Prescot Street, Liverpool L7 8XP
0 195-670 I /99/0302
I9 + 04 $12.00/O
symptoms are often absent* and blood cultures are frequently the arbiter of infection in this group. Positive blood cultures without bacteraemia (pseudobacteraemia) is a common, although probably under-reported, problem.3 Many sources have been cited (reviewed in reference 3) including the skin, mouth and gloves of phlebotomists, haematological sampling bottles, paediatric mist tents, blood gas analyzers, laboratory personnel, contaminated blood culture media, antiseptics and soap. We present three babies with pseudobacteraemia derived from a blood glucose/lactate analyzer. The particular problems associated with pseudobacteraemia in neonates are discussed.
0 1999The
Hospital
Infection
Society
11. Neal
220
Patients
and methods
Patient one was a term neonate (birth weight 3.1 kg) who suffered severe hypoxia during delivery as a consequence of placental abruption. There were no features of sepsis at delivery but blood cultures were drawn on the first day of life due to persistent respiratory distress. Empirical antibiotic therapy was commenced with ampicillin and gentamicin according to the neonatal intensive care unit (NICU) protocol. The blood culture grew a Gram-negative bacillus within 24 h of collection. A lumbar puncture was performed to ensure there was no central nervous system involvement in what, on the basis of blood culture, appeared to be a Gram-negative septicaemia. Cerebral spinal fluid (CSF) microscopy and biochemistry were unremarkable and culture was sterile. The blood culture isolate was identified as Sevrutiu marcescens resistant to ampicillin, co-amoxyclav and gentamicin, sensitive to ciprofloxacin and cefotaxime. Therapy was changed to cefotaxime and ciprofloxacin and continued for seven days. There had been no deterioration in the clinical condition of the baby and the C-reactive protein (CRP) remained normal (< 4 mg/L). Routine surveillance cultures (throat and rectal swabs) from the baby collected on admission and weekly thereafter did not demonstrate evidence of colonization with serratia. Patient two was born at 24 weeks gestation, 4 days after patient one, and weighed 0.73 kg. Blood cultures drawn on admission to the NICU grew two Gram-negative organisms, subsequently identified as Klebsiella pneumoniae; resistant to ampicillin, co-amoxyclav, gentamicin and trimethoprim, sensitive to ciprofloxacin and cefotaxime, and S. marcescens with a sensitivity pattern indistinguishable to that isolated from patient one. Antimicrobial therapy was changed from ampicillin and gentamicin to ciprofloxacin and cefotaxime. There was no rise in CRP. CSF and repeat blood cultures were both sterile. This baby had a protracted course, complicated by intestinal perforation and sepsis, requiring repeated courses of antibiotics which included ciprofloxacin as a consequence of the original isolation of an organism resistant to gentamicin.
et al.
He eventually died of candida septicaemia. Surveillance cultures did not reveal evidence of colonization with either of the two organisms grown from the original blood culture. The third episode occurred several weeks after the first two cases. This baby was born at 24 weeks gestation and weighed 0.67 kg. He developed ventilator dependent chronic lung disease and had repeated coagulase-negative staphylococcal bacteraemia. On day 44 of life a septic screen was performed because of persistent desaturations. Blood cultures grew a S. marcescens phenotypically indistinguishable from the first cases. There was no rise in CRP and surveillance cultures did not demonstrate colonization with Serrutia sp.
Environmental
somp/ing
As invasive infections with S. mawescens are unusual on our unit, an external source of the organism was suspected. Environmental sampling was undertaken, following the first two cases, including both the NICU and the delivery ward. Direct questioning of the healthcare worker (HCW) collecting both blood cultures revealed a practice in one instance of filling bottles for haematological and biochemical investigations before inoculation of the blood culture. Environmental samples collected from resuscitation equipment, haematological and biochemical sample bottles were sterile. Specimens collected from the sampling probe of a blood analyzer (Yellow Springs glucose/lactate Instruments, Ohio, USA) situated on the neonatal unit grew a scanty growth of S. marcescens with a sensitivity pattern indistinguishable from the blood culture isolates. Further sampling of this machine revealed contamination at several sites with S. marcescens, K. pneumoniae and a Pseudomonas sp.
Wing Genotyping of test isolates was performed by pulsed-field gel electrophoresis (PFGE). As S. marcescens produces extracellular deoxyribonuclease (DNase), extracted chromosomal DNA is liable to auto-digestion. Therefore all serratia
Pseudobacteraemia
in neonates
examined were pre-incubated at 72°C for 30 min in an attempt to suppress DNase activity. Briefly, chromosomal DNA was extracted by lysozyme-detergent lysis of bacteria suspended in 1 .O% agarose. Digestion of the chromosomal DNA was performed with the restriction endonuclease Spe 1 (Gibco BRL), 20 Units per agarose plug, at 37°C. Electrophoretic parameters were as follows; initial switch time 2 seconds with a linear increase to 40 seconds after 20 hours, buffer temperature 14”C, 6 volts/ cm with a pulsing angle of 120”. PFGE results were confirmed by subjecting the isolates to a second molecular typing scheme; PCR based ribotyping” using primers (Gibco BRL) complementary to the conserved regions of the 16s and 23s regions of the rRNA gene. This process amplifies the 16S23s intergenic spacer region, differences in the size of products obtained have been used to type different bacterial species.*
Results Isolates from the blood cultures of the first two cases and the glucose/lactate analyzer were indistinguishable by PFGE (Figure 1). Ribotyping by PCR confirmed the relatedness of the isolates. Contra/
measures
Attention to general infection control procedures on the unit was heightened with particular emphasis on handwashing after the use of ward-based laboratory equipment. Cleaning of the glucose/lactate analyzer was performed immediately, as results suggested a link with this machine. However superficial cleaning was ineffective and clearance was eventually achieved by recalling the manufacturer to dismantle and decontaminate the equipment.
Discussion Pseudobacteraemia, the isolation of an organism from blood culture which was not in the
221
1
2
3
4
5
Figure I PFGE of Serrotia marcescens cut with Spe I. Lanes I and 2 blood culture isolates from patients I and 2 respectively, lanes 3,4 and 5 environmental isolates from blood glucose/lactate analyzer.
patients bloodstream, is a frequent problem,3 however to our knowledge this is the first report of a blood glucose/lactate analyzer being implicated as a source. Pseudobacteraemia associated with an external source on neonatal units demonstrates particular problems. Empirical antibiotic therapy has to be adequate to cover common pathogens and be administered before microbiological results are available. In general antibiotics are stopped if blood cultures are negative and continued if they are positive i.e., the blood culture result defines the condition. Several factors suggest that the blood culture results in the cases described an environmental organism were erroneous; cultured from unrelated babies (within 24 h of birth in the first two instances), no baby was colonized with the organism, no baby showed a rise in CRP, and no infant demonstrated a deterioration in clinical condition despite being inadequately treated for the 48 h while the blood cultures were being processed. If the blood culture result is false, antibiotics are continued inappropriately and this increases antibiotic pressures for the individual and the unit which may increase the risk of infection with more resistant organisms e.g., yeasts.
T.J. Neal
222
A further consequence of the isolate involved in these cases was the decision to treat with ciprofloxacin which is contra-indicated in children due to a risk of arthropathy.6 Although the blood cultures were contaminated, the precise mechanism by which this occurred is not known. However despite warnings advising care when using the glucose machine, the HCW involved in the latest incident revealed a practice of using the blood sample for glucose analysis prior to inoculating the blood culture bottle. As a result, prompt action was possible to curtail inappropriate therapy in the third baby. This emphasizes the need to provide and enforce appropriate procedures when taking blood cultures. In particular the skin must be cleaned well before venepuncture and the sample drawn must be used only for inoculating blood culture bottles. If it is essential that other tests are performed on the same sample of blood the blood culture bottles must always be inoculated first to diminish the ris’k of erroneous results. The interpretation of blood culture results must always be considered with care, this is especially true when managing neonates in whom the clinical picture may not always provide confirmatory evidence of infection.2 The isolation of unusual organisms should alert laboratory and clinical staff to a potential false
et al.
positive result. Attempts to confirm such a false positive should be undertaken in order to prevent inappropriate use of antibiotics and also to prevent continuing symptoms being ascribed to non-existent sepsis. Strict attention to infection control measures should always be employed particularly when using ward-based laboratory equipment situated close to clinical areas.
References 1. Goldman DA, Durbin WA, Freeman J. Nosocomial infections in a neonatal intensive care unit. J If Dis 1981; 144: 449-459. 2. Hensey OJ, Hart CA, Cooke RWI. Serious infection in a neonatal intensive care unit: a two year survey. J Hyg Camb 1985; 95: 289-297. 3. Jumaa PA, Chattopadhyay B. Pseudobacteraemia. J Hasp Inf 1994; 27: 167-177. 4. Kostman JR, Edlind TD, LiPuma JJ, Stull TL. Molecular epidemiology of Pseudomonas cepacia determined by polymerase chain reac3 C&n Microbial 1992; 30: tion ribotyping. 2084-2087. 5. Kostman JR, Alden MB, Mair M, Edlind TD, LiPuma JJ, Stull TL. A universal approach to bacterial molecular epidemiology by polymerase chain reaction ribotyping. J Inf Dis 1995; 171: 204-208. 6. Ciproxin Data Sheet, Bayer plc. ABPI Compendium of Data Sheets 1998-99.
of Hospital infection ( I 999) 4 I : 2 I 9-222
Serrcltio marcescens pseudobacteraemia in neonates associated with a contaminated blood glucose/lactate analyzer confirmed by molecular typing. TJ. Neal*, J.E. CorkiIl*,
K.J. Bennettt,
and C.W.Yoxall$
*Department o f Medical Microbiology, Royal Liverpool University Hospital, fDepartment of C/inical Chemistry, Alder Hey Childrens Hospital, Liverpool, and #Department ofhediatrics, Liverpool Womens Hospital.
Summary:
Three episodes of Serratia mavcescens pseudobacteraemia occurred on a neonatal intensive care unit. Following the first two cases, one full term and one pre-term infant, the source was identified as a glucose/lactate analyzer. Blood culture and environmental isolates of the organisms involved were indistinguishable when subjected to pulsed-field gel electrophoresis of Spe 1 digests and PCR ri b o t yping. Failure to recognize pseudobacteraemia in neonates results in inappropriate therapy for the individual and increased antibiotic pressures on the unit. Attention to the possibility of cross infection when using automated analyzers is required to minimize the risks of true or pseudoinfection to patients. Kq)
words: Pseudobacteraemia;
neonate;
molecular
typing.
Introduction Infants requiring neonatal intensive care are particularly susceptible to nosocomial infection as a consequence of immature defence mechamonitoring and therapeutic nisms, invasive pr0cedures.l Diagnosis of infection in the neonate may be difficult as classical signs and
Received 8 October 1998; revised manuscript accepted 2.5 November 1998 Address correspondence to: TJ Neal, Department of Medical Microbiology 8th Floor Duncan Building Royal Liverpool University Hospital, Prescot Street, Liverpool L7 8XP
0 195-670 I /99/0302
I9 + 04 $12.00/O
symptoms are often absent* and blood cultures are frequently the arbiter of infection in this group. Positive blood cultures without bacteraemia (pseudobacteraemia) is a common, although probably under-reported, problem.3 Many sources have been cited (reviewed in reference 3) including the skin, mouth and gloves of phlebotomists, haematological sampling bottles, paediatric mist tents, blood gas analyzers, laboratory personnel, contaminated blood culture media, antiseptics and soap. We present three babies with pseudobacteraemia derived from a blood glucose/lactate analyzer. The particular problems associated with pseudobacteraemia in neonates are discussed.
0 1999The
Hospital
Infection
Society
11. Neal
220
Patients
and methods
Patient one was a term neonate (birth weight 3.1 kg) who suffered severe hypoxia during delivery as a consequence of placental abruption. There were no features of sepsis at delivery but blood cultures were drawn on the first day of life due to persistent respiratory distress. Empirical antibiotic therapy was commenced with ampicillin and gentamicin according to the neonatal intensive care unit (NICU) protocol. The blood culture grew a Gram-negative bacillus within 24 h of collection. A lumbar puncture was performed to ensure there was no central nervous system involvement in what, on the basis of blood culture, appeared to be a Gram-negative septicaemia. Cerebral spinal fluid (CSF) microscopy and biochemistry were unremarkable and culture was sterile. The blood culture isolate was identified as Sevrutiu marcescens resistant to ampicillin, co-amoxyclav and gentamicin, sensitive to ciprofloxacin and cefotaxime. Therapy was changed to cefotaxime and ciprofloxacin and continued for seven days. There had been no deterioration in the clinical condition of the baby and the C-reactive protein (CRP) remained normal (< 4 mg/L). Routine surveillance cultures (throat and rectal swabs) from the baby collected on admission and weekly thereafter did not demonstrate evidence of colonization with serratia. Patient two was born at 24 weeks gestation, 4 days after patient one, and weighed 0.73 kg. Blood cultures drawn on admission to the NICU grew two Gram-negative organisms, subsequently identified as Klebsiella pneumoniae; resistant to ampicillin, co-amoxyclav, gentamicin and trimethoprim, sensitive to ciprofloxacin and cefotaxime, and S. marcescens with a sensitivity pattern indistinguishable to that isolated from patient one. Antimicrobial therapy was changed from ampicillin and gentamicin to ciprofloxacin and cefotaxime. There was no rise in CRP. CSF and repeat blood cultures were both sterile. This baby had a protracted course, complicated by intestinal perforation and sepsis, requiring repeated courses of antibiotics which included ciprofloxacin as a consequence of the original isolation of an organism resistant to gentamicin.
et al.
He eventually died of candida septicaemia. Surveillance cultures did not reveal evidence of colonization with either of the two organisms grown from the original blood culture. The third episode occurred several weeks after the first two cases. This baby was born at 24 weeks gestation and weighed 0.67 kg. He developed ventilator dependent chronic lung disease and had repeated coagulase-negative staphylococcal bacteraemia. On day 44 of life a septic screen was performed because of persistent desaturations. Blood cultures grew a S. marcescens phenotypically indistinguishable from the first cases. There was no rise in CRP and surveillance cultures did not demonstrate colonization with Serrutia sp.
Environmental
somp/ing
As invasive infections with S. mawescens are unusual on our unit, an external source of the organism was suspected. Environmental sampling was undertaken, following the first two cases, including both the NICU and the delivery ward. Direct questioning of the healthcare worker (HCW) collecting both blood cultures revealed a practice in one instance of filling bottles for haematological and biochemical investigations before inoculation of the blood culture. Environmental samples collected from resuscitation equipment, haematological and biochemical sample bottles were sterile. Specimens collected from the sampling probe of a blood analyzer (Yellow Springs glucose/lactate Instruments, Ohio, USA) situated on the neonatal unit grew a scanty growth of S. marcescens with a sensitivity pattern indistinguishable from the blood culture isolates. Further sampling of this machine revealed contamination at several sites with S. marcescens, K. pneumoniae and a Pseudomonas sp.
Wing Genotyping of test isolates was performed by pulsed-field gel electrophoresis (PFGE). As S. marcescens produces extracellular deoxyribonuclease (DNase), extracted chromosomal DNA is liable to auto-digestion. Therefore all serratia
Pseudobacteraemia
in neonates
examined were pre-incubated at 72°C for 30 min in an attempt to suppress DNase activity. Briefly, chromosomal DNA was extracted by lysozyme-detergent lysis of bacteria suspended in 1 .O% agarose. Digestion of the chromosomal DNA was performed with the restriction endonuclease Spe 1 (Gibco BRL), 20 Units per agarose plug, at 37°C. Electrophoretic parameters were as follows; initial switch time 2 seconds with a linear increase to 40 seconds after 20 hours, buffer temperature 14”C, 6 volts/ cm with a pulsing angle of 120”. PFGE results were confirmed by subjecting the isolates to a second molecular typing scheme; PCR based ribotyping” using primers (Gibco BRL) complementary to the conserved regions of the 16s and 23s regions of the rRNA gene. This process amplifies the 16S23s intergenic spacer region, differences in the size of products obtained have been used to type different bacterial species.*
Results Isolates from the blood cultures of the first two cases and the glucose/lactate analyzer were indistinguishable by PFGE (Figure 1). Ribotyping by PCR confirmed the relatedness of the isolates. Contra/
measures
Attention to general infection control procedures on the unit was heightened with particular emphasis on handwashing after the use of ward-based laboratory equipment. Cleaning of the glucose/lactate analyzer was performed immediately, as results suggested a link with this machine. However superficial cleaning was ineffective and clearance was eventually achieved by recalling the manufacturer to dismantle and decontaminate the equipment.
Discussion Pseudobacteraemia, the isolation of an organism from blood culture which was not in the
221
1
2
3
4
5
Figure I PFGE of Serrotia marcescens cut with Spe I. Lanes I and 2 blood culture isolates from patients I and 2 respectively, lanes 3,4 and 5 environmental isolates from blood glucose/lactate analyzer.
patients bloodstream, is a frequent problem,3 however to our knowledge this is the first report of a blood glucose/lactate analyzer being implicated as a source. Pseudobacteraemia associated with an external source on neonatal units demonstrates particular problems. Empirical antibiotic therapy has to be adequate to cover common pathogens and be administered before microbiological results are available. In general antibiotics are stopped if blood cultures are negative and continued if they are positive i.e., the blood culture result defines the condition. Several factors suggest that the blood culture results in the cases described an environmental organism were erroneous; cultured from unrelated babies (within 24 h of birth in the first two instances), no baby was colonized with the organism, no baby showed a rise in CRP, and no infant demonstrated a deterioration in clinical condition despite being inadequately treated for the 48 h while the blood cultures were being processed. If the blood culture result is false, antibiotics are continued inappropriately and this increases antibiotic pressures for the individual and the unit which may increase the risk of infection with more resistant organisms e.g., yeasts.
T.J. Neal
222
A further consequence of the isolate involved in these cases was the decision to treat with ciprofloxacin which is contra-indicated in children due to a risk of arthropathy.6 Although the blood cultures were contaminated, the precise mechanism by which this occurred is not known. However despite warnings advising care when using the glucose machine, the HCW involved in the latest incident revealed a practice of using the blood sample for glucose analysis prior to inoculating the blood culture bottle. As a result, prompt action was possible to curtail inappropriate therapy in the third baby. This emphasizes the need to provide and enforce appropriate procedures when taking blood cultures. In particular the skin must be cleaned well before venepuncture and the sample drawn must be used only for inoculating blood culture bottles. If it is essential that other tests are performed on the same sample of blood the blood culture bottles must always be inoculated first to diminish the ris’k of erroneous results. The interpretation of blood culture results must always be considered with care, this is especially true when managing neonates in whom the clinical picture may not always provide confirmatory evidence of infection.2 The isolation of unusual organisms should alert laboratory and clinical staff to a potential false
et al.
positive result. Attempts to confirm such a false positive should be undertaken in order to prevent inappropriate use of antibiotics and also to prevent continuing symptoms being ascribed to non-existent sepsis. Strict attention to infection control measures should always be employed particularly when using ward-based laboratory equipment situated close to clinical areas.
References 1. Goldman DA, Durbin WA, Freeman J. Nosocomial infections in a neonatal intensive care unit. J If Dis 1981; 144: 449-459. 2. Hensey OJ, Hart CA, Cooke RWI. Serious infection in a neonatal intensive care unit: a two year survey. J Hyg Camb 1985; 95: 289-297. 3. Jumaa PA, Chattopadhyay B. Pseudobacteraemia. J Hasp Inf 1994; 27: 167-177. 4. Kostman JR, Edlind TD, LiPuma JJ, Stull TL. Molecular epidemiology of Pseudomonas cepacia determined by polymerase chain reac3 C&n Microbial 1992; 30: tion ribotyping. 2084-2087. 5. Kostman JR, Alden MB, Mair M, Edlind TD, LiPuma JJ, Stull TL. A universal approach to bacterial molecular epidemiology by polymerase chain reaction ribotyping. J Inf Dis 1995; 171: 204-208. 6. Ciproxin Data Sheet, Bayer plc. ABPI Compendium of Data Sheets 1998-99.