Mokhtar E; El-Shereef A; Abdel-Kader A; Al-Tounisy A; Karam El-Din A

Research Article

Austin J Public Health Epidemiol. 2014;1(2): 1006.

Early Diagnosis of Neonatal Sepsis Caused by Yeast Infection

Mokhtar E¹, El-Shereef A², Abdel-Kader A³, Al-Tounisy A⁴ and Karam El-Din A¹*

¹Departments of Microbiology, Ain-Shams University, Egypt

²Department of Microbiology and Immunology, Ain-Shams University, Egypt

³Department of Clinical Pathology, Ain-Shams University, Egypt

⁴Department of Pediatrics, Al-Azhar University, Egypt

*Corresponding author: Al-Zahraa A Karam El-Din, Department of Microbiology, Ain-Shams University, Abasia Cairo, Egypt

Received: June 20, 2014; Accepted: July 18, 2014; Published: July 21, 2014

Abstract

Early diagnosis of neonatal sepsis is lifesaving to the neonates. The present study was conducted to clarify the rate of incidence of neonatal early and late onset sepsis caused by yeast infection. Conventional methods, Buffy coat examination and molecular (PCR) methods were adopted and compared for the isolation of etiologic agents. In addition, other tests were also carried out to complete our data. One hundred and twenty neonates suspected of having sepsis were identified. One hundred and ten cases were found positive using blood cultures and PCR, and 20 neonates were negative. Torch antibodies were detected for the negative cases confirming viral septicemia. The remaining 100 positive cases were classified as bacterial (88%) and yeast infections (12%). Candida albicans was isolated from 11 cases (91.7%) while Debaryomyces hansenii was isolated from one case only, representing 8.3% of the positive yeast isolates. The analysis of neonatal cases showed that there is a perfect correlation between molecular and microbiological data. PCR for the 12 cases having yeast infection gave positive results at 615 bp, and that for C. albicans was observed at 156 bp. In conclusion PCR is the best and rapid method for early detection of neonatal yeast infection.

Keywords: Neonatal sepsis; Candidemia; Laboratory diagnosis

Introduction

Neonatal infections remain a major cause of morbidity and mortality and death in newborn infants [1]. Septicemia is a systemic illness caused by spread of microbes or their toxins via the blood stream [2]. The reported incidence of neonatal sepsis varies from 7.1 to 38 per 1000 live births in Asia [3,4], from 6.5 to 23 per 1000 live births in Africa [5,6] and from 3.5 to 8.4 per 1000 live births in South America and the Caribbean [7,8]. By comparison, rates reported in the United States and Australia range from 6-9 per 1000 [9,10]. Neonatal sepsis classified according to the time of onset of the disease: early onset (EOS) and late onset (LOS). The distinction has clinical relevance, as "EOS" disease is mainly due to pathogens acquired before and during delivery, and "LOS" disease to pathogen acquired after delivery (nosocomial or community source). Some reports distinguish between early onset (within 24 hours), "EOS" (24 to 6 days), and "LOS" (more than 6 days) sepsis [4,11]. Clinical diagnosis of sepsis is not easy, because, symptoms and signs are not specific and dramatic deterioration of clinical conditions can supervene rapidly long before blood cultures results are available even in asymptomatic newborn infants [12]. Predisposing risk factors associated with neonatal sepsis include neonatal group B streptococcal infection (GBS) colonization, premature rupture of membrane (PROM), and chorioamionitis12. Neonatal candidiasis can be subdivided into two categories, catheter related candidemia and disseminated or invasive candidiasis [13]. Candida spp are the common cause of nosocomial infections in neonatal intensive care units (ICUs). Although C. albicans has historically been the most frequently species isolated, infections caused by other species of Candida have been diagnosed with increased frequency [14,15]. Diagnosis of sepsis is difficult and there are no laboratory tests with 100% specificity and sensitivity, (with the exception of blood cultures which needs at least 48-72 hours). PCR methodology has been used to diagnose different infections, and the possibility of amplifying the DNA region common for all microorganisms could represent an optimal method for the diagnosis of sepsis [9,10]. PCR also allows rapid onset of treatment [1,15].

Materials and Methods

Patients

This study was carried out at the neonatal intensive care unit (during 2004) of Al-Azhar University Hospital, Cairo, Egypt on 120 neonates of an age group ranging from one day to four weeks suspected to have neonatal sepsis.

Sample collection and pre-analytical preparation

Two ml. Of venous blood were drawn in sterile EDTA-treated tubes (Bekton Dickinson Vaccutainer system, Europe, UK). One ml. of venous blood was used for blood culture and one ml. was used for molecular analysis. The blood for molecular analysis was stored at -20OC until use. A complete blood count was carried out using Hemat 8 (Radium Group) and the detection of C-reactive protein level. A Gram stained smear of the plasma buffy coat layer, obtained by centrifuging anti-coagulated capillary was examined and a rapid diagnosis of bacteremia in neonates was carried out [16].

Identification of the isolates

Venous blood samples were inoculated on Sabouraud dextrose agar (SDA) and Sabouraud-Brain Heart Infusion (BHI) broth and incubated for 2 weeks at 25°C prior to the identification of fungal isolates. The isolates were then identified using two techniques, ApI 20C Aux (Bio Mérieux SA, Marcy-L'Eliale, France) and the Micro-scan 4-Hour Rapid Yeast Identification panel (YIP, Baxter- Microscan, W. Sacramento, Calif.).

Polymerase chain reaction (PCR)

DNA Extraction from whole blood was carried out using "QIAamp DNA blood kit" (Quiagen, Inc.), this was followed by detection of yeast DNA using the universal primers "F-5'-GCETATCAATAAGCGGAGGAAAA-'3" and "R-5'- GGTCCGTGTTTCAGAAG-'3" that amplifies the V3 region of the large subunit of the ribosomal DNA [17]. For identification of Candida albicans, the following specific PCR primers were used "F-5'-TTGGAGCGGCAGGATAATCG-'3" and "R-5'GGTCCGTGTTTCAAGACG-'3" [18]. Detection of the amplified.

PCR product was carried out using gel electrophoresis. The fragments were separated in 2% agarose gel stained with ethidium bromide and visualized using ultraviolet transillumination.

Statistical analysis

The data of the study were analyzed using "SPSS" version 12 and this data was represented in a descriptive and analytical form. Appropriate statistical tests were chosen for each table and figure, depending on the type of data present, either Chi square (X2) and Fisher exact test (if cells are less than 5). The accepted levels of significance were 0.05 or less.

Ethical issues

The Ethics Committees of Al-Azhar University Hospital Cairo, Egypt approved the study. All patient information and test results were kept confidential.

Results

In the present study, one hundred neonates were positive for blood cultures and PCR, Table 1 shows the number of cases and percentage of each etiologic agent.

Table 1: Number and percentage of the etiologic agents causing sepsis.



  
    Causal agent
    No.
    %Total cases
    % Positive cases
  
  
    Bacteria
      Viruses
      Yeasts
    88
      20
      12
    73.33
      16.67
      10.00
    88.00
      -
      12.00
  
  
    Total
    130
    100
    100



Table 1:  Number and percentage of the etiologic agents causing sepsis.

Two yeast species were isolated from blood of 12 neonates. Candida albicans was the predominant species, and isolated from 11 neonates representing 91.7% of the total yeast cases, while D.hansenii (C. famata) was isolated from one case only, representing 8.3% of the total yeast isolates (Table 2).

Table 2: The isolated yeast species from blood of neonates.



  
    Yeast isolates
    No = 12
    %
  
  
    Candida albicans
      Debaryomyces hansenii
    11
      1
    91.7
      8.3
  
  
    Total
    12
    100



Table 2:  The isolated yeast species from blood of neonates.

The different methods adopted for the identification of yeast infections were compared. There was no difference between the sensitivity of blood cultures and PCR results (Table 3). It was observed that male neonates weighing less than 2.5kg are highly affected as in Table 4.

Table 3: Sensitivity comparison between the different methods used in the identification of yeast isolates.



  
    Method
    No = 12
    %
  
  
    Blood culture
      PCR
      Buffy Coat
    12
      12
      8
    100
      100
      66.7



Table 3:  Sensitivity comparison between the different methods used in the identification of yeast isolates.

Table 4: Relation between different risk factors and the isolated yeast species.



  
    Risk factors
    C. albicans
    D. hansenii 
    Total
    X²
    P
  
  
    Onset sepsis
    Early
    8
    -
    8
    0.14
    0.71
  
  
    Late
    3
    1
    4
  
  
    Birth weight
    ≤ 2.5
    7
    1
    8
    0.14
    0.71
  
  
    ≥ 2.5
    4
    -
    4
  
  
    Gestational ages (weeks)
    Preterm(37)
    3
    1
    4
    0.14
    0.71
  
  
    Term >37
    8
    -
    8
  
  
    Gender
    Males
    10
    -
    11
    2.48
    0.11
  
  
    Females
    1
    1
    1



Table 4:  Relation between different risk factors and the isolated yeast species.

Complete blood count revealed that, 80% of the positive neonates had normal HB level, platelets, HCT and MCHC, 50% had normal WBCs count, 70% normal RBCs while 40% suffered from leukocytosis, 70% had high MCH, 60% had high MCV and most of the studied neonates had thrombocytopenia.

The multiplex PCR performed for universal yeast DNA showed positive results of the test sampler with the positive control at 615 bp. Also PCR for C. albicans showed positive result of the test samples with positive control at 156 bp (Figure 1 & 2).

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Citation: Mokhtar E, El-Shereef A, Abdel-Kader A, Al-Tounisy A and Karam El-Din A. Early Diagnosis of Neonatal Sepsis Caused by Yeast Infection. Austin J Public Health Epidemiol. 2014;1(2): 1006. ISSN 2381-9014

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