CHANGES IN LEVEL OF C-REACTIVE PROTEIN AT 0 HOURS AND AT 72 HOURS AMONG NEONATES WITH SUSPECTED SEPSIS AT NEONATAL INTENSIVE CARE UNIT OF BIRAT MEDICAL COLLEGE TEACHING HOSPITAL 1636

Changes in Level of C-Reac ve Protein at 0 Hours and at 72 Hours among Neonates with Suspected Sepsis at Neonatal Intensive Care Unit of Birat Medical College Teaching Hospital. Ramesh Yadav, Hem Sagar Rimal, Santosh Upadhyaya Kafle . BJHS 2021;6(3)16. 1636-1641. Introduc on Neonatal sepsis s ll remains serious and poten ally lifethreatening events with a mortality rate of up to 50% in very premature infants. Efforts were made to improve laboratory sepsis diagnosis. C-reac ve protein (CRP) is the most extensively studied acute phase reactant so far. Very few studies have been done to see the varia on of serial measurement of CRP ter.


Introduc on
Neonatal sepsis s ll remains serious and poten ally lifethreatening events with a mortality rate of up to 50% in very premature infants. Efforts were made to improve laboratory sepsis diagnosis. C-reac ve protein (CRP) is the most extensively studied acute phase reactant so far. Very few studies have been done to see the varia on of serial measurement of CRP ter.

Objec ves
To find out the level of CRP ter at 0 hour and at 72 hours among neonates with suspected sepsis at the neonatal intensive care unit of Birat Medical College Teaching Hospital.

Methodology
It was a cross-sec onal descrip ve study from 26 March 2021 to 25 July 2021, to see serial CRP tre among the sepsis suspected neonates at the Neonatal intensive care unit of Birat Medical College Teaching Hospital. A Total of 95 cases of neonates suspected sepsis were enrolled and their serial CRP tre at 0 hour and 72 hours were studied. The data was entered into Microso office excel and analyzed using sta s cal package for social sciences (SPSS 20.0)

Results
There was no significant associa on of gender, birth weight, mode of delivery and gesta onal age with an increase of CRP at 0 to 72 hours a er birth. Among the enrolled neonates 34 had posi ve blood culture while 61 had blood culture which was sterile. It showed that, 70.6% who had blood culture posi ve had increased CRP level at 0 to 72 hours whereas only 29.4% had not no increment in CRP despite posi ve blood culture.

Conclusions
CRP tre increment at 72 hours a er the first one correlated be er with culture proven sepsis in comparison to CRP ter increment at 0 hours a er birth. The sensi vity, specificity, posi ve and nega ve predic ve values as calculated in this study are not high enough to make it a good screening test. The test is not specific enough to rely upon as the sole indicator. The clinical judgment along with other hematological parameters and diagnos c markers along with serial CRP should be considered in evalua ng a neonate for sepsis.

INTRODUCTION
During the last decades advances in neonatal intensive care have led to an impressive decrease of neonatal mortality and morbidity. However, infec ous episodes in the early postnatal period s ll remain serious and poten ally lifethreatening events with a mortality rate of up to 50% in very 1 premature infants. The signs and symptoms of neonatal sepsis is clinically indis nguishable from various noninfec ous condi ons such as respiratory distress or maladapta on. Therefore, rapid diagnosis is crucial for preven ng the child from an adverse outcome. The current prac ce of star ng empirical an bio c therapy in all neonates showing infec on-like symptoms results in their exposure to adverse drug effects, nosocomial complica ons, 2 and in the emergence of resistant strains. Laboratory sepsis markers represent a helpful tool in the evalua on of a child with clinical signs and complement the evalua on of a neonate with a poten al infec on. The tests used include the white blood cell count (WBC) and assays for markers of inflammatory reac on in serum, such as, Creac ve protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and procalcitonin. During the last decades efforts were made to improve laboratory sepsis diagnosis and a variety of the markers and more were studied with different success. Despite the promising results for some of them, current evidence suggests that none of them can consistently diagnose 100% of infected cases. C-reac ve protein (CRP) is the most extensively studied acute phase reactant so far and despite the ongoing rise (and fall) of new infec on markers it s ll remains the preferred index in many 3 neonatal intensive care units. Suspected neonatal sepsis were considered if neonate had features of perinatal risk factors i.e., maternal pyrexia (within 1 week prenatal and/or 48 hours postnatal), prolonged rupture of membranes (18 hours), foul smelling vaginal discharge or/and maternal urinary tract infec on diagnosed in last month. Neonates having unexplained hypothermia/hyperthermia, lethargy, irritability, poor feeding or milk intolerance, tachypnoea (>60breaths/ minute), cardiovascular dysfunc on such as persistent tachycardia (>160 beat/min) or bradycardia (<100beats/min), poor peripheral circula on, hypotonia or circumoral cyanosis or pallor, birth asphyxiated neonates, low birth weight <2500, very low birth weight <1500 grams and extremely premature <32 weeks gesta on. A newborn will suffer if infec on is not diagnosed properly: under diagnosed or over diagnosed. For early diagnosis of newborn sepsis, combined and/or alone interleukin-6 (IL-6) and C-reac ve protein (CRP), has a recognized role. CRP produc on is a non-specific response to disease and cannot be used alone as a diagnos c test for sep cemia. Along with clinical evidence of the disease, CRP provides good ideas regarding sep cemia diagnosis. In many centers only one CRP test is done on admission and if it comes out to be nega ve, an bio cs are stopped and later on the baby comes back with severe sepsis. CRP serial measurement in infec on progress is helpful, also in infec on diagnosis. Very few studies have been done to see the varia on of serial measurement of CRP tre with blood culture. Checking the varia on of quan ta ve CRP tre when done twice, one at admission and second at 72 hours a er the first one with 4 blood culture seems essen al. Hence, we aimed to find out the level of CRP tre and how it varies according to gesta onal age, gender, birth weight, types of sepsis, modes of delivery, different laboratory parameters at 0 to 72 hours among sepsis suspected neonates in the Neonatal intensive care unit of Birat Medical College Teaching Hospital. We also aimed to assess the differences in level of CRP tre among sepsis suspected neonates at admission and a er 72 hours with blood culture proven sepsis.

METHODOLOGY
It was a cross-sec onal descrip ve study from 26 March 2021 to 25 July 2021, to see varia on in CRP ter with blood culture among the neonates suspected of sepsis at the Neonatal intensive care unit of Birat Medical College Teaching Hospital. A total 95 sample sizes were calculated by using expected prevalence 62%, desired precision 10% and confidence level 95%. A er taking informed and wri en consent from parents and approval from the ins tu onal review commi ee, neonates brought to the neonatal intensive care unit of BMCTH were selected by non-probability consecu ve sampling techniques. Neonates suspected of sepsis and planned to treat with injectable an bio cs by the trea ng pediatrician were included. Neonates who already had taken an bio cs prior to admission and parents unwilling to give wri en consent were excluded. All pa ents included were started on empirical an bio cs a er drawing samples for blood cultures and CRP along with other inves ga ons advised by the trea ng physician and sent to the laboratory. Strict asep c measures were taken while taking blood for inves ga ons. For CRP blood was collected in plain vial without any an coagulants and the tests were performed by Huma Tex CRP latex agglu na on slide test. A second sample for determina on of CRP was drawn 72 hours a er the first one. For blood culture 1 ml of blood were drawn. Blood culture bo les were checked for expiry date printed on each bo le. 1ml each of blood was inoculated in a blood culture bo le containing media i.e., Blood Agar or MacConkey Agar media. Blood culture bo les were clearly labeled with the name of the pa ent and date and me of collec on of blood before sending to the laboratory. Blood culture and sensi vity were performed by BD BACTEC. If the first CRP tre was non-significant, the an bio c therapy was con nued and if the second CRP was also nonsignificant, the decision to con nue or discon nue an bio c was decided by the trea ng physician. But, if there was significant rise in the second CRP tre, the an bio cs were con nued or changed, looking at the clinical status of the pa ent and decided by the trea ng physician. If first and second CRP both were posi ve, the therapy was con nued and the culture and sensi vity report were awaited. CRP was read as nega ve when level was < 6mg/dl and posi ve when level > or = 6 mg/dl and ter were recorded. Blood culture was followed for growth up to 7 days. Data collec on was done using a specifically designed ques onnaire. All the data were entered in excel sheets and converted into SPSS version 20. The descrip ve and inferen al sta s cs were used for data analysis. The test of significance was done by the chi-square test. The sensi vity, specificity, nega ve predic ve value and posi ve predic ve value of CRP to diagnose neonatal sepsis at 0 hours and at 72 hours were calculated separately taking blood culture as standard.

RESULT
This study included 95 neonates who met the inclusion criteria. Among them 74(77.9%) were male and 21(22.1%) were female. 20(21.1%) were of <37 weeks gesta on and 75(78.9%) were of >37 weeks gesta on. Among 95 neonates 31(33.7%) were of birth weight <2.5kg, 59(62.1%) were of normal birth weight (2.5 -3.9 Kg) and 4(4.2%) were of birth weight (>3.9kg). Among the study popula on 40 (42.1%) were delivered by SVD, 52 (54.7%) were delivered by LSCS and 3 (3.2%) by SVD and instrument assisted (Table1). There was no significant associa on of gender, birth weight, mode of delivery and gesta onal age of the newborn with an increase of CRP at 0 to 72 hours.  Table 2 shows different reasons for which LSCS was the preferred mode of delivery among the included neonates. There was no significant difference in level of CRP ter at 0 hours to 72 hours a er birth according to mode of delivery among the study popula on.  Among the study popula on 66 (69.5%) did not have any adverse perinatal events while 29(31.5%) had some adverse perinatal events (Table 4). There was no significant associa on of adverse perinatal events on increment of CRP at 0 hours to 72 hours a er birth.     Table 6, shows that the sensi vity of CRP in the diagnosis of culture proven sepsis increased from 35% at the ini al sepsis work-up to 82.22% when CRP determina on was performed at 72 hours following the first one while we found specificity ranging only from 35.565 to 58.43%. The posi ve predic ve value of CRP in iden fying sepsis at birth was 38.33% which was similar when calculated for CRP ter at 72 hours 38.95%. The nega ve predic ve value of CRP ter in iden fying sepsis at birth was 70.27% which increased to 80.00% when calculated for CRP at 72 hours a er birth. Similarly, accuracy of the CRP ter at birth in iden fying sepsis was 55.97% which calculated at 72 hours a er birth was 51.11%.

DISCUSSION
The signs and symptoms of neonatal sepsis can be clinically indis nguishable from various noninfec ous condi ons such as respiratory distress syndrome or maladapta on. Therefore, rapid diagnosis is crucial for preven ng the child from an adverse outcome. Based on clinical pictures alone the diagnosis of neonatal infec on is difficult to establish, yet it is crucial that treatment is ins tuted early because of the high mortality associated with neonatal infec on. Clinical suspicion along with various laboratory sepsis markers represents a helpful tool in the evalua on of a child with clinical signs and complement the evalua on of a neonate with a poten al infec on. Variety of sepsis markers were studied with different success to aid diagnosis of neonatal sepsis but none of them was able to consistently diagnose 100% of infected cases. Moreover, newer markers studied are expensive to perform and not easily accessible to economically constrained countries like ours. C-reac ve protein is the easily available and cheap sepsis marker useful for diagnosis of neonatal sepsis. A limited number of studies have been conducted to see whether single CRP tre or serial measurement of CRP ter at 72 hours a er the first one correlates be er with the blood culture. So, we aimed to study how the value of CRP ter changes at birth and at 72 hours according to demographic, laboratory and blood culture among neonatal sepsis suspected neonates at the neonatal intensive care unit of BMCTH. In our study we also noted that there was no significant associa on of mode of delivery with increment of CRP at 0 hours to 72 hours a er birth among the study popula on. Also, we noted in our study 84(88.4%) were neonates with early onset neonatal sepsis and 11(11.6%) were neonates suspected of late onset neonatal sepsis. There was no significant associa on of different types of sepsis and the various underlying causes for sepsis suspicion with increment of CRP at 0 hours to 72 hours a er birth. In our study, 66 (69.5%) of the study popula on did not have any adverse perinatal events while 29(31.5%) had some adverse perinatal events. There was no significant associa on of adverse perinatal events on increment of CRP at 0 hours to 72 hours a er birth. Also, we noted that none of the inves ga on's parameters had associa on with increment of CRP from 0 hours to 72 hours a er birth. Among them 34(35.7%) had posi ve blood culture while 61(64.2%) had blood culture which was sterile. The blood culture report had significant associa on with 72 hours CRP increment. It showed that 24(70.6%) who had blood culture posi ve had increased CRP level at 72 hours whereas only10 (29.4%) had not increased. The p -value was <0.05 and was significant. Hisamuddin et. al in his study also has reported that the diagnos c accuracy of CRP in diagnosis of neonatal 4 sepsis was 70.07% which is similar to finding in our study. In our study we also noted that the sensi vity of CRP in the diagnosis of culture proven sepsis increased from 35% at the ini al sepsis work-up to 82.22% when CRP determina on was performed at 72 hours following the first one. In a similar study done by Benitz et al. found that the sensi vity in the diagnosis of culture proven early onset sepsis increased from 35% at the ini al sepsis work-up to 79% and 89% when CRP determina on was performed on the two 8 following days. In a large series of 689 neonates (187 with sepsis) Pourcyrous et al. reported a higher sensi vity for CRP levels determined at least 12 hours a er the ini al 9 evalua on compared to the first value (54% vs. 74%). In general, the sensi vity substan ally increases with serial determina ons 24 to 48 hours a er the onset of symptoms, and several studies reported on sensi vi es and specifici es 10 ranging from 78% to 98% and from 81% to 97%, respec vely. But in our study, we found specificity ranging only from 35.565 to 58.43% which was very low. So, it shows that serial CRP ters had greater sensi vi es for diagnosing neonatal sepsis in comparison to single CRP ters in comparison to our study measured at 72 hours a er the first. Specificity was found lesser in our studies in comparison to previous studies which may be because in previous serial CRP ters were measured earlier within 48 hours. This needs further studies measuring CRP ter earlier within 48 hours which may have greater specificity. In our study we noted that there was no significant associa on of gender, birth weight, mode of delivery and gesta onal age with an increase of CRP at 0 to 72 hours a er birth. But Ishibashi et al found that in 110 uninfected symptoma c neonates gesta onal age and birth weight significantly influence CRP concentra on within 48 hours a er birth. Neonates with low gesta onal age and low birth 11 weight had lower CRP concentra on.

CONCLUSION
Level of CRP tre increment from 0 hours to 72 hours a er birth did not vary with gesta onal age, gender, birth weight, different reasons for cesarean sec on, different laboratory parameters, change of an bio cs for management, adverse perinatal events and types of sepsis. Blood culture report had significant associa on with 72 hours CRP increment. It showed that 24(70.6%) who had blood culture posi ve had increased CRP level at 72 hours whereas only10(29.4%) had not increased. CRP es ma on does have a role in the diagnosis of neonatal sepsis but serial CRP tre increment at 72 hours of life correlates more with proven sepsis and has greater sensi vity in diagnosing neonatal sepsis. The test is not specific enough to be relied upon as the only indicator. The sensi vity, specificity, posi ve and nega ve predic ve values as calculated in this study are not high enough to make it a good screening test. The clinical criteria along with other hematological parameters and diagnos c markers along with serial CRP should be considered in evalua ng a neonate for sepsis.

LIMITATION OF THE STUDY
The study was done only for a period of 6 months dura on. S ll, further research is needed on the topics for longer Original Research Ar cle Poudel B et al dura on of me with greater sample size to see the influence of gesta onal age on CRP kine cs in infec on, noninfec ous confounders, and the evalua on of dynamic and gesta onal age dependent reference values, could have be er external validity.

ACKNOWLEDGMENT
I would especially like to thank Professor Dr. Hemsagar Rimal and Professor Dr. Santosh Upadhyaya Kafle for their con nuous guidance and expert opinion throughout the study period.

CONFLICT OF INTEREST
None