Candidemia has been identified as a public health problem in HIV-infected patients. The evaluation of CD4 count, transaminases and blood glucose, are being used as a means to monitor the health of HIV-infected patients, without excluding the diagnosis of candidemia and other opportunistic infections. In order to contribute in improving the care of HIV-infected patients attending Dschang District Hospital and later on, in other hospitals in Cameroon, we conducted from June to September 2014 a cross-sectional study, with general objective; to determine the association between candidemia and selected biochemical and haematological parameter changes in HIV-infected patients, as a possible indicator in monitoring HIV disease progression. To do this, blood samples were collected from HIV-infected patients assigned to the UPEC of Dschang District Hospital for follow up, and haemogram report, CD4 counts, ALAT level, ASAT level, and glucose level in blood were evaluated by cytometric and spectrophotometric assays. Candida species were isolated from some blood samples, and then identified using CHROMagar Candida culture medium. The broth microdilution method was afterwards used to test the susceptibility of the fungal isolates vis-a-vis three conventional antifungal agents. Mycological analysis of blood samples showed that eight (08) patients had candidemia, a prevalence of 6.11%. Eight (08) isolates were obtained from these eight (08) candidemic HIV-infected patients; this consisted of 4(50%) Candida albicans, 3(37.5%) Candida parapsilosis and 1(12.5%) Candida glabrata. All these isolates were resistant (MICs ranged from 2 to >256 µg/mL) to the antifungals used, that is, ketoconazole, amphotericin B and nystatin. A significant correlation was found between candidemia and white blood cell count, with a correlation coefficient of r = 0.240 (p < 0.05). Based on the results obtained, the systematic diagnosis of candidemia should be performed in patients infected with HIV in Cameroon in order to improve on their care. Key words: Candidemia, HIV, biochemical parameters, hematological parameters, Antifungals activities.

1. 1
DEPARTMENT OF BIOCHEMISTRY
DEPARTEMENT DE BIOCHIMIE
Laboratory of Microbiology and Antimicrobial Substances (LAMAS)
Option: Clinical Biochemistry
Presented by:
NANGWAT Claude
BSc in Science
CM04-08SCI 1673
Master of Science Thesis
FACULTE DES SCIENCES
****************
FACULTY OF SCIENCE
************
ECOLE DOCTORALE
UNIVERSITE DE DSCHANG
****************
UNIVERSITY OF DSCHANG
************
POST GRADUATE COLLEGE
June 2015

2. 2
TITLE:
Candidemia in HIV-infected patients attending
Dschang District Hospital: effects on the level of
some biochemical and hematological parameters
and the sensitivity of the etiological agents to
some antifungals.

3. PLAN
INTRODUCTION
METHODOLOGY
RESULTS AND DISCUSSION
CONCLUSION AND PERSPECTIVES
3

4. 4

5. Complex immune
disorder
responsible for increased
morbidity and a mortality rate
estimated at 30-50% within 5-10
years (Lohoue et al., 2005).
HIV
Infections
(Opportunistic)
5

6. 6
Candida
Kotthoff-Burrell, 2012
Candidiasis
affects more than 80% of
HIV infected subjects
(Lohoue et al., 2005) .
It is the single most
important cause of fungal
infections worldwide
Also one of the most important
causes of bloodstream infection
that can develop while one is in
the hospital
&

7. 7
Candida infection can occur in almost
any part of the body. Usually it develops
on mucous membranes (such as in the
mouth and genitals),
BUT

8. 8
Other parts of the
body
Invasive
Candidiasis
Organs like the
kidneys and liver may
become Affected
Modification in the level of some
biochemical parameters(e.g ALAT, ASAT)
and hematological parameters(e.g WBCs,
RBCs)
(Kotthoff-Burrell, 2012).
Bloodstream (candidemia)

9. Concurrent with the increase in fungal infections such
as candidemia in HIV-infected patients, a large variety
of antifungal drugs are available with different spectra
of activity (Khan et al., 2006).
There is therefore a need to determine the antifungal
susceptibility of isolates to available drugs
(Khan et al., 2006).
9

10. Our research was focused on determining the
association between candidemia and selected
biochemical and haematological parameter
changes in HIV-infected patients, as a possible
indicator of monitoring HIV disease progression.
10

11. Hypothesis
The presence of Candida species in the bloodstream of HIV-
infected patients affects the level of some biochemical and
hematological parameters.
General objective
To determine the prevalence of HIV/candidemia coinfection and
the pattern of hematological and biochemical abnormalities
associated with such co-infection.
11

12. Isolate and identify Candida species from the blood samples of
HIV-infected patients,
Perform the biochemical blood analysis for; hemogram, CD4
counts, ALAT level, ASAT level and glucose level,
Perform an antifungal susceptibility test of Candida species
isolated.
12

13. 13

14. 14
 Culture media; Sabouraud Dextrose Broth (SDB), Sabouraud Dextrose
Agar (SDA) and CHROMagar Candida (CHROMagar TM Candida, Paris).
Reagents for CD4 counts and haemogram
ALAT, ASAT and blood glucose Kits (HOSPITEX DIAGNOSTICS (ITALY))
 Apparatus; Spectrophotometer (BIOSYSTEM BTS-310), Centrifuge (NF
200, ERMA INC PCE-210), BD FACS counting system.
Antifungals; ketokonazole and nystatin (Sigma Aldrich, Milan, Italy),
amphotericin B (Bristol Myers Squibb 3, rue Joseph Monier),
Dimethylsulfoxide (DMSO)

15. 15
Study periodType of study
Inclusion Criteria
Exclusion criteria
Cross-sectional
study
10th of June to the 18th of
September 2014
131 patients of all ages and of both sexes
Patients under antifungal therapy and
those with any form of hepatitis

16. 16
Ethical considerations
Hospital administration
CBI/298/ERCC/CAMBIN
230/AR/MSP/DRO/DSD/HDD
CAMBIN ERCC
Authorizations
A volume of 5 mL of blood was collected per patient by well trained
personnel of the Hospital Laboratory under aseptic conditions.

17. Blood samples were collected
And used for
a) Haematological and
blood biochemical
analyses for: hemogram,
CD4 counts, ALAT, ASAT
and glucose levels.
17
c) Antifungal
susceptibility test
b) Candida species
Isolation and identification
EDTA tubesPlain tubes

18. Collection of blood samples
18
preparation of culture media
standard methods

19. Identified colonies
Storage in 50%
glycerol/SDB, at
O°C
19
Blood in EDTA tube
a) Isolation and identification of Candida species from blood samples
Streak samples onto the Petri
dishes containing CHROMagar
 Inoculation (Beighton et al.,1995)
Incubation at 30-37 °C
under aerobic conditions

20. b) Haematological and blood biochemical analyses for:
haemogram, CD4 counts, ALAT, ASAT and glucose.
Haemogram
(as directed by manufacturer)
AUTOMATE (ERMA INC)
20

21. 21Spectrophotometer
Centrifugation
R1 + R2 =
samplestandardblank
Working
reagent
Biochemical blood analysis for ALAT, ASAT, and glucose (Reitman
and Frankel (1957); Bergmeyer (1972)).

22. 22
 Evaluation of CD4 counts ((Janossy et al., 2000; Jani et al., 2001))
BD FACS counting system
50 μL of blood
reagent tubes
30 mins
Incubation
Vortex mixing
Addition of 50 μL
Fixative solution
Vortex mixing
and 1 hr
incubation

23. Two fold
serial dilution
•100 µl / well of inoculum 2x104 CFU/ml
•Incubation at 35 0C/48 hrs
MICs read by observation of the tubidities at the base of wells
Highest
concentration
100 µl
Keto
-ve control+ve
control
100 µl
Nys
100 µl
AMB
100μl
culture
medium
Lowest
concentration
23
1 2 3 4 5 6 7 8 9 10 11 12
2
3
4
5
6
7
8
(NCCLS, document
M-27 A2)

24. 24
The table for
Standard threshold values of MICs provided by
Thérèse et al. (2006)
Permitted us
to classify isolates as
S, I or R

25. 25
Prevalence=
persons with a given health indicator during a specified time period
population during the same time period
X 100
 Microsoft Excel 2007
 SPSS version 16.0
(CDC, 2010)
Tabulate results
Plot graphs
with Pearson’s correlation Correlation
The p value < 0.05 was considered statistically significant.
 Prevalence

26. 26

27. Three Candida species were isolated from these patients
27
Candida albicans
Candida glabrata
Candida parapsilosis
 Isolation and Identification of Candida species
Out of the 131 patients recruited for the study, 8 patients had candidemia

28. 28
PREVALENCE
prevalence of Candidemia = 6.11 %
50%
37.50%
12.50%
C. albicans C. parapsilosis C. glabrata
C. albicans C. parapsilosis
C. glabrata
This result accords with the results of other
studies, including that of Godoy et al (2003),
which showed that Candida albicans was the
common cause of bloodstream infection in 42% of
cases in Latin America.

29. 29
Male pop. Female pop.
Number of
candidemic patients
3 5
Percentage 37.5 % 62.5 %
Variation of Candidemic patients per gender

30. 30
Age groups
(years)
Males Percentage
(%)
Females Percentage
(%)
6 - 21 0 0 0 0
22 - 30 0 0 2 25
31 - 60 3 37.5 3 37.5
> 60 0 0 0 0
Variation of Candidemic patients per age group

31. 31
CD4 counts (counts/µl)
<200 200 - 500 >500
Number of
candidemic
patients
Males Females Males Females Males Females
3 3 0 1 0 1
Percentage (%) 37.5 37.5 0 12.5 0 12.5
Variation of CD4 counts in Candidemic patients
75% of patients had less than 200 CD4 counts/µL

32. 32
These results suggest that age, sex and CD4 counts, have an
effect on Candida infection in HIV-infected patients.
According to MSF (2014),
CD4 counts less than 200 counts/µL , in HIV infected patients, favour
opportunistic infections like candidemia.

33. 33
More over, elderly women in the same age group with less than 200
CD4 counts/µL are more exposed to candidemia than men of the same
age group and CD4 counts.
Also, elderly HIV-infected patients aged between 31 and 60 years,
precisely, greater than 40 years, and having less than 200 CD4
counts/µL, are more exposed to candidemia than those who are less
than 31 years old and having less than 200 CD4 counts /µL.
Our results therefore confirm these findings

34. 34
Hemogram report
1 (12.5%)
male
2 (25%)
females
Anemia
3/8 patients (37.5%)
41/131 patients -were anaemic (31.30%);
 7(5.34%) males # 34 (25.95%) females
 1 female (0.76%) had thrombocytopenia.
HIV-infected
patients
Candidemic
patients

35. 35
WBCs RBCs PLT Hgb
Candidemia
( r )
0.240* - 0.083 - 0.082 - 0.083
( P ) 0.006 0.346 0.349 0.344
* Correlation significant at the 0.01 level (2-tailed).
A Weak significant
correlation
Correlations
And, no significant correlation between candidemia
and other selected haematological parameters.

36. (Coyle, 1997)
36
haematological abnormalities are
among the most common
complications of infection with
the HIV virus
Anaemia,
thrombocytopenia
(Enawgaw, 2012)
secondary effects of opportunistic infections like candidemia,
coexisting medical problems that may be prevalent in the HIV-infected population
is caused by the

37. 37
The insignificant correlations between candidemia and; RBCs and Hemoglobin clearly
confirm the findings of Coyle (1997), which showed that Anaemia is generally caused
by inadequate production of RBCs because of suppression of the bone marrow by the HIV
infection through abnormal cytokine expression and alteration of the bone marrow
microenvironment.
The weak significant correlation between candidemia and the WBCs observed, can
be explained by the fact that; the immune system tends to produce more of its cells to
help combat Candida infection as the Candida species increase in blood due to the
weak immune system.

38. 38
ALAT ASAT Glucose
Females 2 (25%) 4 (50%) 3 (37.5%)
Males 0 (0%) 1 (12.5%) 2 (25%)
Total 2 (25%) 5 (62.5%) 5 (62.5%)
Number and percentage of patients with high selected biochemical parameters
BIOCHEMICAL ANALYSIS

39. 39
ALAT ASAT Glucose
Candidemia
( r )
0.42 - 0.035 0.037
( P ) 0.638 0.705 0.676
No significant correlation between candidemia
and biochemical parameters.
Correlations between candidemia and selected biochemical parameters

40. 40
The increase in the level of ALAT and ASAT in 25% and 62.5% of candidemic patients
respectively, was attributed to HIV, ARVs and probably other opportunistic
infections.
Since no correlation existed between candidemia and; ALAT and ASAT,
Reiner and Spivak, 1988
Gathe et al., 2006
The presence of HIV in the liver provokes
macrophage activation and lysis of liver
infected cells by the virus
Antiretrovirals are usually primarily metabolized
by the liver, resulting frequently to mitochondrial
toxicity marked by lysis of liver cellsKenneth and Sherman, 2011
These two mechanisms thus lead to an increase in transaminases activities
in the blood

41. 41
correlation between candidemia
and glucose,
According to them, Hyperglycaemia is thought to impair several nonspecific
mechanisms of the host defense and to increase virulence of C. albicans.
Considering the
hyperglycaemia influences the development
of candidemia.
(Nieto-Rodriguez et al., 1996).
Previous studies including that of
Hyperglycemic environment
C. albicans
C3-receptor-like protein
impairs phagocytic recognition
promotes the adhesion of the fungus to
endothelial mucosal or foreign surfaces
&

42. All Candida isolates were resistant to the antifungals used, that is,
ketoconazole, amphotericin B and nystatin.
42
MICs ranged from 2 to >256 µg/mL
Sanglard and Odds (2002);
blood stream Candida isolates have developed a
high resistance to antifungals over time.
Antifungal susceptibility test

43. 43

44. 44
 This study showed a 6.11 % prevalence of candidemia during
the study period, with a greater prevalence observed among patients
(especially women) older than 40 years, and having less than 200
CD4 counts/µL.
certain factors were identified that favoured Candida bloodstream
infection. These factors were; advanced age (> 40 years), high blood
glucose level and very low CD4 counts (< 200 counts/µL).

45. 45
Candida albicans, Candida parapsilosis and Candida glabrata, were identified as
the major causes of candidemia in our patients. Candida albicans was the most
prevalent specie (50%), followed by Candida parapsilosis (37.5%).
All our isolates proved to be resistant to all the tested antifungals, namely,
nystatin, ketokonazol and amphotericin B with respective MICs of 8 - >256
µg/mL, 2 - >256 µg/mL and > 256 µg/mL.
A Weak significant correlation was found between candidemia and WBCs,
while insignificant correlations were found between candidemia and other
biochemical and hematological parameters

46. 46

47. 47
We intend to increase:
 the number of hospitals (i.e Hospitals with a unit of care for HIV-positive
patients)
and the number of patients, in order to better assess the correlation
between selected biochemical and haematological parameters, and
candidemia.
Do a susceptibility test of isolates to a combination of the antifungals used
in order to know which combination of these antifungals will be able to
overcome resistance.

48. 48
Health personnel responsible for monitoring the
health status of HIV-infected patients should do
mycological examination of their blood samples at
regular intervals, in order to check for the presence or
absence of candidemia.

49. 49
health personnel should avoid admistering
antifungals to patients without doing an
antifungal susceptibility test

50. 50
THANKS
FOR
YOUR
KIND ATTENTION
Your critics, suggestions and appraisals are welcomed, for they will help us improve on the quality of our document

51. 51
Fig 1: Anatomy of HIV (Bonsor, 2001)

52. 52
Fig 2: HIV life cycle (MSF, 2014).

53. 53
Fig 3: Course of HIV infection (MSF, 2014)

54. 54
Fig 4: Targets of antiretroviral drugs in the HIV life cycle.
(Palmisano and Vella, 2010).

55. 55
Fig 5: Structure of the liver (Kapoor, 2013)

56. 56
C. parapsilosis
Fig 6: Some Candida species that could be found in blood (Sahand et al, 2005)

57. 57
Candida glabrata

58. 58
Fig i: BD FACS counting system

59. 59
Fig iii: ERMA INC PCE-210
Fig iv: Centrifuge of trademark NF 200

60. 60
Antifungal Sensible Intermediary Resistant
Ketoconazole (µg/mL) ≤ 0,125 0,25 - 0,5 ≥ 1
Amphotericine B (µg/mL) ≤ 1 > 4
Nystatine (µg/mL) ≤ 1 2 - 4 > 4
Table 1: Standard threshold values of MIC (Thérèse et al., 2006)