Distribution,antimicrobial resistance and predictors of mortality in neonatal sepsis

Abbreviations

1 Introduction

One-fifth of global live births and more than a twenty five percent of neonatal deaths occur in India [1]. Nearly, 0.75 million neonates died in 2013, the highest for any country in the world [2]. The current neonatal mortality rate (NMR) is 28 per 1000 live births [3]. Given the infant and under-five child mortality rates of 40 and 49 per 1000 live births, respectively, 70% of total infant deaths and more than half of under-five deaths fall in the neonatal period. The major causes of neonatal deaths in India are prematurity, sepsis and perinatal asphyxia [1].

Neonatal sepsis is defined as a multisystem disorder with positive blood culture during the 1st month of life [4]. It contributes to one of the most common causes of neonatal deaths globally as well as in India. Hospital based studies in India suggest an incidence of 30 per 1000 live births, where nearly 20% die in the hospital; the figure rises to up to 50% for those with culture positive sepsis [5, 6]. Not only they are subjected to increased mortality but they also have longer hospital stays, consume more resources and are also at a higher risk of adverse neurodevelopmental outcomes [7].

In contrast to the neonatal intensive care units (NICUs) in developed countries where gram positive organisms still remain the predominant pathogens in both early as well as late onset sepsis and the spectrum of pathogens infecting neonates in NICUs in developing countries is altogether different [8, 9]. In a recent multi-center cohort study from India, the pathogen mix in early onset sepsis (EOS) did not differ from late onset sepsis (LOS), with gram negative pathogens contributing almost two thirds of isolates [10].

Recently, there has been an upsurge and spread of infection due to resistant organisms in developing countries. This is secondary to misuse of antibiotics, over-the-counter and parallel market access, and counterfeit or poor quality drugs, combined with substandard hygiene and living conditions [11, 12]. In a recent multi-center cohort study from India, isolated pathogens showed a high degree of antimicrobial resistance, not only to commonly used antibiotics but also to so-called reserve antibiotics such as extended-spectrum cephalosporins and carbapenems [10]. The World Health Organization (WHO) has recently heightened awareness of this important issue and calls for action to contain antibiotic resistance on a global scale [13].

Periodic surveillance of organisms causing neonatal sepsis, which not only differ from one place to another but also differ in the same place over a time period as well as their antimicrobial sensitivity and resistance pattern can guide the appropriate empirical treatment and prevent emergence of the resistant flora. While this essential information is collected and updated regularly in developed countries, data remains scarce from developing countries, where the majority of neonatal deaths due to sepsis occur. Therefore, this study was carried out to identify the distribution of pathogens and their antimicrobial resistance patterns in the neonates admitted to the NICU of a tertiary care hospital of northern India. This hospital caters a large population of neonates in its region, being a referral center and also is equipped with facilities required for the adequate care of sick newborns.

2 Methods

3 Results

A total of 183 case sheets of babies diagnosed with culture positive sepsis were included in this study. Table 1 shows the baseline characteristics of study participants. Approximately 57.4 % (105) were male. The median birth weight of enrolled neonates was 1340 g (IQR1000-1920 g), and median gestation age was 33·0 weeks (IQR 29– 36 weeks). Approximately, 76 % of neonates (139) were low birth weight, and nearly 25.8 % were small for gestational age (48). Nearly, 77 % (141) were born preterm. About 44% (81) of enrolled neonates were born by caesarean section.

EOS occurred in 59% (108) whereas LOS occurred in 40.9% (75) of study subjects. Meningitis and pneumonia occurred in 39.9% (73) & 40.9% (75) of neonates, respectively.

Breast milk was the predominant form of feeding at 73.8% (135) with the median age of initiation of feeding being 2days (IQR 2– 5) and the amount of time required to achieve full feeds was at a median of 6 days (IQR 0– 12). Median duration of hospital stay was 22 days (IQR 7– 36).

Maternal and perinatal variables posing risk to sepsis were maternal fever within 7 days before delivery (22, 12 %), vaginal examination (≥3) (4, 2.2 %), prolonged rupture of membranes (≥18 hours) (57, 31.1 %), prolonged labor (≥24 hours) (3, 1.6 %), chorioamnionitis (4, 2.2 %), and urinary tract infection (7, 3.8 %).

Central catheters were placed in 63.9 % of neonates with umbilical venous cannulation (96, 52.5 %) being more common than umbilical arterial cannulation (21, 11.4 %). Peripheral catheters were placed in 34.4 % of neonates (63). Vasopressors were used in 61.2 % of neonates (112) with the median duration of use being 2 days (IQR 2– 4).

Surfactant was administered to 21.9 % of neonates due to respiratory distress syndrome (40). Median duration of oxygen use was 4 days (IQR 0– 12). Respiratory support in any form (invasive or non-invasive) was used in 76 % of neonates (n = 139). Non-invasive ventilation was used most commonly with continuous positive airway pressure (CPAP) being the most common mode in 56.2 % (n = 103). Median duration of CPAP use was 1 day (IQR 0– 4). Invasive ventilation was used in 51.9 % (95), with a median duration of invasive ventilation use of 1day (IQR 0– 4).

Neonatal morbidities noted were hemodynamically significant PDA (hsPDA) seen in 25.1 % of neonates (46) with closure required in 76 % of cases. Ibuprofen (29, 15.8 %) was the most common drug used for the closure of hsPDA followed by indomethacin (6, 3.3 %). Other morbidities included Meconium aspiration syndrome, hypoxic ischemic encephalopathy and intraventricular hemorrhage seen in 5.4% (10), 11.5% (21) and 28.4% (52), respectively.

The overall case fatality rate was 29.5% (54/183) for culture positive sepsis cases. Case fatality rate was not significantly different between EOS and LOS (30.5% vs 28%, respectively).

The median duration of time to detection of positive blood culture was 13 hours (IQR 6– 24 hours) with the median duration of time to positivity being significantly less in EOS as compared to LOS; 12 hours (IQR 5– 24 hours) vs 14 hours (IQR 10– 24 hours) respectively, p = 0.001.

Table 2 shows the distribution of microbial isolates along with their case fatality rates. Of the total 183 isolates, about 56.1% were gram positive and 43.3% Gram negative pathogens. The predominant gram-positive pathogens were Staphylococcus aureus, coagulase-negative staphylococcus and enterococcus spp. in that order. The predominant gram-negative pathogens were klebsiella spp., Escherichia coli, acinetobacter spp., pseudomonas spp., and citrobacter spp. The case fatality rate in neonates with sepsis due to gram-positive pathogens was not different as compared to the gram-negative pathogens (29% vs 30% respectively). The case fatality rates were highest for citrobacter spp. followed by enterococcus spp., klebsiella spp. and Staphylococcus aureus.

Table 3 shows the distribution of microbial isolates according to the time of infection. Majority of culture proven sepsis were of early onset (108 vs 75). The pathogen mix in early-onset sepsis did not differ much from that of late-onset sepsis (i.e. sepsis after 72 h). However, infection due to klebsiella spp. was more common in early onset sepsis cases whereas Staphylococcus aureus was more common in late onset cases.

Table 4 shows isolation of resistant isolates and their case fatality rates. Most of the pathogens isolated showed antimicrobial resistance, not only to commonly used antibiotics but also to reserve antibiotics such as carbapenems and extended-spectrum cephalosporins. Majority of gram-negative isolates were multidrug resistant (klebsiella spp. 49.1%, Escherichia coli 50%, citrobacter spp. 50%, acinetobacter spp. 28.5%, pseudomonas spp 100%). Colistin resistance was detected in one (1.2%) of the gram-negative isolates. Among gram-positive pathogens, methicillin resistance was detected in 16.6% (7/42) of coagulase-negative staphylococci, 24.4% (11/45) of Staphylococcus aureus and 62.5% (10/16) of enterococcus spp. isolates. Resistance to vancomycin was observed in 25% of enterococcus spp. isolate whereas it was only 4.4% and 7.1% for Staphylococcus aureus and coagulase-negative staphylococci respectively. The case fatality due to resistant organisms is markedly higher amongst both Gram-positive as well as Gram-negative organisms

Tables 5 and 6 shows univariate followed by multivariate analysis of the factors significantly associated with increased mortality amongst culture positive isolates. The data shows that earlier gestational age, lower birth weight, use of invasive mode of ventilation, vasopressor use, infection with multidrug resistant organisms and early onset sepsis to be associated with increased risk of mortality, and feeding with human breast milk to be associated with decreased risk of mortality in culture positive neonatal sepsis. Furthermore, multivariate analysis of the data shows that use of invasive mode of ventilation and early onset neonatal sepsis are independently associated with increased risk and that feeding with human breast milk is independently associated with decreased risk of mortality in culture positive sepsis.

The clinical signs and symptoms of neonatal sepsis are subtle and nonspecific, making its early diagnosis difficult. Blood culture is still the gold standard for definitive diagnosis of neonatal sepsis, in spite of some drawbacks of blood cultures [16].

This retrospective study recorded a high case fatality rate of culture positive sepsis amongst preterm and low birth weight neonates, accounting for nearly one third of deaths in enrolled neonates. Most infections occurred early, and klebsiella spp. emerged as the predominant causative organisms. The majority of pathogens showed an alarming degree of antimicrobial resistance.

Most episodes of sepsis occurred in preterm (77 % of enrolled cases) and low birth weight (76 % of enrolled cases) neonates at a quite early age, within 72 hours of life. This pattern is in sharp contrast with that reported from other Asian countries, where most infections are of late onset [17, 18] as well as those from developed countries [19]. However, the data is consistent with 3308 isolates reported by the NNPD (National Neonatal Perinatal Database) Network as well as reports from other developing countries [20, 21] in which two thirds of cases are of early onset sepsis [5]. Nearly one third of neonates with culture-positive sepsis in our study died despite early detection, supportive care and antibiotic therapy. This finding can be explained by a high degree of antimicrobial resistance as well as high virulence of isolated pathogens in this study. The majority of babies with culture positive sepsis were males and born vaginally.

Only 40% of neonates with culture positive sepsis have ≥1 antenatal risk factors with prolonged rupture of membranes (18 hours or more) and maternal fever within 7 days prior to delivery being the most common antenatal risk factors. This is in concordance to the study done by Gebremedhin D. et al. [22].

In the present study, the pathogen mix in both early and late onset sepsis are similar. Isolation rates of gram positive organisms (53.7%) are higher as compared to gram negative organisms (46%) in early onset neonatal sepsis whereas in late onset neonatal sepsis gram positive organisms (60%) are more frequently isolated as compared to the gram negative organisms (39.8%).The predominant organism causing EOS was klebsiella spp whereas it was Staphylococcus aureus for LOS.

In a recent multicentric trial from India, klebsiella spp., Escherichia coli and acinetobacter spp. emerged as the most common gram negative isolates whereas coagulase-negative staphylococcus, Staphylococcus aureus, and enterococcus spp. were the most common gram positive isolates [10]. Although, acinetobacter spp. emerged as the most common isolated organism in the entire study cohort, klebsiella spp. was the predominant isolate in one of the centers enrolled in the study. According to 1248 isolates reported by the NNPD Network, klebsiella spp. (32%), Staphylococcus aureus (13.6 %) and Escherichia coli (10.6%) were the leading pathogens, with the proportion of acinetobacter spp. at a low of 3% [5] as seen in our study. Similarly, Waters et al in their review of the etiology of community acquired neonatal sepsis in low and middle income countries found klebsiella spp. to be highly prevalent in South-East Asia [23]. Tallur et al., also reported klebsiella spp. as the most common organism in their study [24]. Vishwanathan R et al., in their study in a rural NICU set up, reported 46.3% blood culture positivity with predominant gram negative isolates. Klebsiella spp. was the most common organism followed by E coli. They also noted that profile of organisms causing early and late onset sepsis was similar in their study [25].

The median time to positivity for culture proven sepsis in the present study was 13 hours (IQR 6– 24 hours) with the median time to positivity being significantly earlier in EOS as compared to the LOS 12 hours(IQR 5– 24 hours) vs 14 hours (IQR 10– 24 hours). This result is in contrast to the previous studies done by Guerti et al., and Jardine et al., who found time to positivity in both EOS and LOS to be similar [26, 27]. However, these differences in results could be explained by altogether different spectrums of isolated organisms, higher incidence of LOS and lesser virulence of isolated organisms seen in those studies as compared to our study.

In the present study, case fatality rates did not differ between early-onset and late-onset sepsis. The case fatality rates in culture proven sepsis in developed countries [28] is higher in cases of LOS as compared to EOS whereas a recent multicenter trial from developing countries indicated that mortality rates do not differ between EOS and LOS in accordance with our study [10].

Our study has also shown an alarmingly high degree of antimicrobial resistance to reserve antibiotics in both gram positive as well as gram negative isolates. The definition of multidrug resistance amongst gram negative isolate was more rigorous in our study as we have defined resistance against reserve group of antibiotics. The high rates of multidrug resistance in the Gram-negative isolates is a real threat in the choice of antibiotics in Indian NICUs. New Delhi metalloβlactamase-1(NDM-1 is a carbapenemase produced by certain gram negative organisms capable of inactivating almost all β- lactams such as penicillin’s, cephalosporin’s and the carbapenems. The gene encoding for NDM-1 is called bla_{NDM - 1} and has been identified on both chromosomes and plasmids. Plasmids also carry genes conferring resistance to other groups of antibiotics making it multidrug resistant or, because of other non-plasmid mediated resistances, resistance in some cases to all antibiotics [29]. These plasmid mediated resistance is also transferrable to other bacteria resulting in extremely drug- resistant phenotypes.

Different factors were found to contribute to increased mortality rate in the present study. Factors which were independently found to predict death and survival in this study were invasive ventilation, EOS and breast feeding respectively. Previous observational studies have identified risk factors for sepsis such as prematurity, low birth weight, premature membrane rupture, maternal pyrexia, poor intra- and postpartum hygiene, invasive medical procedures, and hospital stay [30, 31].

These results are in agreement with previous reports that associate these factors with poor prognosis and death in the neonate with culture proven sepsis [32, 33].

Our study has several limitations. First, this was a retrospective study and included a cohort from a single center, therefore, the general validity may be inevitably limited. Second, we may have overestimated the incidence of coagulase- negative staphylococci. However, in our NICU we follow stringent, protocol-driven practices for skin preparation before obtaining all blood culture specimens, and the diagnosis of coagulase-negative staphylococci sepsis was made only when the clinical picture is compatible with sepsis along with a positive blood culture. Third, sample size was small and study period was short.

5 Conclusion

The present study describes the profile of neonatal sepsis along with the degree of antimicrobial resistance as well as predictors of mortality in culture positive sepsis in a level III NICU in northern India. Two areas of concern are the emergence of gram positive organisms as the major cause of neonatal sepsis and the high degree of antibiotic resistance observed for reserve antibiotics in both gram positive and gram negative organisms. This perhaps suggests that the time may have come to review the empirical antibiotics recommended by WHO for neonatal sepsis, as well as to rationalize the use of antibiotics in the NICU to avoid the emergence of MDR organisms, avoid invasive procedures to the maximum extent and promote early breast milk feeding in neonates that have culture positive sepsis.

Funding

None

Conflict of interest

None