DIAGNOSIS OF FUNGEMIA USING BLOOD-CULTURE TECHNIQUES

IN IMMUNOSEPRESSED PATIENTS

Dr. Ahmad MoussaMicrobiology Department

Medical Research Institute

Alexandria University

INTRODUCTION:

Fungemia Fungemia (also known as Candidemia, Candedemia, and

Invasive Candidiasis) is the presence of fungi or yeasts in the blood.

Invasive fungal infection is a major cause of morbidity and mortality in immunocompromised patients.

Fungemia is most commonly seen in:

Immunosuppressed patients with severe neutropenia. Patients with intravenous catheters. Oncology patients.

The two most important risk factors are:

Use of broad-spectrum antibiotics. Colonization by fungi (e.g. candidiasis).

Other risk factors:

Dialysis Diabetes Lowered intestinal flora Suppressed Immune system Central venous catheter High severity of illness Chemotherapy Malignancy Multiple abdominal surgeries Use of steroids Surgery and burns Intensive care unit stay.

The most commonly known pathogens involved in fungemia:

Candida albicans, causing roughly 70% of fungemias Candida glabrata causing 10%, and Aspergillus causing 1%.

Candidosis and aspergillosis remain the most significant problems in the United Kingdom.

The precise prevalence of disease is not known, but population-based surveillance estimates it at 12–17 / 100.000 populations.

TYPES OF FUNGEMIA:

A. Invasive Candidiasis:

The term "Invasive Candidiasis" is equivalent to "Disseminated Candidiasis", "Systemic Candidiasis," and "Hematogenous Candidiasis". We preferentially use the first term, but you will see the others used as well.

Candida spp. can invade and cause disease in virtually any organ of the body. However, there are many natural barriers to this form of disease, and it only occurs in individuals who have reduced or altered host defenses.

Invasive Candida infections are most commonly seen in critically ill patients in intensive care units (ICUs) and very low birth weight infants.

Reported mortality in patients with candidaemia ranges from 36% to 63%, although mortality in ICU patients has decreased in recent years, possibly due to more prompt initiation of antifungal therapy.

Epidemiology:

Candidemia has become one of the most important nosocomial infections over the last two decades.

An increment in its frequency of more than 400 times as agent causing bloodstream infections, was noted during the 1980‘s.

During the 1990's Candida spp. were the fourth most common agent causing nosocomial bloodstream infection.

B. Invasive Aspergillosis:

Aspergillosis is the most significant fungal infection in immunosuppressed patients with haematological malignancy, and patients undergoing haematopoietic stem cell transplantation (HSCT).

Despite the increasing incidence, mortality from invasive aspergillosis (IA) has decreased over recent years, due partly to the improvements in diagnosis and more effective antifungal drugs.

DIAGNOSIS OF FUNGEMIA:

The diagnosis is complicated, as routine blood cultures have poor sensitivity. Recently, diagnosis became reliant on traditional culture methods that have improved through the introduction of larger volume lytic-automated culture systems.

Recovering Candida from the blood - While candidemia would seem to be a key element of essentially all cases of invasive candidiasis, retrospective studies have shown that blood cultures are positive in less than 50% of patients with autopsy-proven invasive candidiasis.

The best sensitivity reported to date has been achieved with the lysis-centrifugation method, and even then it was only 58%.

While this technique reduces the time needed for Candida spp. and to allow estimation of the number of colonies forming units of that organism per milliliter of blood, it is quite expensive and labor intensive.

Other techniques with at least equal sensitivity include the BACTEC high-blood-volume fungal media (HBV-FM) and the BacT/Alert system.

TYPES OF BLOOD CULTURE SYSTEMS:

Numerous blood culture systems are available for the optimum recovery of fungi; however all systems must be vented to atmospheric air and incubated at 30 C to maximize the rate and time of recovery of fungal organisms.

Aseptically collect 10 ml of blood and prepare several smears for Giemsa, Gram and PAS staining.

Culture the remaining specimen by one or more of the following methods.

With bone marrow aspirates the initial material is generally used for making smears for Giemsa staining, the remaining 3-5 ml of marrow and blood may be cultured on the media listed in slide number (14).

1. DIRECT CULTURE METHOD:

Inoculate 0.5-1.0 ml of buffy coat, prepared by centrifuging 5-10 ml of blood, onto the surface of the media listed in slide number (14).

This inoculum can then be spread over the surface of the agar with a sterile inoculating loop and the plate incubated aerobically at 30C.

This method is suitable for small low volume laboratories where there are few requests for fungal blood cultures.

Cultures should be maintained for 4 weeks.

2. BIPHASIC CULTURE BOTTLE:

The recovery of fungi from blood may be enhanced by using a biphasic bottle containing a slant of brain heart infusion agar and 60-100 ml of BHI broth.

A ratio of 1:10 to 1:20 (blood to broth) is recommended, a minimum of 5.0 ml of blood is required for each culture bottle.

The biphasic culture bottle is kept vented and is tilted daily to allow broth to flow over the agar surface.

These cultures must be carefully checked daily for growth.

Because fungi will not turn the broth very cloudy it is imperative to frequently Gram stain the bottle contents to detect fungal elements.

Cultures should be incubated at 30C and maintained for 4 weeks.

3. MEMBRANE FILTER TECHNIQUE:

This is a superior technique to the vented biphasic blood bottle used to concentrate and culture specimens of blood and CSF. Briefly, specimens are treated sequentially with Triton-X and sodium carbonate solutions to lyse blood cells and then filtered by vacuum through a 0.45 um membrane. This membrane is then placed onto the media listed below.

*** Primary isolation media for blood and bone marrow culture: 

(a) Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26oC and 35oC; and

(b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35C.

Maintain cultures for 4 weeks.

4. LYSIS CENTRIFUGATION METHOD The Wampole Isolator system has been found to significantly

improve the recovery of fungi from blood and is strongly recommend by Koneman and Roberts (1985) as the method of choice for processing blood cultures from patients with suspected fungal septicemia.

The Isolator utilizes a tube that contains components that lyse leukocytes and erythrocytes and also inactivate plasma complement and certain antibiotics.

Once lysed, the cells release the microorganisms contained within them, and the centrifugation step in the procedure serves to concentrate the organisms in the blood sample. This concentrate is then inoculated onto the surface of appropriate culture media listed below. Ten milliliters of blood are required for each tube and cultures should be incubated at 30 C and maintained for 4 weeks.

The lysis-centrifugation blood culture technique offers some advantages over traditional broth techniques in detecting Candida fungemia.

5. BACTEC AUTOMATED SYSTEM

The BACTEC blood culture system is a fully automated microbiology growth and detection system designed to detect microbial growth from blood specimens.

A selective fungal medium, BACTEC Mycosis IC/F (Becton Dickinson, Sparks, Md.), was developed to achieve optimum recovery of yeasts and fungi from blood.

Once again, blood cells are lysed by the medium to enhance recovery of fungi. Note antimicrobials have also been added to limit the growth of bacteria.

The BACTEC™ FX and the BACTEC™ 9000 family of continuous monitoring blood culturing instruments offer performance, safety, reliability, ease of use, media quality and service.

Continuous Monitoring Technology:

BACTEC 9000 Systems feature the unique BACTEC 9000 fluorescent sensor technology that allows for fully-automated, walk-away testing using a continuous-monitoring instrument that agitates and incubates BACTEC/F blood culture bottles, resulting in earlier detection of positives.

The BACTEC instrument also provides advanced algorithms for individual bottle types, for special circumstances such as low blood volume, pediatric specimens, or to detect slow growing organisms such as Haemophilus and Neisseria. These algorithms provide rapid detection of pathogens in blood culture.

The BACTEC™ 9000 Fluorescent Series is available in connectable modules to accommodate any laboratory’s blood culture test volume:

BACTEC 9240 System accommodates 240 test bottles

BACTEC 9120 System accommodates 120 test bottles. (Up to 5 BACTEC 9240/9120 System instruments can be networked together

through a single PC module, for ease-of-use in large laboratories.)

BACTEC 9050 System accommodates 50 test vials, features an extremely small “foot print” (Only 4 1/4 square feet of tabletop needed — and no

external computer required!). It is suitable for the smaller laboratory: one per forming less than 150 blood cultures per month.

HOW DOES BACT/ALERT- 3D WORK?

Here's how it works:

1. Microorganisms multiply in the media, generating carbon dioxide. As the carbon dioxide increases, the sensor in the bottle turns yellow.

2. Measuring reflected light, the BacT/ALERT 3D monitors and detects color changes in the sensor.

3. Algorithms analyze the data to determine positivity, and the laboratory is notified immediately with visual and audible alarms.

4. Changes in the sensor are permanent and visible to the unaided eye, unlike any other method.

BACTEC™ MYCOSIS-IC/F CULTURE VIALS

Mycosis-IC/F culture vials are for aerobic blood cultures. Principal use is with the BACTEC fluorescent series instruments for the selective culture and recovery of yeasts and fungi from blood.

The sample to be tested is inoculated into one or more vials which are inserted into the BACTEC fluorescent series instrument for incubation and periodic reading. Each vial contains a chemical sensor which can detect increases in CO2 produced by the growth of microorganisms. The sensor is monitored by the instrument every 10 minutes for an increase in its fluorescence, which is proportional to the amount of CO2 present. A positive reading indicates the presumptive presence of viable microorganisms in the vial.

The BACTEC Mycosis-IC/F culture vials contain the following reactive ingredients prior to processing: