Eur J Clin Microbiol Infect Dis (2009) 28:11911198 DOI 10.

1007/s10096-009-0762-0

ARTICLE

Bacteriological investigationsignificance of time lapse after death

I. V. K. Lobmaier & . Vege & P. Gaustad & T. O. Rognum

Received: 2 December 2008 / Accepted: 13 May 2009 / Published online: 6 June 2009 # Springer-Verlag 2009

Abstract In cases of sudden unexpected death in infants and children (SUDI), microbiological investigation has been an important part of the autopsy protocol at the University of Oslo for the last 15 years. The purpose of this study was to compare the microbiological findings in samples taken at hospital admittance shortly after death and at autopsy. Blood cultures and cerebrospinal fluid (CSF) were collected both at the hospital and at autopsy; organ samples were additionally collected at autopsy. Hospital samples were collected at a median of 4.5 h (95% confidence interval [CI] 3.255) and autopsy samples at a median of 24.25 h (95% CI 2225.5) after death. The proportion of positive cultures was stable over time; the post mortal time had no influence on bacterial growth. As long as the autopsy is performed within 48 h after death, prior microbiological examination is unnecessary. Blood culture, CSF and lung specimens are the best predictors in our study.

admittance shortly after death has been performed in addition to the sampling during autopsy. The object of our study was firstly to examine the significance of microbiological findings for the final diagnosis. Secondly, we wanted to determine the importance of the post mortal time by comparing the results obtained in the samples taken by admittance at hospital with those obtained at the autopsy. Finally, we wanted to evaluate the need for sampling specimens from the lung, liver, spleen and kidney in addition to cerebrospinal fluid (CSF) and heart blood and the usefulness of obtaining specimens at admittance to hospital before the autopsy.

Subjects and methods Subjects Between 1994 and 2005, 181 cases of SUDI in infants and small children aged 7 days to 1 year were autopsied according to the standardised protocol used at the Institute of Forensic Medicine in Oslo [3, 4]. Forty-two percent of the cases were diagnosed as unexplained SUDI, 26% as borderline SUDI [4], 13% as infectious deaths, 11.5% as non-infectious disease and the rest (7.5%) were due to home accidents, neglect, maltreatment and homicide (Table 1). Borderline SUDI is used as defined in the Nordic SIDS study [7]. According to the San Diego definition, category II sudden infant death syndrome (SIDS) represents similar changes [8]. However, the Nordic classification of borderline changes in the central nervous system (CNS), lungs and heart are precisely specified and proven to be reproducible [9]. In addition, 61 cases of sudden unexpected deaths in children (SUDC) under the age of 7 days and older than 1

Introduction Microbiology is an integrated part of the autopsy protocol in sudden unexpected death in infants and children (SUDI) [16]. At the Institute of Forensic Medicine, University of Oslo, bacteriological sampling has been part of the standard protocol since 1984. From 1994, sampling at hospital

This project has been financed with the aid of EXTRA funds from the Norwegian Foundation for Health and Rehabilitation and the Norwegian SIDS and Stillbirth Society. I. V. K. Lobmaier (*) : . Vege : P. Gaustad : T. O. Rognum University of Oslo, Oslo, Norway e-mail: ilobmaier@gmail.com

1192 Table 1 Distributions of the included sudden unexpected death in infancy (SUDI) cases SUDI (N = 181) Unexplained SUDI Borderline SUDI Infectious death Non-infectious disease Accidents Neglect/maltreatment Homicide No. (%) 77 (42) 47 (26) 23 (13) 21 (11.5) 7 (4) 5 (3) 1 (0.5)

Eur J Clin Microbiol Infect Dis (2009) 28:11911198 Median age in days (range) 107 (7345) 70 (9291) 92 (7362) 48 (7302) 247 (29348) 169 (50248) 263 Male/female 43/34 30/17 17/6 10/11 3/4 5/0 0/1

year were included in the study (Table 2). Twelve percent of these children succumbed to infection, 38% to noninfectious disease, 18.5% to accidents, 3% due to home accidents, neglect and maltreatment, whereas 17.5% were unexplained. Clinical examination and microbiological sampling at the hospital shortly after death According to the Norwegian regulations, infants and toddlers who suffer sudden, unexplained death are admitted to the nearest hospital. In our study, all SUDI cases were examined by a paediatrician on arrival at the hospital, and signs of illness or injuries as well as recent and prior medical history were recorded. Microbiological samples from all of the cases could, therefore, be collected both on arrival at the hospital shortly after death and during the autopsy. Heart blood was obtained by heart puncture and CSF by lumbar puncture. Also, nasopharyngeal aspiration for virus detection and bladder puncture for the bacterial examination of urine was included in the protocol. The time of death was estimated using classical signs of death (rigor mortis, livores mortis), as well as information about the circumstances of death, such as when the infant/child was

last seen alive and when they were put to sleep. The time of sampling was recorded in all cases. Pure growth of potentially pathogenic bacteria in blood culture was considered to be important for the final diagnosis when it agreed with the history of disease and/or signs of inflammation were found by the histological examination. In cases with no signs of illness or infection prior to death, either pure growth of the same bacteria in two different samples, or growth of the same bacteria in combination with normal flora in addition to signs of inflammation by the histological examination, was required. Autopsy procedure All cases were submitted to a complete external examination, documenting injuries and signs of illness. Microbiological samples were obtained at the autopsy from CSF by sub-occipital puncture and from heart blood (one set of aerobic and one set of anaerobic) by heart puncture after opening the thorax and the pericardial sack. Lung, liver, kidney and spleen were seared at the surface and samples were carefully collected with a sterile knife and forceps. All tissue samples were immediately brought to the Department of Bacteriology at the National Hospital in separate, sterile containers without medium. In addition, all inner organs were sampled for histological examination, including ten samples from the lungs, i.e. central and distal parts of all lobes. Equipment Both at the hospital and during the autopsy, Bactec Peds Plus blood culture bottles for aerobic growth and Bactec Plus Anaerobic for anaerobic growth (Beckton, Dickinson and Company, USA) were used for sampling the heart blood and CSF. Procedures in the microbiological laboratory The blood culture bottles were incubated for 5 days at 37C in a Bactec 9240 incubator.

Table 2 Distributions of included neonates, children aged 13 years and older children Neonates (N = 26) Small children 1 3years) (N = 67) 13 11 9 19 13 1 1 Children (> 3years) (N = 31) 6 0 4 11 10 0 0

Unexplained SUDC Unexplained* SUDC Infectious death Non-infectious disease Accidents Neglect/maltreatment Homicide

3 1 2 17 0 3 0

SUDC = sudden unexpected death in children *Post mortem findings not severe enough to explain the cause of death

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When growth was detected in the aerobic bottle, the content was spread onto Petri dishes with blood (with aztreonam [30 g] antibiotic disc), chocolate, lactose and Sabouraud agar. The Petri dish with Sabouraud agar was incubated in aerobic atmosphere by 2630C for 4048 h and checked for growth daily. The rest were incubated in humid aerobic atmosphere with 46% CO2 for 4048 h and checked for growth daily. When growth was detected in the anaerobic bottle, the content was spread onto Petri dishes with blood, chocolate and lactose agar and incubated at 3537C in humid, aerobic atmosphere with 46% CO2 for 4048 h and checked for growth daily. In addition, it was spread onto a Petri dish with HMO agar (with metronidazol [2.5 g] antibiotic disc) and incubated at 3537C in anaerobic atmosphere for 6472 h and its growth checked after 3 days. The tissue samples arrived in sterile containers and were crushed and mixed with saline and spread onto Petri dishes with brown and lactose agar and incubated at 3537C in aerobic atmosphere with 46% CO2 for 4048 h and checked for growth daily. Statistical analysis Calculations and evaluation of the growth results are done with the groups as a whole, i.e. 305 cases. This was done because the bacterial growth is the main focus of this study, not the age of the included subjects. Only infectious deaths were looked at separately; 38 of the 305 cases. We performed logistic binomial regression with each organ sample collected at autopsy separately, with general growth/no growth as the categorical dependent and time set as a continuous variable.

Two hundred and twenty-four blood cultures and 213 CSF cultures were collected at hospital admittance, and growth results were available to us in 109 and 94 cases, respectively (Table 3). Out of the 109 blood cultures, 64% were sterile and only two cases had pure growth of potentially pathogenic bacteria; Escherichia coli and Streptococcus pneumoniae (Table 4). In the case with E. coli, the same bacteria was also found in samples collected at autopsy, but the case was diagnosed as a borderline SIDS, i.e. the finding was not considered to be the cause of death. The case with S. pneumoniae was diagnosed as pneumonia, mainly based on the histological findings in the lungs. From the 94 CSF cultures, 84% were sterile (Table 3) and there was no pure growth of potentially pathogenic bacteria. Except for the E. coli growth in one blood culture, the bacterial growth was mainly considered to be contamination, predominately of skin flora. We obtained information about resuscitation attempts and duration from the hospital journals. One hundred and eighty-seven cases (61.5%) had undergone resuscitation, 108 cases (35.5%) had not. In ten cases (3%), we have no information available concerning resuscitation. We found no relationship between attempted resuscitation and bacterial growth (P=0.47) nor between the duration of resuscitation and bacterial growth (P=0.8) with regard to post mortem growth as well as the hospital growth. Autopsy samples The median time between death and autopsy sampling was 24.25 h (95% CI 2225.5 h). In most cases, the subjects had been kept at 6C the last 12 h before the autopsy. The ambient temperatures during the first 12 h after death may differ; however, in most cases, the subjects had been kept at room temperature. We registered only bacterial growth and the number of cases with pure growth of potentially pathogenic bacteria in the samples; the amount of bacteria was not taken into consideration. The general growth of bacteria is stable over time; the percentage of cases with bacterial growth is stable in each group, regardless of when the sample is collected (Fig. 1). Only 19 cases had pure growth of potentially pathogenic bacteria in more than one sample collected at autopsy, and

Results Hospital samples The median time between death and sampling on arrival at the hospital was 4.5 h (95% confidence interval [CI] 3.255 h). Between death and hospital examination, most subjects had been kept at room temperature and some had been found dead under the covers.
Table 3 Results of hospital sampling and bacterial growth in SUDI cases Source Total no. (% of total) 196 (64) 211 (69) Results (%)

Median post mortem time (h) 4.5 (95% CI 3.255) 4 (95% CI 3.55)

Sterile samples no. (%) 64 (59) 84 (89)

Single isolates no. (%) 34 (31) 7 (1)

Mixed growth no. (%) 11 (10) 3 (0.3)

Heart blood, hospital CSF, hospital

109 (56) 94 (44)

1194 Table 4 Distribution of selected isolates in all samples Bacteria Heart blood, hospital SI MG SI MG SI MG SI MG SI MG SI MG SI 1 1 1 1 CSF, hospital 1 1 2 1 Heart blood, autopsy 7 18 3 17 6 4 4 4 1 1 3 3 10 9 5 CSF, autopsy 4 2 2 1 3 3 1 2 1 1 3 9 3

Eur J Clin Microbiol Infect Dis (2009) 28:11911198

Lung tissue 6 11 20 4 4 4 1 1 1 1 6 5 16 3 1

Liver tissue 7 2 1 3 1 1 10 4 -

Kidney tissue 23 10 5 5 2 1 1 2 1 12 4 -

Spleen tissue 12 1 2 1 9 3 -

E. coli S. aureus S. pneumoniae Group B streptococci Group A streptococci H. influenzae K. pneumoniae S. epidermidis

MG SI 8 MG -

SI = single isolates;MG = isolates occurring in mixed growth

13 of them (68%) were collected more than 24 h after death (range 2696.5 h). Within 48 h of death, 67% of the heart blood samples and 31.5% of the CSF samples collected at autopsy showed bacterial growth (Table 5). Twenty-four heart blood cultures had pure growth of potentially pathogenic bacteria (Table 4), but only 13 of them (54%) had pure growth of potentially pathogenic bacteria in at least one other sample; six E. coli, three S. pneumoniae, two -haemolytic streptococci group A, one S. aureus and one Klebsiella species. Out of these 13, two were diagnosed with S. pneumoniae sepsis, one with Klebsiella sepsis, one with E. coli sepsis and one with
Fig. 1 Bacterial growth in autopsy specimens according to time after death grouped in 6-h intervals, i.e. < 12 means 612 h etc. Post mortal time has no significant influence on the results during the first 42 h
100 90 80 percent of cases (305) 70 60 50 40 30 20 10 0 < 12 < 18

E. coli pyelonephritis. Six of these samples were collected more than 24 h after death (range 2633.5 h) and five of them had bacterial growth considered as diagnostic. In 13 (5%) cases, cerebrospinal cultures had a potentially pathogenic bacteria growing (Table 4), but only ten (4%) had potentially pathogenic bacteria in the CSF and at least one other sample. Eight (3%) had pure growth in the blood culture sample (four of which were diagnosed as sepsis). The ninth case had group B streptococci (GBS) growing in the CSF. We do not know the results of the blood culture samples collected at autopsy, but we found GBS in other samples collected during autopsy. Six out of ten (60%) of the samples with pure growth in CSF were collected more

< 24

< 30

< 36 hours

< 42

< 48

< 54

> 54

csf

blood

lung

liver

kidney

spleen

Eur J Clin Microbiol Infect Dis (2009) 28:11911198 Table 5 Table of autopsy sampling and bacterial growth in all cases Source Total no. (305) (% of total) 287 260 302 296 297 300 (94) (85) (99) (97) (97) (98) Median post mortem time (h) 24.25 (95% CI 2226) 24.25 (95% CI 2226) 24.5 (95% CI 2226) 24.25 (95% CI 2226) 24.5 (95% CI 2226) 24.5 (95% CI 2226) Sterile samples no. (%) 94 (33) 177 (68) 94 (31) 225 (76) 168 (57) 228 (76) Single isolates no. (%) 76 60 88 42 69 50 (26) (23) (29) (14) (23) (16)

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Mixed growth no. (%) 117 (41) 23 (9) 120 (40) 29 (10) 60 (20) 24 (8)

Heart blood, autopsy CSF, autopsy Lung tissue Liver tissue Kidney tissue Spleen tissue

than 24 h after death (range 27.533.5 h) and in five of these cases, the bacterial growth was considered to be contributable for the final diagnosis. Only one of the four sepsis cases had samples collected within 24 h of death. Organ samples The bacterial growth range in the lung, liver, kidney and spleen samples collected at autopsy was roughly 70, 20, 50 and 20%, respectively, throughout the sampling period. Airway flora was found in 22.5% of the lung samples, but gastrointestinal flora was also found, which was the most frequent in the other organ samples. The type of bacteria growing in all samples is randomly distributed throughout the sampling period. There was pure growth of potentially pathogenic bacteria in 54 lung, 45 kidney, 23 spleen and 20 liver samples (Table 4), but looking at growth in more than one sample, or in a mix with normal flora, there was growth in 12 of 19 (63.1%) liver samples, ten of 19 (52.6%) kidney samples and nine of 19 (47.4%) lung and spleen samples collected at autopsy. Four of the cases with growth in the lung samples were diagnosed as sepsis, three of which were collected more than 24 h after death. For lungs, the odds ratio (OR) for bacterial growth versus no growth for a 1-h time increase was 0.997 (0.985, 1.009), P=0.594, which was not significant. For spleen, the OR for bacterial growth versus no growth for a 1-h time increase was 0.994 (0.979, 1.009), P=0.408, which was not significant. For kidneys, the OR for bacterial growth versus no growth for a 1-h time increase was 0.996 (0.984, 1.007), P=0.476, which was not significant. For livers, the OR for bacterial growth versus no growth for a 1-h time increase was 1.011 (0.999, 1.023), P= 0.074, which was not significant at the 0.05% level. Infectious disease From the total group of 305 cases, there were 38 cases of infectious deaths. There were 17 cases of pneumonia, 12 cases of sepsis, three cases of myocarditis, two cases of

meningitis and four cases of peritonitis due to volvulus. The samples were collected between 6.25 and 142 h after death and the bodies had, in most cases, been kept at (6C) within 12 h after death. Fifty-five percent (21/38) of the cases had samples collected within 24 h after death. Only 12 cases (32%) had bacterial growth of potentially pathogenic bacteria that were considered to be of importance for the final diagnosis. From the 12 cases of sepsis, eight cases had growth of potentially pathogenic bacteria in pure culture or mixed with other bacteria in blood culture, six in liver and lung tissue, five in kidney and four in CSF and spleen (Table 6). In five out of eight cases of sepsis in which blood cultures showed relevant bacterial growth, the blood samples had been collected more than 24 h after death. In two sepsis cases, there are no blood culture results, but there are potentially pathogenic bacteria growing in other organ samples. There were 17 cases of pneumonia, four of them with viral agents. In the remaining 13 cases, there was pure growth of bacteria known to induce pneumonia or such bacteria mixed with the respiratory tract flora in six cases (46%). In two of these cases, the samples were collected more than 24 h after death.

Discussion The main finding of the present paper is that the results of the microbiological investigation of samples obtained at autopsy are constant during the first 48 h after death. The hospital samples were collected from 15 h before to 14 h after death, median 3.5 h, whereas the autopsy samples were collected between 6.25 and 146.5 h after death, median 24.5 h. Most (88.5%) of the autopsies were conducted within 48 h after death. No bacteria of diagnostic significance were found in the hospital samples that could not be traced in the autopsy samples. Thus, in no case was pure growth of bacteria that were essential to the cause of death lost by postponing heart puncture and lumbar puncture from hospital admittance to the autopsy. Surpris-

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Table 6 Infectious deaths: number of cases with pure bacterial growth, fever and virus infection Infectious deaths (no. 38) Pure bacterial growth in blood culture from hospital (no.) Pure bacterial growth in CSF from hospital (no.) 0 0 0 0 0 Pure bacterial growth in blood culture from autopsy (no.) Pure bacterial growth in CSF from autopsy (no.) 0 4 0 0 1 Pure bacterial growth in lung from autopsy (no.) 6 6 0 0 1 Pure bacterial growth in liver from autopsy (no.) 0 6 0 0 1 Pure bacterial growth in kidney from autopsy (no.) Pure Diagnosed bacterial as virus growth in infection spleen from autopsy (no.)

Pneumonia (no. 17) Septicaemia (no. 12) Myocarditis (no. 3) Meningitis (no. 2) Volvulus/ peritonitis (no. 4)

1 1 0 0 0

2 8 0 0 1

2 5 0 0 0

1 4 0 0 1

4 1 3 0 0

ingly, even the frequency of mixed bacterial growth was not influenced by the post mortal time within 48 h. Three main ways of post mortal bacterial spread are discussed in the literature; invasion in the agonal phase due to reduced circulation, bacterial growth at the mucosal surface invading blood and tissue after death, and contamination during the sampling [1012]. The degree of contamination was greater in the autopsy samples, as might have been expected. Especially, the increased amount of gastrointestinal flora growing in several samples could be a result of mucosal spread after death. Skin flora in the CSF is more likely a result of contamination during puncture of the skin and may represent an introduction of contaminative bacteria. It is argued that low temperatures, around 6C, will harm temperature-sensitive bacteria like meningococci, pneumococci and GBS, leading to a loss of these bacteria in samples taken after cool storage [13]. This problem is avoided by obtaining samples at the hospital shortly after death, before the bodies were stored at 6C. We only had one case in which GBS in mixture with other streptococci was found in the hospital blood culture but not in the autopsy samples. Here, we only found other types of streptococci, probably masking the GBS. In this case, GBS sepsis was probably not the cause of death, because the victim was too old for a late-onset GBS sepsis. In conclusion, the early sampling in our study did not contribute to a more accurate final diagnosis. The temperature-sensitive bacteria (pneumococci and GBS) found at the autopsy were grown in samples collected 28 and 27.5 h after death, after cool storage. Nevertheless, based on our relatively large series of subjects, it is impossible to know whether thermo-sensitive bacteria might have disappeared if they were present at the beginning. The question, thus, remains open. Sixty-eight

percent of the autopsy samples in our study with significant bacterial growth were collected more than 24 h after death, but within 48 h. The observation indicates that the autopsies should be conducted within 48 h after death. It still remains important to conduct the autopsy as soon as possible after death. A further advantage by not sampling the victims of sudden death at hospital admittance is the avoidance of unnecessary delay, implying that the autopsy may take place sooner after death. Furthermore, not opening the natural barriers through sampling will eliminate one possible source of contamination. Focusing on sterile sampling at autopsy will reduce another possible source of contamination. Conducting the autopsy at an early stage will reduce the mucosal invasion, and, thus, reduce the risk that the normal flora masks significant bacteria. Since autolysis and bacterial invasion through the deteriorating mucosal barriers start immediately after death and is temperature-dependent [14], it is important to keep the body at a low temperature as soon as possible after admittance to the hospital. Such an approach interacts with the need of the family to have time together with the deceased child, which is important for the process of mourning. However, if the autopsy is performed properly, the family may have the child back directly afterwards. Given good cooperation between the involved personnel, doctors, nurses, pastors and pathologists, a reasonable procedure is possible. Interpreting the bacterial findings Four percent of the collected samples showed bacterial growth of diagnostic significance. Pure growth of potentially pathogenic bacteria in the different types of samples differed between 0 (CSF) and 54 (lung) cases (Table 4). In

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the kidneys, E. coli was the most frequent potentially pathogenic bacteria growing, which is not very surprising, considering the close proximity to the colon and the fact that E. coli is the most frequent bacteria causing lower urinary tract infections. Only in one case did histological examination of the kidneys and bladder disclose signs of inflammatory reaction, a case of pyelonephritis, where E. coli was found in pure growth in many samples collected at autopsy. Respiratory tract flora was frequently found in the lungs (22.5%). The bacterial growth was only considered of clinical significance when there was evidence of inflammation in the histological samples. This combination was only found in six out of 13 cases of bacterial pneumonia, whereas the remaining cases were diagnosed based on histological examination alone. Pneumonic infiltrates can be focal, as can the bacterial growth. Thus, not having bacterial growth in one sample from the lung does not exclude bacterial growth in the entire lung. Roberts [12] argue that the samples for histological examination have to be taken from the same area as the sample for bacteriology, to ensure corresponding findings. In the present study, samples for histological examination were not obtained at exactly the same place as those for the bacteriology. However, according to the standard autopsy protocol, ten samples were collected for histological examination, central and peripheral in all five lobes, and the possibility of missing pneumonia is minimised. The extensive lung histology increases the possibility of detecting a correlate to the findings. Rationale for sampling Besides testing the importance of the post mortal time when sampling for microbiology, one main purpose was to test the diagnostic significance of extensive, time-consuming and costly sampling of four internal organs besides heart blood and CSF. The study shows that blood culture, CSF and lung tissue are the most informative concerning the final diagnosis. Tsokos and Pschel [11] argue that blood and spleen should be sampled, using the spleen as an internal control for the blood culture. Based on a study from 1905 [15], he and several other authors argue that the bacterial growth in the lungs are often unreliable. Still, there is a broad international agreement that bacteriology is an important tool in any thorough post mortal examination of infants and small children [1621] In the present study, pure growth in blood culture was considered to be diagnostic if the circumstances of death, the history of disease and/or histological examination supported the diagnosis. In 66% of the sepsis cases, there was positive blood culture, in 50% positive lung culture, in 41% positive CSF and liver culture, in 33% positive

kidney culture and in 25% positive spleen culture. In our study, the lung, liver and CSF are the best indicators for sepsis and internal control to the blood culture. It is, of course, recommended to always collect CSF, to disclose the relevant bacteria for meningitis. It may be concluded that sampling the spleen, liver and kidney adds little independent information. Pneumonia is the most frequent cause of infectious death in the present study and relevant bacterial growth was found in almost 50% of the cases. Possibly, it is useful to take samples for bacteriology and histology at the same location, to assure corresponding findings. In conclusion, the microbiological procedures with the highest independent significance were blood culture, CSF culture and bacteriological examination of lung samples. These procedures should, therefore, always be included when autopsying sudden deaths in infants and small children. By macroscopical evidence of infection in other organs or bodily fluids, microbiological testing of the respective site should be included in addition. In countries with well organised forensic and paediatric pathological services, the sampling may wait until the autopsy, given that the body is kept in a cold storage (< 6C) and that the autopsy is performed within 48 h after death.

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