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International Journal of Recent Scientific Research
Vol. 2, Issue, 6, pp. XXX -XXX, June, 2011
ISSN: xxxxx


Vinodkumar C.S1, Umakanth Patil2, Kalapanavar N.K3, Basavarajappa K.G4,
Department of Microbiology, S. S. Institute of Medical Sciences and Research Centre, Davangere-577005, Karnataka, INDIA
Department of Pharmacology, S. S. Institute of Medical Sciences and Research Centre, Davangere-577005, Karnataka, INDIA
Department of Paediatrics, S. S. Institute of Medical Sciences and Research Centre, Davangere-577005, Karnataka, INDIA

Septicemia is the leading cause of morbidity and mortality in neonates. In this study, 1647 consecutive neonates suspected of having
septicemia from various neonatal intensive care unit hospitals in central Karnataka were investigated for isolation of microorganisms.
Two samples of blood were collected aseptically for isolating the etiology. The cultures were positive in 877 (53.2%) cases for aerobic
bacteria and 96 (10.9%) for Candida species. Among Candida species, C.tropicalis- 39(40.6%) was the predominant organism
followed by C.albicans 22(22.4%) and C.guillermondi 17(17.7%). The standard macrobroth dilution method was carried out to
determine the minimum inhibitory concentration (MIC); C.krusei ATCC 6258 standard strain was included for quality control
purpose. 4(25.0%) stains of C.albicans were resistant to amphotericin-B, 5-fluorocytosine respectively and 2(12.5%) to fluconazole.
High-level resistance to fluconazole, ketoconozole and 5-fluorocytosine was observed in C.krusei.

© 2010 IJRSR. All rights reserved.

Key words: Neonatal septicemia, Candida species, Antifungal agents
Neonatal mortality rate is one of the indicators measuring Drug resistance is the major cause of increase in
the health status of a nation. There could be various morbidity and mortality in neonates. Among the different
reasons for neonatal mortality but septicemia continues to antifungal agents, resistance to the polyenes compounds
be a major cause of neonatal mortality and morbidity has remained an uncommon problem, but resistance to
worldwide1. Neonatal septicemia refers to infection of flucytosine and azoles now appears to be increasing in
infants during first month of life,2. According to National importance, especially after the wide spread use of
Neonatal Perinatal Database (NNPD) 2002, the incidence fluconazole for extended period12,13. The aim of the study
of neonatal septicemia has been reported to be 30/1000 is to determine the incidence and antifungal susceptibility
live births in India2-4. A very wide spectrum of organisms of Candida species in neonatal septicemia.
has been described to cause neonatal septicemia, and most
predominant organisms are Klebsiella pneumoniae, MATERIAL AND METHODS:
Staphylococcus aureus, Group B and D Streptococci,
Escherichia coli, Pseudomonas aeruginosa, Coagulase A total of 1647 clinically diagnosed cases of septicemia
negative Staphylococci and Candida species2-5. Candida were studied prospectively for over the period of 5 years
species are the most common fungal pathogens isolated Neonatal sepsis were suspected when any of the signs and
from blood cultures of neonates5,6. Numbers of risk systems or predisposing factors such as reduced activity,
factors are associated with the development of neonatal fever, refusal of feed, seizures, prolonged jaundice, birth
candidaemia, such as very low birth weight, prematurity, asphyxia, umbilical sepsis, prematurity, abdominal
prolonged antibiotic therapy, prolonged use of fat distensions and history of premature rupture of membrane
emulsion in total parenteral nutrition and use of artificial were noted in the newborns. Two samples of blood were
ventilation7-9. Though majority of nosocomially acquired collected from each case using aseptic precautions. About
Candidaemia is due to C.albicans10, the emergence of 2 ml of blood was added immediately into 20 ml of brain
non- albicans Candida species like C.tropicalis, heart infusion broth with 0.025% sodium polyethol
C.glabroata, C.krusei and C.parapsilosis as predominant sulphonate as anticoagulant. The bottles were incubated
species causing blood stream infection in premature for seven days and subcultures were done appropriately2.
newborns in neonatal intensive care units11-13 are also Candida species were isolated from 96 cases, which were
reported. further confirmed by germ-tube test, urease test, reduction
of tetrazolium media, different conidial arrangement on
corn meal agar, sugar assimilation and fermentation test14-
* Corresponding author: +91 9964402525 .
E-mail address:
International Journal of Recent Scientific Research, Vol. 2, Issue, 5, pp. XXX -XXX, June, 2011

The antifungal susceptibility for yeasts was tested using

broth macrodilution technique recommended by National
Committee for Clinical Laboratory Standards 16. Media 45 39(40.6%)
used was RPMI-1640 with glutamine, buffered to pH 7.0 se 35
at 250C with morpholinopropane sulphonic acid buffer t 30
os 25
(final concentration 0.165 mol/1). Serial two-fold dilution I
f 20 17(17.7%)
o 14(14.5%)
of antifungal agents was prepared (0.1ml of various re 15
antifungal concentrations in 12 x 75 mm tubes). For b 10 4(4 .01%)
u 5
inoculum preparation, 5 colonies of  1mm diameter from N
24 hours old cultures of Candida species were suspended
in 5ml of normal saline and mixed well for 15sec. The
cell density was spectrophotometrically adjusted to 0.5 Candida species
McFarland standards.
FIGURE 1 Prevalence of Candida Species In Neonatal Septicemia
This procedure yielded a stock suspension of 1.5 x 106
cells / ml. A working suspension was made by a 1:100 The test organisms were interpreted depending on
dilution followed by 1:20 dilution of the stock suspension minimum inhibitory concentration (MIC) by broth
with RPMI 1640 broth medium which results in 5.0 x 102 dilution against different antifungal agents. In case of 5-
to 2.5 x 103 cells /ml. A volume of 0.9ml of inoculum was
Fluorocytosine,  4g/ml (Sensitive), 4 – 16 intermediate
added to each tube. The tubes were incubated at 350C for
and  32g/ml (Resistant); for amphotericin-B, 1g/ml
48 hours. Plate counts were performed on representative
inocula to check viability of Candida. The lowest (Sensitive), and 1g/ml (Resistant); for Ketoconazole, 
concentration of an antifungal that substantially inhibited 0.125g/ml (Sensitive), and  1g/ml (Resistant) and for
growth of the organism was detected visually. The end Fluconazole, 8g/ml (Sensitive), 16-32g/ml
point of amphotericin B was easily defined but in case of (intermediate), 64g/ml (Resistant) In-vitro antifungal
5-fluorocytosine and azoles, minimum 80 percent susceptibility of 96 Candida species for antifungal agents
inhibition was taken as the end point standard (standard like amphotericin-B, fluconazole, ketoconazole and 5-
was prepared by diluting 0.2ml of drug free control fluorocytosine is depicted in table-2 & table-3. Among
growth with 0.8ml of media).C.krusei ATTC 6258 Candida albicans, 4(18.2%) strains were resistant to
standard strain was included for quality control purpose. amphotericin-B, 4(18.2%) to 5-fluorocytosine, 2(9.7%) to
The range of concentration tested for fluconazole was fluconazole and none of the isolates were resistant to
0.125-64g/mL and for amphotericin B was 0.03- ketoconazole
High-level resistance was seen in Candida krusei to
RESULT fluconazole 6(42.9%), followed by 4(28.6%) to
amphotericin-B, 3(21.4%) to ketoconazole and 2 (14.3%)
Out of 1647 clinical suspected septicemia, 96 infants had to 5-fluorocytosine. Resistance to Candida tropicalis was
candidaemia. The predisposing factors that possibly led to observed in 4(10.3%), 6(15.4%), 4(10.3%) and 4(10.3%)
neonatal candidaemia are depicted in table-1. All the 96 to amphotericin-B, ketoconazole, 5-fluorocytosine and
infants received systemic broad spectrum antibiotic fluconazole respectively. Among C.guillermondi,
therapy, 91(94.7%) were low birth weight and 72(75%) 3(17.6%) were resistant to amphotericin-B, 2(11.8%) to
were preterm babies [Table-1]. The percent prevalence of fluconazole, 1(5.9%) to 5-FC and none of the isolates
Candida species in neonatal septicemia is 10.9%, with were resistant to ketoconazole. In Candida parapsilosis, 2
Candida tropicalis 39(40.6%) being the predominant isolates of each were resistant to amphotericin-B and
isolate followed by C.albicans 22(22.4%), C.guillermondi fluconazole.
17(17.7%), C.krusei 14(14.5%) and C.parapsilosis
04(4%) [fig-1]

Table 1 Clinical features in neonates with systemic Candida species are normally found on skin and mucous
candidiasis (n=96) membrane of healthy individuals and therefore
candidaemia is generally an endogenous infection 18-20.
Clinical features Incidence
Candidaemia is the most frequently encountered fungal
No. (%)
infection especially in those babies with the predisposing
Antibiotic therapy 96(100)
Low birth weight 91(94.7) factors like prolonged antibiotic therapy, intravascular
Preterm babies 72(75.0) catheterization, endotracheal intubation, parenteral
Respiratory distress syndrome 62(64.5) nutrition and artificial ventilation7-9. In the present study,
Bacterial sepsis 45(45.8) the factors that possibly led to candidaemia were broad-
Ventilator therapy 34(35.4) spectrum antibiotic therapy, low birth weight and
Skin lesions 16(16.6)
Central line 08(8.3)
prematurity. Parenteral nutrition was not common as the
Neurological features 05(5.2) babies were breast-fed.
International Journal of Recent Scientific Research, Vol. 2, Issue, 5, pp. XXX -XXX, June, 2011

Table 2 Minimum inhibitory concentration of candida species by broth dilution method for amphotericin- b and

Amphotericin- B 1g/mL (Sensitive), 1g/mL (Resistant)

Fluconazole  8g/mL (Sensitive), 16-32g/mL (Dose depedendent Susceptibility),
64g/mL (Resistant)

Table 3 Minimum inhibitory concentration of candida species by broth dilution method for
5-fluorocytosine and ketoconazole

The candidaemia in neonates is most commonly due to studies elsewhere, we predict that clinicians will
C.albicans. The present study shows non- albicans encounter increasing episodes of candidaemia due to non-
Candida has emerged as important nosocomial pathogens. albicans Candida species with the risk of increased
Over all isolation of non- albicans Candida species are antifungal resistance.
higher (76.5%) as compared to C.albicans (23.5%). Thus
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