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ORIGINAL ARTICLE
Year : 2014  |  Volume : 27  |  Issue : 2  |  Page : 284-289

Neutrophil expression of CD64 in the diagnosis of early-onset neonatal sepsis


Department of Clinical Pathology, Medical School, Menoufia University, Menoufia, Egypt

Date of Submission05-May-2013
Date of Acceptance26-Jun-2013
Date of Web Publication26-Sep-2014

Correspondence Address:
M A Bassuoni
MSc, Sabry Abo Allam St., Shebin El-Kom, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.141677

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  Abstract 

Objective
The aim of the study was to determine the role of CD64 expression as a neutrophil surface marker in the early diagnosis of neonatal sepsis.
Background
Neonatal sepsis is a life-threatening disease with an incidence of 1-10 per 1000 live births and a mortality rate of 15-50%. The clinical signs are nonspecific and indistinguishable from those caused by a variety of neonatal noninfectious disorders.
Patients and methods
The studied population comprised 62 neonates with gestational ages of 26-41 weeks who were suspected to have sepsis (poor suckling, fever, vomiting, diarrhea, pallor, tachycardia, and others) in the first 15 days of life, and 18 healthy age and sex-matched neonates. Complete blood count analysis, C-reactive protein determination, blood culture, and flow cytometric analysis of CD64 expression on the neutrophil surface were carried out.
Results
CD64 expression was significantly higher in the group with sepsis than in the control groups (P < 0.001). Sensitivity and specificity of CD64 were 95 and 98.3%, respectively. The negative and positive predictive values of CD64 for identifying sepsis were 95 and 98.3%, respectively.
Conclusion
A change in cell surface expression of CD64 on peripheral blood neutrophils may be considered a sensitive marker for the detection of neonatal sepsis if used in combination with other laboratory parameters.

Keywords: CD64, C-reactive protein, neonatal sepsis, neutrophil


How to cite this article:
Bassuoni M A, Helwa M A, Donia MM. Neutrophil expression of CD64 in the diagnosis of early-onset neonatal sepsis. Menoufia Med J 2014;27:284-9

How to cite this URL:
Bassuoni M A, Helwa M A, Donia MM. Neutrophil expression of CD64 in the diagnosis of early-onset neonatal sepsis. Menoufia Med J [serial online] 2014 [cited 2020 Feb 20];27:284-9. Available from: http://www.mmj.eg.net/text.asp?2014/27/2/284/141677


  Introduction Top


Sepsis in neonates hospitalized in the neonatal intensive care unit (NICU) is a global problem and a significant contributor to morbidity and mortality. Most studies focus on infants of very low birth weight, given their immature immune system and the added contribution of a variety of risk factors [1]. It is important to realize that even late-preterm infants have a compromised immune system and are susceptible to infections in the NICU [2].

Isolation of bacteria from a central body fluid (usually blood) is the standard and most specific method for diagnosing neonatal sepsis; however, positivity rates vary widely, ranging from 30 to 87%. Physicians must therefore rely on a variety of nonspecific laboratory tests to assist in and perhaps speed up the diagnosis of sepsis. The microorganisms most commonly associated with early-onset infection include  Escherichia More Details coli, Klebsiella spp., coagulase-negative Staphylococcus, Haemophilus influenzae, group B Streptococci, Listeria monocytogenes, and Candida spp., as well as mixed infections [3].

C-reactive protein (CRP) is probably the single most commonly used laboratory test for early diagnosis of neonatal sepsis. It is, however, well known that the CRP concentration does not increase very early during an infection and a low value does not rule out sepsis. The production of CRP does not start until 6 h after the onset of the infection [4].

One of the effects of inflammatory cytokines on the innate immune response is the rapid upregulation of CD64 expression on the neutrophil membrane. When neutrophils detect an infection they become activated and produce a protein called CD64 (a cell marker) on their surface. Neutrophils produce CD64 within 1 h of detecting an infection [5].

CD64 is considered one of the new generations of tests to detect early systemic infections. It measures the upregulation of an Fc receptor (Fc-g R1, or CD64) on neutrophils. The Fc receptors on white blood cells are very important for effective phagocytosis of bacteria and are upregulated during infection [6].


  Patients and methods Top


Patients

In the current study, 62 at-risk neonates in the first 15 days of life who were admitted to the NICU at Benha Teaching Hospital were studied. Selection criteria were the presence of at least one clinical sign (temperature instability, grunting, apnea, cyanosis, tachycardia, or bradycardia) or perinatal risk factor (prematurity, low birth weight, prolonged ruptured membranes of >24 h, maternal peripartum fever, infection, or urinary tract infection) suggesting infection. Selected neonates were classified into four groups:

(1) Proven sepsis (n = 30) (group I): this group comprised neonates who had positive blood cultures and clinical symptoms/signs.

(2) Infection free (n = 12) (group II): this group comprised neonates who had diseases other than sepsis, such as physiologic hyperbilirubinemia, respiratory distress, or neonatal diarrhea.

(3) Sepsis suspected (n = 20) (group III): this group consisted of babies who had clinical symptoms/signs but negative blood cultures.

Control

Eighteen healthy term age-matched and sex-matched neonates were selected as the control group (group IV).

Laboratory investigations

Complete blood count

0Complete blood count was taken using a cell counter (Sysmex KX-21N), with examination of Leishman-stained peripheral blood smears for differential leukocytic count.

C-reactive protein assay

Plasma CRP concentrations were measured immunoturbidimetrically (detection limit 5 mg/l). Any levels greater than 5 mg/l were defined as abnormal.

Blood culture

Appropriate samples for bacteriological cultures were taken from neonates and inoculated on suitable culture media.

Assay for CD64

Flow cytometric determination of neutrophil CD64 expression was performed. Samples were processed within 24 h of collection, and whole blood samples were diluted with PBS. For each sample, two tubes were prepared (one for test and the other for the isotypic control); 100 ml of the diluted sample was added to 10 ml MoAbs (MCA756F) mouse anti-human CD64 : FITC, vortexed, and incubated at room temperature for 15 min in the dark. Three milliliters of the Lyse solution was added to each tube, vortexed, and then incubated for 15 min at room temperature. The tubes were centrifuged at 3000 rpm for 5 min, and then the supernatant was discarded. The cells were washed twice with 2 ml PBS, with repeating centrifugation, followed by discarding of the supernatant. Samples were then ready for processing on the flow cytometer.

Statistical analysis

The collected data were tabulated and analyzed using SPSS (version 16; SPSS Inc., Chicago, Illinois, USA). Categorical data were presented as number and percentages, whereas quantitative data were expressed as mean and SD. The c2 -test, analysis of variance followed by Bonferroni's correlation for multiple comparisons, and Spearman's correlation coefficient were used as tests of significance. The receiver operating characteristic (ROC) curve was used to determine the cutoff values of CD64 with optimum sensitivity and specificity.


  Results Top


Eighty neonates were enrolled in this study and divided into four groups: the first group (group I) included 30 neonates with early-onset neonatal sepsis (EONS) (20 male and 10 female); the second group (group II) included 12 patients without sepsis (six male and six female); the third group (group III) included 20 clinically suspected cases of EONS (six male and 14 female); and the fourth group (group IV) included 18 controls (nine male and nine female). Altogether there were 41 male and 39 female patients, with a male to female ratio of 1 : 1. Demographic data of the study group are presented in [Table 1]. Patients and controls were comparable with respect to age and sex.
Table 1: Description of the demographic data of the studied groups

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Highly significant differences were observed in certain laboratory data. The platelet count was significantly lower (P = 0.001), whereas white blood cells, CRP, and CD64 were significantly higher (P < 0.001) in cases than in controls. No significant difference as regards hemoglobin (Hb) was seen (P > 0.05) [Table 2] and [Figure 1].
Figure 1:

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Table 2: Laboratory data of the studied groups (mean ± SD)

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There was a highly significant difference between the studied groups as regards blood culture (P < 0.001) [Table 3]. Positive correlations were found between CD64 and CRP and total leukocyte count of patients. Significant negative correlations were present between CD64 and platelet count, as shown in [Table 4] and [Table 5]. [Figure 2] shows data obtained from the ROC curve in which the cutoff value of CD64, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve were 86.9, 95%, 98.3%, 95%, 98.3%, and 0.99, respectively, whereas the corresponding values for CRP were 42.5 mg/l, 90%, 81.7%, 62.1%, 96.1%, and 0.88, respectively, and for TLC were 11.9 × 10 3 /mm 3 , 60%, 60%, 33.3%, 81.8%, and 0.69. Thus, it is seen that CD64 is more accurate than CRP and TLC in the diagnosis of sepsis.
Figure 2:

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Table 3: Comparing the studied groups regarding blood culture

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Table 4: Spearman's correlation between CD64 and C-reactive protein, TLC, and platelets among the confirmed sepsis groups

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Table 5: Data obtained from the receiver operating characteristic curve

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  Discussion Top


The diagnosis of sepsis remains one of the most difficult tasks for physicians and other medical staff. Blood cultures often remain negative in the presence of pneumonia, meningitis, and even fulminant blood-borne septicemia. Identifying the specific organism in a small sample of venous blood remains difficult. A rapid laboratory test with high specificity for neonatal sepsis would be a valuable tool in therapeutic decision making, avoiding the unnecessary use of antibiotics in patients with clinical signs and symptoms of sepsis but having a negative blood culture [7].

There is a clear need for improved indicators of neonatal sepsis to increase the sensitivity and specificity of both diagnosis and therapeutic monitoring. One of the effects of inflammatory cytokines on the innate immune response is the rapid upregulation of CD64 expression on the neutrophil membrane. This rapid upregulation of CD64 expression on PMNs and the normal 6-h half-life of blood PMNs make the determination of the PMN CD64 expression a true indication of the current status of neonatal sepsis. Thus, neutrophil CD64 expression provides improved diagnostic detection of infection/sepsis compared with the standard diagnostic tests used in current medical practice [5].

The high affinity for CD64 mainly indicates phagocytosis and intracellular killing of pathogens, but it is also expressed at very low levels on the surface of unstimulated neutrophils [8]. In addition, it has been shown that neutrophils of preterm infants express CD64 antigen to a similar extent as that of older children and adults. Thus, this specific marker can be used for the identification of life-threatening infections in preterm infants [9].

For this study we selected 80 neonates up to 15 days of age who were admitted to the NICU in Banha Teaching Hospital: 30 cases with proven EONS, 20 cases with clinical suspicion of EONS, and 12 cases admitted for other reasons (such as neonatal jaundice), and 18 healthy age-matched and sex-matched controls.

In this study, we tried to determine the CD64 expression as an immunological marker for the rapid diagnosis of neonatal sepsis. We tried to elucidate the value of CD64 expression on the neutrophil surface in patients with EONS using flow cytometry. The significance of CD64 expression was investigated in relation to various clinical, laboratory, and standard prognostic factors.

In our study, there were statistically significant differences between the studied groups with respect to gestational age. This is in agreement with the study by Bizzarro et al. [1], who stated that the majority of cases of early-onset sepsis had a gestational age of less than 30 weeks, and with the study by Stall et al. [10], who stated that the risk of early-onset sepsis increases with decreasing gestational age because of the inability of white blood cells to phagocytose, immaturity of the immune system, low complement levels, and hypogammaglobulinemia.

In this work, no statistically significant correlation was noted between the ages of patients, which ranged from 1 to 15 days, and those of infants who were positive for CD64 and those negative for it. This is in concordance with the study performed by Christian et al. [11] and that by Horns et al. [12].

No male predominance in EONS was seen in this study (male to female ratio 1 : 1). A similar finding was recorded by Shaheen [13] but Klein [14] recorded that male infants had increased risk of developing sepsis compared with female infants. A gene located on the X chromosome and involved with the function of the thymus or with synthesis of immunoglobulin has been postulated to exist; thus, female infants have a greater resistance to infection.

There was a high statistically significant difference in mean CD64 expression levels among the studied groups (P < 0.001) (Table 15). These findings are in agreement with those of a previous study by Espinosa et al. [15], which showed increased rates of CD64+ cells in a group with clinically proven sepsis. Similar results have been reported in adults by Lino et al. [16] and Fjaertoft et al. [8] and in neonates by Herra et al. [17]. A study by Qureshi et al. [18] revealed that patients with sepsis had a greater number of circulating CD64-positive PMNs (mean 98%) compared with healthy controls (mean 10%). This means that there is an increase in the number of CD64 molecules expressed on the PMN surface compared with healthy individuals.

There were no significant correlations between the hematological data (red blood cell counts, Hb, CT, MCV, MCH, eosinophils, basophils, lymphocytes, TLC, Seg, Staff, and immature forms) of the patients and CD64 expression in this study. These results are in agreement with those reported by Espinosa et al. [15] and Livditi et al. [19], who found no significant association between CD64 expression and red blood cell count, Hb percentage, MCV, MCH, eosinophils, basophils, lymphocytes, TLC, Seg, Staff, and immature forms.

Thrombocytopenia is one of the most common complications of neonatal sepsis; this may be attributed to bone marrow depression, consumption coagulopathy, platelet sequestration, or a combination of these processes [20]. Thrombocytopenia is considered one of the hematological parameters of severity of neonatal sepsis, but a normal platelet count does not exclude sepsis [21].

The present study showed a significant negative correlation between CD64 and platelet count, which deems CD64 as a parameter of the severity of sepsis. This is in agreement with a previous study by Gonzalez et al. [22].

In the current study, CD64 expression had a positive correlation with CRP level, which is a laboratory marker of neonatal sepsis, indicating its usefulness as an additional marker of sepsis. This is in agreement with previous studies performed by Kantar et al. [23] and Martin et al. [24], who found that CD64 was positively correlated with CRP.

In this work, the ROC curve shows a greater area under the curve (accuracy) for CD64 than for CRP. This implies the greater discriminatory power of CD64 compared with CRP for the early detection of EONS. This is in agreement with previous studies by Lichtman et al. [25] and Lee et al. [26]. A study by Vercauteren et al. [27] on 92 EONS patients found that CD64 expression had a greater discriminatory power compared with CRP.

In our work, there were no significant differences between CD64 and culture results in the study group with proven EONS (positive culture), nor were there significant differences between CRP and culture results. However, there was a highly significant difference between CD64 and culture results in the study group with clinical suspicion of EONS (negative culture), although there were no significant differences between CRP and culture results in the same study group. This is in contrast to the results of Ng et al. [28], who found no significant difference between CD64 and culture results in septic neonates. The discrepancy between the results may be attributed to patients being at different stages of the infectious process, especially at resolution of infection, which results in a decline in the percentage of CD64-bearing blood PMNs. Moreover, the administration of antibiotics might also have affected the level of CD64 expression in such patients (our own explanation). In our study, we found 95% sensitivity and 98.3% specificity for CD64 expression, in agreement with the results of Ng et al. [28], who showed very high sensitivity and specificity for CD64 in early-onset sepsis (about 97 and 89%, respectively). Another study showed that sensitivity and specificity of CD64 in early-onset sepsis was 97 and 72%, respectively [17].

In addition, we obtained the PPV and NPV of CD64 as 95 and 98.3%, respectively. This is in agreement with the results of Herra et al. [17] and Lichtman et al. [25], who showed high PPV and NPP for CD64 in early-onset sepsis (about 87 and 72%, respectively).

It is worth mentioning that the analysis of CD64 expression is performed with a small sample (25-50 ml of whole blood), is very rapid (<20 min), and highly reliable. In addition, our data indicate that the high sensitivity of elevated levels of CD64 for neonatal sepsis is achieved through a single determination and the high percentage of CD64+ cells seen in EONS is maintained in these patients for at least 6 h [7], The results of the present study and those of previous studies show that measurement of neutrophil surface marker CD64 can be useful for the diagnosis of sepsis in early phase.


  Conclusion Top


A change in cell surface expression of CD64 on peripheral blood neutrophils can be considered a sensitive marker for the detection of EONS if used in combination with other laboratory parameters. CRP has good diagnostic properties during later phases of sepsis but not for early detection of it.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.Bizzarro MJ, Raskind C, Baltimore RS, Gallagher PG. Seventy-five years of neonatal sepsis at Yale: 1928-2003. Pediatrics 2005; 116:595-602.  Back to cited text no. 1
    
2. Benjamin DK. Infection in late preterm infants. Clin Perinatol 2006; 33:871-882.  Back to cited text no. 2
    
3. Caldas JP, Oliveira RT. Accuracy of white blood cell count and C-reactive protein, for diagnosing early neonatal sepsis. J Pediatr (Rio J) 2008; 84:536-542.  Back to cited text no. 3
    
4. Sabel KG, Wadsworth C. C-reactive protein (CRP) in early diagnosis of neonatal septicemia. J Acta Paediatr Scand 1999; 88:825-831.  Back to cited text no. 4
    
5. Davis B, Karen C, Rinder H. CD64 expression in early onset sepsis. J Crit Care 2008; S4:20-26.  Back to cited text no. 5
    
6. Bhandari V, Wang C, Rinder C, Henry H. Hematologic profile of sepsis in neonates: neutrophil CD64 as a diagnostic marker. Pediatrics 2008; 121:129-134.  Back to cited text no. 6
    
7. Baranda L, Layseca-Espinosa E, Perez-Gonzalez LF, Rosenstein Y et al. Expression of CD64 as a potential marker of neonatal sepsis. Pediatr Allergy Immunol 2010; 13:319-327.  Back to cited text no. 7
    
8. Fjaertoft G, Pauksen K, Hankansson L, Xu S, Venge P. Cell surface expression of CD64 on neutrophils and monocytes in patients with influenza A with or without complications. Scand J Infect Dis 2010; 37:882-889.  Back to cited text no. 8
    
9. Rosales WC, Akisu M, Cetingul N, Caglayan S. Reactive hyperemia and interleukin 6, interleukin 8, and tumor necrosis factor-a in the diagnosis of early-onset neonatal sepsis. Pediatrics 2011; 108:E61.  Back to cited text no. 9
    
10.Stall BJ, Gordon T, Korones SB, Shankaran S, Bauer CR. Late-onset sepsis in very low birth weight neonates. A report from the National Institute of Child Health and Human Development Neonatal Research Network. J Pediatr 2006; 129:63-71.  Back to cited text no. 10
    
11.Christian W, Richard R, Gunther S, Claudia S, Jochen H, Susanne S, et al. Impact of CD64 expression analysis for early onset neonatal sepsis immunophenotyping. Haematologica 2009; 86:154-141.  Back to cited text no. 11
    
12.Horns KM, Torres-Monies A, Fuente H. Neoteric physiologic and immunologic methods for assessing early-onset neonatal sepsis. J Perinat Neonatal Nurs 2010; 13:50-66.  Back to cited text no. 12
    
13.Shaheen A. Plasma concentration of defensin in neonatal septicemia [MD thesis]; Benha Faculty of Medicine; 2008.  Back to cited text no. 13
    
14.Klein JO. Bacterial sepsis and meningitis. In: Klein Remington, editors. Infectious diseases of fetus and neonates. 4th ed. NB Saunders 2007; 835-890.  Back to cited text no. 14
    
15.Espinosa E, Perez-Gonzalez LF, Torres-Montes A, Baranda L, de la Fuente H, Rosenstein Y et al. Expression of CD64 as a potential marker of neonatal sepsis. Pediatr Allergy Immunol 2009; 13:319-327.  Back to cited text no. 15
    
16.Lino S, Ravetch R, Kint JP, Perussia F, Bazil V. Cell adhesion molecules in inflammatory diseases. Drugs 2009; 56:977-988.  Back to cited text no. 16
    
17.Herra G, Groselj-Grenc M, Jhan A, Derganc M. Neutrophil and monocyte CD64 and CD163 expression in critically ill neonates and children with sepsis: comparison of fluorescence intensities and calculated indexes. Mediators Inflamm 2008; 2008:2026-2046.  Back to cited text no. 17
    
18.Qureshi S, Lewis M, Gant V, et al. Increased distribution and expression of CD64 on blood polymorphonuclear cells from patients with the systemic inflammatory response syndrome (SIRS). Clin Exp Immunol 2011; 125:258-265.  Back to cited text no. 18
    
19.Livaditi O, Kotanidou A, Psarra A, Dimopoulou I, Sotiropoulou C, Augustatou K, et al. Neutrophil CD64 expression and serum IL-8: sensitive early markers of severity and outcome in sepsis. Cytokine 2010; 36:283-290.  Back to cited text no. 19
    
20.Wong W, Glader B. Approach to the newborn who has thrombocytopenia. Neoreviews 2010; 5:444-450.  Back to cited text no. 20
    
21.Aird WC. The hematologic system as a marker of organ dysfunction in sepsis. Mayo Clin Proc 2009; 78:869-881.  Back to cited text no. 21
    
22.Gonzalez BE, Mercado CK, Johnson L, Brodsky NL Bhandari V. Early markers of late onset sepsis in premature neonates: clinical, hematological and cytokine profile. J Perinat Med 2009; 31:60-68.  Back to cited text no. 22
    
23.Kantar M, Kultursay N, Kutukculer N, Akisu M, Cetingul N Caglayan S. Plasma concentrations of granulocyte-macrophage colony-stimulating factor and interleukin-6 in septic and healthy preterms. Eur J Pediatr 2009; 159:156-157.  Back to cited text no. 23
    
24.Martin H, Olander B, Norman M. Reactive hyperemia and interleukin 6, interleukin 8, and tumor necrosis factor-alpha in the diagnosis of early-onset neonatal sepsis. Pediatrics 2010; 108:E61.  Back to cited text no. 24
    
25.Lichtman MA, Liesveld JL. Comparison of procalcitonin with CD64, C-reactive protein and differential white blood cell count for the early diagnosis of bacterial infections in newborn infants. Pediatr Infect Dis J 2009; 18:666-671.  Back to cited text no. 25
    
26.Lee M, Tan T, Feng A. Clinicopathologic analysis of early onset neonatal sepsis in a single institution. J Chin Med Assoc 2010; 70:269.  Back to cited text no. 26
    
27.Vercauteren SM. Sutherland predictive value of soluble immunological mediators in neonatal infection. Clin Sci (Lond) 2011; 87:165-171.  Back to cited text no. 27
    
28.Ng PC, Li G, Chui KM, Chu WC, Li K, Wong RP. Neutrophil CD64 is a sensitive diagnostic marker for early-onset neonatal infection, Pediatr Res 2009; 5:796-803.  Back to cited text no. 28
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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