Late Onset Neonatal Sepsis in Sudan: Incidence, Bacteriological Profiles, Patterns of Antimicrobial resistance and Fatality

Neonatal sepsis refers to systemic and generalized bacterial infection of newborns, documented by a positive blood culture in the first 4 weeks of life with high mortality rates in developing countries. It is characterized by fever, hypothermia, malaise, tachycardia, hyperventilation, toxicity and/or prostration which results from circulating multiplying bacteria. Neonatal sepsis is an important cause of mortality and morbidity and is a life-threat ening emergency where prompt antibiotics treatment is essential for favorable outcomes. It accounts for 30-50% of total neonatal deaths each year. Neonatal sepsis is classified into early (EOS) and late onset (LOS) on the basis of presentation within 72 hours or after 72 hours to 30 days of life respectively. The timing of the transition from EOS to LOS is not clear-cut and depends on the causative pathogen. The causative organisms of neonatal sepsis vary from Academic Journal of Pediatrics & Neonatology ISSN 2474-7521

nursery to nursery and depend on prenatal factors, quality and quantity of the health care personnel, flora of the delivery rooms and availability of antibiotics. EOS is usually due to microorganisms that are acquired from the mother during the ante-partum or intra-partum periods, while pathogens causing LOS are generally acquired in the post-natal environment. Infections with group B Streptococci presenting within the first 7 days of life are usually regarded as early-onset and therefore of maternal origin. Whereas, infections with coagulase negative Staphylococci presenting at any age are likely to be hospital acquired. The usual mode of transmission of microbes in the nursery is by direct physical contact or indirectly from another infant through hands of facility personnel. Maternal factors like fever, pre-mature rupture of membranes, premature onset of labor, chorio-amnionitis, urinary tract infections and group B Streptococcus colonization play important roles in development of neonatal sepsis. Laboratory-confirmed bloodstream infection (LCBI) in neonates must meet the following criteria: patient ≤ 1 year of age and has at least 1 of the following: fever or hypothermia (rectal temperature >38 ℃ or <37 ℃) respectively), apnea or bradycardia and positive laboratory results that are not related to an infection at another site. The gold standard for diagnosis of neonatal septicemia is the isolation of bacterial agents from blood culture from one or two blood cultures with other laboratory evidence of sepsis, such as an elevated serum C-reactive protein level (CRP). Gram-negative bacteria [E. coli, P. aeruginosa, K. pneumonae, H. influenzae], Staphylcoccus spp and β hemoliticus Streptococcus remain the major cause of infection with increasing resistance to commonly used antibiotics in developing countries. As many as 60% of blood cultures would be falsely negative for common neonatal pathogens due to a number of causes. In cases of clinically suspect culture-negative neonates, adjunct laboratory tests like serum pro-calcitonin, C-reactive protein, IL-6/IL-8 levels can be helpful. The polymerase Chain Reaction (PCR) is emerging as an important technique for accurate and rapid diagnosis of neonatal septicemia [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15].
Management of Neonatal Sepsis is challenging due to several physiological peculiarities, different neonatal antibiotic pharmacokinetics compared to older children. The knowledge of anti-microbial susceptibility patterns of common causative pathogens of neonatal sepsis helps to inform antibiotics choice provided periodic active surveillances are carried out. Antibiotics are used prophylactically to manage asymptomatic newborns when the mother has risk factors for infection or has a confirmed infection. WHO recommendation, of Ampicillin and Gentamicin combination for treatment of neonatal sepsis may no longer be effective in treating many neonates with sepsis. This is due to the fact that the majority [71%] of Klebsiella and 50% of E. coli are reportedly resistant to Gentamicin, leading to increase mortality due to neonatal sepsis in developing countries [16][17][18][19][20][21].
This study aimed to determine the bacteriological profiles, antibiotics susceptibility patterns of isolates, risk factors and mortality rates of late neonatal septicemia in intensive care units in a central maternal referral hospital in Sudan.

Ethical Considerations
The research proposal was reviewed and approved by the Scientific and Ethics Committees of the Federal Ministry of Health, Sudan. Parents were enlightened and asked to sign a consent form if they agree to enroll their neonates in the study.

Study Design and site
This was a prospective, cross-sectional and facility-based study that was conducted at the Intensive Care Unit of Omdurman Maternity Hospital Sudan, during the period of January to April 2014. Three hundred and seventeen neonates of 0-28 days of age were enrolled. Inclusion criteria included: neonates suspected of having septicemia on clinical grounds [fever/hypothermia, rectal temperature >38 ℃, <37 ℃ respectively); apnea; bradycardia; tachycardia; hyperventilation; toxicity and/or prostration]. Exclusion criteria were out-patient born babies, birth weight <1500 grams and presence of apparent congenital anomalies.

Sampling: Sample frame: Sample size:
The study sample size was calculated as follows: To adjust for non-responses, 10% of the calculated sample size will be added to n as follows: The Total sample size = 288 + 28.8 = 316.8 Therefore, n was set as = 317 neonates

Sampling technique
Systematic random sampling was chosen as the sampling technique, where the first one was selected randomly and another one will be after k interval which was set as equal to 3.

Study variables
Dependent variables: septicemia.
Independent variables: age, sex, birth weight, mood of delivery, Apgar score, duration of mechanical ventilation, intra-vascular catheterization, antibiotic usage, duration of hospitalization, profile of infecting bacteria and its antibiotic susceptibility patterns.

Identification of organisms
Culture and gram staining: Blood cultures that showed signs of microbial growth were sub-cultured onto blood, chocolate and MacConkey agar. The blood, MacConkey and chocolate agar plates were incubated in aerobic in micro-aerophilic atmospheres using a candle jar at 37 °C for 24-48 hours. All positive blood cultures were identified by their characteristic appearance, gram-staining and confirmation by the pattern of biochemical reactions using standard methods. Cultures that showed no microbial growth within 7 days were reported as negative.

Antimicrobial susceptibility testing
Small filter paper disks (6mm) impregnated with a standard amount of antibiotic (commercially available) were placed onto a Muller Hinton agar plates to which test bacteria had been swabbed. The plates were incubated overnight at 37 ℃ and the zone of inhibition of bacterial growth was used as a measure of susceptibility. Interpretation of results was done as suggested by Clinical Laboratory Standards Institute (CLSI) [23].

Data management and analysis
Data was tabulated, entered into Microsoft excel sheets and uploaded to SPSS (Statistical Package for Social Science) software. Frequencies with 95% confidence interval (CIs) and Odds Ratio were calculated.

Discussion
Neonatal sepsis poses great mortality risk in developing countries, regular bacterial resistance surveillances are lacking making empirical antibiotics unsatisfactory. It is a fairly neglected disease that did not benefit much from recent advances in medical care. The incidence, clinical features, risk factors [low birth weight, prematurity] and mortality rates of neonatal septicemia in Sudan were found to be similar to those previously reported in other developing nations [1,3,4,7,17]. Clinical assessment using a combination of symptoms and signs was shown to be an inadequate guide to provisional diagnosis of neonatal sepsis, a finding in agreement with previous studies. This inadequacy is probably due to lack of consensus on the clinical definition of neonatal septicemia. In addition, these definitions are inappropriate for pre-terms who constitute considerable numbers of live births. Blood culture positivity rates in our study are comparable to reports from other developing nations but are higher than those reported from others. Causative organisms' profiles reported in this study were similar to those reported from other parts of the developing world. K. pneumoniae, P. aeruginosa and E. coli were the most resistant pathogens to routinely used antibiotics, findings concordant with previous studies [24][25][26][27][28][29][30].

Conclusion
In conclusion, incidence and mortality rates of neonatal septicemia were high in neonatal ICUs in Sudan. Prematurity, low birth weights and prolonged hospital stay [>7 days] are important risk factors. Causative organisms [K. pneumoniae, Pseudomonas aeruginosa, S. aureus and E. coli] are considerably sensitive to Meropenium, Vancomycin, Genamicin and Amikacin, with marked resistance to Penicilin G. There is a pressing need to conduct regular anti-microbial sensitivity patterns to better guide empirical antibiotic use and update existing guidelines.