Neonatal Sepsis Diagnosis: ; PCR Commercial Technique and Blood Culture
Condition: Sepsis
MeSH terms: Sepsis; Toxemia; Neonatal Sepsis
Study type: Observational

Summary: 
      Although advances in neonatal care have improved survival and reduced complications in
      preterm infants, sepsis still contributes significantly to mortality and in Neonatal
      Intensive Care Units (NICUs), in particular for very-low-birth-weight (VLBW, <1500 g) and
      extremely-low-birth-weight (ELBW, <1000g). Based on the timing of the infection neonatal
      sepsis has been classified into early-onset sepsis (EOS) and late-onset sepsis (LOS), with
      differences in the mode of transmission and predominant organisms. EOS is defined as onset in
      the first 3 days of life generally due to vertical transmission of bacteria from mothers to
      infants during the intrapartum period. LOS occurs after 3 days of life and it is attributed
      to pathogens acquired postnatally (horizontal transmission). Considering generally neonatal
      sepsis in Europe, 90% of the responsible bacteria resulted to be: Streptococcus agalactiae,
      Escherichia coli, Klebsiella pneumoniae, e Listeria monocytogenes. The diagnosis is difficult
      because clinical signs, particularly early in the course of disease, are subtle and
      nonspecific, and laboratory tests and blood culture are not always reliable. Moreover. blood
      culture (considered the 'gold standard) takes 48-72 hours for result. In fact the cultural
      method requires the presence of living and vital germs, depends on the volume of the sample -
      serious problem in neonatal population -, several hours are needed to process the sample,
      possibly resulting falsely negative in subjects undergoing concomitant antibiotic treatment
      or a false positive result can be found by contamination. The method based on molecular
      biology does not require living germs and, therefore, is not characterised by the sensitivity
      limitations. Such method can result to be extremely effective in patients receiving
      antibiotic therapy.

      In the present study, when an infant has to undergone blood sample for bacteria culture to
      verify a possible sepsis, a residual blood (200µl) is processed in the same time using a kit
      based on molecular biology.

      This kit is designed to obtain the highest sensitivity and specificity in the determination
      of most invasive bacterial diseases (meningitis, sepsis, pneumonia, etc.) affecting
      full-term, preterm infants to determine any presence of bacterial DNA belonging to all
      serotypes of Klebsiella pneumoniae, Escherichia coli, Streptococcus agalactiae and Listeria
      monocytogenes.

      The target bacteria have been chosen on the basis of the current Italian epidemiological
      context, so as to include germs causing about 90% of the meningitis/sepsis cases among the
      neonatal population. The detection system can unmistakably identify the germ against which it
      is directed and without causing any cross-reaction with other germs or human DNA..

      The results obtained with this method have demonstrated a 100% specificity (no false positive
      result) The sensitivity of this method compared with the cultural method has turned out to be
      twice as high.

      The aim of the present study is to compare the efficacy of the blood culture method and the
      kit for molecular detection of bacterial DNA (all serotypes of Klebsiella pneumoniae,
      Escherichia coli, Streptococcus agalactiae and Listeria monocytogenes) considering the
      relevant epidemiology of our NICU, in order to verify the relative frequency of sepsis (EOS
      and LOS) caused by the target bacteria on the whole frequency of the bacteria responsible of
      all the sepsis in our ward.
    

Detailed description: 
      Neonatal sepsis remains one of the leading causes of morbidity and mortality both among term
      and preterm infants. Although advances in neonatal care have improved survival and reduced
      complications in preterm infants, sepsis still contributes significantly to mortality and in
      Neonatal Intensive Care Units (NICUs).in particular for very-low-birth-weight (VLBW, <1500 g)
      and extremely-low-birth-weight (ELBW, <1000g).The signs and symptoms of neonatal sepsis are
      non specific. These include fever or hypothermia, respiratory distress including cyanosis and
      apnea, feeding difficulties, lethargy or irritability, hypotonia, seizures, bulging
      fontanel... Based on the timing of the infection neonatal sepsis has been classified into
      early-onset sepsis (EOS) and late-onset sepsis (LOS), with differences in the mode of
      transmission and predominant organisms. EOS is defined as onset in the first 3 days of life
      generally due to vertical transmission of bacteria from mothers to infants during the
      intrapartum period. LOS occurs after 3 days of life and it is attributed to pathogens
      acquired postnatally (horizontal transmission). The epidemiology of neonatal sepsis is a
      changing landscape, nevertheless an increasing rates of EOS Escherichia coli sepsis,
      particularly among preterm infants, and Streptococcus group B (GBS), in particular S.
      agalactiae, - resulted to be 70% of the responsible microorganisms, In a study on 6215
      infants admitted to National Institute of Child Health and Human Development (NICHD) Neonatal
      Research Network (NRN) centres, 70% of first episode late-onset infections were caused by
      gram-positive organisms, with coagulase-negative staphylococci accounting for 48% of the
      infections. Considering generally neonatal sepsis in Europe, 90% of the responsible bacteria
      resulted to be: Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, e Listeria
      monocytogenes. The diagnosis of neonatal sepsis is difficult because clinical signs,
      particularly early in the course of disease, are subtle and nonspecific, and laboratory tests
      and blood culture are not always reliable, moreover blood culture, (the 'gold standard) takes
      48-72 hours or more for result. In fact the cultural method shows a number of weak points: it
      requires the presence of living and vital germs, it depends on the volume of the collected
      sample - serious problem in neonatal population -, several hours are needed to process the
      sample, possibly resulting falsely negative in subjects undergoing concomitant antibiotic
      treatment or a false positive result can be found by contamination. The methods based on
      molecular biology do not require living germs and, therefore, are not characterised by the
      sensitivity limitations that are typical of cultural methods. For this reason, such methods
      can result to be extremely effective in patients who have already received an antibiotic
      therapy, and are not affected by rapid transport (so much so that samples can be stored at
      room temperature even for a few days) and, finally, do not require any culture medium.

      In the present study, when an infant has to undergone blood sample for bacteria culture to
      verify a possible sepsis, a residual blood (200µl) is processed in the same time using a kit
      based on molecular biology. This kit is designed to obtain the highest sensitivity and
      specificity in the determination of most invasive bacterial diseases (meningitis, sepsis,
      pneumonia, etc.) affecting full-term, preterm infants to determine any presence of bacterial
      DNA belonging to all serotypes of Klebsiella pneumoniae, Escherichia coli, Streptococcus
      agalactiae and Listeria monocytogenes The target bacteria have been chosen on the basis of
      the current Italian epidemiological context, so as to include germs causing about 90% of the
      meningitis/sepsis cases among the neonatal population. The detection system can unmistakably
      identify the germ against which it is directed and without causing any cross-reaction with
      other germs or human DNA.

      The results obtained with this method have demonstrated a 100% specificity (no false positive
      result). The sensitivity of this method compared with the cultural method has turned out to
      be twice as high.

      The aim of the present study is to compare the efficacy of the blood culture method and the
      kit for molecular detection of bacterial DNA (all serotypes of Klebsiella pneumoniae,
      Escherichia coli, Streptococcus agalactiae and Listeria monocytogenes) considering the
      relevant epidemiology of our NICU, in order to verify the relative frequency of neonatal
      sepsis (EOS and LOS) caused by the target bacteria on the whole frequency of the bacteria
      responsible of all the sepsis in our ward.

      The observational estimated period to collect 100 measurable samples, is 18 months from the
      beginning of the enrolment.

      Samples correspond to the enrollment of term o preterm neonates who needed a blood withdrawal
      for a suspect of sepsis, that are processed by both mothods.
    

Intervention name(s): molecular assay; Blood culture
Intervention type(s): Diagnostic Test; Diagnostic Test
Intervention description(s): performed with blood culture simultaneously; performed with molecular assay simultaneously
Other intervention name(s): "EuSepScreen lattanti" diagnostic kit
Observational model: Cohort
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Neonates (full-term or pre-term) requiring to be sampled for suspected sepsis (EOS or LOS)
      
Biospecimen description: 
      blood samples
    
Keywords: neonatal sepsis; Blood culture; molecular assay