Investigating the Current Blood Culture System

for Possible Improvements in Isolation of

Fungal Pathogens

Nicole Spiteri

May 2014

A Dissertation presented to the Faculty of Health Sciences in part-

fulfilment of the requirements for the Degree of Bachelor of Science

(Honours) (Applied Biomedical Science) at the University of Malta

Supervisor: Mr. Stephen Decelis

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i

Declaration

I declare that the work presented in this thesis is original and not copied from other

sources. References to other works are well documented.

______________________

Nicole Spiteri

ii

Dedication

Dedicated to my beloved late grandparents who were always there for

me and supported me all the way

iii

Acknowledgements

First of all I would like to thank my supervisor Mr. Stephen Decelis for all his

patience, help and guidance throughout this project.

I would also like to thank Dr. Christopher Barbara and the Pathology Department for

allowing me to utilise the Mycology laboratory as well as the necessary equipment

required for this project.

My gratitude also goes to Mr. Kenneth Mallia at the Mycology Laboratory, Mr.

Mario Camilleri Brennan and Mr. Jean-Pierre Gialanze at the Microbiology

Laboratory for their help during the practical work; Mrs. Monique Borg Inguanez for

helping me out with the statistical part of the project.

I also thank the Dean of the Faculty of Health Sciences Profs. Angela Xuereb,

lecturers and staff, for their constant support and guidance throughout these past four

years.

Last but not least, I would like to show my gratitude towards my family, friends,

classmates and especially my fiancé, for their constant support and encouragement

throughout the course.

iv

Abstract

Invasive fungal infections are the cause of morbidity and mortality in developed

countries. Unfortunately blood culture systems possess several limitations in the

isolation of fungi. The lack of ability to culture Aspergillus species from blood in

invasive aspergillosis is probably the most important limitation in the diagnosis of

fungal infections. The aim of this project was to evaluate the blood culture system

(BacT/Alert) for possible improvements in the isolation of fungi. In the first part of this

project, the growth of four Aspergillus species was evaluated in the Standard Aerobic

and FAN Aerobic blood culture media. Terminal subcultures were then prepared using a

‘novel’ subculture technique. In the second part of the project false negatives were

tested by preparing terminal subcultures from automated negative blood cultures, which

contained the blood of neutropenic and immunocompromised patients. All four species

were detected visually in both bottles between 24 and 48 hours, at the highest conidial

concentration. Growth was less successful in the bottles inoculated with the hyphal

suspensions due to serious limitations in the technique used for their preparation. The

novel subculture technique was also successful in the recovery of Aspergillus on solid

media, even at the lowest inocula concentrations. After performing terminal subcultures,

Candida tropicalis was recovered from one automated negative bottle, thus identifying

a false negative. The results obtained were compared with similar studies which utilised

the BACTEC blood culture system. The latter produced better results. This study has

shown that blood culture media support the growth of Aspergillus. This is concordant

with similar studies performed on other blood culture systems. The challenge is

therefore finding the cause for the lack of positive blood cultures in patients with

invasive aspergillosis. This study has also shown the importance of terminal subcultures

in cases of suspected Candidaemia.

v

List of Tables

Table 3.1: Average number of colonies counted from SDA plates inoculated

with the conidial and hyphal concentrations ............................................ 47

Table 3.2: Results showing time of growth at different conidial concentrations

in both SA and FAN bottles & results of terminal subcultures ............... 53

Table 3.3: Results showing time of growth at different hyphal concentrations

in both SA and FAN bottles & results of terminal subcultures ............... 54

Table 3.4: P-values obtained when testing the effect of concentration on time

for both the conidial and hyphal inocula .................................................. 60

Table 3.5: P-values obtained when testing the difference in the time of growth

between the highest and lowest conidial and hyphal concentrations ....... 61

vi

List of Figures

Figure 1.1: Infectious life cycle of Aspergillus spp. (Dagenais & Keller, 2009) ......... 8

Figure 1.2: Distribution of isolated Candida spp. CALB: Candida albicans;

CDUB: Candida dubliniensis; CGLB: Candida glabrata; CGUI:

Candida guillermondii; CKRU: Candida krusei; CLUS: Candida

lusitaniae; CPAR: Candida parapsilosis; CTRO: Candida

tropicalis (Horn et al., 2009) ................................................................... 10

Figure 1.3: BacT/ALERT 3D's colorimetric sensor-and-detection technology

detects microorganisms by tracking CO2 production (BioMérieux,

n.d) ........................................................................................................... 25

Figure 1.4: Schematic diagram of the BacT/Alert blood culture bottle showing

the: a) Bottle wall; b) Semi-permeable membrane; c) Carbon

dioxide sensor; d) Light emitting diode; e) Light absorbing

photodiode; f) Amplifier; g) Block (Thrope et al., 1990) ........................ 26

Figure 2.1: Densimat readings and corresponding spore counts for a) A.

fumigatus and b) A. flavus (Araujo, Rodrigues & Pina Vaz, 2004) ......... 34

Figure 2.2: Preparation of conidial suspensions for A. flavus and A. niger using

a prepared 2McF conidial suspension and sterile distilled water

(SDW) (Figure created from data in: SciQuip Ltd., n.d) ......................... 35

Figure 2.3: Preparation of conidial suspensions for A. fumigatus and A. terreus

using a prepared 2McF conidial suspension and sterile distilled

water (SDW) (Figure created from data in: SciQuip Ltd., n.d) ............... 36

vii

Figure 2.4: Standard Aerobic (blue cap) and FAN aerobic (green cap)

BacT/Alert blood culture bottles inoculated with the prepared serial

dilutions .................................................................................................... 38

Figure 3.1: Detection of growth of A. fumigatus in the BacT/Alert standard

aerobic blood cultures after 7 days .......................................................... 45

Figure 3.2: Detection of growth of A. fumigatus in the BacT/Alert FAN aerobic

blood cultures after 7 days ....................................................................... 46

Figure 3.3: Graph showing the linear relationship between the average number

of colonies and different conidial concentrations .................................... 49

Figure 3.4: Subcultures of A. fumigatus (left) and A. flavus (right) on

Sabouraud Dextrose Agar ........................................................................ 51

Figure 3.5: Subcultures of A. niger (left) and A. terreus (right) on Sabouraud

Dextrose Agar .......................................................................................... 52

Figure 3.6: Graph comparing the detection of growth of A. fumigatus by the

BACTEC automated system; the grey, white and black bars

represent the BACTEC Plus Aerobic/F, Mycosis-IC/F and Myco/F

Lytic bottles respectively, as presented in the study by Rosa et al.

(2011) ....................................................................................................... 55

Figure 3.7: Graph comparing the detection time of growth of A. fumigatus

using the BacT/Alert SA and FAN aerobic bottles .................................. 55

Figure 3.8: Average time of growth for each Aspergillus spp. in the SA bottles ....... 62

Figure 3.9: Average time of growth for each Aspergillus spp. in the FAN

bottles ....................................................................................................... 63

viii

List of Abbreviations

c.f.u Colony-forming unit

IA Invasive Aspergillosis

DNA Deoxyribonucleic acid

KOH Potassium hydroxide

McF McFarland

MDH Mater Dei Hospital

NAC Non-albicans Candida

PCR Polymerase Chain Reaction

SA Standard Aerobic

SDA Sabouraud Dextrose Agar

SDW Sterile distilled water

SOP Standard Operating Procedure

Spp Species

ix

Table of Contents

Declaration ...................................................................................................................i

Dedication ................................................................................................................... ii

Acknowledgements ................................................................................................... iii

Abstract… ...................................................................................................................iv

List of Tables ............................................................................................................... v

List of Figures .............................................................................................................vi

List of Abbreviations .............................................................................................. viii

CHAPTER 1: LITERATURE REVIEW ................................................................. 1

1.1. Introduction ....................................................................................................... 2

1.1.1. Characteristics of Fungi .......................................................................... 2

1.1.2. Genus Aspergillus ................................................................................... 3

1.1.3. Genus Candida ....................................................................................... 4

1.2. Systemic Fungal Infections ............................................................................... 6

1.2.1. Introduction to Fungaemia ...................................................................... 6

1.2.2. Invasive Aspergillosis ............................................................................. 7

1.2.3. Systemic Candidiasis .............................................................................. 9

1.2.4. Other fungi that can cause systemic fungal infections ......................... 11

x

1.3. Diagnosis of fungal infections ......................................................................... 12

1.3.1. Culture and microscopy ........................................................................ 13

1.3.2. Blood Cultures ...................................................................................... 15

1.3.3. Galactomannan Antigen Assay ............................................................. 17

1.3.4. β-D- glucan assay.................................................................................. 18

1.3.5. The Polymerase Chain Reaction ........................................................... 19

1.3.6. Radiology .............................................................................................. 20

1.4. Methodology ..................................................................................................... 21

1.4.1. Blood Culture Systems ......................................................................... 21

1.4.1.1. Manual Blood Culture Systems ....................................................... 21

1.4.1.2. Automated Blood Culture Systems .................................................. 22

1.4.1.2.1. The BACTEC blood culture system ....................................... 23

1.4.1.2.2. The BacT/Alert Blood Culture System ................................... 24

1.5. Sub-Culture Techniques from Mycology ...................................................... 27

1.5.1. Routine Sub-Culture Technique ........................................................... 27

1.5.2. ‘Novel’ Sub-Culture Technique ............................................................ 28

1.5.3. Terminal Sub-cultures ........................................................................... 28

1.6. Limitations ....................................................................................................... 29

1.7. Statement of Problem ...................................................................................... 30

1.8. Objectives and Aims of Project ...................................................................... 31

xi

CHAPTER 2: METHODOLOGY .......................................................................... 32

2.1. Ethics Approval ............................................................................................... 33

2.2. Fungal Species .................................................................................................. 33

2.3. Inoculation of conidial and hyphal suspensions in blood culture bottles ... 33

2.3.1. Preparation of conidial suspensions ...................................................... 33

2.3.2. Preparation of hyphal suspensions ........................................................ 36

2.3.3. Inoculation of suspensions into BacT/Alert blood culture bottles ........ 37

2.4. Incubation of blood culture bottles ................................................................ 38

2.5. Preparation of terminal sub-cultures: the ‘Novel’ sub-culture technique . 39

2.6. Preparation of terminal sub-cultures from negative blood cultures .......... 39

2.6.1. Collection of blood culture bottles ........................................................ 39

2.6.2. Terminal sub-cultures ........................................................................... 40

CHAPTER 3: RESULTS ......................................................................................... 41

3.1. Results involving visual readings ................................................................... 42

3.1.1. Detection of Aspergillus spp. In BacT/Alert blood cultures ................. 42

3.1.1.1. Conidial inoculum ............................................................................ 42

3.1.1.2. Hyphal inoculum .............................................................................. 43

3.1.2. Results of growth after 7 days .............................................................. 44

3.1.2.1. Conidial inoculum ............................................................................ 44

3.1.2.2. Hyphal Inoculum ............................................................................. 45

xii

3.1.3. Validation of inocula ............................................................................ 47

3.1.3.1. Number of colonies grown from plates inoculated with prepared

concentrations .................................................................................. 47

3.1.4. Recovery of Aspergillus spp. on solid culture medium ........................ 49

3.1.4.1. Conidial inoculum ............................................................................ 49

3.1.4.2. Hyphal inoculum .............................................................................. 50

3.1.5. Recovery of yeast from negative blood culture .................................... 52

3.2. Statistical analysis ............................................................................................ 55

3.2.1. Comparison of results with results of published study ......................... 55

3.2.2. Testing for normality – The Shapiro Wilk’s Test ................................. 57

3.2.3. Comparison of results: The Kruskal-Wallis Test ................................. 58

3.2.3.1. Comparison of SA and FAN BacT/Alert bottles ............................. 58

3.2.3.2. Comparing results of conidial and hyphal inocula .......................... 59

3.2.3.3. Comparing the effect of concentration on time ............................... 60

3.2.3.4. Comparing the time of growth for each Aspergillus spp. ................ 62

CHAPTER 4: DISCUSSION & CONCLUSION ................................................... 65

4.1. Detection of fungi in automated blood culture systems: Comparison with

published studies.............................................................................................. 66

4.2. The Type of Inoculum ..................................................................................... 70

4.3. The ‘Novel’ (Rosa et al., 2011) subculture technique ................................... 71

xiii

4.4. The importance of terminal subcultures from negative blood cultures ..... 72

4.5. Limitations ....................................................................................................... 75

4.6. Conclusion ........................................................................................................ 78

REFERENCES .......................................................................................................... 80

APPENDIX ................................................................................................................ 91

Preparation of Culture Media ......................................................................... 92 I.

Quality Control of Media ............................................................................... 94 II.

Tables of Results ............................................................................................ 95 III.

Statistical Analysis ....................................................................................... 103 IV.

Costs……. .................................................................................................... 108 V.

Permissions to carry out study ..................................................................... 109 VI.

1

1. CHAPTER 1

LITERATURE REVIEW

2

1.1. Introduction

1.1.1. Characteristics of Fungi

The fungal kingdom includes a spectrum of eukaryotic organisms which

mainly consist of yeasts and moulds (Blackwell, 2011). Fungi are more complex than

bacteria (Fridkin & Jarvis, 1996). They are ubiquitous in nature since they are very

fit for survival. Even though thousands of fungal species have been identified, only

approximately two hundred of these species are considered to be able to cause human

disease. However, studies have shown an increased incidence in systemic diseases

caused by several different fungal species (Chakrabarti, 2005). Due to their

ubiquitous nature, several other fungi capable of causing infections and their

potential fungal habitat are yet to be discovered.

Fungi can exist in three different forms:

a) Unicellular organisms, such as yeasts, which reproduce by simple budding.

b) Filamentous fungi, which include moulds and are composed of thread-like

hyphae. These reproduce by means of specialised structures which produce

conidia. The latter are the source of fungal infections. Conidia can survive

several extreme environmental conditions, such as heat and dehydration. They

are present in air, soil and water.

c) Certain fungi can also exist as both forms and are thus referred to as

dimorphic. (Law, 2010).

3

Fungi are heterotrophic organisms and thereby feed by absorption. They

consist of several membrane bound organelles including ribosomes, mitochondria as

well as an endoplasmic reticulum and Golgi apparatus. A polysaccharide cell wall

consisting of chitin and glucan is also present. Reproduction can be sexual i.e. by

meiosis, or asexual i.e. by mitosis. Due to their ubiquitous nature, fungi may survive

in different habitats, however, they are mainly found in soil or decomposing material,

with the main role of recycling nutrients in the environment (Webster & Weber,

2007).

1.1.2. Genus Aspergillus

Aspergilli fall under the Phylum Ascomycota which includes most medically

important genera. There are over two hundred and fifty Aspergillus species all of

which play an important role, both clinically and commercially (Anzai et al., 2008).

Aspergilli are filamentous, saprophytic fungi which primarily grow on organic debris

or decaying vegetation. They produce conidia which are easily dispersed into the air

and can survive several environmental conditions. (Dagenais & Keller, 2009).

Aspergilli consist of branching hyphae with the conidia arranged on conidiophores.

Different species can be classified on different conidiophore structures as well as

conidial arrangement (Ryan, Ray, Ahmad, Drew & Plorde, 2010). Upon germination,

Aspergillus spp. grow as a mycelium exhibiting dichotomous branching. The hyphae

are able to invade tissue and cause disease in susceptible patients (Reichenberger,

Habicht, Gratwohl &, Tamm, 2002).

4

The genus Aspergillus plays an important role in the production of several food

products such as fermented foods (Anzai et al., 2008) as well as in the production of

pharmaceutical products. For example, Aspergillus niger is used in the production of

proteases and amylases. However, these can also have a negative impact on public

health. Medically important Aspergillus species include: Aspergillus fumigatus,

Aspergillus terreus, Aspergillus niger, Aspergillus flavus as well as Aspergillus

nidulans (Dagenais & Keller, 2009).

Aspergillus fumigatus is not the most prevalent fungus in nature. It is highly

ubiquitous and produces abundant conidia which are small enough to enter the

alveoli of the lungs. Once they are dispersed in the air, humans are at risk of inhaling

hundreds of these spores daily. Disease will thus develop in the lungs; however,

immunocompetent individuals can easily remove the conidia through efficient

immune mechanisms of the body. Aspergillus fumigatus is thought to be responsible

for approximately 90% of human Aspergillus infections especially in

immunocompromised hosts, even though Aspergillus terreus, Aspergillus niger,

Aspergillus flavus and Aspergillus nidulans are also responsible for causing human

disease (Latgé, 1999).

1.1.3. Genus Candida

The Genus Candida involves yeast-like organisms which consist of round to

elongated budding cells, also known as blastoconidia. Overall, Candida spp. are able

to grow at any temperature and under most conditions. The majority of these

5

organisms possess pseudohyphae which involve branches of elongated cells held

together in a chain-like pattern (Martinez-Anaya, Dickinson, Sudbery, 2003). Apart

from morphological characteristics, different species can be distinguished on the

basis of biochemical characteristics such as fermentation and carbohydrate

assimilation. Most Candida species can be grown on Sabouraud Dextrose agar as

well as an enriched medium such as blood agar (Ryan et al., 2010).

Candida spp. are the fourth most common pathogens causing bloodstream

infections in hospitalised patients (Horvath, George, Murray, Harrison, Hospenthal,

2004) with Candida albicans being the primary agent responsible for causing most

opportunistic infections in humans. It is commonly found in the gastrointestinal tract

as part of the normal commensal flora. It is able to produce hyphae depending on

different environmental conditions such as temperature, pH as well as the availability

of nutrients. For example, at 37oC, Candida albicans forms elongated hyphae known

as germ tubes. It can produce thick-walled chlamydoconidia which can be easily

distinguished from other Candida spp. Also, under different conditions the

composition of the cell wall components differs. The cell wall usually consists of

different polysaccharides which mainly include: mannan, glucan, and chitin (Ryan et

al., 2010).

Other Candida species including Candida tropicalis, Candida glabrata,

Candida parapsilosis and Candida krusei are also common yeasts responsible for

causing bloodstream infections and account for approximately 98% of all the blood

6

yeasts (Chang, Leaw, Huang, Wu, Chang, 2001). However, there is a possibility that

rare species of this genius can be encountered.

1.2. Systemic Fungal Infections

1.2.1. Introduction to Fungaemia

Fungaemia can be defined as the presence of fungi in blood. It has long been

recognised that the presence of such organisms in the bloodstream of a patient can be

highly fatal and bring about an increased morbidity and mortality (Reimer, Wilson &

Weinstein, 1997). Advancements in medicine and technology, such as organ

transplants and chemotherapeutic agents have led to an increase in

immunocompromised patient populations. These individuals have become

susceptible to opportunistic infections, including those caused by fungi. In the past,

such infections were considered as questionable, however their importance is now

recognised as these can become rapidly progressive, severe and difficult to manage

(Fridkin & Jarvis, 1996).

The increased incidence of fungaemia was first demonstrated in Denmark in

2003 where retrospective information was collected regarding fungaemia in

immunocompromised patients. (Arendrup et al., 2011). Other studies have also

shown that the etiology of fungaemia is changing and evolving (Nucci & Marr,

2005). Most of these infections are caused by Candida and Aspergillus spp. however

new fungal pathogens are being recognised such as non-albicans Candida spp.

7

(NAC) and Fusarium spp. (Fridkin & Jarvis, 1996). The rapid and reliable detection

of these organisms causing fungaemia has become a challenging problem. The

increased recognition of fungaemia has led to the development of newer diagnostic

systems however each system possesses several limitations (Thrope, Wilson, Turner,

DiGuiseppi, Willert, Mirrett & Barth Reller, 1990).

1.2.2. Invasive Aspergillosis

A wide spectrum of diseases are caused by Aspergillus species (Perfect et al.,

2001), however one of the most severe Aspergillus-related diseases is Invasive

Aspergillosis where neutropenia is considered to be the primary risk (Reichenberger

et al., 2002). Aspergilli begin their life cycle by the production of airborne conidia

which are dispersed into the air. Aspergillus fumigatus produces conidia with an

average size of 2-3µm and are thus capable of infiltrating the alveoli of the lungs,

unlike Aspergillus flavus and Aspergillus terreus which produce larger conidia.

(Dagenais & Keller, 2009).

If immunocompromised patients come in contact with these conidia and inhale

them, they will be deposited in the alveolar spaces and bronchioles of the individual

resulting in conidial germination. Several host defences come into action in the case

of immunocompetent individuals. The primary defence mechanisms for the removal

of these conidia include alveolar macrophages, which phagocytose and kill the

invading conidia. Also, neutrophils, such as polymorphnuclear cells (PMN)

accumulate at the site of infection resulting in a pro-inflammatory response. Those

8

conidia that are not destroyed by macrophages become targeted by neutrophils

instead, in order to destroy the invading hyphae. Invasive aspergillosis results when

one of these host defences malfunctions leading to uncontrolled hyphal growth

(Dagenais & Keller, 2009).

Figure 1.1: Infectious life cycle of Aspergillus spp. (Dagenais & Keller, 2009)

Several epidemiological studies have shown an increased incidence of this

disease which has substantially increased the cost in hospitalisations associated with

aspergillosis. As a result patients are staying longer in hospitals thus leading to an

increase in the mortality rate (Perfect et al., 2001). The risk factors associated with

invasive aspergillosis can be several and these include: haematological malignancies

and transplants; medications such as chemotherapy; nosocomial risk factors such as

water and air filtration. Upon identifying these risk factors, as well as risk groups,

improvement in the diagnosis and management of patients with invasive

aspergillosis, such as antifungal therapy and new diagnostic methods, can be

implemented (Baddley, 2011).

9

1.2.3. Systemic Candidiasis

Yeasts have become one of the most important agents of blood stream

infections (Chang et al., 2001). Systemic candidiasis is an infection caused by the

presence of Candida in the blood (Law, 2010). Patients with invasive candidiasis

primarily present with acute sepsis (Cheng et al., 2005). Several risk factors

constitute to this infection and these include: patients in the Intensive Care Unit,

patients with intravascular catheters and patients on chemotherapy. Also, patients in

which anti-fungal therapy was delayed showed an increased risk of death due to

candidaemia (Søgaard, Hjort, Højbjerg & Schønheyder, 2006).

Studies have shown an increase in the number of reported cases of

candidaemia, especially in patients at Intensive Care units (ICU). Lengthy stays at

hospitals were shown to increase the risk of candidaemia and provide a poor outcome

for patients (Dupont, Lortholary, Ostrosky-Zeichner, Stucker & Yeldandi, 2009). An

increased prevalence of non-albicans Candida (NAC) species has also been recorded

in recent years where in some hospitals most cases of candidaemia reported were

caused by NAC species (Cheng et al., 2005). A study was conducted in 2009 in order

to evaluate the epidemiology and outcome of this disease in several American

centres. Results have shown that 50% of patients with invasive fungal infections had

candidaemia. Candida albicans was isolated in approximately 45% of the patients

however, 54% of other Candida spp. were isolated from blood cultures (Horn et al.,

2009). The distribution of Candida spp. isolated can be observed in Figure 1.2.

10

Figure 1.2: Distribution of isolated Candida spp. CALB: Candida albicans; CDUB:

Candida dubliniensis; CGLB: Candida glabrata; CGUI: Candida guillermondii; CKRU:

Candida krusei; CLUS: Candida lusitaniae; CPAR: Candida parapsilosis; CTRO:

Candida tropicalis (Horn et al., 2009)

The distribution of Candida species amongst different patients were due to

several factors including: antifungal treatment, organ transplantation, use of

catheters, presence of bacterial infections, neutropenia and other immunosuppressive

agents. The mortality rate amongst these patients was 35% with the highest mortality

being observed in patients infected with Candida krusei and the lowest mortality was

observed amongst patients infected with Candida parapsilosis. Even though Candida

albicans is the predominant species isolated worldwide, a surveillance program that

was carried out between 1997 and 2003 has shown a decrease in Candida albicans

and an increase in Candida parapsilosis and Candida tropicalis, with different

species distributed amongst several countries (Horn et al., 2009).

Possible increase in the prevalence of NAC species could be due to the use of

Fluconazole as a prophylactic agent (Charlier, Hart, Lefort, Ribaud, Dromer,

Denning & Lortholary, 2006). Candida albicans tends to be susceptible to this drug,

however the NAC species tend to have higher Minimum Inhibitory Concentrations

(MIC’s). According to the study carried out by Horn et al. (2009), there has been an

11

improvement in the survival rate in patients with invasive candidiasis and this can be

due to the fact that knowledge on the risk factors has improved as well as better

diagnostic tests and the introduction of newer antifungals such as Echinocandins

(antifungal drugs responsible for inhibiting the production of glucan in the cell wall).

1.2.4. Other fungi that can cause systemic fungal infections

The epidemiology of invasive fungal infections has developed over the years

and is still evolving. Yeasts other than Candida spp. and moulds other than

Aspergillus spp. are known to be the cause of fungal infections, some of which can

be very serious. Also, newly emerging fungi have been identified to cause invasive

mycoses especially in immunocompromised patients (Nucci & Marr, 2005).

One other yeast responsible for causing severe systemic mycosis is

Cyrptococcus neformans. The latter is a yeast-like fungus responsible for causing

life-threatening infections namely cryptococcosis and fungal meningitis (Law, 2010).

The latter is the commonest cause of infection and specifically affects patients with

AIDS (Mukherjee, Pirofski, Scharff & Casadevall, 1993). Spores formed by this

organism are less than 2µm in diameter and are thus able to enter the alveolar spaces

of the lung when inhaled. The virulence of Cyrptococcus neformans is primarily due

to the presence of a polysaccharide capsule surrounding its cell wall. This structure

acts as a barrier and will prevent phagocytosis from occurring (Bose, Reese, Ory,

Janbon & Doering, 2003). Rapid diagnosis is thus required in order to provide

immediate antifungal therapy.

12

A small number of infections can also be caused by other filamentous fungi

namely Fusarium and Scedosporium species (Law, 2010). Fusarium species can

cause a spectrum of infections in humans, most of which include disseminated

infections in immunocompromised patients. They are widely distributed in soil and

plant debris. They can also grow on a wide range of organic substrates and have

evolved several efficient mechanisms of dispersal, thus contributing to their

widespread distribution. Little information is provided on the pathogenesis of

invasive fusariosis; however, it shares several features with other mould infections

such as invasive aspergillosis (Nucci & Anaissie, 2007).

Organisms of the Genus Scedosporium include ubiquitous filamentous fungi

which can be found in the soil and sewage as well as polluted water (Cortez et al.,

2008). Scedosporiosis is the term used to describe a wide spectrum of fungal

infections caused by Scedosporium species. Such infections can be localised or

disseminated to several organs. Due to the resistance to several anti-fungal agents,

treatment of scedosporiosis can be challenging (Cortez et al., 2008).

1.3. Diagnosis of fungal infections

The diagnosis of invasive fungal infections has become a great challenge due

to a low sensitivity in the gold standard tests, these being, culture and histopathology

(Barnes & Marr, 2007). Clinicians must rely on clinical signs and symptoms as well

as laboratory investigations including culture, direct microscopy, histopathology,

antigen tests, PCR and other diagnostic tests. Radiology is also a very important tool.

13

There is no single best method for diagnosing invasive fungal infections, but a

combination of tests. Delayed diagnosis will only lead to an increase in the mortality

rate due to such infections (Posteraro, Torelli, Carolis, Posteraro, Sanguinetti, 2011).

1.3.1. Culture and microscopy

Culture, microscopy and identification still remain the current conventional

methods for the diagnosis of invasive fungal infections (Cuenca-Estrella, Bassetti,

Lass-Flörl, Rácil, Richardson & Rogers, 2011). However, culture and microscopy

are limited in the diagnosis of such infections and this can be due to the fact that

microbiological cultures are not very sensitive (Posteraro et al., 2011). Blood

cultures are primarily used for the diagnosis of invasive candidiasis, however studies

have shown that they are approximately 50% sensitive to Candida spp. (Pasqualotto

& Denning, 2005) and are hardly of use in detecting Aspergillus spp. Also, in cases

where fungal infections are suspected, blood cultures may need to be incubated for

longer periods in order to obtain positive growth (Pasqualotto & Denning, 2005).

Microscopy is very useful in the detection of fungi and is a faster diagnostic

method than culture. This rapid technique can provide a quick result upon receiving

the sample. It is also useful in presumptively identifying the fungal species present

based on morphological features, as well as aid in confirming the species isolated

through culture. However, sometimes it might be difficult to distinguish hyphal

elements from other cellular material (Law, 2010). Current microscopic techniques

involve the preparation of potassium hydroxide mounts. The latter involves the

14

addition of approximately 20-30% potassium hydroxide on a slide followed by

addition of the specimen. The prepared mounts can then be visualised under the

microscope for the presence of thread-like hyphae or spores (Shenoy, Teerthanath,

Karnaker, Girisha, & Pinto, 2008). Recently the use of Calcofluor white, a

fluorescent dye, has also been employed in addition to KOH preparations. This dye

binds to chitin present within fungal cell walls, resulting in fluorescence. Upon

examining under UV light, bright fluorescing fungal elements can be observed and

identified (Law, 2010). Direct microscopy can lead to diagnosis of invasive fungal

infections using specimens such as urines, aspirates and bronchoalveolar lavages.

The primary isolation of Candida spp. can be performed using Candida

Chromogenic Agar, which is the medium employed in the isolation of clinically

important Candida spp. and is able to identify Candida albicans, Candida tropicalis

and Candida krusei. Other species can be identified using rapid tests, biochemical

tests and morphological studies (Posteraro et al., 2011). Filamentous fungi can be

identified through macroscopic and macroscopic examination. Through macroscopic

analysis, identification of fungi includes colonial form, pigmentation as well as

surface colour.

The India ink stain can be utilised in order to visualise the capsule present in

the cell wall of Cryptococcus neoformans. This will prevent the ink particles from

entering the capsule thus forming a halo around the organism allowing easy detection

and visualisation by microscopy (Bose, Reese, Ory, Janbon & Doering, 2003).Also

15

the detection of cryptococcal antigen in serum or CSF can aid in providing a definite

diagnosis of cryptococcal meningitis.

1.3.2. Blood Cultures

Automated blood culture systems are routinely being used in order to diagnose

bloodstream infections even though sensitivity is low. Different blood culture

systems have been developed over the years in order to enhance the detection of

these organisms. The contents of the culture media used for detection differ between

one system and the other; however, the same standard procedure is used. Upon

obtaining a blood sample from the patient suspected of systemic mycoses, the blood

is inoculated into the blood culture bottle after which it is incubated for

approximately 5 days, or until growth is observed (Borst, Leverstein-Van Hall,

Verhoef & Fluit, 2000).

There are several factors which affect the sensitivity of blood cultures some of

which include the following:

a) Number of blood cultures used: Studies have shown that, in order to detect

septicaemia at least two blood cultures should be prepared for each sample.

Single blood cultures will not be sensitive enough for detecting organisms and

identification will be difficult (Weinstein, 1996).

b) Volume of blood required: In the case of adults with bloodstream infections,

relatively few organisms can be present in the blood thus requiring a larger

volume of blood. Studies have shown that the recommended volume of blood

16

required per culture is that of 10-30ml. However, a blood volume above 30ml

will not enhance the sensitivity and yield, and such volume may cause the blood

to clot in the syringe (Weinstein, 1996). For fungaemias, the amount of blood

culture does have an effect on outcome. To increase the blood volume cultured,

several bottles can be inoculated at the same time.

c) Different culture media: Different blood culture systems utilise different culture

media. Different manufacturers utilise supplements and additives in order to

enhance growth. One commonly used supplement is soybean casein digest broth

(Weinstein, 1996).

d) Dilution of blood sample: Another way to optimise the yield of growth is to

dilute the blood in the culture broth since blood contains substances such as

lysozymes and white blood cells which can inhibit microbial growth. By diluting

the blood, the effect of these substances is reduced (Weinstein, 1996).

e) Length of incubation: Studies have shown that an incubation of 5 days is

adequate for detecting and identifying pathogens since after 5 days any positive

blood cultures are most likely to be due to contamination. A study was conducted

by Wilson et al. (1993), which showed that after a 5-day incubation blood culture

bottles can be discarded with a minimal chance of obtaining true positive isolates

and maximal reduction in the presence of contaminants (Wilson, Mirrett, Reller,

Weinstein & Reimer, 1993). However an extended incubation period of more

than 5 days has been reported to be necessary in cases of fungaemia (Weinstein,

1996), even though several authors declare that clinically important organisms

will grow at a fast rate as opposed to slow growing organisms which are usually

considered contaminants (Borst et al., 2000).

17

1.3.3. Galactomannan Antigen Assay

One of the most useful methods in the diagnosis of invasive aspergillosis is the

galactomannan antigen detection method using an enzyme immunoassay.

Galactomannan is a polysaccharide component present in the cell wall of Aspergillus

species (Pfeiffer, Fine & Safdar, 2006). This polysaccharide consists of a mannan

core as well as group residues consisting of galactofuranosyl units. This test involves

a double sandwich enzyme immunoassay which detects the presence of this

polysaccharide in serum samples (Verdaguer, Walsh, Hope & Cortez, 2007). This

test has been made available since 2003 and is one of the diagnostic methods for

invasive aspergillosis. It has proved to be highly sensitive and specific even though

there is a risk of false-positivity in certain cases. This test can also aid in monitoring

immunocompromised patients at risk of developing the galactomannan antigen, a

marker for invasive aspergillosis.

The galactomannan assay utilises specific monoclonal antibodies which act as

both a detector and acceptor of galactomannan (Pfeiffer, Fine & Safdar, 2006). Even

though this assay can be quite sensitive and accurate, it is time consuming and takes

approximately four hours to complete. Results are usually expressed using the

galactomannan index (GMI). Since Aspergillus is a common pathogen found in

hospitals as well as laboratory environments, there is a risk of contamination of

serum samples through collection, transport or processing of the sample and thus

result in a false positive result. Thus, this test should be restricted to patients who are

at high risk of invasive aspergillosis (Wheat & Walsh, 2008).

18

Even though this assay has proved to be highly sensitive and specific, there are

certain factors which can cause false positives or negatives, thus affecting the

accuracy of the test. These factors include:

a) Presence of antibiotics: studies have shown that antibiotics such as amoxicillin

are prone to cause false-positives. However, even though patients are receiving

such drugs, a positive result should not be disregarded since it can ultimately be

caused by invasive aspergillosis and thus further tests should be performed

(Wheat & Walsh, 2008);

b) Treatment with antifungal drugs: studies have shown that antifungal drugs tend

to reduce sensitivity of the test (Marr, Laverdiere, Gugel & Leisenring, 2005).

Thus, negative results obtained from patients taking antifungal therapy should be

further investigated before confirming the presence of a false negative;

c) Cross-reactivity: organisms such as Penicillium, Trychophyton, Histoplasma and

Cladosporium may contain a cross reactive galactomannan. The monoclonal

antibody will react with the β-1-5 galactofuranose present in the galactomannan

of these organisms resulting in a false positive result (Wheat & Walsh, 2008).

1.3.4. β-D- glucan assay

β-D- glucan is another fungal cell wall component and can be found in several

species such as Aspergillus and Candida spp. The β-D- glucan detection assay can

also be performed in order to confirm the presence of these organisms. However,

when compared to the galactomannan antigen assay, the sensitivity of the β-D-glucan

test is less. Also, it is not specific for invasive aspergillosis. Patients suffering from

19

this disease are also prone to the yeast infection candidiasis and this test will not be

useful in differentiating between the two fungal infections. Therefore, for such

reasons, the galactomannan test is preferred when diagnosing patients with invasive

aspergillosis. The test is highly useful in detecting fungaemias with other fungi

including Fusarium and Scedosporium.

1.3.5. The Polymerase Chain Reaction

The Polymerase Chain reaction (PCR) can also be useful in detecting fungi

causing systemic infections. Such molecular techniques are highly sensitive and

specific. PCR allows the amplification of specific target DNA sequences (Harwood,

2010). The ribosomal RNA gene is cluster is a popular target gene for PCR since

different fungi contain multiple copies of this cluster and can thus aid in increasing

the sensitivity of this assay. Examples of the target genes responsible for the

identification of fungi of the Aspergillus and Candida genus include the 18S rRNA,

28S rRNA, ITS-1 and ITS-2 (Khot & Fredricks, 2009).

Primers and probes can be designed so that the target DNA can be refined to a

taxonomic level i.e. to the genus or species level. In the case of real time PCR this

can provide rapid detection and confirmation of the fungus using a ‘taxon’-targeted

assay (Khot & Fredricks, 2009). There are several detection methods for the

amplified DNA sequences produced by the PCR assay. One of the most sensitive,

specific and utilised methods is gel electrophoresis where the PCR products are

separated according to size and charge after which they can be analysed.

20

The polymerase chain reaction also possesses certain limitations in the

detection of fungi since it differs from the detection of other microorganisms and can

thus be challenging (Khot & Fredricks, 2009). This can be due to the complicated

extraction of fungal DNA as a result of the presence of a durable cell wall, as well as

due to low amounts of DNA. (Dornbusch, Groll & Walsh, 2010). False positivity is

also an issue as a result of a high risk of contamination from the environment (Khot

& Fredricks, 2009). In addition, PCR is a more complex and expensive method when

compared to antigen detection techniques (Wheat & Walsh, 2008).

1.3.6. Radiology

The diagnosis of invasive fungal infections can also be based on radiological

methods since this can aid in early diagnosis of the disease (Nurzyńska-Flak,

Zawitkowska-Klaczyńska & Kowalczyk, 2004). CT scans have been proven to be

useful in the diagnosis of invasive aspergillosis. This can be characterised by the

presence of a ‘halo sign’ which mainly consists of a dense fungal ball. As the disease

progresses a ‘nodular appearance’ can be observed. However, chest radiography can

be slightly insensitive for the diagnosis of invasive fungal infections since at very

early stages of the disease the halo sign might not be visible even though nodular

lesions would already be present. Overall a CT scan can be considered as a sensitive

radiological method for the early detection of invasive aspergillosis (Reichenberger

et al., 2002). Radiological techniques must be performed in conjunction with other

laboratory tests as well as based on clinical observations in order to diagnose

invasive fungal infections.

21

1.4. Methodology

1.4.1. Blood Culture Systems

1.4.1.1. Manual Blood Culture Systems

Before the introduction of automated blood culture systems, the traditional

manual systems were utilised. Such systems use bottles filled with liquid broth

medium which are inoculated with the patient’s blood sample. The bottles are

incubated aerobically or anaerobically for 7 days where each bottle is examined daily

for the presence of microbial growth (Weinstein, 1996). Growth can be identified

either by haemolysis, an increase in turbidity as well as gas production. Commonly

used blood culture media are the ‘Castenada’ bottles which incorporate plastic

paddles containing an agar slant within the bottle. Such bottles are advantageous

since they do not require venting to room air, however, they are relatively expensive

(Reimer, Wilson & Weinstein, 1997).

Manual systems are labour intensive since frequent checking of the bottle for

growth is required. Thus, they are not very practical, especially for laboratories

where large numbers of blood cultures are analysed daily (Reimer, Wilson &

Weinstein, 1997). Apart from frequent checking of the bottles, blind cultures are

usually prepared at the end of the incubation period. There is also a high risk of

contamination upon opening the bottles as well as risk of infection to the individual

22

analysing the bottles (Law, 2010). Processing of several blood cultures will also

increase laboratory costs.

1.4.1.2. Automated Blood Culture Systems

During the past few decades several automated systems have been developed

and introduced in order to make the processing of blood cultures more efficient.

These systems vary in the method to detect growth of microorganisms, as well as

other factors, such as the type of culture media and supplements used, the volume of

blood required to inoculate the media etc. (Wilson, Weinstein, Reimer, Mirrett &

Reller, 1992). Before the introduction of continuous-monitoring blood culture

systems, automated detection systems were utilised. Examples include the BACTEC

radiometric system, which was the first commercial blood culture system employed,

and the BACTEC non-radiometric system (Reimer, Wilson & Weinstein, 1997).

Advancements in blood cultures resulted in the introduction of the continuous-

monitoring blood culture systems which are commonly employed nowadays. These

systems differ from the automated detection systems due to continuous monitoring of

the blood culture bottles for the detection of microbial growth. Examples include the

BacT/Alert blood culture systems, BACTEC 9000 series blood culture systems, the

Vital blood culture system and the Signal Oxoid blood culture system (Reimer,

Wilson & Weinstein, 1997). However, the BACTEC and BacT/Alert are the most

commonly utilised systems. Most of these systems are based on carbon dioxide

detection. This is due to the fact that growth of organisms will increase the

23

concentration of carbon dioxide inside the blood culture bottle (Rohner, Pepey &

Auckenthaler, 1995) and thus growth can be detected by monitoring changes in

carbon dioxide concentration.

1.4.1.2.1. The BACTEC blood culture system

Amongst the commonly used blood culture systems is the BACTEC (BD

Diagnostics Systems, Sparks, Md.) blood culture system which is a continuously

automated monitoring system (Mirrett, Reller, Petti, Woods, Vazirani, Sivadas &

Weinstein, 2003) and is designed purposely to detect microorganisms from blood.

BACTEC systems include the BACTEC FX system and the BACTEC

9000 series

both of which are based on fluorescent technology. Blood cultures are monitored

through a fluorescent sensor and the production of carbon dioxide is detected,

indicating the growth and presence of microorganisms (Shigei, Shimabukuro, Pezzlo,

De La Maza & Peterson, 1995). These systems are highly reliable and safe and offer

high performance as well as quality media. Amongst the BACTEC 9000 series

include the BACTEC 9240, BACTEC 9120 and the BACTEC 9050 which

accommodate 240, 120 and 50 blood culture bottles respectively (Becton Dickinson

and Company, 2012).

Media of the BACTEC blood culture systems are particularly designed for

inoculation using a small volume of blood, allowing the effective growth of

microorganisms in order to identify bacteria, yeasts and fungi present in the

bloodstream. BACTEC blood culture media include:

24

BACTEC™ Plus Aerobic/F and Plus Anaerobic/F

BACTEC Peds Plus™/F Medium

BACTEC™ Lytic/10 Anaerobic/F Medium

The BACTEC Mycosis-IC/F is a selective fungal medium which is useful in

recovering yeasts or moulds from blood specimens (Rosa, Araujo, Rodrigues, Pinto-

de-Sousa & Pina-Vaz, 2011). Studies have concluded that it is a more effective and

reliable medium in the recovery of fungi than other standard media (Meyer,

Letscher-Bru, Jaulhac, Waller & Candolfi, 2004).

1.4.1.2.2. The BacT/Alert Blood Culture System

The BacT/Alert 3D automated blood culture system (BioMérieux, Inc.,

Durham, N.C.) was introduced by the Organon Teknika Corporation in 1990

(Wilson, Weinstein, Mirrett, Reimer, Feldman, Chuard & Reller, 1995) with the

purpose of identifying pathogens causing bloodstream infections (Rohner, Pepey &

Auckenthaler, 1995). This system consists of an incubator with an adjusting

temperature of between 35 and 37oC ±0.5

oC, a shaker which continuously mixes the

bottles, and a detector. It is based on the colorimetric detection of carbon dioxide

which is produced by microorganisms growing in the blood culture bottles (Thrope

et al, 1990). The following is a brief description of the features making up this

system:

25

Carbon Dioxide sensor: a sensor is attached to the bottom of the blood culture

bottle and this is separated from the culture medium by a semi-permeable membrane.

This acts as a barrier and is impermeable to ions, water, blood and other media

components. However, it is permeable to carbon dioxide which, upon production by

the growing microorganisms, it will cross the membrane and into the sensor. This

will lead to the dissolution of water, generating free hydrogen ions. The latter will

interact with the sensor producing a colour change from blue to green, due to a

decrease in the pH. As more carbon dioxide is produced, the green colour eventually

turns yellow (Thrope et al., 1990).

Figure 1.3: BacT/ALERT 3D's colorimetric sensor-and-detection technology detects

microorganisms by tracking CO2 production (BioMérieux, n.d)

Colorimetric detection system: within the system are blocks which consist of a

number of wells. Each well contains a colorimetric detector which consists of a red

light emitting diode. The latter emits light onto an absorbing photodiode. This will

result in the production of a voltage signal which is directly proportional to the

concentration of carbon dioxide produced in the bottle (Thrope et al., 1990).

26

Computer driven algorithm: Each voltage signal produced is then amplified

and filtered after which it is transmitted to a computer for analysis. The data acquired

is then plotted resulting in the production of a growth curve (Thrope et al., 1990).

Figure 1.4: Schematic diagram of the BacT/Alert blood culture bottle showing the: a)

Bottle wall; b) Semi-permeable membrane; c) Carbon dioxide sensor; d) Light emitting

diode; e) Light absorbing photodiode; f) Amplifier; g) Block (Thrope et al., 1990)

The BacT/Alert blood culture system utilises two types of media: one for the

growth of aerobic, microaerophlic bacteria and yeasts and another for the growth of

anaerobes. Blood culture systems differ in the type of supplement media utilised. In

the case of the BacT/Alert system, the culture media contain a tryptic soy broth

supplement. The following are the blood culture media utilised by the BacT/Alert

blood culture system:

BacT/Alert SA: Standard Aerobic medium

BacT/Alert SN: Standard Anaerobic medium

BacT/Alert FA: FAN Aerobic

BacT/Alert FN: FAN Anaerobic

BacT/Alert PF: Pediatric FAN

BacT/Alert MB: Mycobacteria Blood

27

The FAN aerobic blood cultures are useful in enhancing the recovering of

microorganisms from blood in order to detect fungaemia, as well as bacteraemia, in

patients taking antibiotics. It contains charcoal which plays an important role in

inactivating any anti-microbial agents thus allowing growth. Studies were conducted

where the standard aerobic and anaerobic BacT/Alert media were compared with the

FAN aerobic and anaerobic media and results showed a greater recovery of yeasts in

the FAN bottles (Weinstein et al., 1995; Wilson et al., 1995). Apart from isolating

Mycobacteria, the BacT/Alert MB medium is also designed in order to support the

growth of yeasts and other fungi (Mattei, Savarino, Fabbri, Moneta & Tortoli, 2009).

1.5. Sub-Culture Techniques from Mycology

1.5.1. Routine Sub-Culture Technique

In order to recovery microorganisms from blood cultures, sub-cultures onto

solid media must be prepared. This is performed in order to further characterise and

identify the species isolated within the blood culture medium and also perform

susceptibility testing. In a study performed by Rosa, Araujo et al. (2011), two sub-

culture techniques were performed in order to recover moulds from BACTEC blood

culture bottles, since the sensitivity of blood cultures for moulds is extremely low.

The first technique utilised was the routine sub-culture technique which involves the

collection of approximately 25µl (2-3 drops) of culture medium using a sterile

airway needle (Rosa et al., 2011). This technique is widely used for sub-culturing

blood culture bottles both for bacteriology and mycology.

28

1.5.2. ‘Novel’ Sub-Culture Technique

In the study performed by Rosa et al. (2011), a new sub-culture technique was

introduced and this involved the collection of a higher volume of culture medium

(approximately 100µl) using a tuberculin syringe. Before collection, the blood

culture bottle was vigorously agitated for approximately 10 seconds. In the presence

of fungi, this will allow the fragmentation of any fungal clumps, known as fungal

balls (aspergilloma), present within the medium (Rosa et al., 2011). When comparing

these two sub-culture techniques, the novel technique was more successful at

isolating Aspergillus spp., even in blood culture bottles inoculated with low conidial

concentrations.

1.5.3. Terminal Sub-cultures

Terminal subcultures are performed at the end of the incubation period onto

solid media, regardless of whether the blood culture reads positive or negative.

Several studies have been performed which determined the value of terminal sub-

cultures, especially in patients with invasive candidiasis. Isolation of yeasts from

blood cultures is considered the Gold Standard in diagnosis of disseminated yeast

infections. However, the sensitivity of blood culture systems for the isolation of

yeasts needs improvement since undiagnosed cases of invasive candidiasis have been

also recorded (Søgaard et al., 2006). A study was conducted by Borst et al. (2000)

which determined the value of terminal sub-cultures using blood cultures in patients

with invasive candidiasis. In this study it was recommended that sub-cultures are

29

prepared from both positive and negative blood cultures where disseminated fungal

infection is suspected. Results showed that by performing terminal sub-cultures on

negative bottles, additional important and relevant information can be obtained.

1.6. Limitations

Even though blood cultures are nowadays being considered the gold standard

in the diagnosis of bloodstream infections, they possess several limitations. There

hasn’t been a single blood culture system which has proven to be hundred percent

sensitive in the detection of bloodstream pathogens since certain organisms grow

poorly in blood cultures (Weinstein, 1996) and this results in the detection of several

false negatives. In the case of yeasts, studies have shown that the sensitivity of blood

cultures for such organisms need improvement since several undiagnosed cases of

invasive candidiasis have been identified (Søgaard et al., 2006). This has led to the

importance of performing terminal sub-cultures. If sensitivity of these blood culture

systems can be improved then terminal sub-culture would not be necessary and thus

cut down on costs as well as provide better use of laboratory resources (Shigei et al.,

1995). In addition, the sensitivity of blood culture for detecting moulds is extremely

low. Rosa et al. (2011) speculate that when Aspergillus is cultured in blood cultures,

it is many times considered as a contaminant and thus other investigations should be

carried out in order to confirm or disprove this.

30

1.7. Statement of Problem

Blood culture systems have been found to be very useful in the detection of

bloodstream infections, however, when it comes to fungal pathogens their sensitivity

is not as high as with bacterial pathogens. Blood culture systems using culture bottles

are not considered useful for culturing moulds. Therefore, other serological tests, and

sometimes invasive procedures, must be performed instead. Culturing such an

organism from blood in blood culture bottles is highly desirable. Sensitivity of blood

culture bottles for Aspergillus is extremely low, and this is attributed to the fact that

Aspergillus produces airborne conidia rather than waterborne ones. In fact, systemic

infections caused by Fusarium and Scedosporium, which both produce waterborne

conidia; can lead to positive blood cultures.

Aspergillus is highly angioinvasive; therefore it is argued that other

structures, such as pieces of viable hyphae, can be present in the bloodstream. Other

reasons for the lack of positive blood cultures with Aspergillus may play a role, but

these are still not clearly identified (Rosa et al., 2011). If Aspergillus cultures could

be cultured in blood culture bottles, adequate and useful information on the mould

such as the identity of the species and material for susceptibility testing can be

obtained and therefore aid in the diagnosis and management of IA.

The detection of yeasts is also crucial in the diagnosis of systemic

candidiasis, which has become increasingly common. Automated blood culture

systems are routinely utilised in order to diagnose this infection (Borst et al, 2000).

31

Isolation of yeasts from blood cultures is considered the Gold Standard in diagnosis

of disseminated yeast infections. However, the sensitivity of blood culture systems

for the isolation of yeasts needs improvement. A study was conducted by Borst et al.

(2000) which determined the value of terminal sub-cultures using blood cultures in

patients with candidaemia.

1.8. Objectives and Aims of Project

The aim of this project is to enhance the blood culture system for Mycology by

evaluating the already existing system (BacT/Alert) for possible improvements. The

system will be tested to see whether it supports the growth of Aspergillus spp. This

part of the project will be based on a study performed by Rosa et al. (2011) using the

novel technique as the standard technique is not used at Microbiology Department

MDH. The system will also be evaluated to see whether the detection of yeast

infections can be improved. This will be done by preparing terminal sub-cultures

from all blood cultures containing the blood of neutropenic patients at Mater Dei

Hospital, in order to test for the potential growth of yeasts but also, not excluding

moulds. All this will be performed to see whether the changes mentioned will lead to

results that can be used to modify the current blood culture system for Mycology,

and therefore update the SOP for blood cultures for Mycology.

32

2. CHAPTER 2

METHODOLOGY

33

2.1. Ethics Approval

Before carrying out this project, ethics approval was obtained from the

University Research Ethics Committee (UREC). Attached in the Appendix (Section

VI) are copies of all the permissions required to carry out this study.

2.2. Fungal Species

Four clinical isolates of Aspergillus spp. belonging to the Mycology

Laboratory of Mater Dei Hospital (MDH) were utilised in this study. The four

Aspergillus spp. used were Aspergillus fumigatus, Aspergillus flavus, Aspergillus

niger and Aspergillus terreus.

2.3. Inoculation of conidial and hyphal suspensions in blood culture bottles

2.3.1. Preparation of conidial suspensions

Serial suspensions of 2.5, 25, 250 and 2500 c.f.u per 10ml of the four

Aspergillus spp. were prepared. An initial concentration of 2McF was utilised for

each organism. This is equivalent to 1x107 c.f.u per ml for A. flavus and A. niger, and

2x107 c.f.u per ml for A. fumigatus and A. terreus. The higher value for the latter is

due to the fact that these organisms have smaller conidia than the former ones. These

concentrations were determined using the graphs in Figure 2.1 which indicate the

optical density values and their equivalent cell counts for each organism. The agar

34

cultures containing the Aspergillus spp. were first flooded in approximately 1ml of

1% Tween 80. Using a sterile pipette, the conidial suspension created was collected

and mixed together in sterile distilled water until a concentration of 2McF was

obtained. This was determined using a Densitometer (Densimat, BioMérieux). The

vial was mixed well using a vortex mixer in order to obtain a homogenous

suspension.

Figure 2.1: Densimat readings and corresponding spore counts for a) A. fumigatus and

b) A. flavus (Araujo, Rodrigues & Pina Vaz, 2004)

A rack with 5 conical-bottomed test tubes was set up for each organism and

each test tube was labelled with each dilution and the species’ name. An initial

concentration of 1x106 c.f.u per 10ml of sterile distilled water for A. flavus and A.

niger was prepared in the first test tube. This was done by mixing 100µl, using a

35

micropipette, from the 2McF conidial suspension previously prepared, together with

10mL distilled water. The tube was then vortexed. Twenty-five microliters of the

latter were transferred to the tube labelled with the highest conidial concentration

(2500 c.f.u/10ml) together with 10ml of sterile distilled water and vortexed. Further

dilutions were prepared by pipetting 1ml from the previous dilution to 9ml distilled

water in the next tube. After each dilution was prepared, the tubes were vortexed

well in order to obtain an even suspension. The preparation of the conidial

suspensions for A. flavus and A. niger can be summarised in Figure 2.2.

Figure 2.2: Preparation of conidial suspensions for A. flavus and A. niger using a

prepared 2McF conidial suspension and sterile distilled water (SDW) (Figure created

from data in: SciQuip Ltd., n.d)

In the case of A. fumigatus and A. terreus an initial concentration of 2x10-6

c.f.u/10ml, equivalent to 2McF, was prepared. Two hundred microliters of the latter

was pipetted in a tube containing 10ml of sterile distilled water thus diluting the

solution ten-fold. The tube was vortexed. The highest dilution was prepared by

pipetting 25µl of the latter suspension together with 20ml distilled water and

36

vortexed. The rest of the dilutions were then prepared. The process was repeated

until a total of 60ml for each dilution was prepared since each blood culture bottle

was inoculated with 10ml of each dilution. The preparation of the conidial

suspensions for A. fumigatus and A. terreus can be summarised in Figure 2.3.

Figure 2.3: Preparation of conidial suspensions for A. fumigatus and A. terreus using a

prepared 2McF conidial suspension and sterile distilled water (SDW) (Figure created

from data in: SciQuip Ltd., n.d)

2.3.2. Preparation of hyphal suspensions

All four Aspergillus spp. were previously cultured in Sabouraud broth in order

to allow the growth of hyphae. The media were incubated at 30oC and at intervals

they were mixed well on a magnetic stirrer to avoid the formation of a mycelium on

the surface. After approximately 5 days of allowing sufficient growth, the culture

media containing the hyphae were centrifuged in conical-bottomed universal

containers in order to deposit the hyphae. Following centrifugation, the hyphae were

37

washed three times with sterile distilled water (Wells, Dickson & Lester, 1996; Liu et

al., 2010). The hyphae were then utilised in order to prepare hyphal suspensions.

The same concentrations were used as for the conidial suspensions. In this

case, when preparing a 2McF concentration using the hyphae, the vial was vortexed

well using glass beads in order to produce hyphal fragments.

The concentrations of the serial suspensions were validated by cultures on

Sabouraud Dextrose agar. This was done by pipetting 100ul of the each prepared

dilution and inoculating separate Sabouraud Dextrose plates. These were incubated

for 3 days and the colonies were counted.

2.3.3. Inoculation of suspensions into BacT/Alert blood culture bottles

The standard aerobic (SA) and FAN aerobic BacT/Alert blood cultures were

utilised in this study. The bottles were first labelled with the species’ name,

concentration and date. Starting from the smallest dilution (2.5 c.f.u/10ml), 10mls

was first aspirated using a 10mL sterile syringe and the each bottle was inoculated

with 10mL of the chosen concentration. Care was taken so as not to handle the blood

culture bottles at the site of inoculation as this could easily result in contamination.

Each concentration was tested in triplicate. A set of 3 blood culture bottles was

inoculated using sterile distilled water and this served as a negative control.

38

A total of 30 bottles was thus required for each organism: 15 SA and 15 FAN

BacT/Alert blood cultures which were inoculated as follows:

o 3 of each bottle inoculated with the 2500 c.f.u/10ml suspension

o 3 of each bottle inoculated with the 250 c.f.u/10ml suspension

o 3 of each bottle inoculated with the 25 c.f.u/10ml suspension

o 3 of each bottle inoculated with the 2.5 c.f.u/10ml suspension

o 3 of each bottle inoculated with sterile distilled water which served as a negative

control

Figure 2.4: Standard Aerobic (blue cap) and FAN aerobic (green cap) BacT/Alert blood

culture bottles inoculated with the prepared serial dilutions

2.4. Incubation of blood culture bottles

Due to the lack of space in the BacT/Alert blood culture analyser of the

Microbiology laboratory, the blood culture bottles were incubated at 35oC for 7 days

39

using Mycology Laboratory incubator. The contents of the bottles were mixed every

day and observed for growth.

2.5. Preparation of terminal sub-cultures: the ‘Novel’ sub-culture technique

After 7 days, the bottles were observed visually for the presence of growth.

Terminal sub-cultures were prepared in a safety cabinet and Sabouraud Dextrose

agar plates were utilised. The plates were labelled with the species’ name,

concentration and date. The ‘novel’ sub-culture technique, as proposed by Rosa et al.

(2011), was carried out. The blood culture vials were vigorously agitated for

approximately 10 seconds in order to fragment the fungus. Using a 21G Vacuette®

blood collection needle, the culture medium was aspirated into a red-capped

vacutainer. One hundred microliters of the culture medium was used to inoculate the

corresponding agar plate which was then streaked accordingly. After all sub-cultures

were prepared, the plates were incubated at 30oC for 3 days. The plates were then

checked for growth.

2.6. Preparation of terminal sub-cultures from negative blood cultures

2.6.1. Collection of blood culture bottles

Approximately twice a week negative blood cultures inoculated with the blood

of neutropenic and immunocompromised patients, were collected from the blood

culture laboratory at the Microbiology department of MDH. Other negative blood

40

cultures which gave a positive galactomannan test were also utilised. The clinical

details were checked from the request form and any sample with the required criteria

was taken note of. If the specimen was issued as negative after 5 days incubation in

the BacT/Alert automated system, the blood culture bottle was collected and

incubated for another 5 days in the Mycology lab incubator since studies have shown

that an extended incubation period might be required in cases of suspected

fungaemia (Weinstein, 1996).

2.6.2. Terminal sub-cultures

After a total of 10 days incubation, terminal sub-cultures were performed from

the negative blood cultures. The ‘novel’ sub-culture technique (Rosa et al., 2011)

was performed again. The bottles were agitated vigorously for approximately 10

seconds before preparing the sub-cultures, in order to fragment the fungus or yeast, if

present. After 3 days the plates were observed for the presence or absence of growth.

41

3. CHAPTER 3

RESULTS

42

3.1. Results involving visual readings

3.1.1. Detection of Aspergillus spp. In BacT/Alert blood cultures

3.1.1.1. Conidial inoculum

Inoculated bottles were checked for growth every 24 hours. The growth of

Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus was detected

visually within 24 hours in the BacT/Alert Standard Aerobic (SA) blood culture

bottles at the highest concentration of 2500 c.f.u/10ml. Aspergillus niger was

detected after 48 hours at conidial concentrations of 2500 and 250 c.f.u/10ml. The

growth of A. flavus was detected after 24 hours at a concentration of 250 c.f.u/10ml

whilst A. niger was detected after 48 hours. The growth of A. fumigatus and A.

terreus was detected after 48 hours. Growth was identified by the presence of a

white fungal ball floating within the culture medium. This was more visible in the

standard aerobic bottles due to the light colour of the medium.

Growth could also be observed after 24 hours in the BacT/Alert FAN aerobic

blood culture bottles; however, due to the dark nature of the FAN aerobic culture

medium, growth was more difficult to detect visually and could only be confirmed

through the preparation of terminal subcultures. All four Aspergillus spp. were also

detected at the lowest conidial concentration of 2.5 c.f.u/10ml after 72 hours for A.

fumigatus and A. terreus, and 120 hours for A. flavus and A. niger. Table 3.2

43

summarises the time of growth detected for each organism, at different conidial

concentrations.

Since the growth of each organism was detected visually and not by the

BacT/Alert automater, there might have already been growth several hours before,

which would have been difficult to detect visually.

3.1.1.2. Hyphal inoculum

Once again, inoculated bottles were checked for growth every 24 hours.

Slightly different results were produced for the hyphal concentrations. A. fumigatus

and A. niger were detected soon after 48 hours at concentrations of 2500 and 250

c.f.u/10ml whilst A. flavus and A. terreus were detected soon after 24 hours at a

concentration of 2500 c.f.u/10ml in the standard aerobic BacT/Alert bottles. It was

difficult to detect any growth at the lower concentrations of 25 and 2.5 c.f.u/10ml

for A. niger. On the other hand growth was detected soon after 48 hours for A.

terreus at a concentration of 25 c.f.u/10ml and after 96 hours for A. fumigatus.

Growth was detected after 120 hours at the lowest hyphal concentration of 2.5

c.f.u/10ml for A. terreus and A. fumigatus. Growth could be identified by the

presence of a white fungal ball in the blood culture medium.

As mentioned previously, the detection of growth in the BacT/Alert FAN

bottles was not easy to detect visually due to dark colour of the culture medium.

However, in the case of A. fumigatus growth was detected after 2 days at

44

concentrations of 2500 and 250 c.f.u/10ml and after 6 days in the 25 c.f.u/10ml

bottles. Table 3.3 summarises the time of growth for each organism at different

hyphal concentrations.

3.1.2. Results of growth after 7 days

3.1.2.1. Conidial inoculum

Since the blood culture bottles were incubated for a total of 7 days, each

bottle was observed for growth after the total incubation period. Growth was

observed in all standard aerobic bottles inoculated with concentrations of 2500, 250

and 25 c.f.u/10ml, for all four organisms. However, in the case of A. flavus, one of

the 25 c.f.u/10ml bottles was contaminated due to the presence of turbidity in the

blood culture medium. Contamination was also observed in the A. terreus SA

bottles inoculated with the lowest conidial concentration, as well as the negative

controls. Contamination could not be identified in the FAN bottles due to the dark-

coloured medium. After a total of 7 days, the white fungal clumps could be

visualised more clearly upon mixing of the bottles.

45

Figure 3.1: Detection of growth of A. fumigatus in the BacT/Alert standard aerobic

blood cultures after 7 days

3.1.2.2. Hyphal Inoculum

Due to the dark nature of the FAN blood culture medium, it was difficult to

identify whether any growth was present just through visual observation. However,

growth was observed in almost all the bottles with the highest conidial

concentrations, mainly those bottles inoculated with conidial concentrations of

2500 and 250 c.f.u/10ml. Growth was confirmed through the preparation of

terminal subcultures. All negative controls produced no growth.

46

Figure 3.2: Detection of growth of A. fumigatus in the BacT/Alert FAN aerobic blood

cultures after 7 days

In the case of the hyphal concentrations, less growth could be observed in the

bottles after 7 days. At a concentration of 2500 c.f.u/10ml growth was observed in all

bottles for all four organisms. No growth was observed at any other concentration for

A. flavus. The growth of A. fumigatus was only observed in one bottle at the lowest

concentration of 2.5 c.f.u/10ml whilst no growth was observed for the other three

Aspergillus spp.

In the case of the FAN aerobic bottles, growth was observed at the highest

concentrations (2500 and 250 c.f.u/10ml) in almost all FAN aerobic bottles. Only the

growth of A. fumigatus and A. terreus was observed at a concentration of 25

c.f.u/10ml unlike the remaining Aspergillus spp. It was difficult to identify any

growth at a concentration of 2.5 c.f.u/10ml.

47

3.1.3. Validation of inocula

3.1.3.1. Number of colonies grown from plates inoculated with prepared

concentrations

Inoculum Organism Concentration

(c.f.u/10ml)

Average number

of colonies

Conidial

Inoculum

A. fumigatus 2500 15

250 2

25 1

A. flavus 2500 6

250 2

25 1

A. niger 2500 5

250 1

25 0

A. terreus 2500 16

250 2

25 0

Hyphal

Inoculum

A. fumigatus 2500 1

250 0

25 0

A. flavus 2500 0

250 0

25 0

A. niger 2500 1

250 0

25 0

A. terreus 2500 0

250 0

25 1

Table 3.1: Average number of colonies counted from SDA plates inoculated with the

conidial and hyphal concentrations

48

In order to validate the prepared concentrations, SDA plates were inoculated

with 100µl of the prepared suspensions.

Table 3.1 summaries the average number of colonies obtained after 3 days of

incubation. Since there is a ten-fold difference between each concentration, the

average number of colonies obtained is correct for the conidial suspensions and this

is clearly evident in the results of the conidial concentrations of A. fumigatus and A.

terreus. A graph of ‘concentration’ and ‘average number of colonies’ was plotted

proving the linear relationship between the two variables and thus confirming that

the conidial concentrations prepared were correct.

Due to limitations in the procedure utilised in preparing the hyphal

concentrations, the number of colonies obtained were not correct and some plates did

not produce any growth. As a result, a graph could not be plotted to show the

linearity between the two variables.

The same method for the preparation of conidial suspension was utilised for the

preparation of the hyphal concentrations. However, there was difficulty in breaking

the hyphal fragments, even when using glass beads. Thus, large hyphal clumps

remained and the concentrations prepared were not equal. As a result, lack of growth

was observed when validating the hyphal concentrations on solid media. Therefore

the hyphal suspensions could not be validated.

49

Figure 3.3: Graph showing the linear relationship between the average number of

colonies and different conidial concentrations

3.1.4. Recovery of Aspergillus spp. on solid culture medium

3.1.4.1. Conidial inoculum

Terminal subcultures were prepared after 7 days of incubation of the blood

culture bottles. The novel subculture technique (Rosa et al., 2011) was carried out

on each blood culture bottle respective of whether growth was observed or not. This

proposed sub-culture technique was successful in recovering all four Aspergillus

species even at low conidial concentrations of 2.5 c.f.u/10ml.

All four species were recovered at concentrations of 2500 and 250 c.f.u/10ml

from almost all three SA and FAN bottles. Aspergillus spp. were also recovered

from positive BacT/Alert bottles at lower conidial concentrations of 25 and 2.5

c.f.u/10ml from both the SA and FAN bottles.

0

2

4

6

8

10

12

14

16

18

0 1000 2000 3000

Aver

age

nu

mb

er o

f co

lon

ies

Concentration (c.f.u/10ml)

A. fumigatus

A. flavus

A. niger

A. terreus

Linear (A. fumigatus)

Linear (A. flavus)

Linear (A. niger)

Linear (A. terreus)

50

For the negative controls, no fungal growth was observed however, in the case

of A. fumigatus and A. flavus contamination was observed in some of the plates

from both the SA and FAN bottles. Contamination was also observed in some sub-

cultures of A. terreus from both blood cultures which were inoculated at low

conidial concentrations. Since the growth of Aspergillus is not affected by bacterial

growth, terminal subcultures were not repeated.

Contamination was also observed in some of the bottles inoculated with

lowest conidial concentrations of A. flavus and A. terreus and this was confirmed

after the preparation of terminal subcultures. It is important to note that slight

turbidity was also observed in the bottles inoculated with the highest concentrations

(2500 and 250 c.f.u/10ml). However, Aspergillus spp. grew just the same and were

still recovered through terminal subculture on SDA. Thus, the presence of bacterial

contamination will not prevent the growth of Aspergillus.

3.1.4.2. Hyphal inoculum

Recovery of Aspergillus spp. by terminal subculture was less successful in

the case of the hyphal concentrations, especially at lower concentrations (25 and 2.5

c.f.u/10ml). The recovery of all four species was successful at the highest

concentration of 2500 c.f.u/10ml from both the SA and FAN bottles. A. flavus was

not recovered at any other hyphal concentration from the SA bottles. In the case of

the FAN aerobic bottles A. flavus was also recovered at a concentration of 250

c.f.u/10ml but not at the lowest hyphal concentrations (25 and 2.5 c.f.u/10ml).

51

A. fumigatus was recovered at all hyphal concentrations from both bottles,

except from the FAN aerobic bottles inoculated at a concentration of 2.5 c.f.u/10ml.

In contrast, A. terreus was recovered at all concentrations from the FAN bottles

except from the SA bottle inoculated at the lowest concentration. A. niger was

recovered from both bottles at the highest concentrations and at a concentration of

2.5 c.f.u/10ml for the FAN aerobic bottle but not at a concentration of 25

c.f.u/10ml. No growth was observed from the negative controls.

Lack of growth in the bottles inoculated with the hyphal concentrations is

likely due to the limitation of the procedure carried out for the preparation of the

hyphal concentrations. The ideal method for the preparation of the hyphal inocula

was described by Sande, Travakol, Vianen & Bakker-Woudenberg (2009) in which

a sonicator was utilised. Since the latter instrument was not available this method

could not be applied. The likely cause of this problem is the presence of uneven

concentrations due to errors in fragmenting the hyphal clumps.

Figure 3.4: Subcultures of A. fumigatus (left) and A. flavus (right) on Sabouraud

Dextrose Agar

52

Figure 3.5: Subcultures of A. niger (left) and A. terreus (right) on Sabouraud Dextrose

Agar

3.1.5. Recovery of yeast from negative blood culture

A total of hundred negative blood cultures were sub-cultured using the novel

sub-culture technique (Rosa et al., 2011) onto Sabouraud Dextrose agar. These blood

cultures were incubated for 10 days and the machine (BacT/Alert automater) did not

detect any growth in them, therefore these are normally issued as negative. One of

these blood cultures resulted in growth on terminal sub-culture. Yeast-like colonies

were observed after 24 hours incubation at 30oC. These appeared to be white/cream

in colour and smooth. The yeast was sub-cultured on Brilliance Candida Agar

(Oxoid) and identified as Candida tropicalis which is commonly known to cause

septicaemia.

53

Table 3.2: Results showing time of growth at different conidial concentrations in both SA and FAN bottles & results of terminal subcultures

Organism Concentration

(c.f.u/10ml)

Time of growth

(Hours)

SA

Time of growth

(Hours)

FAN

Terminal

Subculture

SA

Terminal

Subculture

FAN

A. fumigatus 2500 24 24 P P

250 48 48 P P

25 48 96 P P

2.5 72 120 P P

NC NG NG NG NG

A. flavus 2500 24 24 P P

250 24 24 P P

25 48 48 P P

2.5 120 72 P NG

NC NG NG NG NG

A. niger 2500 48 48 P P

250 48 48 P P

25 72 96 P P

2.5 120 96 P NG

NC NG NG NG NG

A. terreus 2500 24 48 P P

250 48 48 P P

25 48 72 P P

2.5 72 96 P P

NC NG NG NG NG

Legend:

NG = No Growth

NC = Negative Control

P = Positive growth

54

Table 3.3: Results showing time of growth at different hyphal concentrations in both SA and FAN bottles & results of terminal subcultures

Organism Concentration

(c.f.u/10ml)

Time of growth

(Hours)

SA

Time of growth

(Hours)

FAN

Terminal

Subculture

SA

Terminal

Subculture

FAN

A. fumigatus 2500 48 48 P P

250 48 96 P P

25 96 120 P P

2.5 120 NG P NG

NC NG NG NG NG

A. flavus 2500 24 48 P P

250 NG 48 NG P

25 NG NG NG NG

2.5 NG NG NG NG

NC NG NG NG NG

A. niger 2500 48 48 P P

250 48 48 P P

25 NG NG NG NG

2.5 NG NG NG P

NC NG NG NG NG

A. terreus 2500 24 48 P P

250 48 48 P P

25 48 96 P P

2.5 120 NG NG P

NC NG NG NG NG

Legend:

NG = No Growth

NC = Negative Control

P = Positive growth

N = Negative growth

55

3.2. Statistical analysis

3.2.1. Comparison of results with results of published study

Since no raw data was presented in the study by Rosa et al. (2011), no

statistical tests could be carried out to compare the studies. Instead, a similar graph

was plotted and compared visually, as can be seen in the figures below:

Figure 3.6: Graph comparing the detection of growth of A. fumigatus by the BACTEC

automated system; the grey, white and black bars represent the BACTEC Plus

Aerobic/F, Mycosis-IC/F and Myco/F Lytic bottles respectively, as presented in the

study by Rosa et al. (2011)

Figure 3.7: Graph comparing the detection time of growth of A. fumigatus using the

BacT/Alert SA and FAN aerobic bottles

56

When comparing the results of growth in the Aerobic/F, Mycosis-IC/F

BACTEC bottles (Rosa et al., 2011), with the SA and FAN aerobic BacT/Alert

bottles used in this study, similar results were obtained in both studies at the highest

conidial concentrations (2500 and 250 c.f.u/10ml). However, at a concentration of

250 c.f.u/10ml, growth was detected after 25 hours for the BACTEC bottles, whilst

for the BacT/Alert bottles growth was detected after 48 hours.

In the case of the lower concentrations (25 and 2.5 c.f.u/10ml), both the

Aerobic/F and Mycosis-IC/F BACTEC vials produced similar results when

compared to the BacT/Alert vials. In the latter, growth was more easily detectable in

the SA BacT/Alert bottles at low concentrations, when compared to the FAN aerobic

bottles.

The overall time of growth of A. fumigatus ranged between 20 and 99 hours for

the BACTEC vials, whilst for the BacT/Alert vials, the time of growth ranged

between 24 and 120 hours. On the other hand, A. flavus and A. niger were detected

after approximately 50 and 37 hours respectively in the study by Rosa et al. (2011),

whereas in this study all four organisms were detected at similar times, either 24 or

48 hours after inoculation.

Overall, when comparing both blood culture systems, the BACTEC produced

better results when compared to the BacT/Alert system. This can be easily identified

from the graphs in figures 3.6 and figure 3.7 where the overall detection time of

growth in the BacT/Alert vials is greater than in the BACTEC vials. Thus, the

57

Aspergillus spp. required less hours for growth in the BACTEC vials than in the

BacT/Alert vials indicating that the BACTEC system is the better choice, although

this could not be statistically verified.

3.2.2. Testing for normality – The Shapiro Wilk’s Test

We are interested in testing the effect of different types of blood culture media

on the time of growth using statistical tests.

However before deciding which statistical test can be used it is important to

check if the dependent variable ‘time’ follows a normal distribution. This is done

using the Shapiro Wilk’s test which tests the following hypotheses.

Ho: The variable ‘Time’ is normally distributed

H1: The variable ‘Time’ is not normally distributed

The following output is obtained:

Tests of Normality

Kolmogorov-Smirnova Shapiro-Wilk

Statistic df Sig. Statistic df Sig.

Time in hours .321 52 .000 .835 52 .000

a. Lilliefors Significance Correction

58

As can be seen in the table above, the p-value from the Shapiro-Wilk’s test

obtained is 0.000 which is smaller than 0.05 hence we reject the null hypothesis Ho

and accept H1. Therefore we can conclude that the variable ‘time’ is not normally

distributed hence non-parametric tests will be used.

3.2.3. Comparison of results: The Kruskal-Wallis Test

3.2.3.1. Comparison of SA and FAN BacT/Alert bottles

Since the variable ‘time’ is not normally distributed the Kruskal-Wallis test

will be used. The following hypotheses can be tested for the results of the conidial

inocula:

Ho: Average time of growth is the same for each bottle type

H1: Average time of growth is not the same for all bottle types

After performing the test, the following SPSS output was obtained:

Ranks

BacT/Alert Blood

Culture Bottle

N Mean

Rank

Time in

hours

SA 16 14.97

FAN 16 18.03

Total 32

Test Statisticsa,b

Time in hours

Chi-Square .932

Df 1

Asymp. Sig. .334

a. Kruskal Wallis Test

b. Grouping Variable:

BacT/Alert Blood Culture

Bottle

59

Since the p-value obtained is 0.334 and greater than 0.05, then we accept H0 showing

that the type of bottle does not affect the time of growth.

The same hypotheses can be tested for the results of the hyphal inocula. The

following output is obtained:

Since the p value obtained is also greater than 0.05, then we accept H0 showing

that the type of bottle does not affect the time of growth even in the case of the

hyphal inocula.

3.2.3.2. Comparing results of conidial and hyphal inocula

The following hypotheses can be tested:

Ho: The type of inoculum does not affect the time of growth

H1: The type of inoculum affects the time of growth

Ranks

BacT/Alert Blood

Culture Bottle

N Mean

Rank

Time in

hours

SA 10 9.35

FAN 10 11.65

Total 20

Test Statisticsa,b

Time in hours

Chi-Square 1.047

Df 1

Asymp. Sig. .306

a. Kruskal Wallis Test

b. Grouping Variable:

BacT/Alert Blood Culture

Bottle

60

The p value 0.864 is greater than 0.05, thus showing that the type of inoculum

does not affect the time of growth.

3.2.3.3. Comparing the effect of concentration on time

The effect of different conidial and hyphal concentrations on the time of

growth was determined using the Kruskal-Wallis test. The null and alternative

hypothesis will be as follows:

Ho: Concentration does not affect time

H1: Concentration has an effect on time

The following p-values were obtained:

P-Value

Conidial Inoculum SA 0.01

FAN 0.012

Hyphal Inoculum SA 0.116

FAN 0.043

Table 3.4: P-values obtained when testing the effect of concentration on time for both

the conidial and hyphal inocula

Ranks

Type of Inoculum N Mean Rank

Time in hours

Conidial Inoculum 32 26.23

Hyphal Inoculum 20 26.93

Total 52

Test Statisticsa,b

Time in

hours

Chi-Square .030

df 1

Asymp. Sig. .864

a. Kruskal Wallis Test

b. Grouping Variable: Type

of Inoculum

61

All the p-values obtained, except for the hyphal concentrations in the SA

bottles, are smaller than 0.05. Thus we reject H0 and accept H1 showing that

concentration affects the time of growth.

The test was also carried out in order to test the difference in the time of

growth between the highest (2500 and 250 CFU/10ml) and lowest concentrations (25

and 2.5 CFU/10ml). Table 3.5 illustrates the p-values obtained when comparing the

time of growth between the highest conidial and hyphal concentrations in the SA and

FAN aerobic bottles:

Conidial Inoculum Hyphal Inoculum

SA FAN SA FAN

High

Concentrations 0.186 0.495 0.180 0.317

Low

Concentrations 0.04 0.278 0.221 N/A

Table 3.5: P-values obtained when testing the difference in the time of growth between

the highest and lowest conidial and hyphal concentrations

All p-values obtained are greater than 0.05 except for the lowest conidial

concentrations in the SA bottles. The latter signifies that at lower concentrations

there is a significant difference in the time of growth. On the other hand the p-values

which are greater than 0.05 show that at higher concentrations the time of growth

does not vary significantly. This test could not be carried out for the results hyphal

62

concentrations in the FAN bottles since some bottles did not produce any growth and

no data was available.

3.2.3.4. Comparing the time of growth for each Aspergillus spp.

The line graphs below illustrate the time at which growth was detected at

different conidial concentrations for the SA and FAN aerobic BacT/Alert bottles.

Since no growth was observed at some hyphal concentrations, the graphs could not

be plotted.

Figure 3.8: Average time of growth for each Aspergillus spp. in the SA bottles

63

Figure 3.9: Average time of growth for each Aspergillus spp. in the FAN bottles

As can be observed from the graphs in Figure 3.8 and Figure 3.9, all four

organisms seem to have a similar average time of growth except for A. niger in the

SA bottles and A. flavus in the FAN bottles. The Kruskal-Wallis test was carried out

in order to confirm whether there is a significant difference in the time of growth

between all four species. According to the following hypotheses:

Ho: Different species produce similar results

H1: Different species produce different results

64

The following output was obtained:

Since the p-value obtained is greater than 0.05, then we accept H0, showing

that there isn’t a significant difference in the time of growth between the four

Aspergillus spp. even though A. niger had a greater average time of growth in the SA

bottles, compared to the other three species.

Ranks

Aspergillus species N Mean Rank

Time in

hours

A. fumigatus 4 8.00

A. flavus 4 6.63

A. niger 4 11.38

A. terreus 4 8.00

Total 16

Test Statisticsa,b

Time in hours

Chi-Square 2.440

df 3

Asymp. Sig. .486

a. Kruskal Wallis Test

b. Grouping Variable:

Aspergillus species

65

4. CHAPTER 4

DISCUSSION & CONCLUSION

66

4.1. Detection of fungi in automated blood culture systems: Comparison with

published studies

Several studies have been conducted in order to compare the BACTEC and

BacT/Alert blood culture systems. However, most of these studies were performed

solely for the detection of bacteria and yeasts. The reason behind the lack of such

studies for the detection of moulds such as Aspergillus spp. in blood culture systems

is due to the fact that the sensitivity of blood cultures for Aspergillus is extremely

low, most likely as a result of the production of airborne conidia rather than

waterborne ones.

Other structures, such as hyphae, can also be present in the bloodstream;

however it is argued that these may not proliferate in blood cultures thus reducing the

sensitivity of blood cultures for the detection of Aspergillus (Kami, Murashige,

Fujihara, Sakagami & Tanaka, 2005). It is also argued that in cases of disseminated

aspergillosis, hyphae are endocytosed by endothelial cells and the endocytosed

hyphae are not able to grow within blood cultures (Simoneau, Kelly, Labbe, Roy &

Laverdière (2005). Another factor which may play a role is the lack of carbon

dioxide production by certain fungal species in the blood culture vials (Fricker-

Hidalgo, Lebeau, Pelloux & Grillot, 2004).

Since Aspergillus is angioinvasive, it is argued that hyphal structures should be

found in the blood stream of patients with IA especially because haematogenous

spread to other organs occurs.

67

After carrying out this study, as well as comparing the results obtained with

those of the published study by Rosa et al. (2011), results have shown that it is, in

fact, possible to culture Aspergillus in both the BACTEC as well as the BacT/Alert

blood culture systems. Results have shown that most species are detected after

approximately 24 hours of incubation. When comparing both systems, the BACTEC

seemed to perform better. However, it must be taken into consideration that the

BacT/Alert automated machine was not utilised in this study, but rather, growth was

detected visually in order not to interfere with the workflow of the blood culture

laboratory. As a result, the time for the detection of growth might not have been very

accurate, that is growth could have been detected earlier if automation was used.

Most published works have tested the difference in the detection time of

growth between the two systems. One of the earliest studies carried out by Wilson et

al., (1992) (two years after the introduction of the BacT/Alert automated blood

culture system) demonstrated that the BacT/Alert system detects fungi (mainly

yeasts) much earlier than the BACTEC system. Mirrett et al., (2003), as well as

Hovarth et al. (2004) have also shown that the BacT/Alert system is better in the

detection of yeasts.

When comparing the BacT/Alert SA and FAN aerobic media statistically, no

significant difference (p>0.05) was observed between the two media for both the

conidial and hyphal inocula. The FAN aerobic medium is advantageous especially

when inoculating the media with the blood of patients taking antimicrobial agents. It

has been speculated that the Ecosorb supplement present within the FAN medium

68

might bind and inactivate antimicrobial agents thus reducing their inhibitory effects

and enhance growth (Smith, Bryce, Ngui-Yen & Roberts, 1995). Also, the use of

brain heart infusion broth present within the FAN medium, as opposed to the tryptic

soy broth in the SA medium, can enhance the recovery of such organisms (Weinstein

et al., 1995).

The BACTEC blood culture system includes a selective fungal medium: the

BACTEC Mycosis IC/F medium. The latter was developed in order to enhance the

detection of fungi from blood. This medium contains a lysing agent, chloramphenicol

in order to inhibit bacterial growth and other components, all of which enhance

fungal growth (Fricker-Hidalgo et al., 2004).

Unfortunately this type of medium also has its limitations such as the low

sensitivity for the detection of Aspergillus. This may lead to the conclusion that the

hypothesis of Aspergillus not growing would not be valid as this medium has a lytic

agent. Other limitations of this medium include additional volume of blood for

inoculation and it is very costly. As a result, most laboratories do not utilise this

medium and instead rely on terminal subcultures (Fricker-Hidalgo et al., 2004).

A study was also carried out in order to compare the BACTEC Mycosis IC/F

medium with the BacT/Alert FAN aerobic medium for the detection of fungaemia

(McDonald, Weinstein, Fune, Mirrett, Reimer & Barth Reller, 2001). Results have

shown that the FAN medium is more reliable in the detection of fungaemia due to

69

reasons mentioned previously such as the medium supplements which inactivate

antimicrobial agents.

A higher tendency of contamination with non-significant organisms in the FAN

aerobic media has also been described (Jorgensen et al., 1997). Similarly,

contamination was also observed in some of the FAN aerobic bottles in this study

and this was identified by terminal sub-culture. It is important to note that bacterial

contamination was also observed in the both the SA and FAN aerobic media. In fact,

a total of 8 SA and 6 FAN bottles were contaminated. However, contamination was

easily detected in the SA bottles as a result of turbidity within the medium. This was

not possible to detect in the dark-coloured FAN medium. In this case, the most

probable cause of contamination could be the result of a poor inoculation technique.

In order to solve this problem it was assumed that venting the FAN blood

cultures might reduce contamination. However, Weinstein et al. (1995) compared

unvented BacT/Alert FAN media to the SA BacT/Alert media and contamination

was observed in both media. This was also the case in this study, where bacterial

contamination most probably occurred during the inoculation of the bottles. The

latter is a problem which commonly results from skin commensals and is observed in

several microbiology laboratories (Weinstein, 2003). It is important to note, that

contamination did not, in any way, interfere with the results obtained, since fungal

growth was still observed in most bottles.

70

When taking into consideration the time of growth for all four Aspergillus spp.

no statistically significant difference (p>0.05) was observed in the detection time of

growth for both bottles. However, it is known that the conidia of A. fumigatus

germinate much faster than the other Aspergillus spp. Since this was not the case in

this study, the results obtained may be attributed to the fact that visual detection of

growth rather than detection via the automated system was performed.

With regards to the time of incubation, a total of 7 days may be required for the

growth of both moulds and yeasts. Several episodes of candidaemia have been

missed when a 5-day incubation period was applied (Hovarth et al., 2003) even

though a longer incubation period is more likely to result in contamination (Reisner

& Woods, 1996).

4.2. The Type of Inoculum

This study also tested whether growth is affected by the type of inoculum.

From the statistical analysis performed, there was no significant difference in the

time of growth (p>0.05) between the two inocula, taking into consideration the

limitation for the preparation of the hyphal inocula. Rosa et al. (2011) have also

tested the difference in the time of growth between the two inocula. The hyphal

inocula produced similar results to those of the conidial inocula. Therefore, the

detection time of growth was similar for both inocula and in both studies.

71

4.3. The ‘Novel’ (Rosa et al., 2011) subculture technique

In 2011, Rosa et al. proposed a new subculture technique which involves the

collection of a larger volume of culture medium (approximately 100µl), after

vigorously mixing the vial in order to dislodge the fungal ball. During this study, all

terminal subcultures were prepared using this technique and the latter was successful

in the recovery of all four Aspergillus spp. even at the lowest concentration (2.5

c.f.u/10ml). The routine method of subculture usually involves the collection of one

or two drops (25µl) of culture medium. Rosa et al (2011) compared the routine

technique with the ‘Novel’ technique. Results have shown that at a concentration of

25 c.f.u/10ml and lower concentrations, no Aspergillus spp. were recovered by the

classic subculture technique.

Overall, most laboratories often consider Aspergillus spp. as a contaminant

since Aspergilli are rarely recovered from blood cultures (Rosa et al., 2011). As a

matter of fact these results are, in most cases, interpreted as false-positives. Thus,

determining the presence of Aspergillus fungemia in immunocompromised patients

may be a limiting factor of blood culture systems as a result of what is falsely

interpreted as contamination (Girmenia, Gucci & Martino, 2001). Since this issue

was not properly being handled and blood cultures were being carelessly analysed, a

new subculture technique was proposed which was successful in the recovery of

Aspergillus even at very low concentrations (3 c.f.u/10ml) (Rosa et al., 2011).

72

In certain cases, during this study, it was difficult to visually identify growth,

especially in the FAN bottles due to the dark-coloured medium. Growth was thus

confirmed through the preparation of terminal subcultures using the ‘novel’ method

(Rosa et al., 2011). When taking into consideration the conidial inocula, this

proposed subculture technique was successful in the recovery of all four species,

even at the lowest conidial concentrations of 2.5 c.f.u/10ml. In fact, the growth of A.

flavus was not visually detected after 7 days, in any of the 3 bottles inoculated with a

concentration of 2.5 c.f.u/10ml. However, after performing terminal subculture, A.

flavus was recovered from one of the bottles.

When looking at the results of the hyphal inocula, growth could not be detected

in any of the FAN bottles inoculated with the lowest concentration of 2.5 c.f.u/10ml.

Upon preparing terminal subcultures, A. terreus and A. niger were recovered on solid

media from one out of the three bottles. Thus, this subculture technique has

overcome the limitations of the routine method. If this technique is applied, then

there is a greater probability of increasing the detection of Aspergillosis (Rosa et al.,

2011).

4.4. The importance of terminal subcultures from negative blood cultures

The value of terminal sub-cultures on negative blood cultures was evaluated in

this study. Several studies have been carried out and questioned the necessity of

performing terminal subcultures. From this study the importance of terminal

subcultures performed on negative blood cultures, especially in patients with

73

suspected fungaemia has been confirmed. Terminal sub-cultures were performed on

one hundred negative blood cultures which contained the blood of neutropenic,

septic and immunosuppressed patients. Out of the hundred samples, a positive

terminal sub-culture was obtained. The organism identified was the yeast Candida

tropicalis. During the period of the study there were no confirmed cases of invasive

aspergillosis, therefore the technique of terminal subculture applied in this study

could not be applied to blood cultures taken from such patients.

From the early 1980’s, studies were carried and evaluated the necessity of

terminal subcultures from negative blood cultures. The following findings were

obtained from these studies:

Campbell and Washington II (1980) were the first to test the value of terminal

subcultures. Two terminal subcultures yielded significant growth from 2780

negative bottles;

In the study by Gill (1981) 12 significant organisms were identified from

14,000 negative bottles;

Araj, Hopfer, Wenglar & Fainstein (1981), identified 15 positive cultures from

5354 previously negative blood cultures. The only fungus which was identified

is Cryptococcus neoformans.

The majority of these studies have shown that the isolates detected through

terminal subcultures had already been previously isolated from another bottle of the

same pair or a matching bottle belonging to the same set (Campbell & Washington

II, 1980; Gill, 1981).

74

Therefore, from these studies the results suggest that even though the blood

culture media, subculture methods as well as the time of incubation varies between

different laboratories, terminal subcultures might be of little value. However, one

particular study showed that the major false-negatives involved patients with

Candida infections (Shigei et al., 1995). Borst Leverstein-Van Hall, Verhoef and

Fluit (2000) also proved the importance of terminal subcultures from blood cultures

in patients with invasive candidiasis.

A more recent study conducted by Hovarth et al. (2004) detected Candida spp.

from 65 false negatives. Sixty-five out of 300 bottles showed no growth but after

performing terminal subcultures, all 65 bottles demonstrated the growth of pure yeast

colonies (Hovarth et al., 2004). Fifty of these bottles were of the BACTEC system,

however only 5 of these were aerobic; the other 15 bottles were of the BacT/Alert

system. Thus, this study also compared the BACTEC and BacT/Alert blood culture

systems.

At MDH, terminal subcultures are only performed on blood cultures sent for

mycology investigations, which include 2 aerobic bottles with a request for

mycology investigations. This could mean that some cases of fungaemia are being

missed as not all patients at risk have blood cultures taken for mycology

investigations.

Overall, the preparation of terminal subcultures has proved to be of vital

importance, especially in the detection of yeasts from previously negative bottles

75

following the incubation period. The detection of Aspergilli from immunosuppressed

and neutropenic patients must not be excluded and terminal subcultures should also

be performed in cases where patients have a positive galactomannan antigen.

4.5. Limitations

Unfortunately, this study faced several limitations. One of the main limitations

was the fact that the blood culture bottles were not incubated in the BacT/Alert

automated system in order to detect growth, due to the lack of space in the machine

utilised in the blood cultures laboratory at the Microbiology department of MDH and

to avoid disturbing the workflow of the blood culture laboratory. As a result, the

detection of growth was detected visually and thus lacked accuracy.

The BacT/Alert blood culture system is a continuous-monitoring device

(Weinstein, 1996) where positive cultures are detected upon the production of carbon

dioxide. The system monitors both the initial as well as the increased concentrations

of carbon dioxide (Kocoglu, Bayram & Balct, 2005) and is thus accurate in detecting

the time of growth. Since the bottles were checked every 24 hours, it could be the

case that there was growth beforehand, especially at the highest concentration (2500

c.f.u/10ml).

In order to minimise the effects of this limitation, similar conditions were

applied including the temperature of incubation (35oC), as well as agitation of the

blood culture vials. The automated system provides constant agitation of the bottles

76

and thus, in order to provide the same conditions, the bottles were agitated once a

day. Unfortunately, these could not be constantly agitated.

Early studies show that agitation decreases the detection time of growth

(Hawkins, Peterson de la Maza, 1986) due to an increase in the oxygen concentration

within the medium. However, others argue that if this principle was correct, then

hardly any organisms would grow in anaerobic blood cultures. This was evaluated in

several studies which have shown that automated systems that do not agitate

anaerobic bottles also recover anaerobic microorganisms from anaerobic blood

cultures (Wilson & Weinstein, 1994; Reimer, Wilson & Weinstein, 1997). Thus,

agitation may improve the yield and detection time of positive cultures but lack of

agitation will not affect positivity of growth.

Another important limitation with regards to the visual detection of growth is

the FAN aerobic blood culture bottle. The latter posed a problem as the dark colour

of the medium made it difficult to visually detect the presence of growth. Due to this,

recorded time for growth could have been greater than the actual time as there might

have already been positive growth within the bottle. In studies where the BacT/Alert

FAN aerobic and the BacT/Alert SA media were compared, the detection time of

growth of most organisms was shorter in the FAN aerobic medium (Weinstein et al.,

1995). However, it is important to note that such study did not test for the growth of

moulds such as Aspergillus spp. but was mainly based on bacteria and yeasts. The

new FAN bottles have been recently changed, and are now utilising a resin. The

contents are now clear, therefore growth would be easily visualised.

77

The method utilised in order to prepare the hyphal concentrations was not ideal

for this study. The method for the preparation of hyphal inocula was described by

Sande et al. (2009), where the hyphal clumps were homogenised using a sonicator at

a wavelength of 660nm. Through this method, the amount of hyphae present within

the hyphal clumps can be known (Sande et al., 2009). Unfortunately, a sonicator was

not available at the Microbiology or Mycology laboratories of MDH and this

technique could not be carried out.

The hyphal suspensions were thus prepared in the same way as the conidial

suspensions. Glass beads were used to break the hyphal clumps during vortexing, but

this technique was not successful at doing that. Due to the presence of large hyphal

fragments, it was difficult to break them up. As a result, these fragments were present

and the suspensions prepared were of unequal concentrations. This is the most

probable reason behind the lack of growth observed in most blood culture bottles

inoculated with the hyphal suspensions. However this may shed some light on the

fact that although Aspergillus is angioinvasive and spreads via the blood stream, it is

not found in blood cultures. It could be the case that clumps of hyphae are very few

in the blood stream, for the simple reason that hyphae do not break up easily into

smaller fragments.

During the preparation of the terminal subcultures, a 21G Vacuette® blood

collection needle was utilised. However, when aspirating the culture medium, the

fungal clumps blocked the needle and thus it was difficult to aspirate the medium

into the vacutainer. This could be due to the attachment of the fungus to the Ecosorb

78

particles present within the medium. As a result the process was time consuming.

The bottles required long periods of agitation in order to try and dislodge the fungal

balls, even though most of the time, the fungal clumps remained intact. Ultimately, a

small volume of the medium was aspirated.

4.6. Conclusion

The Galactomannan antigen assay still remains the Gold Standard test in the

diagnosis of Invasive Aspergillosis (IA) and thus cannot replace the detection of

Aspergillus by blood cultures systems. However, it would be ideal if such systems

could be utilised as a diagnostic test. Culture means that the identity of the fungus

can be obtained and material for susceptibility testing is available. From this study

and that by Rosa et al. (2011), it can be concluded that both the BACTEC as well as

the BacT/Alert blood culture systems support the growth of Aspergillus spp.

Most studies have shown that the BacT/Alert is more efficient and detects

organisms at an earlier stage. Since the two systems could not be compared

statistically, due to lack of data presented in the study by Rosa et al. (2011), results

were only compared visually. Also, if the BacT/Alert automated machine was

utilised, it is most likely that the detection time of growth would be shorter than

those obtained in this study. Ideally, a study should be carried out to confirm the

comparison between the BACTEC and BacT/Alert systems solely for the detection

of moulds using the automated system in order to produce more accurate results.

79

In order to confirm whether Aspergillus spp. can be isolated from

immunocompromised hosts with invasive aspergillosis, a study can be carried out

where blood samples are collected from patients already diagnosed with IA,

inoculated in both the SA and FAN blood culture media and incubated for a total of 7

days. This will allow the direct detection of Aspergillus from infected hosts and thus

conclude whether blood culture systems can be useful for the detection of such

species. Since Aspergillus may be found as a few clumps, multiple blood culture

bottles may be needed to culture a relatively large volume of blood. Other studies can

also be carried out in order to compare the time and cost for other methods of

identification including molecular based techniques such as PCR.

The ‘novel’ subculture technique should also be applied in laboratories as it has

been concluded that this newly proposed technique enhances the recovery of fungi

when compared to the routine sub-culture technique. Until further improvements are

made, it is still suggested that clinical laboratories perform terminal subcultures on

negative blood cultures, especially in immunocompromised and neutropenic hosts

where bloodstream infections caused by fungi are suspected (Hovarth et al., 2004).

Due to the increased morbidity and mortality as a consequence of neutropenia

and immunosuppression, it would be ideal if such fungi can be isolated through rapid

and reliable techniques such as blood culture systems. As a result this will provide an

early diagnosis and improve the management of patients with IA as well as

candidaemia. Cultures are important material both for identification of fungi and also

for susceptibility testing if required.

80

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91

APPENDIX

92

Preparation of Culture Media I.

Sabouraud Dextrose Agar

Sixty-five grams of Sabouraud Dextrose Agar were weighed for each litre of

agar prepared. After weighing, the powder was transferred into a clean conical flask

and topped with 1 litre of distilled water. The contents were mixed thoroughly

allowing the powder to dissolve. Each flask was then placed in an autoclave and

sterilized at 121oC for 15 minutes. After allowing the mixture to cool and reach

50oC, 25ml of the medium was poured into sterile Petri dishes. This was carried out

in a laminar flow hood so as to prevent any contamination. After allowing the media

to solidify the plates were labelled with the batch number and expiry date.

Figure I: Media Preparation & Quality Control form showing the materials used as

well as the preparation procedure of Sabouraud Dextrose Agar

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Sabouraud broth

Ten grams of peptone and 40g of glucose were weighed in order to prepare one

litre of Sabouraud broth. A total of 4 litres were prepared, one litre in each separate

flask. The contents were transferred into each flask and mixed well with one litre of

distilled water. The prepared media were then autoclaved at 121oC for 15 minutes.

Figure II: Media Preparation & Quality Control form showing the materials used as

well as the preparation procedure of Sabouraud Broth

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Quality Control of Media II.

Sabouraud Dextrose Agar

The pH of the medium was checked using a pH meter. The intended pH of the

medium is 5.6 ± 0.2. A sterility check was performed by incubating three plates at

30oC for 3 days. Positive controls were prepared from clinical isolates of Candida

albicans (ATCC 102131), Aspergillus niger (ATCC 28191) and Trichophyton

rubrum (ATCC 16404).The plates were inoculated with the organisms and incubated

at 28oC for three days after which growth was observed.

Figure III: Media Preparation & Quality Control form showing the QC procedure

carried out on the prepared Sabouraud Dextrose Agar plates

95

Tables of Results III.

Table I: Results showing number of bottles (out of 3) with positive growth after 7 days

in SA bottles (conidial inoculum)

FAN Concentration (c.f.u/10ml)

2500 250 25 2.5 Negative Control

A. fumigatus 3 3 3 2 0

A. flavus 3 3 2 1 0

A niger 3 2 1 0 0

A. terreus 3 3 2 1 0

Table II: Results showing number of bottles (out of 3) with positive growth after 7 days

in FAN bottles (conidial inoculum)

Cont. = contaminated

SA Concentration (c.f.u/10ml)

2500 250 25 2.5 Negative Control

A. fumigatus 3 3 3 1 0

A. flavus 3 3 1

(1 cont.)

0 1 cont.

A niger 3 3 3 1 1 cont.

A. terreus 3 3 3 1

(1 cont.)

0

96

Table III: Results showing number of bottles (out of 3) with positive growth after 7

days in SA bottles (hyphal inoculum)

FAN Concentration (c.f.u/10ml)

2500 250 25 2.5 Negative Control

A. fumigatus 3 2 3 0 0

A. flavus 2 1 0 0 0

A niger 3 3 0 0 0

A. terreus 3 3 1 1 0

Table IV: Results showing number of bottles (out of 3) with positive growth after 7

days in FAN bottles (hyphal inoculum)

SA Concentration (c.f.u/10ml)

2500 250 25 2.5 Negative Control

A. fumigatus 3 3 1 1 0

A. flavus 3 0 0 0 0

A niger 3 2 0 0 0

A. terreus 3 3 2 0 0

97

Table V: Results of terminal sub-cultures of SA bottles (out of 3) showing number of

plates with positive growth (conidial inoculum)

FAN Concentration (c.f.u/10ml)

2500 250 25 2.5 Negative Control

A. fumigatus 3 3 3 1 0

A. flavus 3 3 3 1 cont. 2 cont.

A niger 3 3 2 0 1 cont.

A. terreus 3 3 1

(1 cont.)

1 cont. 0

Table VI: Results of terminal sub-cultures of FAN bottles showing number of plates

(out of 3) with positive growth (conidial inoculum)

Cont. = contaminated

SA Concentration (c.f.u/10ml)

2500 250 25 2.5 Negative Control

A. fumigatus 3 2 2 1 0

A. flavus 3 3 2 1 1 cont.

A niger 3 3 3 1 1 cont.

A. terreus 3 3 1

(1 cont.)

1

(1 cont.)

0

98

Table VII: Results of terminal sub-cultures of SA bottles showing number of plates (out

of 3) with positive growth (hyphal inoculum)

FAN Concentration (c.f.u/10ml)

2500 250 25 2.5 Negative Control

A. fumigatus 3 3 3 0 0

A. flavus 2 1 0 0 0

A niger 3 3 0 1 0

A. terreus 3 3 2 1 0

Table VIII: Results of terminal sub-cultures of FAN bottles showing number of plates

(out of 3) with positive growth (hyphal inoculum)

SA Concentration (c.f.u/10ml)

2500 250 25 2.5 Negative Control

A. fumigatus 3 2 1 1 0

A. flavus 3 0 0 0 0

A niger 3 2 0 0 0

A. terreus 3 3 2 0 0

99

Automated negative blood culture samples and the results of terminal subcultures

Sample Clinical Details Growth

1 Immunosuppressed; neutropenia NG

2 Immunosuppressed; neutropenia NG

3 Immunosuppressed; neutropenia NG

4 Immunosuppressed; neutropenia NG

5 Immunosuppressed; neutropenia NG

6 Immunosuppressed; neutropenia NG

7 Febrile neutropenia NG

8 Febrile neutropenia NG

9 Neutropenia NG

10 Neutropenia NG

11 Febrile neutropenia NG

12 Neutropenia NG

13 Neutropenia NG

14 Febrile neutropenia NG

15 Neutropenia NG

16 Sepsis on chemotherapy NG

17 Febrile neutropenia NG

18 Febrile neutropenia NG

19 Febrile neutropenia NG

20 Febrile neutropenia NG

21 Lethargy; immunosuppression NG

22 Lethargy; immunosuppression NG

23 Immunosuppression with lymphadenopathy NG

24 Neutropenia NG

25 Febrile neutropenia NG

26 Febrile neutropenia NG

27 Febrile neutropenia NG

28 Febrile neutropenia NG

100

29 Febrile neutropenia NG

30 Febrile neutropenia NG

31 Neutropenia; sepsis NG

32 Neutropenia; sepsis NG

33 Neutropenia; fever NG

34 Neutropenia; fever NG

35 Febrile neutropenia NG

36 Febrile neutropenia NG

37 Immunosuppressed NG

38 Neutropenia; sepsis NG

39 Hypotension in immunocompromised patient NG

40 Hypotension in immunocompromised patient NG

41 Hypotension in immunocompromised patient NG

42 Hypotension in immunocompromised patient NG

43 Neutropenia; fever NG

44 Neutropenia; fever NG

45 Neutropenia; fever NG

46 Neutropenia; fever NG

47 Neutropenia; fever NG

48 Neutropenia; fever NG

49 Neutropenia & sepsis in tALL patient NG

50 Neutropenia & sepsis in tALL patient NG

51 Neutropenia & sepsis in tALL patient NG

52 Neutropenia & sepsis in tALL patient NG

53 Neutropenia & sepsis in tALL patient NG

54 Neutropenia & sepsis in tALL patient NG

55 Febrile neutropenia NG

56 Febrile neutropenia NG

57 Febrile neutropenia NG

58 Febrile neutropenia NG

59 Neutropenia; sore throat NG

101

60 Neutropenia; sore throat NG

61 Febrile neutropenia NG

62 Febrile neutropenia NG

63 Sepsis P

64 Neutropenia; fever NG

65 Neutropenia; fever NG

66 Neutropenia; fever NG

67 Neutropenia; fever NG

68 Neutropenia; fever NG

69 Neutropenia; fever NG

70 Neutropenia; fever NG

71 Neutropenia; fever NG

72 Neutropenia; fever NG

73 Neutropenia; fever NG

74 Neutropenia; fever NG

75 Neutropenia; fever NG

76 Neutropenia; fever NG

77 Neutropenia; fever NG

78 Neutropenia; fever NG

79 Neutropenia; fever NG

80 Neutropenia; fever NG

81 Neutropenia; fever NG

82 Neutropenia; fever NG

83 Neutropenia; fever NG

84 Neutropenia; fever NG

85 Neutropenia; fever NG

86 Neutropenia; fever NG

87 Neutropenia; sepsis NG

88 Neutropenia; sepsis NG

89 Febrile neutropenia NG

90 Febrile neutropenia NG

102

Table IX: Results of terminal subcultures from negative blood cultures

91 Febrile neutropenia NG

92 Febrile neutropenia NG

93 Febrile neutropenia NG

94 Febrile neutropenia NG

95 Febrile neutropenia NG

96 Febrile neutropenia NG

97 Febrile neutropenia NG

98 Febrile neutropenia NG

99 Febrile neutropenia NG

100 Febrile neutropenia NG

101 Febrile neutropenia NG

102 Febrile neutropenia NG

Legend:

NG: No growth

P: Positive growth

tALL: T-cell acute lymphoblastic leukaemia

103

Statistical Analysis IV.

SPSS output results comparing the effect of concentration on time

Ranks

Concentration

in c.f.u/10ml

N Mean Rank

Time in hours

2.5 4 14.13

25 4 9.38

250 4 6.63

2500 4 3.88

Total 16

Test Statisticsa,b

Time in

hours

Chi-Square 11.387

df 3

Asymp. Sig. .010

a. Kruskal Wallis Test

b. Grouping Variable:

Concentration in

c.f.u/10ml

Test Statisticsa,b

Time in

hours

Chi-Square 11.018

df 3

Asymp. Sig. .012

a. Kruskal Wallis Test

b. Grouping Variable:

Concentration in

c.f.u/10ml

Ranks

Concentration

in c.f.u/10ml

N Mean Rank

Time in hours

2.5 4 14.13

25 4 9.38

250 4 6.63

2500 4 3.88

Total 16

104

Test Statisticsa,b

Time in

hours

Chi-Square 5.913

df 3

Asymp. Sig. .116

a. Kruskal Wallis Test

b. Grouping Variable:

Concentration in

c.f.u/10ml

Ranks

Concentration

in c.f.u/10ml

N Mean Rank

Time in hours

2.5 1 10.00

25 2 7.25

250 3 5.50

2500 4 3.50

Total 10

Ranks

Concentration

in c.f.u/10ml

N Mean Rank

Time in hours

25 2 9.25

250 4 5.13

2500 4 4.00

Total 10

Test Statisticsa,b

Time in

hours

Chi-Square 6.281

df 2

Asymp. Sig. .043

a. Kruskal Wallis Test

b. Grouping Variable:

Concentration in

c.f.u/10ml

105

SPSS output results comparing high and low concentrations

Ranks

Concentration

in c.f.u/10ml

N Mean Rank

Time in hours

250 4 5.50

2500 4 3.50

Total 8

Test Statisticsa,b

Time in

hours

Chi-Square 1.750

df 1

Asymp. Sig. .186

a. Kruskal Wallis Test

b. Grouping Variable:

Concentration in

c.f.u/10ml

Test Statisticsa,b

Time in

hours

Chi-Square .467

df 1

Asymp. Sig. .495

a. Kruskal Wallis Test

b. Grouping Variable:

Concentration in

c.f.u/10ml

Ranks

Concentration

in c.f.u/10ml

N Mean Rank

Time in hours

250 4 5.00

2500 4 4.00

Total 8

Test Statisticsa,b

Time in

hours

Chi-Square 1.800

df 1

Asymp. Sig. .180

a. Kruskal Wallis Test

b. Grouping Variable:

Concentration in

c.f.u/10ml

Ranks

Concentration

in c.f.u/10ml

N Mean Rank

Time in hours

250 3 5.00

2500 4 3.25

Total 7

106

Ranks

Concentration

in c.f.u/10ml

N Mean Rank

Time in hours

250 4 5.00

2500 4 4.00

Total 8

Test Statisticsa,b

Time in

hours

Chi-Square 1.000

df 1

Asymp. Sig. .317

a. Kruskal Wallis Test

b. Grouping Variable:

Concentration in

c.f.u/10ml

Ranks

Concentration

in c.f.u/10ml

N Mean Rank

Time in hours

2.5 4 6.13

25 4 2.88

Total 8

Test Statisticsa,b

Time in

hours

Chi-Square 4.225

df 1

Asymp. Sig. .040

a. Kruskal Wallis Test

b. Grouping Variable:

Concentration in

c.f.u/10ml

Ranks

Concentration

in c.f.u/10ml

N Mean Rank

Time in hours

2.5 4 5.38

25 4 3.63

Total 8

Test Statisticsa,b

Time in

hours

Chi-Square 1.175

df 1

Asymp. Sig. .278

a. Kruskal Wallis Test

b. Grouping Variable:

Concentration in

c.f.u/10ml

107

Ranks

Concentration

in c.f.u/10ml

N Mean Rank

Time in hours

2.5 1 3.00

25 2 1.50

Total 3

Test Statisticsa,b

Time in

hours

Chi-Square 1.500

df 1

Asymp. Sig. .221

a. Kruskal Wallis Test

b. Grouping Variable:

Concentration in

c.f.u/10ml

108

Costs V.

Reagents/Materials Cost

Sabouraud Dextrose Agar €60

Standard Aerobic (SA) BacT/Alert blood culture bottles €200

FAN Aerobic BacT/Alert blood culture bottles €250

21G Vacuette®

blood collection needle €210

Total €720

Table X: Reagents and materials utilised in this project and their respective cost

The SA and FAN Aerobic BacT/Alert blood culture bottles as well as the 21G

Vacuette®

blood collection needles were supplied by the Microbiology laboratory of

MDH. The only reagent purchased was the Sabouraud Dextrose Agar. Disposable

equipment including sterile loops, conical-bottomed test tubes and sterile plastic

pipettes were supplied by the Mycology laboratory.

109

Permissions to carry out study VI.

Within this section please find all the permissions required for carrying out this

project.